The present invention relates to methods for the treatment of diseases associated with the secretion of HMGB1.
The high mobility group box 1 (HMGB1) protein, a highly conserved, ubiquitous protein, was first purified almost 30 years ago as a nuclear protein. HMGB1 is involved in nucleosome stabilization and gene transcription (Lotze, M. T., and K. J. Tracey. Nat. Rev Immunol 5:331-342 (2005)), and it can also localize to the cell membrane of neurites for outgrowth and to the cell membranes of tumor cells for metastasis. HMGB1 is passively released by necrotic cells, though not by apoptotic cells, and triggers inflammation. HMGB1 also functions as a late mediator of endotoxemia, sepsis, and hemorrhagic shock in animals and human patients (Wang, H., O. et al, Science 285:248-251 (1999); Sunden-Cullberg, J. et al, Crit Care Med 33:564-573 (2005); Ombrellino, M., et al, Increased serum concentrations of high-mobility-group protein 1 in haemorrhagic shock. Lancet 354:1446-1447 (1999)). Specific inhibition of endogenous HMGB1 could reverse the lethality of established sepsis with HMGB1 antagonists (Yang, H. et al, Proc Natl Acad Sci USA 101:296-301 (2004)). HMGB1 is released from activated monocytes and macrophages and natural killer (NK) cells and behaves as a proinflammatory cytokine. Exposure to HMGB1 leads to various cellular responses, including the chemotactic cell movement of smooth muscle cells and monocytes and the release of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and IL-8 (14). NK cells are in close, physical contact with immature dendritic cells (DCs). IL-18, produced by immature DCs, causes NK cells to produce HMGB1. HMGB1, in turn, causes dendritic cell maturation and Th1 polarization, events that initiate the adaptive immune responses.
HMGB1 contains two homologous DNA-binding motifs (HMG boxes A and B) and an acidic tail. It also contains two nuclear localization signals (NLSs) and two putative nuclear export signals (NESs), demonstrating that HMGB1 shuttles between the nucleus and cytoplasm through a tightly controlled mechanism.
WO 2004/044001 describes acetylated HMGB1 and its role as a mediator of the late phases of inflammation. However, the phosphorylation of HMGB1 has not been previously shown to have any regulatory role in the function of HMGB1 as a mediator of the inflammation. No evidence of phosphorylation, methylation, or glycosylation has previously been found in HMGB1 from calf thymus, mouse thymus, and activated human monocytes (Bonaldi, T. et al, Embo J 22:5551-5560 (2003)).
In this background, the present inventors surprisingly found that HMGB1 is phosphorylated in the animal cells and the translocation of the protein between the nucleus and the cytoplasm is regulated by the phosphorylation. The present invention thus provides a method of treating diseases associated with the secretion of HMGB1 protein.
According to one aspect of the present invention there is provided pharmaceutical composition comprising an antagonist of the phosphorylation of HMGB1, and a pharmaceutically acceptable carrier, excipient or diluent The antagonist may regulate the phosphorylation process or it may affect the phosphorylated HMGB1, e.g. by removing the phosphorylation from the HMGB1 through phosphatase pathway. In one embodiment, the antagonist is the PKC inhibitors.
According to another aspect of the present invention there is provided a method for treating a condition in a subject wherein the condition is characterized by activation of an inflammatory cytokine cascade, comprising administering an effective amount of an antagonist of HMGB1 phosphorylation.
The present invention also provides a method of identifying an agent that regulates the phosphorylation of HMGB1, comprising the steps (a) determining the level of the phosphorylated HMGB1 in the presence and absence of said agent; (b) comparing the level of the protein determined in step (a); and (c) identifying said agent as a regulator by the differences in the phosphorylation of HMGB1 activity in the presence or absence of said compound. In one embodiment, the regulator is an antagonist of the HMGB1 phosphorylation. In another embodiment, the regulator is an agonist of the HMGB1 phosphorylation.
