Patent Application
20180171015
This application incorporates by reference the contents of a 48.4 kb text file created on Jan. 24, 2018 and named “14033_165_ST25.txt,” which is the sequence listing for this application.
N.A.
The present invention relates to a method of increasing GiPCR signalization in the cells of a scoliotic subject.
Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named 14033_165_ST25.txt, that was created on Jan. 22, 2018 and having a size of 49 kilobytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.
Idiopathic Scoliosis is a spine deformity of unknown cause generally defined as a lateral curvature greater than 10 degrees accompanied by a vertebral rotation1. Adolescent Idiopathic Scoliosis (AIS) is one of the most frequent childhood deformities worldwide, characterized by a 3D spinal deformity with unknown cause, and represents both an immediate medical challenge and a chronic condition affecting individuals throughout their lives. It is the most common orthopedic condition requiring surgery in adolescents and affects 4% of this population. This condition is most commonly diagnosed between the ages of 9 to 13 years2,3,4. The diagnosis is primarily of exclusion and is made only after ruling out other causes of spinal deformity such as vertebral malformation, neuromuscular or syndromic disorders. Traditionally, the truncal asymmetry is revealed by Adams forward bending test and measured with scoliometer during physical examination. The diagnosis can then be confirmed by radiographic observation of the curve and the angle measurement using the Cobb method.
Once diagnosed, the primary concern for physicians in managing scoliotic children is whether the curve will progress. Indeed, the curve progression is often unpredictable and is more frequently observed among girls than in boys. If untreated, the curve can progress dramatically, creating significant physical deformity and even cardiopulmonary problems. These manifestations become life threatening when the curve exceeds 70 degrees. The current treatment options to prevent or stop curve progression include bracing and surgery. In general, bracing is recommended for curves between 25 and 40 degrees, while surgery is reserved for curves greater than 45 degrees or curves that are unresponsive to bracing. Today in the United States there are approximately one million children between ages 10 and 16 with some degree of IS. Approximately, 10% of children diagnosed with idiopathic scoliosis have curve progression requiring corrective surgery. About 29,000 scoliosis surgeries are done every year in North America, resulting in significant psychological and physical morbidity. (Goldberg M S, Mayo N E, Poitras B et al. The Ste-Justine Adolescent Idiopathic Scoliosis Cohort Study. Part I: Description of the study. Spine 1994; 19:1551-61; Poitras B, Mayo N E, Goldberg M S et al. The Ste-Justine Adolescent Idiopathic Scoliosis Cohort Study. Part IV: Surgical correction and back pain. Spine 1994; 19: 1582-8).
Currently, there is no proven method or test available to identify subjects at risk of developing IS to predict which affected individuals require treatment to prevent or stop progression of the disease so that appropriate treatment can be early provided and prevent surgical complications and cardiac and/or respiratory problems. (Weinstein S L, Dolan L A, Cheng J C et al. Adolescent Idiopathic Scoliosis. Lancet 2008; 371:1527-37).
Therefore, the application of current treatments, such as bracing or surgical correction, is delayed until a significant deformity is detected or until a significant progression is clearly demonstrated, resulting in a delayed, less than optimal treatment and often important psychological sequels (Society SR. Morbidity & Mortality Committee Annual Report 1997).
Currently, in order to detect the deformity, diagnosed children are subjected to multiple radiographs over several years, usually until they reach skeletal maturity. It is estimated that the typical patients with scoliosis will have approximately 22 radiological examinations over a 3-year period. There are potential risks in multiple radiographic examinations. For this reason also, alternative approaches that could allow performing the prognosis of idiopathic scoliosis are strongly desirable.
The major limitation in developing prognostic tests that could facilitate treatment choices for patients is the heterogeneous nature of AIS. At the clinical level, the heterogeneity of AIS is clearly illustrated by the variability of curve patterns, localisations and curve magnitude even in families with multiple affected members.
In absence of reliable AIS phenotypes, there is a need to understand better the molecular changes associated with disease onset and spinal deformity progression. Molecular definition of disease is rapidly replacing traditional pathology-based disease descriptions in part because of its utility in identifying the optimal treatment regimen for patients.
To this effect, the existence of a differential melatonin signaling dysfunction was reported among AIS patients leading to their stratification into three functional groups or biological endophenotypes (Moreau et al., 2004); (Azeddine et al., 2007); (Letellier et al., 2008) and WO2003/073102 to Moreau. More particularly, AIS patients were stratified into three functional groups (FG1, FG2 and FG3) representing distinct biological endophenotypes. According to this stratification, scoliotic patients and children more at risk of developing scoliosis are less responsive to Gi protein stimulation when compared with healthy control subjects, and the classification is based on the percentage of degree of reduction relative to the control group. The classification ranges were fixed between about 10 and 40% of reduction of response relative to control group for FG3, between about 40 and 60% for FG2 and between about 60 and 90% of reduction of response relative to control group for FG1.
More recently, using the cellular dielectric spectrometry (CDS) technique, which is a label-free method for the functional evaluation of G proteins and endogenous receptors coupled to those proteins (Verdonk et al., 2006), it was found that the cellular response following melatonin receptor stimulation by melatonin was mainly Gi-dependent in normal osteoblasts and was reduced to different extents in osteoblasts derived from AIS patients (Akoume et al., 2010). Approximately 33% of asymptomatic children diagnosed with a defective Gi protein function have developed scoliosis many years later (Akoume et al., 2010).
Early detection/prognosis of scoliosis is not only critical to successful and less invasive clinical outcomes but broadens the range of treatment options for clinicians. Indeed, improving patients' stratification and disease staging represent key steps to select AIS patients for minimally invasive surgeries before their spinal deformity is too advanced. OPN, a multifunctional cytokine, has been identified as a potentially key pathophysiologic contributor in the development of idiopathic scoliosis. Particularly, increased plasma OPN levels in patients with idiopathic scoliosis and in bipedal mice, a well-established animal model of this disease, were correlated with the disease (see WO 2008/119170 to Moreau).
It is commonly accepted that the development of scoliosis is influenced by a postural mechanism. The bipedal condition, naturally present in humans or experimentally induced in animals seems to play an important role in the manifestation of scoliotic deformities (Machida et al., 1999). Importantly, it has been reported that mice on a C57BI/6 or C3HHe background develop scoliosis closely similar to human idiopathic scoliosis when they gain bipedal posture for 40 weeks following amputation of their forelimbs and tails (Machida et al., 2006); (Oyama et al., 2006). Hence, to address the question of whether OPN contributes to the development of idiopathic scoliosis we took advantage of the availability of OPN-deficient mice on a C57BI/6 background.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present invention shows that OPN reduces GiPCR signaling in vitro mainly via α5β1 integrin engagement. Without being bound by such hypothesis, the mechanism by which OPN interferes with signaling of GiPCR is believed to be a depletion of functional Gi proteins necessary for these receptors through the sequestration of a part of Gi proteins by integrin β1 subunit and the inactivation of the remaining Gi proteins following their phosphorylation by various kinases engaged in the signaling cascade of α5β1 integrin. Furthermore, CD44 levels and/or bioavailability to bind to OPN can modulate the inhibitory effect of OPN on GiPCR signaling. The present invention shows that depletion of CD44 exacerbates the inhibitory effect of OPN and that hyaluronic acid (HA), a known CD44 ligand, further potentiates OPN's effect in a dose-dependent manner. HA exhibits a higher binding affinity than OPN toward CD44. Therefore, HA is competing against OPN for the liaison of CD44; thereby increasing OPN's bioavailability to bind α5β1 integrin and to inhibit GiPCR signaling. Finally, the present invention shows that the presence of a mutation in CD44 further decreases GiPCR signaling in osteoblasts of scoliotic subjects.
Accordingly, the present invention concerns a method of increasing GiPCR signalization in the cells of a subject in need thereof comprising administering to a subject in need thereof an effective amount of: (a) an inhibitor of OPN expression and/or activity; (b) an inhibitor of integrin α5β1; or (c) a combination of (a) and (b), whereby GiPCR signalization is increased in the cells of a subject in need thereof.
In accordance with a related aspect, the present invention concerns a method of treating or preventing scoliosis in a subject in need thereof comprising reducing α5β1 expression or activity (e.g., binding to OPN) in the cells of the subject. In an embodiment, the method comprises administering to the subject an effective amount of an inhibitor of integrin α5β1 expression or activity.
