Methods for diagnosing osteoarthritis

Abstract

A method of predicting the risk of developing osteoarthritis (OA) comprising: (a) measuring the nuclear cellular level of prohibitin (PHB-1) in nucleated cells present in a blood sample from a subject having or suspected of having OA; and (b) comparing said nuclear cellular level to that corresponding to a control sample; and (c) identifying the subject as being at risk of developing OA when the nuclear cellular level of said PHB-1 in said blood sample is higher than in the control sample; and a composition for determining the risk of developing osteoarthritis (OA), said composition comprising: a cell sample from a subject; and a non-naturally occurring molecule for detecting nuclear accumulation of PHB1.

Claims

1. A composition for determining the risk of developing osteoarthritis (OA), said composition comprising: (a) a cell sample from a subject suffering from knee or hip OA or at risk of developing early onset knee or hip OA, wherein said cell sample comprises primary cells and wherein said cell sample is an articular chondrocyte sample, a growth plate chondrocyte sample, an osteoblast sample, a skeletal myoblast sample, a synoviocyte sample or a blood cell sample; and (b) a non-naturally occurring antibody which specifically binds to PHB1 for detecting nuclear accumulation of PHB1.

2. The composition of claim 1, wherein said molecule comprises a detectable label.

3. The composition of claim 1, wherein the composition comprises a primary antibody which specifically binds to PHB1 and a detectably-labeled secondary antibody which binds to said primary antibody.

4. The composition of claim 3, wherein said detectably-labeled secondary antibody comprises a fluorescent label.

5. The composition of claim 1, wherein said cell sample is a nuclear fraction or nuclear extract.

6. The composition of claim 5, further comprising (c) an antibody which specifically binds to lamin.

7. The composition of claim 1, wherein said subject is human.

8. The composition of claim 1, wherein said subject has primary OA.

9. The composition of claim 1, wherein said subject has early onset OA.

10. The composition of claim 1, wherein said subject is 40 years old or younger.

11. The composition of claim 1, further comprising (c) an antibody which specifically binds to PAN SUMO, BCoR and/or PHB2.

12. A composition for determining the risk of developing osteoarthritis (OA), said composition comprising: (c) a cell sample from a subject suffering from OA or at risk of developing early onset OA, wherein said cell sample comprises primary cells and wherein said cell sample is an articular chondrocyte sample, a growth plate chondrocyte sample, an osteoblast sample, a skeletal myoblast sample, a synoviocyte sample or a blood cell sample; and (d) a non-naturally occurring antibody which specifically binds to PHB1 for detecting nuclear accumulation of PHB1.

13. The composition of claim 12, wherein said osteoarthritis is knee or hip osteoarthritis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the appended drawings:

(2) FIGS. 1A-D show a bone histology from a distal end of right femur of 7-month old normal mouse (wt) and pitx1+/ (ht). Goldner staining shows in bone and calcified tissues as green and cartilage and bone marrow cells as red. A representative section of subchondral, cortical and trabecular bone thickening observed in pitx1+/ mice (FIG. 1B) as compared to wild-type ones (FIG. 1A) At higher magnification, a substantial increase in fibrillation and calcification is observed in the articular cartilage of heterozygous mice (FIG. 1D) as compared to wild-type ones (FIG. 1D). Note that original magnification of FIG. 1A and FIG. 1B) is 5 and FIG. 1C and FIG. 1D) is 20;

(3) FIGS. 2A-D compare Pitx1 expression in articular chondrocytes from OA patients and matched control subjects. (FIG. 1A) Reverse transcription-polymerase chain reaction for pitx1 gene expression in human articular chondrocytes derived from knee cartilage of control subjects (N, n=4) and patients with osteoarthritis (OA, n=7). Pitx1 specific mRNA transcripts were detected in all control tissues (N1-N4). Loss of the pitx1 gene expression was observed in all examined OA samples (OA1-OA7) and -actin expression was used as internal control. Immunodetection of Pitx1 proteins in human control (FIG. 1B) and OA (FIG. 1C) cartilage tissues was performed using a specific antibody against human Pitx1 protein. In control cartilage sections (n=8), specific immunoreactivity was demonstrated by intense brown staining in nucleus of chondrocytes of the superficial and deep zones (arrow). In OA cartilage (n=8), only a few cells stained specifically for Pitx1. These were located mainly in the superficial layer (arrow). The specificity of staining was evaluated by omission of the primary antibody and by substitution of the primary antibody with non-immune IgG (Nordic Immunology, Tilburg, The Netherlands) following the same experimental protocol. No staining was observed. (Original magnification 20). (FIG. 1D) Cell scores for Pitx1-positive chondrocytes, indicating significant differences between OA and normal (control) cartilage and between the deep zones for each cartilage type, by Mann-Whitney U test. Values are the mean+SEM of 8 normal and 8 OA specimens;