The present invention also provides a method for diagnosis and/or prognosis of the conditions associated with the activation of the inflammatory cascade comprising measuring the concentration of phosphorylated protein HMGB1 in a sample, and comparing that concentration to a standard for phosphorylated protein representative of a normal concentration range of phosphorylated HMGB 1 in a like sample, whereby higher levels of phosphorylated HMGB1 are indicative of the disease. In one embodiment the sample is a serum sample.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.
The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein.
The term “regulate” as used herein refers to a change or alteration in the phosphorylation of HMGB1. The “regulation” therefore includes inhibition of phosphorylation, e.g. by compounds which block the protein kinases. The “regulation” also includes the activation of the HMGB1 phosphorylation, e.g. by compounds which stimulate the activity of protein kinases or by compounds which inhibit protein phosphatase pathway. The “regulator” of the present invention could be in the form of a chemical compound, or an mixture (for example, extract made from biological materials), proteins, carbohydrates, small molecules, or nucleic acids. Regulators are evaluated for their activity as antagonist or agonist of the HMGB1 phosphorylation. Such regulators can be screened using the methods described herein.
The term “antagonist”, is intended to refer broadly to any agent which lowers the level of phosphorylation of HMGB1 protein, and it is used interchangeably with “inhibitor”. “Agonist” is used to refer to any agent which increases the level of phosphorylation of HMGB1 protein, and it is used interchangeably with “activator”
In one embodiment the present invention provides a pharmaceutical composition and method for treating diseases characterized by activation of an inflammatory cytokine cascade, particularly sepsis, including septic shock and ARDS (acute respiratory distress syndrome), comprising administering an effective amount of an antagonist to HMGB1 phosphorylation.
A pharmaceutical composition of the present invention comprises an antagonist of the phosphorylation of HMGB1, and a pharmaceutically acceptable carrier, excipient or diluent. The antagonist may regulate the phosphorylation process or it may affect the phosphorylated HMGB1, e.g. by removing the phosphorylation from the HMGB1 through phosphatase pathway. In one embodiment, the antagonist is the PKC inhibitors. In another embodiment, the antagonist is the activator or protein phosphatases.
HMGB1 is a member of the B family of HMG proteins. cDNAs coding for HMGB1 have been cloned from human, rat, mouse, mole rat, trout, hamster, pig and calf cells, and is believed to be abundant in all vertebrate cell nuclei. The protein is highly conserved.
The inventors discovered that in the cells, the HMGB1 is phosphorylated at Ser residue and the phosphorylated HMGB1 was relocated to the cytoplasm. In most cells HMGB1 shuttles continually from the nucleus to the cytoplasm, but the equilibrium is almost completely shifted towards a nuclear accumulation. Treatment of cells with phosphatase inhibitor (OA) caused phosphorylation of the protein HMGB1 (Example 2). The phosphorylation also blocked re-entry of the protein to nucleus (Example 4), which suggests that the phosphorylation plays an important role in the controlled shuttling mechanism of the HMGB1. The inventors also showed that Protein Kinase C phosphorylates HMGB1 (Example 8 and Example 9).
HMGB1 can be phosphorylated at multiple sites. HMGB 1 contains two independent NLSs, and the phosphorylation of both NLS regions of HMGB1 was required for its relocation to the cytoplasm. Example 5 shows that HMGB1 binds to KAP-α1, a shuttling receptor protein, and the binding was affected by the phosphorylation of the HMGB1.
Phosphatase inhibitor (for example, OA) caused hyperphosphorylation of HMGB1 and the relocation of the protein to the cytoplasm. It was demonstrated that Protein Kinase C but neither Casein Kinase II nor cdc2 phosphorylated HMGB1 (Example 8). When the cells were treated with Protein Kinase C inhibitor, Go6983 (2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) Maleimide), the secretion of HMGB1 was inhibited.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art. Where appropriate, the pharmaceutical compositions can be administered by any one or more of administration route known in the art.
The formulation of therapeutic compounds is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 0.05 μg to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intra nasal, intradermal or suppository routes or implanting (eg using slow release molecules by the intraperitoneal route or by using cells e.g. monocytes or dendrite cells sensitised in vitro and adoptively transferred to the recipient). Depending on the route of administration, the peptide may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
For example, the low lipophilicity of the peptides will allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer peptides by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation. For example, peptides may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.