In a specific embodiment, the inhibitor of OPN activity is an OPN antibody. In another specific embodiment, the inhibitor of OPN expression is a siRNA specific to OPN. In another specific embodiment, the inhibitor of α5β1 is (a) an antibody that binds specifically to integrin subunit α5; (b) an antibody that binds specifically to integrin subunits β1; or (c) a combination of (a) and (b). In a specific embodiment, the inhibitor of α5β1 is an siRNA specific to α5 or β1.
In accordance with another aspect of the present invention, there is provided a method of selecting an agent as a potential candidate for the reduction or prevention of GiPCR signaling inhibition induced by OPN comprising contacting a candidate agent with a cell expressing (i) OPN; (ii) integrin a5β1, and/or (iii) CD44/sCD44, wherein (a) when OPN expression or activity is decreased in the presence of the candidate agent as compared to in the absence thereof; (b) when integrin α5β1 expression or activity (e.g., binding to OPN) is decreased in the presence of the candidate agent as compared to in the absence thereof, (c) when sCD44 and/or CD44 expression (level) or binding to OPN is increased in the presence of the candidate agent as compared to in the absence thereof; or (d) any combination of at least two of (a), (b) and (c), the candidate agent is selected.
In accordance with another aspect of the present invention, there is provided a method of selecting an agent as a potential candidate for the reduction or prevention of GiPCR signalization comprising contacting a candidate agent with a cell expressing (a) OPN; and/or (b) integrin α5β1, wherein (a) when OPN expression or activity; and/or (b) when integrin α5/β1 is increased in the presence of the candidate agent as compared to in the absence thereof, the candidate agent is selected.
In accordance with another aspect of the present invention, there is provided (i) an inhibitor of OPN expression or activity; ii) an inhibitor of integrin α5β1 expression or activity; iii) sCD44 or a stimulator of sCD44/CD44 expression; or iv) any combination of at least two of (i) to (iii) for use in increasing GiPCR signalization in cells of a subject in need thereof.
In accordance with another aspect of the present invention, there is provided a use of (i) an inhibitor of OPN expression or activity; ii) an inhibitor of integrin α5β1 expression or activity; iii) sCD44 or a stimulator of sCD44/CD44 expression; or iv) any combination of at least two of (i) to (iii), for the preparation of a medicament for increasing GiPCR signalization in cells of a subject in need thereof.
In accordance with another aspect of the present invention, there is provided a composition for increasing GiPCR signalization in the cells of a subject in need thereof comprising (i) an inhibitor of OPN expression or activity; (ii) an inhibitor of α5β1 expression or activity; (iii) sCD44 or a stimulator of sCD44/CD44 expression; or (iv) any combination of at least two of (i) to (iii).
In a specific embodiment, the subject in need thereof is a subject diagnosed with a scoliosis or at risk of developing a scoliosis.
In accordance with another aspect of the present invention, there is provided a method of increasing GiPCR signalization in the cells of a subject in need thereof comprising administering to the subject an effective amount of an inhibitor of integrin α5β1 expression and/or activity, whereby GiPCR signalization is increased in the cells of the subject.
In a specific embodiment, the method further comprises administering to the subject an effective amount of an inhibitor of OPN expression and/or activity. In another specific embodiment, the inhibitor of OPN activity is an OPN antibody. In another specific embodiment, the inhibitor of OPN expression is a siRNA specific to OPN. In another specific embodiment, the inhibitor of α5β1 activity is (a) an antibody that binds specifically to integrin subunit α5; (b) an antibody that binds specifically to integrin subunit β1; (c) an antibody that binds specifically to integrin subunits α5β1; (d) a molecule that specifically blocks the binding of OPN to α5β1 integrin; or (e) any combination of at least two of (a) to (d). In another specific embodiment, said molecule that specifically blocks the binding of OPN to α5β1 integrin is a RGD peptide or derivative thereof. In another specific embodiment, said molecule that specifically blocks the binding of OPN to α5β1 integrin is a peptide fragment of OPN comprising a RGD motif. In another specific embodiment, the peptide fragment of OPN comprises the amino acid sequence GRGDSWYGLRS (SEQ ID NO: 18). In another specific embodiment, the inhibitor of α5β1 expression is an siRNA specific to α5 or β1. In a specific embodiment, the method further comprises administering to the subject i) an effective amount of sCD44 or a fragment thereof which specifically binds to OPN; or ii) a stimulator of sCD44 and/or CD44 expression.
In accordance with another aspect of the present invention, there is provided a use of an inhibitor of integrin α5β1 expression and/or activity in the preparation of a medicament for inhibiting GiPCR signalization in cells of a subject in need thereof.
In accordance with another aspect of the present invention, there is provided a use of an inhibitor of integrin α5β1 expression and/or activity for inhibiting GiPCR signalization in cells of a subject in need thereof.
In accordance with another aspect of the present invention, there is provided an inhibitor of integrin α5β1 expression and/or activity for use in inhibiting GiPCR signalization in cells of a subject in need thereof.
In a specific embodiment of the use or the inhibitor, said inhibitor of α5β1 activity is (a) an antibody that binds specifically to integrin subunit α5; (b) an antibody that binds specifically to integrin subunit β1; (c) an antibody that binds specifically to integrin subunits α5β1; (d) a molecule that specifically blocks the binding of OPN to α5β1 integrin; or (e) any combination of at least two of (a) to (d). In another specific embodiment of the use or the inhibitor, the molecule that specifically blocks the binding of OPN to α5β1 integrin is a RGD peptide or derivative thereof. In another specific embodiment, the molecule that specifically blocks the binding of OPN to α5β1 integrin is a peptide fragment of OPN comprising a RGD motif. In another specific embodiment, the peptide fragment of OPN comprises the amino acid sequence GRGDSWYGLRS (SEQ ID NO: 18). In another specific embodiment, the inhibitor of integrin α5β1 expression is an siRNA specific to α5 or β1.
In accordance with another aspect of the present invention, there is provided a composition comprising the inhibitor of the present invention, and a pharmaceutical carrier.
In another specific embodiment, the composition is for increasing GiPCR signaling in cells of a subject in need thereof.
In specific embodiments of the method, the use, the inhibitor, or the composition of the present invention, the subject is a subject diagnosed with a scoliosis or at risk of developing a scoliosis. In another specific embodiment, the scoliosis is an idiopathic scoliosis. In another specific embodiment, the scoliosis is adolescent idiopathic scoliosis (AIS).
In accordance with another aspect of the present invention, there is provided a method of determining the risk of developing a scoliosis in a subject comprising: (i) providing a cell sample isolated from the subject; (ii) detecting, in the cell sample from the subject, the presence of at least one copy of a CD44 risk allele which introduces a mutation at amino acid position 230 of CD44 or a marker in linkage disequilibrium therewith; and (iii) determining that the subject is at risk of developing a scoliosis when at least one copy of the risk allele or marker in linkage disequilibrium therewith is detected in the cell sample from the subject.
In a specific embodiment, the risk allele comprises SNP rs1467558 and the mutation changes an isoleucine at position 230 of CD44 to a threonine.
In accordance with another aspect of the present invention, there is provided a method of stratifying a subject having scoliosis (e.g., idiopathic scoliosis such as adolescent idiopathic scoliosis (AIS)) comprising: (i) providing a cell sample isolated from the subject; (ii) detecting, in the cell sample from the subject, the presence of at least one copy of a CD44 risk allele which introduces a mutation at amino acid position 230 of CD44 or a marker in linkage disequilibrium therewith; and (iii) (a) stratifying the subject into a first AIS subclass when at least one copy of the CD44 risk allele or marker in linkage disequilibrium therewith is detected in the cell sample from the subject; or (b) stratifying the subject into a second AIS subclass when the CD44 risk allele is not detected in the cell sample from the subject.
In a specific embodiment, the mutation changes an isoleucine at position 230 of CD44 to a threonine. In another specific embodiment, the at least one copy of the CD44 risk allele consists of two copies of the CD44 risk allele and the subject is homozygote for the mutation. In another specific embodiment, the sample is a nucleic acid sample. In another specific embodiment, said sample is a protein sample.
In accordance with another aspect of the present invention, there is provided a kit for performing the method as defined in any one of claims 24 to 30 comprising: (i) a nucleic acid probe or primer for detecting a CD44 risk allele comprising a mutation in a codon encoding amino acid 230 of CD44; and/or (ii) a protein ligand which specifically detects a mutation at amino acid 230 of CD44.