The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, theomersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When the peptides are suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.
The tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody or a peptide of the invention, care must be taken to use materials to which the protein does not absorb. In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome. In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.
According to another aspect of the present invention there is provided a method for treating a condition in a subject wherein the condition is characterized by activation of an inflammatory cytokine cascade, comprising administering an effective amount of an antagonist of HMGB1 phosphorylation.
Diseases and conditions mediated by the inflammatory cytokine cascade are numerous. An inflammatory condition that is suitable for the methods of treatment described herein can be one in which the inflammatory cytokine cascade is activated. In one embodiment, the inflammatory cytokine cascade causes a systemic reaction, such as with endotoxic shock. In another embodiment, the inflammatory condition is mediated by a localized inflammatory cytokine cascade, as in rheumatoid arthritis. Nonlimiting examples of inflammatory conditions that can be usefully treated using the antibodies and antigen-binding fragments of the present invention include, e.g., diseases involving the gastrointestinal tract and associated tissues (such as ileus, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple's disease); systemic or local inflammatory diseases and conditions (such as asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, and sarcoidosis); diseases involving the urogenital system and associated tissues (such as septic abortion, epididymitis, vaginitis, prostatitis, and urethritis); diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratory distress syndrome, pneumoultramicroscopic silicovolcanoconiosis, alveolitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C virus and herpes), bacteria (such as disseminated bacteremia, Dengue fever), fungi (such as candidiasis) and protozoal and multicellular parasites (such as malaria, filariasis, amebiasis, and hydatid cysts); dermatological diseases and conditions of the skin (such as burns, dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals); diseases involving the cardiovascular system and associated tissues (such as stenosis, restenosis, vasculitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, myocardial ischemia, periarteritis nodosa, and rheumatic fever); diseases involving the central or peripheral nervous system and associated tissues (such as Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); diseases of the bones, joints, muscles and connective tissues (such as the various arthritis and arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune and inflammatory disorders (such as myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, and Reiter's syndrome); as well as various cancers, tumors and proliferative disorders (such as Hodgkins disease); and, in any case the inflammatory or immune host response to any primary disease.
In one embodiment, the condition is selected from the group consisting of sepsis, allograft rejection, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, restenosis, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial ischemia, organic ischemia, reperfusion ischemia, Behcet's disease, graft versus host disease, Crohn's disease, ulcerative colitis, ileus, multiple sclerosis, and cachexia. In another embodiment, the condition is selected from the group consisting of sepsis, arthritis (e.g., rheumatoid arthritis), asthma, lupus, psoriasis, inflammatory bowel disease and Crohn's disease.
As used herein, an “effective amount” or “therapeutically effective amount” is an amount sufficient to prevent or decrease an inflammatory response, and/or to ameliorate and/or decrease the longevity of symptoms associated with an inflammatory response. The amount of the composition of the invention can be determined, for example, by administering the composition to an animal models. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.
Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors that are known to one of ordinary skill in the art. Such factors include, e.g., the condition or conditions to be treated, the severity of the subject's symptoms, the subject's age, the subject's body mass, the subject's immune status, the response of the individual subject, and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.
The present invention also provides a method for diagnosis and or prognosis of the conditions associated with the activation of the inflammatory cascade comprising measuring the concentration of phosphorylated protein HMGB1 in a sample, and comparing that concentration to a standard for phosphorylated protein representative of a normal concentration range of phosphorylated HMGB 1 in a like sample, whereby higher levels of phosphorylated HMGB1 are indicative of the disease. In one embodiment the sample is a serum sample. The diagnostic method may also be applied to other tissue or fluid compartments such as cerebrospinal fluid or urine. The diagnostic assay may use anti-phosphorylated HMGB1 antibodies. Alternatively, the antibodies specific to HMGB1 and anti-p-amino acid antibody, such as anti-pSer antibody, could be used in combination simultaneously or sequentially. The diagnostic procedure can utilize standard antibody-based techniques such as ELISA assays and Western blot techniques.