In a specific embodiment, the nucleic acid probe or primer comprises a nucleic acid sequence which specifically hybridizes to a nucleic acid encoding a threonine at amino acid position 230 of CD44. In another specific embodiment, the protein ligand is an antibody specific for a CD44 protein comprising a threonine at amino acid position 230.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
As used herein the terms “risk of developing scoliosis” refer to a genetic or metabolic predisposition of a subject to develop a scoliosis (i.e. spinal deformity) and/or to develop a more severe scoliosis at a future time (i.e. curve progression). For instance, an increase of the Cobb's angle of a subject (e.g. from 40° to 50°, or from 18° to 25°) is a “development” of scoliosis.
In an embodiment, the above-mentioned subject is a likely candidate for developing a scoliosis, such as idiopathic scoliosis (e.g., Infantile Idiopathic Scoliosis, Juvenile Idiopathic Scoliosis or Adolescent Idiopathic Scoliosis (AIS)). As used herein the terms “likely candidate for developing scoliosis” include subjects (e.g., children) of which at least one parent has a scoliosis (e.g., adolescent idiopathic scoliosis). Among other factors, age (adolescence), gender and other family antecedent are factors that are known to contribute to the risk of developing a scoliosis and are used to a certain degree to assess the risk of developing a scoliosis. In certain subjects, scoliosis develops rapidly over a short period of time to the point of requiring a corrective surgery (often when the deformity reaches a Cobb's angle ≥50°. Current courses of action available from the moment a scoliosis such as AIS is diagnosed (when scoliosis is apparent) include observation (when Cobb's angle is around 10-25°), orthopedic devices (when Cobb's angle is around 25-30°), and surgery (over 45°). A more reliable determination of the risk of progression could enable to 1) select an appropriate diet to remove certain food products identified as contributors to scoliosis; 2) select the best therapeutic agent; and/or 3) select the least invasive available treatment such as postural exercises, orthopedic device, or less invasive surgeries or surgeries without fusions (a surgery that does not fuse vertebra and preserves column mobility). The present invention encompasses selecting the most efficient and least invasive known preventive actions or treatments in view of the determined risk of developing scoliosis.
As used herein the term “subject” is meant to refer to any mammal including human, mouse, rat, dog, chicken, cat, pig, monkey, horse, etc. In a particular embodiment, it refers to a human.
A “subject in need thereof” or a “patient” in the context of the present invention is intended to include any subject that will benefit or that is likely to benefit from the modulation (increase or decrease) of OPN's effect on GiPCR signal transduction. In an embodiment, the subject in need thereof is a subject that will benefit or is likely to benefit from an increase in GiPCR signal transduction and thus from (a) an antagonist (inhibitor) of OPN expression and/or activity; (b) an antagonist (inhibitor) of α5 (e.g., Gene ID 3678, NP_002196) and/or β1 expression and/or activity or (c) an increase in sCD44 or CD44 expression. In a specific embodiment, the subject in need thereof is a subject having high levels of OPN as compared to a control subject. In a specific embodiment, the high level of OPN corresponds to a level ≥ to about 600 ng/ml, ≥ to about 700 ng/ml, ≥ to about 800 ng/ml, ≥ to about 900 ng/ml or ≥ to about 1000 ng/ml of OPN as measured in a blood sample from the subject. In an embodiment, a subject in need thereof is a subject diagnosed with a scoliosis (e.g., AIS). In another embodiment, the subject in need thereof is a subject is likely to develop a scoliosis (e.g., AIS).
As used herein the terminology “biological sample” refers to any solid or liquid sample isolated from a living being. In a particular embodiment, it refers to any solid or liquid sample isolated from a human. Without being so limited it includes a biopsy material, blood, tears, saliva, maternal milk, synovial fluid, urine, ear fluid, amniotic fluid and cerebrospinal fluid. In a specific embodiment it refers to a blood sample.
As used herein the terminology “blood sample” is meant to refer to blood, plasma or serum.
As used herein the terminology “control sample” is meant to refer to a sample that does not come from a subject known to have scoliosis or known to be a likely candidate for developing a scoliosis. In methods for determining the risk of developing scoliosis in a subject that is pre-diagnosed with scoliosis, the sample may however also come from the subject under scrutiny at an earlier stage of the disease or disorder. In a specific embodiment, the control sample can come from another subject diagnosed with scoliosis and belonging to the same functional group (e.g., FG1, FG2 or FG3) at an earlier (or later stage) of the disease or disorder.
As used herein the terminology “control” is meant to encompass “control sample”. In certain embodiments, the term “control” also refers to the average or median value obtained following determination of GiPCR signalization in a plurality of samples (e.g., samples obtained from several subjects not known to have scoliosis and not known to be a likely candidate for developing scoliosis).
As used herein the term “treating” or “treatment” in reference to scoliosis is meant to refer to at least one of a reduction of Cobb's angle in a preexisting spinal deformity, improvement of column mobility, preservation/maintenance of column mobility, improvement of equilibrium and balance in a specific plan; maintenance/preservation of equilibrium and balance in a specific plan; improvement of functionality in a specific plan, preservation/maintenance of functionality in a specific plan, cosmetic improvement, and combinations of any of the above.
As used herein the term “preventing” or “prevention” in reference to scoliosis is meant to refer to a at least one of a reduction in the progression of a Cobb's angle in a patient having a scoliosis or in an asymptomatic patient, a complete prevention of apparition of a spinal deformity, including changes affecting the rib cage and pelvis in 3D, and a combination of any of the above.
The terms “suppressor” “inhibitor” and “antagonist” are well known in the art and are used herein interchangeably. They include intracellular as well as extracellular inhibitors.
The terms “inhibitor of OPN” or “OPN antagonist” include any compound able to negatively affect (i.e., reduce totally or partially) the expression and/or activity of OPN (e.g., Gene ID 6696, NP_001035147.1 (SEQ ID NO: 25) and NM_001040058 (SEQ ID NO:26)) in cells. In a specific embodiment, the activity of OPN in cells is a reduction in GiPCR signaling. Similarly, the terms “inhibitor of α5β1”, “inhibitor of α5” or “inhibitor of β1” and the like include any compound able to negatively affect the expression and/or activity of α5 (e.g., Gene ID 3678, NP_002196 (SEQ ID NO:23) and NM_002205.2 (SEQ ID NO: 24)) and/or β1 (Gene ID 3688, NP_002202 (SEQ ID NO: 21) and NM_002211.3 (SEQ ID NO:22)) in cells. In a particular embodiment, the “activity” of α5 and/or β1 in cells is the transduction of the signal leading to the OPN-dependent inhibition of GiPCR signaling.
The terms “inhibitor of OPN expression” or “inhibitor of α5β1 expression” include any compound able to negatively affect OPN's, α5's and/or β1's expression (i.e., at the transcriptional and/or translational level) i.e. the level of OPN/α5 or β1 mRNA and/or protein or the stability of the protein. Without being so limited, such inhibitors include RNA interference agents (siRNA, shRNA, miRNA), antisense molecules, and ribozymes. Such RNA interference agents are design to specifically hybridize with their target nucleic acid under suitable conditions and are thus substantially complementary their target nucleic acid.
The terms “inhibitor of OPN activity” or “inhibitor of α5β1 activity” refers to any molecules that is able to reduce or block the effect of OPN or α5β1 on Gi-mediated signaling. These molecules increase GiPCR signaling in cells by blocking/reducing totally or partially the inhibitory effect induced by OPN and/or α5β1 activity. Non-limiting examples of inhibitors of OPN's activity include proteins (e.g., dominant negative, inactive variants), peptides, small molecules, anti-OPN antibodies (neutralizing antibodies), antibody fragments, inactive fragments of α5 and/or β1 integrins etc. Non-limiting examples of inhibitors of α5β1 activity include proteins (e.g., dominant negative, inactive variants), peptides (RGD peptides or RGD peptide-derivatives), small molecules, anti α5 and/or β1 antibodies (e.g., neutralizing antibodies such as Volociximab™ M200), antibody fragments, etc. In an embodiment, the RGD peptide is a peptide fragment of OPN comprising a RGD motif comprising the amino acid sequence GRGDSWYGLRS corresponding to amino acid 158 to 169 of OPN (SEQ ID NO:18). In an embodiment, the OPN fragment comprising the RGD motif comprises amino acids 158 to 162, 158 to 165, 158 to 167, 158 to 170, 158 to 175, 158 to 180, 158 to 185, 158 to 190, 158 to 195, or 158 to 200 of OPN (e.g., SEQ ID NO: 25). In an embodiment, peptide fragment of OPN comprising a RGD motif comprises amino acids 158 to 161, 156 to 161, 154 to 161, 152 to 162, 150 to 162, 148 to 162, 146 to 162, 144 to 162, 140 to 162, 159 to 163, 159 to 164, 159 to 162, 159 to 166, 159 to 167, or 159 to 169 of OPN (e.g., SEQ ID NO: 25).
In an embodiment, the “inhibitor of OPN's activity” is a neutralizing antibody directed against (or specifically binding to) a human OPN polypeptide which inhibits its binding to α5β1 (i.e. binding to α5 and/or β1 integrin) In an embodiment, the “inhibitor of α5β1 activity” is a neutralizing antibody directed against (or specifically binding to) a human α5β1 (α5 and/or β1) polypeptide which inhibits the binding of OPN to α5β1 (i.e. binding to α5 and/or β1 integrin). In an embodiment, the antibody binds to the RGD domain of OPN. In an embodiment, the antibody is directed against amino acids 159 to 162, 158 to 162, 158 to 165, 158 to 167, 158 to 170, 158 to 175, 158 to 180, 158 to 185, 158 to 190, 158 to 195, or 158 to 200 of OPN (e.g., SEQ ID NO: 25). In an embodiment, the antibody is directed against amino acids 158 to 161, 156 to 161, 154 to 161, 152 to 162, 150 to 162, 148 to 162, 146 to 162, 144 to 162, 140 to 162, 159 to 163, 159 to 164, 159 to 162, 159 to 166, 159 to 167, or 159 to 169 of OPN (e.g., SEQ ID NO: 25).
The term “stimulator” or “enhancer” of sCD44/CD44 expression (e.g., Gene ID 960, NM_000610 (SEQ ID NO: 19), NP_000601.3, (SEQ ID NO: 20)) refers to an agent able to increase the level or expression of CD44, an agent able to increase CD44 secretion. In an embodiment, the stimulator of sCD44/CD44 is an agent able to increase CD44 affinity toward OPN. Without being so limited, the agent can be a protein, a peptide, a small molecule or a nucleotide.
One possible way of decreasing OPN's effect on GiPCR-signaling is to reduce OPN's interaction with the α5β1 integrin by, for example, increasing or favoring OPN's binding to CD44. Hyaluronic acid (HA) is known to bind to CD44. As shown herein HA potentiates the effect of OPN, probably by increasing OPN's bioavailability to α5β1 (e.g., by sequestering CD44 which is no longer available for binding to OPN). Accordingly, subjects which will benefit from OPN inhibition and increase in GiPCR-signalization should not take HA supplements and/or should decrease their food intake of HA by for example avoiding or decreasing his/her consumption of foods that are particularly rich in HA or that are known to increase HA synthesis. Non-limiting examples of such food include meat and meat organs (e.g., veal, lamb, beef and gizzards, livers, hearts and kidneys), fish, poultry (including meat fish and poultry broths), soy (including soy milk), root vegetables containing starch including potatoes and sweet potatoes. Sweet potatoes particularly rich in HA include satoimo and imoji, two Japanese sweet potatoes.
The present invention also relates to methods for the determination of the level of expression (i.e. transcript (RNA) or translation product (protein)) of OPN, α5β1 integrin and/or CD44. As used herein the term α5β1 is used herein to refer to α5β1 integrin. In specific embodiments, it also includes a method that comprises the determination of the level of expression of one or more other scoliosis markers (see for example WO 2008/119170 to Moreau). The present invention therefore encompasses any known method for such determination including ELISA (Enzyme Linked Immunosorbent Assay), RIA (Radioimmunoassay), immunofluorescence, real time PCR and competitive PCR, Northern blots, nuclease protection, plaque hybridization and slot blots.
The present invention also concerns isolated nucleic acid molecules including probes and primers to detect OPN, α5β1 and sCD44/CD44 (and optionally other scoliosis markers). In specific embodiments, the isolated nucleic acid molecules have no more than 300, or no more than 200, or no more than 100, or no more than 90, or no more than 80, or no more than 70, or no more than 60, or no more than 50, or no more than 40 or no more than 30 nucleotides. In specific embodiments, the isolated nucleic acid molecules have at least 17, or at least 18, or at least 19, or at least 20, or at least 30, or at least 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 300 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 200 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 100 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 90 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 80 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 70 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 60 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 50 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 17 and no more than 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 30 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 17 and no more than 30 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 300 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 200 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 100 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 90 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 80 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 70 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 60 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 50 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 40 nucleotides. It should be understood that in real-time PCR, primers also constitute probe without the traditional meaning of this term. Primers or probes appropriate to detect OPN and/or α5β1 in the methods of the present invention can be designed with known methods using sequences distributed across their respective nucleotide sequence. The probes and/or primers of the present invention are designed to specifically hybridize with their target nucleic acid (α5 (SEQ ID NO:24), β1 (SEQ ID NO:22), CD44 wild type (SEQ ID NO:19) or CD44 risk allele (e.g., comprising 230I→T (SNP (CT)) variant) and/or OPN (SEQ ID NO:26)). In an embodiment, the primers and probes of the present invention are substantially complementary to their target nucleic acid.
Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or at least 99%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. 1990 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Modified sugar-phosphate backbones are generally known. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.
As used herein the terms “detectably labeled” refer to a marking of a probe or an antibody in accordance with the presence invention that will allow the detection of OPN and/or α5β1 in accordance with the present invention. Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well-known methods. Non-limiting examples of labels include 3H, 14C, 32P, and 35S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5′ ends of the probes using gamma 32P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (e.g. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
The present invention also relates to methods of selecting compounds. As used herein the term “compound” is meant to encompass natural, synthetic or semi-synthetic compounds, including without being so limited chemicals, macromolecules, cell or tissue extracts (from plants or animals), nucleic acid molecules, peptides, antibodies and proteins.
The present invention also relates to arrays. As used herein, an “array” is an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, e.g., libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.
As used herein “array of nucleic acid molecules” is an intentionally created collection of nucleic acids which can be prepared either synthetically or biosynthetically in a variety of different formats (e.g., libraries of soluble molecules; and libraries of oligonucleotides tethered to resin beads, silica chips, or other solid supports). Additionally, the term “array” is meant to include those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (e.g., from 1 to about 1000 nucleotide monomers in length) onto a substrate. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Thus, the terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include analogs such as those described herein. These analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleotide sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution. Typically, these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. The changes can be tailor made to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.
As used herein “solid support”, “support”, and “substrate” are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations.
Any known nucleic acid arrays can be used in accordance with the present invention. For instance, such arrays include those based on short or longer oligonucleotide probes as well as cDNAs or polymerase chain reaction (PCR) products. Other methods include serial analysis of gene expression (SAGE), differential display, as well as subtractive hybridization methods, differential screening (DS), RNA arbitrarily primer (RAP)-PCR, restriction endonucleolytic analysis of differentially expressed sequences (READS), amplified restriction fragment-length polymorphisms (AFLP).
The present invention encompasses using antibodies for detecting or determining OPN and/or α5β1 levels for instance in the samples of a subject and for including in kits of the present invention. Antibodies that specifically bind to these biological markers can be produced routinely with methods further described below. The present invention also encompasses using antibodies commercially available. Without being so limited antibodies that specifically bind to OPN and/or α5β1 include those listed in Table 1 below.
Both monoclonal and polyclonal antibodies directed to OPN, α5 and/or β1 are included within the scope of this invention as they can be produced by well established procedures known to those of skill in the art. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.
As used herein, the terms “anti-OPN antibody”, anti-α5 antibody“, “anti-β1 antibody” and “anti-α5β1 antibody” or “immunologically specific anti-OPN antibody”, immunologically specific anti-α5 antibody“, immunologically specific anti-β1 antibody” or “immunologically specific anti-α5β1 antibody” refers to an antibody that specifically binds to (interacts with) an OPN, α5, β1 or α5β1 protein, respectively and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the OPN, α5, β1 or α5β1 protein, respectively. The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies™, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.