The present invention also provides a method of identifying an agent that regulates the phosphorylation of HMGB1, comprising the steps (a) determining the level of the phosphorylated HMGB1 in the presence and absence of said agent; (b) comparing the level of the protein determined in step (a); and (c) identifying said agent as a regulator by the differences in the phosphorylation of HMGB1 activity in the presence or absence of said compound. In one embodiment, the regulator is an antagonist of the HMGB1 phosphorylation. In another embodiment, the regulator is an agonist of the HMGB1 phosphorylation. To determine the level of phosphorylation, antibodies that recognize the phosphorylated HMGB1 could be used. Or, the phosphorylation could be monitored by radiolabeling method, for example, using [32P] orthophosphate. Antibody techniques and radiolabeling techniques are all known in the art.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.
Murine macrophage RAW 264.7 cells (American Type Culture Collection, Manassas, Va.) and HeLa cells were cultured at 37° C. under 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (In vitrogen Life Technologies), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. Human peripheral blood monocytes (PBMO) were harvested from the adhesive cells on the culture flask, after yielding peripheral blood mononuclear cells, by Ficoll-hypaque gradient centrifugation. Human recombinant TNF-α (R&D Systems), OA (Calbiochem), trichostatin A (TSA, Sigma-Aldrich), and cycloheximide (Sigma) were purchased.
To analyze the secretion of HMGB1 in the supernatants, culture media were replaced with serum-free OPTI-MEM (Gibco BRL) medium and concentrated with Amicon Centricon filtration (Millipore) after removing cell debris, and Western blot analysis was performed. The cytoplasmic and nuclear fractions from 5×106 cells were separated using a digitonin-based method (Bird, C. H. et al, Mol Cell Biol 21:5396-5407 (2001)) to observe the levels of HMGB1 in each fraction. The cells were lysed using 1% Nonidet P-40 buffer containing a protease inhibitor cocktail (Sigma), and the protein concentrations were measured by Bradford assay (Biorad) for the analysis of whole cell lysates (WCLs). The protein samples underwent 12% SDS-PAGE and were transferred to a nitrocellulose membrane. Western blot analysis was performed using rabbit anti-HMGB1 (BD Pharmingen) and HRP-labeled goat anti-rabbit Ig as primary and secondary antibodies, respectively. The signals were revealed with enhanced chemiluminescence (ECL, Pierce).
To identify the phosphorylated residues of the HMGB1 protein, TNF-α-treated RAW 264.7 cells were lysed with a protease inhibitor cocktail. Cell homogenates were centrifuged at 20,000 g for 15 min and precleared by incubation with protein G-Sepharose (Amersham) at 4° C. for 30 min. The precleared extracts (500 μg) were incubated with rabbit polyclonal anti-pSer, anti-pTyr and anti-pThr (all from Chemicon) and then protein G-Sepharose was added and incubated for 3 h at 4° C. Immune complexes were collected by centrifugation and washed with lysis buffer. Collected complexes were fractionated by SDS-PAGE, transferred to membranes and blotted with anti-HMGB1 for detection. Anti-pAKT (Cell signaling) was used as a negative control. To investigate the time-dependent phosphorylation of HMGB1, the WCLs of RAW 264.7 cells treated with TNF-α for the indicated time were immunoprecipitated with anti-HMGB1 and subjected to Western blot analysis using anti-pSer.
Cells were cultured in LabTek II chambers (Nalgene) and were fixed in 3.7% paraformaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 4 mM MgSO4, pH 7.0) for 10 min at RT. After fixation, the cells were washed with PBS and incubated for 3 min at 4° C. with HEPES-based permeabilization buffer containing 300 mM sucrose and 0.2% Triton X-100. The cells were blocked with 0.2% BSA in PBS for 15 min and were incubated with rabbit anti-HMGB1 for 1 h at RT. After 3 washes with blocking solution, secondary antibody FITC-conjugated goat anti-rabbit Ig (BD Pharmingen) was added. Cells expressing various HMGB1-EGFP proteins were stained with DAPI and the cells were observed with a BX51 fluorescent microscope (Olympus). Cells expressing HMGB1-GFP and its derivatives were fixed as described above.