In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology”, Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody A Laboratory Manual, CSH Laboratories). The term antibody encompasses herein polyclonal, monoclonal antibodies and antibody variants such as single-chain antibodies, humanized antibodies, chimeric antibodies and immunologically active fragments of antibodies (e.g. Fab and Fab′ fragments) which inhibit or neutralize their respective interaction domains in Hyphen and/or are specific thereto.
Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injections of the relevant antigen with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1 are different alkyl groups.
Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with ⅕ to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293.
In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized (e.g., as hereinabove described) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
As used herein, the term “purified” in the expression “purified antibody” is simply meant to distinguish man-made antibody from an antibody that may naturally be produced by an animal against its own antigens. Hence, raw serum and hybridoma culture medium containing anti-OPN antibody are “purified antibodies” within the meaning of the present invention.
The present invention also encompasses arrays to detect and/or quantify the translation products of OPN and α5β1. Such arrays include protein micro- or macroarrays, gel technologies including high-resolution 2D-gel methodologies, possibly coupled with mass spectrometry imaging system at the cellular level such as microscopy combined with a fluorescent labeling system.
The present invention also encompasses methods to screen/select for potential useful therapeutic agents using whole cells assays, the therapeutic compound being able to increase the transcription and/or synthesis of OPN and/or α5 and/or β1 or to decrease the transcription or synthesis of CD44/sCD44. Cells for use in such methods includes cells of any source (including in house or commercially available cell lines) and type (any tissue). In house cell lines could be made for instance by immortalizing cells from AIS subjects. In specific embodiments, methods of screening of the invention seek to identify agents that inhibit OPN and/or α5β1 expression and/or activity. Useful cell lines for these embodiments include those producing high levels of OPN and/or α5β1 or low levels of CD44/sCD44Non-limiting examples of useful cells include MC3T3-E1 cells and PBMCs.
In a particular embodiment, it includes cells of any cell type derived from a scoliotic patient. (whole cell assay). In specific embodiments, it includes osteoblasts, chondrocytes, myoblasts or blood cells including PBMCs including lymphocytes. As used herein, the term “cell derived from a scoliotic patient” refers to cells isolated directly from scoliotic patients, or immortalized cell lines originating from cells isolated directly from scoliotic patients. In specific embodiments, the cells are paraspinal muscle cells. Such cells may be isolated by a subject through needle biopsies for instance.
The present invention also concerns pharmaceutical compositions for modulating OPN-mediated GiPCR cell signalling and/or for treating or preventing scoliosis in a subject in need thereof (e.g., idiopathic scoliosis or AIS). Such compositions include agents for increasing or decreasing OPN-mediated GiPCR signalling inhibition in a subject in need thereof. For instance, pharmaceutical compositions of the present invention may comprise OPN, α5, β1, and/or α5β1 antibodies (i.e., neutralizing antibodies), RGD peptides or antisense/siRNAs against OPN, α5, β1, and/or α5β1 to decrease OPN and/or α5β1 activity. In an embodiment, the pharmaceutical compositions may comprise sCD44 or a stimulator/enhancer of sCD44/CD44 expression. Pharmaceutical compositions can be administered by any suitable routes such as nasally, intravenously, intramuscularly, subcutaneously, sublingually, intrathecally, or intradermally. The route of administration can depend on a variety of factors, such as the environment and therapeutic goals.
Any suitable amount of a pharmaceutical composition can be administered to a subject. The dosages will depend on many factors including the mode of administration. Typically, the amount of anti-scoliosis composition (e.g., agent that decreases OPN and/or α5β1) contained within a single dose will be an amount that effectively prevents, delays or reduces scoliosis without inducing significant toxicity “therapeutically effective amount”.
The effective amount of the agent that decreases OPN and/or α5β1 may also be measured directly. The effective amount may be given daily or weekly or fractions thereof. Typically, a pharmaceutical and/or nutraceutical and/or dietary supplement composition of the invention can be administered in an amount from about 0.001 mg up to about 500 mg per kg of body weight per day (e.g., 10 mg, 50 mg, 100 mg, or 250 mg). Dosages may be provided in either a single or multiple dosage regimen. For example, in some embodiments the effective amount is a dose that ranges from about 1 mg to about 25 grams of the anti-scoliosis preparation per day, about 50 mg to about 10 grams of the anti-scoliosis preparation per day, from about 100 mg to about 5 grams of the anti-scoliosis preparation per day, about 1 gram of the anti-scoliosis preparation per day, about 1 mg to about 25 grams of the anti-scoliosis preparation per week, about 50 mg to about 10 grams of the anti-scoliosis preparation per week, about 100 mg to about 5 grams of the anti-scoliosis preparation every other day, and about 1 gram of the anti-scoliosis preparation once a week.
By way of example, a pharmaceutical (e.g., containing an agent that decreases OPN and/or α5β1) composition of the invention can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream, nebulae, mist, atomized vapor, aerosol, or phytosome. For oral administration, tablets or capsules can be prepared by conventional means with at least one pharmaceutically acceptable excipient such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Preparations for oral administration also can be suitably formulated to give controlled release of the active ingredients.
In addition, a pharmaceutical (e.g., containing an agent that decreases OPN and/or α5β1) composition of the invention can contain a pharmaceutically acceptable carrier for administration to a mammal, including, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Pharmaceutically acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other known carriers appropriate to specific routes of administration.
An agent that decreases OPN and/or α5 and/or β1 expression or activity or an agent that increases sCD44/CD44 level (e.g., binding to OPN) may be incorporated into dosage forms in conjunction with any of the vehicles which are commonly employed in pharmaceutical preparations, e.g. talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives or glycols. Emulsions such as those described in U.S. Pat. No. 5,434,183, may also be used in which vegetable oil (e.g., soybean oil or safflower oil), emulsifying agent (e.g., egg yolk phospholipid) and water are combined with glycerol. Methods for preparing appropriate formulations are well known in the art (see e.g., Remington's Pharmaceutical Sciences, 16th Ed., 1980, A. Oslo Ed., Easton, Pa.).
In cases where parenteral administration is elected as the route of administration, preparations containing agent that decreases OPN and/or α5β1 may be provided to patients in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like.
These are simply guidelines since the actual dose must be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient or by a nutritionist. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient and other clinically relevant factors. In addition, patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that the agent that decreases OPN and/or α5β1 is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.
The present invention also relates to the detection of a CD44 risk allele (e.g., comprising one or more SNPs) in a subject in need thereof. In accordance with the present invention, the CD44 risk allele comprises a SNP located in the coding regions of CD44 and can thus be detected at the genomic, mRNA or protein level. SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species (e.g., humans). A SNP is a single base pair mutation at a specific locus, usually consisting of two alleles (where the rare allele frequency is >1%). SNP genotyping can be performed using methods well-known in the art such as sequencing (e.g., minisequencing, pyrosequencing), hybridization-based methods (using probes complementary to the SNP site which are hybridized under high stringency conditions), enzyme-based methods, Single strand conformation polymorphism (SSCP), Temperature gradient gel electrophoresis, Denaturing high performance liquid chromatography, High-resolution melting of the entire amplicon, using DNA mismatch-binding proteins, or the SNPlex™ platform. Non-limiting examples of hybridization-based methods include Dynamic allele-specific hybridization and use of Molecular beacons. Non-limiting examples of enzyme-based methods include, Restriction fragment length polymorphism (RFLP), PCR-based methods (e.g., ARMS), Flap endonuclease (e.g., Invader), primer extension, 5′-nuclease (TaqMan™ assay), oligonucleotide ligation assays.
In an embodiment, methods of the present invention comprise methods for i) determining the risk of developing a scoliosis; and ii) methods of stratifying subjects having scoliosis comprising determining the presence of a CD44 risk allele comprising SNP rs1467558 or a mutation/SNP/marker in linkage disequilibrium therewith.
In particular embodiments of the invention, linkage disequilibrium (LD) is defined by a specific quantitative cutoff. As described in detail herein, linkage disequilibrium can be quantitatively determined by measures such as r2 and |D′|. As a consequence, certain embodiments of the invention relate to substitute markers in linkage disequilibrium by a measure within a certain range specified by particular values of r2 and/or |D′|. In an embodiment, LD is characterized by numerical values for r2 of greater than 0.1. In another embodiment, LD is characterized by numerical values for r2 of greater than 0.5. In another embodiment, LD is characterized by numerical values for r2 of greater than 0.8. In another embodiment, LD is characterized by numerical values for r2 of 0.9 or more.