Nuclear import assays were performed with minor modification as previously described (Adam, S. A., R. S. Marr, and L. Gerace. 1990. J Cell Biol 111:807-816 (1990). Briefly, HeLa cell cytosol was first prepared. For this, HeLa cells at a density of 5×105 cells/mL were harvested and washed twice in ice-cold PBS and once in washing buffer [10 mM HEPES, pH 7.3, 110 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT]. They were then homogenized with hypotonic lysis buffer (5 mM HEPES pH 7.3, 10 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT, 20 μM cytochalasin B, 1 mM PMSF, 1 μg/mL each of leupeptin, pepstatin, and aprotinin). The supernatants were sequentially centrifuged at 1,500 g for 15 min, 15,000 g for 20 min, and 100,000 g for 1 h, dialyzed against transport buffer (TB; 20 mM HEPES, pH 7.3, 110 mM KOAc, 2 mM Mg(OAc)2, 5 mM NaOAc, 1 mM EGTA, 2 mM DTT, and 1 μg/mL each of leupeptin, pepstatin, and aprotinin), and frozen in aliquots in liquid nitrogen before storage at −80° C.
For the assays, HeLa cells were washed in TB and permeabilized for 5 min on ice in TB containing 40 μg/mL digitonin. The cells were rinsed for 5-10 min with several changes of TB, and the excess buffer was removed. The cells were incubated with transport mixture for 1 h at 22° C. The transport mixture contained HeLa cell cytosol at a final concentration of 2 mg/mL, which was preincubated for 30 min at room temperature with an ATP-regenerating system (1 mM ATP, 5 mM creatine phosphate, 20 U/mL creatine phosphokinase, 0.5 mM GTP) either with or without 10 μM OA, and 30 μg/mL of each substrate. The cells were fixed with 3.7% formaldehyde for 10 min and immediately examined by fluorescent microscope.
The gene encoding human HMGB1 was cloned upstream of GFP in pEGFP-N1 (BD Biosciences Clontech), and the construct was named pHMGB1-EGFP-N1. For the recombinant HMGB1-GFP protein, a SacI/NotI fragment from pHMGB1-EGFP-N1 was subcloned into pET-28a (Novagen). Six-His-tagged HMGB1-EGFP, GST-EGFP, and EGFP proteins were produced in E. coli BL21(DE3) pLysE (Novagen). The cells transformed with each construct were grown in LB medium containing kanamycin (15 μg/mL) and chloramphenicol (34 μg/mL) to an OD600 of 0.4-0.5 at 37° C., cooled to 25° C., induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and grown overnight at 25° C. The cells were lysed by sonication, and the clear lysate was loaded onto a Ni2+-NTA column. The bound protein was washed with 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH 8, and was eluted in the same buffer supplemented with 200 mM imidazole. All proteins were dialyzed into TB and stored at −80° C.
Site-directed mutations of HMGB1 were generated from pHMGB1-EGFP-N1 as a template using the QuickChange™ Site-directed Mutagenesis Kit (Stratagene). Human KAPs were cloned into BamHI/XhoI (KAPs-α1, α2, α4, α6), EcoRI/XhoI (KAPs-α3, α5) or BamHI/Not I (KAP-β1) sites of pGEX-4T-1 (Pharmacia) to produce GST-fusion proteins. KAP-α1 (accession: AAC60648), α2 (AAA65700), α3 (AAH17355), α4 (AAC25605), α5 (AAH47409), α6 (AAC15233), and β1 (AAH03572) were prepared from PCR amplifications of oligo(dT)-selected HeLa cell-derived cDNA. The primers were as follows:
The nucleotide sequences of restriction enzyme sites are underlined. The GST-KAP fusion proteins were produced in E. coli BL21. Cells were harvested and disrupted by sonication in lysis buffer with 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM DTT, and a protease inhibitor mix (1 μg/mL of leupeptin, pepstatin, and aprotinin and 1 mM PMSF) (Sigma) in PBS. After centrifugation, the supernatants were incubated with glutathione-Sepharose at 4° C. Bound proteins were eluted by incubation at room temperature for 30 min with 10 mM reduced glutathione. SDS-PAGE analysis of each eluted GST-KAP protein revealed a major protein band with the predicted molecular size. For the transfection study, Flag-tagged KAP-α1 was cloned into pCMV-Tag2 (Stratagene). All constructs were confirmed by DNA sequencing (Applied Biosystems). Fugene6 (Roche) was used for the transfection study.