The natural phenomenon of recombination, which occurs on average once for each chromosomal pair during each meiotic event, represents one way in which nature provides variations in sequence (and biological function by consequence). It has been discovered that recombination does not occur randomly in the genome; rather, there are large variations in the frequency of recombination rates, resulting in small regions of high recombination frequency (also called recombination hotspots) and larger regions of low recombination frequency, which are commonly referred to as Linkage Disequilibrium (LD) blocks (Myers, S. et al., Biochem Soc Trans 34:526-530 (2006); Jeffreys, A J., et al., Nature Genet 29:217-222 (2001); May, C. A., et al., Nature Genet 31:272-275(2002)).
Linkage Disequilibrium (LD) refers to a non-random assortment of two genetic elements. Thus, the term “linkage disequilibrium” refers to a non-random genetic association between one or more allele(s) of two different polymorphic DNA sequences, that is due to the physical proximity of the two loci. Linkage disequilibrium is present when two DNA segments that are very close to each other on a given chromosome will tend to remain unseparated for several generations with the consequence that alleles of a DNA polymorphism (or marker) in one segment will show a non-random association with the alleles of a different DNA polymorphism (or marker) located in the other DNA segment nearby. This situation is encountered throughout the human genome when two DNA polymorphisms that are very close to each other are studied. Linkage disequilibrium and the use thereof in inheritance studies is well known in the art to which the present invention pertains as exemplified by publications such as Risch and Merikangas, Science 273: 1516-1517 (1996); Maniatis, Methods Mol Biol. 376: 109-21 (2007) and Borecki et al., Adv Genet 60: 51-74 (2008).
For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.25 (25%) and another element occurs at a frequency of 0.25 (25%), then the predicted occurrence of a person's having both elements is 0.125 (12.5%), assuming a random distribution of the elements. However, if it is discovered that the two elements occur together at a frequency higher than 0.125, then the elements are said to be in linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence (e.g., allele or haplotype frequencies) would predict. Roughly speaking, LD is generally correlated with the frequency of recombination events between the two elements. Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurrence of each allele or haplotype in the population.
Many different measures have been proposed for assessing the strength of linkage disequilibrium (LD). Most capture the strength of association between pairs of biallelic sites. Two important pairwise measures of LD are r2 (sometimes denoted Δ) and |D′|. Both measures range from 0 (no disequilibrium) to 1 (‘complete’ disequilibrium), but their interpretation is slightly different. |D′| is defined in such a way that it is equal to 1 if just two or three of the possible haplotypes are present, and it is <1 if all four possible haplotypes are present. Therefore, a value of |D′| that is <1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause |D′| to be <1, but for single nucleotide polymorphisms (SNPs) this is usually regarded as being less likely than recombination). The measure r2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present.
The r2 measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r2 and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model).
One approach of measuring LD across a region is to use the measure r, which was developed in population genetics. Roughly speaking, r measures how much recombination would be required under a particular population model to generate the LD that is seen in the data. This type of method can potentially also provide a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots. For the methods and procedures described herein, a significant r2 value can be at least 0.1 such as at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1.0. In one preferred embodiment, the significant r2 value can be at least 0.8. Alternatively, linkage disequilibrium as described herein, refers to linkage disequilibrium characterized by values of |D′| of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98 or 0.99. Thus, linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or |D′| (r2 up to 1.0 and |D′| up to 1.0). Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population. In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap™ populations (Caucasian, African, Japanese, Chinese), as defined (http://www.hapmap.org). In one such embodiment, LD is determined in the CEU population of the HapMap™ III samples.
If all polymorphisms in the genome were identical at the population level, then every single one of them would need to be investigated in association studies. However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of polymorphisms that need to be investigated in an association study to observe a significant association. Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes (Risch, N. and Merkiangas, K, Science 273:1516-1517 (1996); Maniatis, N., et al., Proc Natl Acad Sci USA 99:2228-2233 (2002); Reich, D E et al, Nature 411:199-204 (2001)).
It is now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating recombination (see, e.g., Wall., J. D. and Pritchard, J. K., Nature Reviews Genetics 4:587-597 (2003); Daly, M. et al., Nature Genet. 29:229-232 (2001); Gabriel, S. B. et al., Science 296:2225-2229 (2002); Patil, N. et al., Science 294: 1719-1723 (2001); Dawson, E. et al., Nature 4JS:544-548 (2002); Phillips, M. S. et al., Nature Genet 33:382-387 (2003)).
There are two main methods for defining these haplotype blocks: Blocks can be defined as regions of DNA that have limited haplotype diversity (see, e.g., Daly, M. et al., Nature Genet. 29:229-232 (2001); Patil, N. et al., Science 294: 1719-1723 (2001); Dawson, E. et al., Nature 418:544-548 (2002); Zhang, K. et al., Proc. Natl. Acad. Sci. USA 99:7335-7339 (2002)), or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium (see, e.g., Gabriel, S. B. et al., Science 296:2225-2229 (2002); Phillips, M. S. et al., Nature Genet. 33:382-387 (2003); Wang, N. et al., Am. J. Hum. Genet. 7 :1227-1234 (2002); Stumpf, M. P., and Goldstein, D. B., Curr. Biol. 13: 1-8 (2003)). More recently, a fine-scale map of recombination rates and corresponding hotspots across the human genome has been generated (Myers, S., et al., Science 310:321-32324 (2005); Myers, S. et al., Biochem Soc Trans 34:526530 (2006)). The map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD. The map can therefore be used to define haplotype blocks/LD blocks as regions flanked by recombination hotspots. As used herein, the terms “haplotype block” or “LD block” includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions.
Haplotype blocks can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers.
The main haplotypes can be identified in each haplotype block, and then a set of “tagging” SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified. These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. If desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks. Thus, the skilled person can readily identify SNPs/variants/markers in linkage disequilibrium with the CD44 risk allele identified herein and can use this marker in linkage disequilibrium to stratify subjects and to determine risk of developing scoliosis.
When the SNP is translated at the protein level, the mutation/variation can be detected using well-known protein detection methods such as ELISA, immunofluorescence, Western blot, sequencing, electrophoresis, HPLC, MS, etc.
The present invention also relates to kits. Without being so limited, it relates to kits for i) increasing GiPCR signaling in cells; ii) stratifying scoliotic subjects, and/or iii) predicting whether a subject is at risk of developing a scoliosis comprising an isolated nucleic acid (e.g., a primer or probe specific for (which is substantially complementary to or hybridizes to α5 (SEQ ID NO:24), β1 (SEQ ID NO:22), CD44 (wild type (SEQ ID NO:19) or SNP risk allele (e.g., 230|→T (CT) variant) and/or OPN (SEQ ID NO:26)) a protein or a ligand such as an antibody (for α5, β1, CD44 (wild type or SNP risk allele (e.g., 230|→T variant) and/or OPN) in accordance with the present invention as described above. For example, a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the subject sample (DNA genomic nucleic acid, cell sample or blood samples), a container which contains in some kits of the present invention, the probes used in the methods of the present invention, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products. Kits of the present invention may also contain instructions to use these probes and or antibodies to stratify scoliotic subjects or predict whether a subject is at risk of developing a scoliosis.
The articles “a,” “an” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
The term “including” and “comprising” are used herein to mean, and re used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”.
The terms “such as” are used herein to mean, and is used interchangeably with, the phrase “such as but not limited to”.
The present invention is illustrated in further details by the following non-limiting examples.
The Institutional Review Board for the care and handling of animals used in research (CHU Sainte-Justine) has approved the protocol in accordance with the guidelines of the Canadian Council of Animal Care.
Breeding pairs of C57BI/6j mice devoid of either OPN (OPN-null mice) or CD44 (CD44-null mice) were obtained from Dr. Susan Rittling (Rutger University, N.J., USA) and were backcrossed for more than 10 generations in C57BI/6j background to establish our own colonies, while C57BI/6j mice were used as wild-type control mice (Charles-River, Wilmington, Mass., USA). C57BI/6j mouse strain was used because it is naturally deficient in melatonin (Von Gall et al., 2000) and exhibits high circulating OPN levels (Aherrahrou et al., 2004). These mice develop scoliosis when they are maintained in a bipedal state. (Machida et al., 2006). Bipedal surgeries were performed after weaning (5-weeks after birth) by amputation of the forelimbs and tail under anesthesia as reported previously. (Machida et al., 2006). Similarly, bipedal C57BI/6j CD44-null mice were generated.