Two μg of each GST-KAP protein was coupled to glutathione sepharose 4B beads and incubated with 500 μg of WCL from RAW 264.7 cells as a HMGB1 source at 4° C. overnight. WCLs were obtained after incubating cells in lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% NP40, 1 mM EDTA, 50 mM NaF, 1 mM sodium-orthovanadate, 1 mM DTT, 1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 μg/mL pepstatin) for 30 minutes on ice. Extracts were clarified by centrifugation at 20,000 g for 15 min at 4° C. GST complexes were washed and separated by 12% SDS-PAGE. The blots were probed with anti-HMGB1 and the signals were revealed by ECL detection as described above.
To observe the binding of KAP protein to each mutant HMGB1 in the cells, Flag-tagged KAP-α1 and each mutant HMGB1-GFP plasmid were co-transfected into RAW 264.7 cells. Cell homogenates of transfected RAW 264.7 cells were harvested and incubated with mouse anti-Flag (M2, Sigma) and mouse anti-GFP (Santa Cruz) at 4° C. overnight. Immune complexes were collected and the membranes were blotted with anti-Flag and anti-GFP, respectively. The reciprocal experiment was also performed. GST was used as a negative control. And to test the direct binding of HMGB1 to KAP-α1, GST pull-down assay was performed as described above.
Recombinant protein of HMGB1 was incubated with GST-KAP-α1 (10 μg) which was coupled to glutathione sepharose 4B beads. For this study, 6×His-tagged wild type HMGB1 and boxes A (aa 1-87) and B (aa 88-162) of HMGB1 were cloned into pRSETB (Invitrogen) and purified proteins were included in this test. After separating on the gel, the membrane was probed with anti-His and reprobed with anti-GST.
RAW 264.7 cells were cultured in phosphate starved condition of phosphate-free DMEM containing 10% dialyzed FBS (Gibco BRL) for 4 h and further incubated for 4 h by adding 600 μCi of [32P]orthophosphate (Amersham Pharmacia Biotech) per mL to each dish. After 4 h, the cells were stimulated with 100 nM OA for 2 h, 100 ng/mL of LPS and 20 ng/mL of TNF-α for 2 and 8 h. The labeling was terminated by removing the culture medium followed by two immediate washes of the cells with ice-cold PBS. The cells were harvested by scrapping in 0.8 mL of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 50 mM NaF, 10 mM Sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM Sodium orthovanadate, 1 mM DTT, 10 μg/mL aprotinin, 10 μg/mL leupeptin, 5 μg/mL pepstatin, and 0.5 mM PMSF) and centrifuged at 21,000×g for 20 min at 4° C. The concentration of total soluble proteins in the supernatant was quantified using the Bradford reagent (Bio-Rad). Pre-cleared lysates were incubated with 2 μg/mL of rabbit anti-HMGB1 from two different companies of BD PharMingen and Upstate Biotechnology for 2 h at 4° C. Following the addition of protein G-Sepharose the tubes were rocked for an additional 1 h and beads were washed ten times with lysis buffer without SDS. Proteins were eluted in Laemmli sample buffer and separated. The gels were dried and the radioactivity was analyzed by autoradiography.