Bone specimens from mice were obtained from the spine after euthanasia. Bone fragments were reduced to smaller pieces with a cutter in sterile conditions. The small bone pieces were incubated in αMEM medium containing 10% fetal bovine serum (FBS; certified FBS, Invitrogen, Burlington, ON, Canada) and 1% penicillin/streptomycin (Invitrogen) at 37° C. in 5% CO2, in a 10-cm2 culture dish. After one month, osteoblasts emerging from the bone pieces were separated from the remaining bone fragments by trypsinization. RNA was extracted from the osteoblasts using the TRIzol™ method, (Invitrogen). Expression profiles were studied by qPCR. Transcript expression was assessed with the Stratagene™ Mx3000P (Agilent Technologies, La Jolla, Calif.).
The functional evaluation of Gi, Gs and Gq proteins was assessed by CDS assays using osteoblasts cells derived from C57BI/6j WT and C57BI/6j OPN−/− mice (bipedal and quadrupedal) using CellKey™ apparatus, as previously described (Akoume et al., 2010, and WO 2010/040234 to Moreau et al.).
Peripheral blood samples were collected in EDTA-treated tubes and then centrifuged. Plasma samples were derived then aliquoted and reserved frozen at −80° C. until thawed and analyzed. The concentrations of plasma OPN were measured by enzyme-linked immunosorbent assays (ELISA) as said by protocols provided by the manufacturer (IBL, Hamburg, Germany). This OPN ELISA kit measures total concentration of both phosphorylated and non-phosphorylated forms of OPN in plasma. All ELISA tests were performed in duplicate and the optical density was measured at 450 nm using an DTX880 microplate reader (Beckman Coulter, USA).
Quantitative Reverse Transcription-Polymerase Chain Reaction (qPCR)
Thermo-Script™ reverse transcriptase (Invitrogen) was used to reverse transcribe mRNA into cDNA (1 mg total concentration). Several dilutions were tested to choose the concentration that yielded the most efficient amplification. The human primers used were the following:
Each amplification was performed in duplicate using 5 ml of diluted cDNA, 7.5 ml of 3 mM primer solution and 12.5 ml of 2× QuantiTect™ SYBR Green PCR Master Mix (QIAGEN Inc, Ontario, Canada). All reaction mixes were run on Mx3000P™ system from Stratagene (Agilent Technologies Company, La Jolla, Calif.) and analyzed with MxPro™ QPCR Software also from Stratagene. Relative quantification was calculated with the delta CT method using β-actin as the endogenous control.
Cell Lines and siRNA Transfection
MC3T3-E1 cells were used to check the effect of the knockdown of OPN and its receptors. MC3T3-E1 osteoblasts cells were transiently transfected in serum-free medium, using Lipofectamine™ RNAiMAX™ reagent (Invitrogen) according to the manufacturer's instructions and functional experiments were performed 48 h post transfection. The sequence of RNA oligonucleotides used for the knockdown of different genes are, ssp1 (encodes OPN) (CCA CAG CCA CAA GCA GUC CAG AUU A (SEQ ID NO: 9)), integrin β1 (CCU AAG UCA GCA GUA GGA ACA UUA U (SEQ ID NO: 10)), integrin β3 (CCU CCA GCU CAU UGU UGA UGC UUA U (SEQ ID NO: 11)), integrin α5 (CCG AGU ACC UGA UCA ACC UGG UUC A (SEQ ID NO: 12)), integrin α8 (GAG AUG AAA CUG AAU UCC GAG AUA A (SEQ ID NO: 13)), integrin αv (GAC UGU UGA GUA UGC UCC AUG UAG A (SEQ ID NO: 14) and CD44 (GAA CAA GGA GUC GUC AGA AAC UCC A (SEQ ID NO: 15)). Two other siRNAs against integrin α5 were tested (UCC ACC AUG UCU AUG AGC UCA UCA A (SEQ ID NO:16) and UCA GGA GCA GAU UGC AGA AUC UUA U (SEQ ID NO:17)). Comparable results were obtained will all siRNAs.
Gi proteins isoforms expression levels were determined using Western blot technique. MC3T3-E1 cells were treated with PBS or rOPN 0.5 μg/ml overnight (R&D systems Inc., Minneapolis, USA). These cells were washed with cold PBS 1× then lysed in RIPA buffer (25 mM Tris.Hcl pH7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) added with 5 mM NaVO4 and protease inhibitor cocktail (Roche molecular Biochemicals, Mannheim, Germany). Immunoblots were performed with primary antibodies directed specifically against Gi1, Gi2, Gi3, Gs, Phosphoserine, integrin β1 (Santa Cruz Biotechnology, Santa Cruz, Calif.), Mu opioid receptor (MOR) (Abcam Toronto, ON, Canada), Lysophosphatidic acid receptor 1 (LPAR1) (Assaybiotech Inc., USA), melatonin receptor 2 (MT2) and peroxidase-conjugated secondary antibody. Bands were then visualized using SuperSignal™ chemiluminescent substrate (Pierce, Rockford, Ill.).
Effect of OPN on Interaction of Gi Proteins with GPCR and β1 Integrin
MC3T3-E1 cells were treated with PBS or rOPN (0.5 μg/ml) then the whole cell proteins (1 mg) were incubated with anti-MOR (Abcam Toronto, ON, Canada), anti-MT2 or anti-β1 integrin (Santa Cruz Biotechnology), plus protein G beads for immunoprecipitation (IP) at 4° C. overnight. Purified proteins were loaded on 10% gel after IP, then, processed for gel transferring to PVDF membrane and 5% BSA blocking. Membranes were exposed to antibodies specific for Gi1, Gi2, Gi3, MOR, MT2 and β1 integrin at 4° C. overnight and, subsequently, treated with secondary antibody at room temperature for 1 h. Bands were visualized using SuperSignal™ chemiluminescent (Pierce, Rockford, Ill.) and quantified by densitometric scanning.
Whole Exome Sequencing Identification of SNP in AIS Subjects Associated with Increased Risk of Spinal Deformity Progression
A Whole Exome Sequencing (WES) study was performed using Agilent targeted exon capture and Life technologies SOLiD 5500™ for sequencing. GEMINI1™ was used for SNV (Single Nucleotide Variant) annotations. A SNP (rs1467558) was identified in AIS subjects with more severe AIS, which changes the amino acid isoleucine 230 to a threonine in the CD44 coding sequence.
Data are presented as mean±SE, and were analyzed by ANOVA or Student's t test using GraphPad™ Prism 4.0 software. Multiple comparisons of means were performed with one-way ANOVA followed by a post-hoc test of Dunnett. Only P values <0.05 were considered significant.
The bipedal C57BI/6 mice are widely used as animal model to study the pathophysiological events leading to idiopathic scoliosis. These mice rapidly develop spinal deformity following 40 weeks of bipedal ambulation (Oyama et al., 2006). To determine whether OPN is involved in the development of idiopathic scoliosis, female wild-type (WT) and OPN knockout (OPN−/−) C57BI/6 mice were amputated from forelimbs and tails at 1 month of age and subjected to bipedal ambulation for 36 weeks to induce scoliosis. Representative radiographs of the spine as taken at the end of the experiment period are shown in
To provide further evidence for this hypothesis, OPN was measured in plasma from bipedal mice at 12, 24 and 36 weeks after surgery (
It was next determined whether lack of OPN can influence Gi protein-mediated signaling. For this purpose, osteoblasts from WT and OPN−/− mice were screened for their response to DAMGO, somatostatin, oxymetazoline and apelin to activate opioid, somatostatin, alpha2-adrenergic and APJ receptors, respectively. These receptors are well known to mediate signal transduction through Gi proteins. Results illustrated in
To corroborate these results with the function of Gi proteins, osteoblasts were treated with pertussis toxin (PTX), which ADP-rybosylates Gi proteins and disrupts their interaction with receptors (Gilman, 1987); (Moss et al., 1984). Results illustrated in
To determine whether loss of OPN specifically enhances Gi protein signaling, we examined the effect of loss of OPN on signaling initiated by other G proteins such as Gs and Gq. We used isoproterenol and desmopressin to activate Gs through beta-adrenergic and vasopressin receptors, respectively; while bradykinin and endothelin-1 were used to activate Gq through their cognate receptors. Results in
To further investigate the role of OPN in Gi protein-mediated receptor signaling, several lines of experiments were performed using MC3T3-E1 osteoblastic cell line. Since these cells express OPN, was first examined whether depletion of OPN by siRNA influences the capacity of GiPCR ligands to induce cell signaling as measured by CDS in MC3T3-E1 cells. Results illustrated in
OPN is known to function by interacting with a variety of cell surface receptors, including CD44 and many integrins (Weber et al., 1996); (Rangaswami et al., 2006). Since CD44 is considered as a key receptor for OPN, was first explored whether CD44 is responsible for the inhibitory effect of OPN on GiPCR signaling. For this purpose, the interaction between OPN and CD44 was interfered, using CD44 antibody. As shown in
The small interfering RNA (siRNA) approach was further used to knockdown the expression of CD44, and the efficiency of siRNA transfection was demonstrated by quantitative real-time PCR and Western blot analysis (
Further experiments in wild type and CD44−/− cells in the presence or absence of HA confirmed that the inhibitory effect of OPN is exacerbated in the absence of CD44 and intensified in the presence of HA, a known CD44 ligand (
Collectively, these data indicate without ambiguity that CD44 is not involved in the inhibition of GiPCR signaling by OPN and its presence reduces the effect of OPN on GiPCR signaling.