To investigate the enzyme that is involved in HMGB1 phosphorylation, in vitro protein kinase assay was performed. Six His-tagged wild type (WT) HMGB1 and HMGB1 NLS1/2A, both of which were not tagged with GFP, were purified from E. coli. HMGB1 NLS1/2A not tagged with GFP was cloned from the HMGB1 NLS1/2A. For the protein kinase C (PKC) reaction, 2 μg of each protein was incubated with 50 ng of PKC protein (Upstate) and 5 μCi [γ-32P]ATP (Amersham Pharmacia Biotech) in a total volume of 50 μl buffer, which composed of 20 mM MOPS (pH 7.2), 25 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT, 1 mM CaCl2, lipid activator (10 μM PMA, 0.28 mg/ml phosphatidylserine, 0.3% Triton X-100 mixed micelle suspension) and 50 μM ATP. And for casein kinase 11 (CK II) reaction, 500 U of CK II (New England BioLabs) was used in the buffer, which composed of 20 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 50 μM ATP. For the cdc2 reaction, 20 U of Cdc2 kinase (New England BioLabs) was used in the buffer, which composed of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 50 μM ATP. All the reactions were performed at 30° C. for 40 min. Each reaction was terminated by adding 5× sample buffer. The samples were separated with 12% SDS-PAGE and autoradiography was performed after drying.
To observe the inhibition of HMGB1 secretion by PKC inhibitor, RAW 264.7 cells were treated with 200 ng/mL of LPS alone or combined with Gö 6983 (Calbiochem), a conventional PKC (α, β, and γ) inhibitor including PKCδ and PKCζ, at the indicated concentrations for 16 h. Other inhibitors of SB203580 (p38 inhibitor, Calbiochem), PD098059 (ERK1/2 inhibitor, Calbiochem), Bay 11-7082 (NF-κB inhibitor, Calbiochem) were included. The supernatants were harvested and separated with 12% SDS-PAGE and Western blot was performed to observe the level of HMGB1.
To investigate whether HMGB1 is phosphorylated and how its phosphorylation influences its nuclear transport, RAW 264.7 cells were treated with OA, a type 1/2A protein phosphatase inhibitor, to induce forced phosphorylation of HMGB1. OA was used at a low concentration of 100 nM for 8 h or less to minimize the nuclear leakage of HMGB1 and block entry into the cell cycle. Treatment with TNF-α as a positive control cytokine resulted in the translocation of nuclear HMGB1 to the cytoplasm (
Next, to demonstrate the direct evidence of HMGB1 phosphorylation, RAW 264.7 cells were labelled with [32P]orthophosphate and stimulated with 100 nM OA, 20 ng/mL TNF-α and 100 ng/mL LPS for the indicated length of time, and the cell lysates were immunoprecipitated with anti-HMGB1 for autoradiography. We used rabbit anti-HMGB1 from two different vendors for confirmation. HMGB1 was phosphorylated by OA, and by TNF-α and LPS treatments. Phosphorylation was increased by the increment of treatment time from 2 h to 8 h (
To determine which amino acid residue of HMGB1 is phosphorylated, RAW 264.7 cells were treated with 20 ng/mL TNF-α for 16 h. WCLs from treated cells were immunoprecipitated with anti-pSer, anti-pTyr and anti-pThr antibodies, separated, and immunoblotted with anti-HMGB1. Only serine residues of HMGB1 were phosphorylated by TNF-α treatment (
To further examine the effect of phosphorylation on the relocation of HMGB1, RAW 264.7 cells were treated with OA, and indirect immunofluorescent staining was performed. HMGB1 was mostly observed in the nuclei of unstimulated RAW 264.7 cells (
To exclude the possibility of HMGB1 presence in the cytoplasm due to new protein synthesis, HMGB1 relocation was directly observed using HMGB-GFP plasmid after treatment with CHX, an inhibitor of any new protein synthesis. RAW 264.7 cells were transfected with wild-type HMGB1-GFP plasmid, incubated for 24 h, and then treated with 2 μg/mL CHX. One h after CHX treatment, OA was added for 4 h in the presence of CHX. As shown in
HMGB1 can transverse the nuclear membrane in both directions, however, HMGB1 molecules are predominantly in the nucleus in an unstimulated state, indicating that import is much more effective than export. To further demonstrate whether phosphorylation influences nuclear import of HMGB1, a nuclear import assay was performed using a digitonin-permeabilized HeLa cell-free transport system (Adam, S. A., R. S. Marr, and L. Gerace. J Cell Biol 111:807-816 (1990)) Digitonin-permeabilized cells have perforated plasma membranes, which release cytosolic components from cells while the nuclear envelope and other major organelle membranes remain intact. As a source of exogenous HMGB1 protein, recombinant HMGB1-GFP protein was purified from E. Coli (
HMGB1-GFP was observed in the nuclei of digitonin-treated HeLa cells when the cells were incubated for 1 h with the transport mixture that contained HMGB1-GFP but not OA, suggesting that HMGB1-GFP entered the nucleus by default way (
To investigate whether phosphorylation prevents HMGB1 from interacting with the nuclear import protein, it was determined which KAP protein is involved in binding with HMGB1 as its cargo protein and then observed the interaction of p-HMGB1 with the KAP protein. The KAP family proteins act as shuttling receptors and specifically bind the NLS motifs of cargo proteins to facilitate their nuclear import (Rendon-Mitchell, B. et al, J Immunol 170:3890-3897 (2003)]. For this study, GST-KAP fusion proteins of α1, α2, α3, α4, α5, α6, β1 were produced in E. coli. GST-KAP-β1 was included because some proteins directly bind to KAP-β1 for their nuclear transport. For an in vitro protein-protein interaction study, WCLs of unstimulated RAW 264.7 cells, a source of unphosphorylated HMGB1, were incubated with each GST-KAP fusion protein, which was bound to glutathione-Sepharose beads. KAP-α1 was identified as the carrier protein for HMGB1 (
Direct binding of recombinant HMGB1 protein to KAP-α1 was tested to exclude the possibility that other HMGB1-interacting proteins present in cell lysates could have a role in binding. Purified six His-tagged wild type HMGB1 protein and boxes A (aa 1-87) and B (aa 88-162) proteins, which were identified at expected size by Coomassie blue staining (
We next tested the binding of p-HMGB1 to KAP-α1 protein. When the binding of KAP-α1 to OA-treated RAW 264.7 cell lysate was tested, the interaction was not observed while the binding to medium-treated RAW 264.7 cell lysate was clearly seen (
HMGB1 was phosphorylated at serine residues (
To observe the effect of phosphorylation in both NLS regions of HMGB1 on the binding to KAP-α1 and on the subcellular localization of HMGB1 in the transfected cells, a number of site-directed mutations in six serines of NLS1 and NLS2 were generated using a HMGB1-GFP fusion construct plasmid (
The movement of HMGB1 by the state of HMGB1 phosphorylation at either or both NLSs was investigated. RAW 264.7 cells were transfected with each mutant HMGB1-GFP plasmid and cultured for 24 h without any stimulation. Then the fluorescent images were observed. The mutant fusion proteins from the HMGB1 NLS1A, NLS2A, NLS1/2A, NLS1E, and NLS2E constructs, which showed the interaction with KAP-α1 at least about 50% compared to wild-type, were localized to the nuclei 24 h after transfection (
Next, RAW 264.7 cells were treated with OA 24 h after each transfection to further investigate the effect of change of phosphorylation state on HMGB1 relocation. HMGB1 NLS WT, NLS1E, and NLS2E were relocalized to the cytoplasm afterOAtreatmentwhile HMGB1 NLS1A, NLS2A, and NLS1/2A were unaffected (
To investigate the enzyme that is involved in HMGB1 phosphorylation, in vitro protein kinase assay was performed. For this assay, PKC, CK II and cdc2 protein kinases were tested because these enzymes were known to phosphorylate HMG-I of HMG family protein (Xiao D M, et al. J Neurochem 74:392-399 (2000), Nissen M S, et al. J Biol Chem 266:19945-19952 (1991), Reeves R, et al. Proc Natl Acad Sci USA 88:1671-1675 (1991), Palvimo J and Linnala-Kankkunen A, FEBS Lett 257:101-104 (1989)). As shown in
The secretion of HMGB1 was evaluated to determine whether it is inhibited by PKC inhibitor to confirm the involvement of PKC in HMGB1 phosphorylation. RAW 264.7 cells were treated with 200 ng/mL of LPS alone or combined with Go 6983 at the indicated concentrations for 16 h. The levels of HMGB1 in the culture supernatants were decreased to undetectable level by increasing the concentration of Go 6983 from 0.1 to 10 μM (
All of the references cited herein are incorporated by reference in their entirety.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.