Was next examined the possible requirement of integrins. Given that OPN contains an arginine-glycine-aspartate (RGD)-motif that engages a subset of cell surface integrins and a serine-valine-valine-tyrosine-glutamate-leucine-arginine (SVVYGLR)-containing domain that interacts with other integrins, it was of interest to examine whether OPN action required a specific domain-dependent integrin. For this purpose, small synthetic peptides were used to compete with integrin binding of the RGD or SWYLRG sequences in OPN. BIO1211 was used to selectively inhibit α4β1 integrin, the only SWYLRG-containing integrin present in osteoblasts. As shown in
Collectively, these data suggest that the inhibitory effect of OPN on GiPCR signaling is mediated by one or more RGD-dependent integrin expressed in osteoblasts.
Having established a probable role for RGD-dependent integrins in the inhibitory effect of OPN on GiPCR signaling in osteoblasts, specific integrins were examined to determine whether they could mediate this effect. For this 0purpose, antibodies were used to selectively neutralize the subunits of αvβ1, αvβ3, αvβ5, α5β1, and α8β1, the RGD-dependent integrins that have been shown to be expressed in osteoblasts (Hughes et al., 1997); (Gronthos et al., 1997); (Grzesik and Robey, 1994); (Clover et al., 1992); (Moursi et al., 1997); (Pistone et al., 1996).
To further support this hypothesis, the siRNA approach was used. When compared with untreated cells, MC3T3-E1 transfected with scrambled siRNA demonstrated less response, while cells transfected with α5 or β1 integrins siRNA demonstrated high response in the presence of rOPN. Cells transfected with both α5 and β1 integrin siRNA demonstrated a further increase (
Given that the level of GPCRs is critical for their signaling, it was of interest to examine whether the defective GiPCR signaling induced by OPN is the consequence of a quantitative reduction of these receptors. For this purpose, MC3T3-E1 cells were treated with PBS or rOPN and the cell lysates were analyzed by Western blot for the expression of three different GPCRs; including mu-opioid (MOR), lysophosphatidic acid type 1 (LPA1R) and melatonin type 2 (MT2R) receptors. As showed in
Gi proteins have been shown to be recruited by β1 integrins via an adaptor molecule and to mediate integrin signaling (Green et al., 1999). It is notable that when different receptors signal through the same subfamily of G proteins, they share the same limited pool of G proteins. Therefore, if OPN enhances the recruitment of Gi proteins in the β1 integrin complex, reduction in the amount of Gi proteins in GiPCR complex would be expected. To evaluate this possibility, cells lysates were immunoprecipitated with antibodies against MO, MT2 or β1 integrin receptors, and the presence of Gi proteins in each precipitate was examined by Western blot using antibodies specific for Gi1, Gi2 or Gi3 isoform.
Given that the defective GiPCR signaling associated with idiopathic scoliosis has causatively been related to increased phosphorylation of Gi proteins (Moreau et al., 2004), it was of interest to examined whether OPN influences the phosphorylation status of Gi proteins. Accordingly, MC3T3-E1 cells were treated with rOPN, then cell lysates were immunoprecipitated with antibodies against Gi1, Gi2 or Gi3 protein isoforms, and the phosphorylation level was examined by Western blot using anti-phospho serine/threonine or anti-tyrosine antibodies. Results revealed the presence of phosphorylated serine and tyrosine residues in Gi1 precipitates obtained from cells treated with PBS or rOPN. However, band density was higher in OPN-treated cells. Similar observations were noted in Gi2 and Gi3 precipitates (
To support the involvement of α5β1 integrin in this process, cells were pre-treated with antibody against α5β1 integrin prior to rOPN treatment. Western blot analysis revealed that the Gi phosphorylation induced by rOPN treatment was attenuated by anti-α5β1 integrin blocking antibody (
To further associate these findings with the decreased GiPCR signaling in scoliosis, Gi proteins were examined directly to determine whether they undergo increased phosphorylation in osteoblasts from scoliotic mice. The immunoprecipitate of Gi1, Gi2 and Gi3 proteins were probed with an anti-phospho-serine/threonine or an anti-phospho-tyrosine specific antibody and it was observed that the level of phosphorylation of each Gi isoform was significantly greater in osteoblasts from scoliotic bipedal WT or CD44−/− mice compared with those from quadrupedal control mice (
To identify which kinases are involved in the phosphorylation of Gi proteins induced by OPN, FAK, MEK, ERK1/2, P38, JNK, PI3K, Src, PKC and CaMKII were pharmacologically inhibited. Cell extracts prepared from MC3T3-E1 that had been treated with rOPN in the presence or absence of an inhibitor specific for each kinase were subjected to immunoprecipitation and Western blot analysis. Results in
Several reports indicate that inhibition of Gi proteins disrupts signal from GiPCR and enhances GsPCR signaling (Itoh et al., 1984); (Katada et al., 1985); (Wesslau and Smith, 1992). On the other hand, it has been demonstrated that the tyrosine phosphorylation of Gs protein by Src also enhances GsPCR signaling, presumably by increasing activity of Gs protein and its association with receptor (Hausdorff et al., 1992); (Chakrabarti and Gintzler, 2007).
Taking into account these considerations and having observed that genetic deletion of OPN inversely influenced response to Gi and Gs stimulation (
Collectively, these results demonstrate that OPN enhances not only the GsPCR signaling, but also the tyrosine phosphorylation of Gs protein and its association with receptors.
Assuming that inhibition of Gi protein and phosphorylation of Gs protein are responsible for the increased GsPCR signaling associated with rOPN, the next experiments aimed to determine the contribution of each parameter. For this purpose, cells were treated with PTX to mimic disruption action of rOPN and compared to cells treated with rOPN alone or in combination with Src inhibitor (PP2) to prevent tyrosine phosphorylation. Results in
A Whole Exome Sequencing (WES) study was performed on cell samples from AIS subjects and a SNP (rs1467558) was identified in AIS subjects with severe AIS, which changes the amino acid isoleucine 230 to threonine in the CD44 coding sequence.
Next, the effect of the CT SNP on GiPCR signaling was assessed. Osteoblasts from severe AIS patients (who underwent surgery) carrying the CT SNP and osteoblasts from healthy controls with wild type CD44 (CC) were stimulated with LPA in the presence of recombinant OPN (rOPN). The GiPCR defective signaling effect in response to the same rOPN treatment was reduced 50% more in mutated CD44 (CT) osteoblasts as compared to in wild type CD44 (CC) osteoblasts (
Based on findings presented herein and the available evidences, and without being so limited, a hypothetical scenario schematized in
Results presented herein demonstrate that absence of CD44 expression potentiates the effect of OPN and further reduces GiPCR signalization in cells (
The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
This Application is a divisional application of U.S. application Ser. No. 14/898,912 filed on Dec. 16, 2015, which is the National Stage of PCT Application No. PCT/CA2014/050569 filed on Jun. 17, 2014 and published in English under PCT Article 21(2), which itself claims benefit of U.S. Provisional Application No. 61/835,926, filed on Jun. 17, 2013. All documents above are incorporated herein in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61835926 | Jun 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14898912 | Dec 2015 | US |
| Child | 15878538 | US |