(4) FIG. 3 shows an alignment of the human pitx1 E2F-like DNA-binding site with known E2F sites found in mammalian cell cycle regulation promoters. Alignment of different E2F binding sites (E2F core) and conserved adjacent cyclin homology region (CHR) found in promoter of genes involved in the regulation of cell cycle. The presented E2F sites are DNA Pol (SEQ ID NO: 3); p107 (SEQ ID NO: 4); Cdc2 (SEQ ID NO: 5); Adeno E2a (SEQ ID NO: 6); E2F-1 (SEQ ID NO: 7); H2A (SEQ ID NO: 8); DHFR (SEQ ID NO: 9); HsOrc1 (SEQ ID NO: 10); c-Myc (SEQ ID NO: 11); CycA (SEQ ID NO: 12); Cdc25 (SEQ ID NO: 13); TK (SEQ ID NO: 14); B-Myb (SEQ ID NO: 15); Hpitx1 (SEQ ID NO: 16). Positions are indicated and numbered in function of the transcriptional start site while Act and Rep indicate an activating and a repressing site respectively. Note that E2F element found in human pitx1 promoter is more distal than usual cell cycle genes;

(5) FIGS. 4A-D show an electrophoretic mobility shift assay (EMSA) of nuclear extracts prepared with OA articular chondrocytes. Representative EMSA using the wild type probe (FIG. 4A) or the mutant probe (FIG. 4B). Note that attempts to supershift the bound complexes were performed with different antibodies and addition of anti-BCoR antibodies disrupted partially the main complex and generated an additional band suggesting the presence of BCoR in the bound complex. (FIG. 4C) Representative off-rate autoradiograph showing the effect of addition of unlabeled double-stranded oligonucleotide corresponding to E2F-like site found in human pitx1 promoter as a competitor on the E2F complex binding to radiolabeled wild-type E2F site (wild-type probe) versus mutant E2F site found in OA patients (mutant probe). Competitor double-stranded oligonucleotide (70-fold molar excess) was added (t=0), and aliquots of gel mobility shift reaction mixtures were removed at the indicated times after the addition. (FIG. 4D) The dissociation rate of wild-type E2F and the mutant (OA) site were compared by directly counting the radioactivity of each bound E2F complex by cutting the corresponding bands on the dried gel. The half-life of binding for off-rate experiments was computed by plotting the disappearance of 50% when compared to the initial binding of E2F complex. The graph depicts the average half-life value for the E2F complex;

(6) FIGS. 5A-B show in vitro and in vivo characterization of PHB-1, PHB-2 and BCoR interactions in primary OA articular chondrocytes. (FIG. 5A) Co-immunoprecipitation were performed with anti-PHB-1, anti-PHB-2 (as internal control) and anti-E2F1 in OA and normal nuclear extracts obtained from primary human articular chondrocytes cultures. Western blot performed with anti-PHB-2 shows that PHB-2 interacts physically with PHB-1 and E2F1 only in OA nuclear extracts. (FIG. 5B) Chromatin immunoprecipitation (ChIP) assays were performed and showed that PHB-1 and BCoR co-localise in vivo with the distal E2F site identified in the human pitx1 promoter. Amplification of a 372 bp PCR fragment was obtained with input (positive control) and after immunoprecipitation with anti-PHB-1 and anti-BCoR antibodies. Immunoprecipitation with anti-E2F1 did not generate a positive PCR amplification indicating that E2F1 does not co-localise on the pitx1 promoter with PHB-1 and BCoR in spite of the fact that PHB-2 can interact in solution with E2F1 in OA nuclear extracts. The second lane represents a negative control generated by omission of the primary antibody during the immunoprecipitation (Ab);

(7) FIGS. 6A-C show immunodetection of PHB-1 proteins in human articular cartilage. FIGS. 6A and 6B represent cartilage section obtained from control and OA subjects respectively immunostained with anti-human PHB-1 protein (cat #RB-292-PO, Lab Vision Corp., Fremont Calif., USA). Nuclear signal for PHB-1 was increased significantly in superficial and deep zones (arrow) only in OA cartilage (10 and 40 magnifications). A non specific immunostaining was detected also with the ECM in both groups (normal and OA). (FIG. 6C) Cell scores for PHB-1-nuclear chondrocytes, indicating significant differences between OA and normal (control) cartilage and between the zones for each cartilage type, by Mann-Whitney U test. Values are the meanSEM of normal (n=3) and OA (n=3) specimens;

(8) FIG. 7 (Table S2) shows peptides (SEQ ID NOs: 17-27) sequencing results identifying PHB-1, PHB-2 and BCoR as part of the repressing complex that binds to the pitx1 E2F-like site;

(9) FIG. 8 shows EMSA using total nuclear extract (alone or with PHB-1 antibody (Prohibitin Ab-2 (RB-292-P0) Lab Vision Corp., Fremont, Calif.)) and PHB-1-depleted nuclear extract. The use of a PHB-1 antibody did not allow any supershift but depletion of PHB-1 in OA nuclear extract abrogated all the complexes formation bound to the wild-type radiolabeled probe;

(10) FIG. 9 shows an expression analysis by semi-quantitative RT-PCR of PHB2, PHB-1 and BCoR in human articular chondrocytes (normal vs. OA);

(11) FIG. 10 shows the nuclear localization of PHB-1 in OA patients by immunohistochemistry using an anti-PHB-1 (Santa Cruz, sc-18196);

(12) FIGS. 11A-C. FIG. 11A shows a western blot of PHB-1 expressed in cytoplasm (c) and nucleus (n) of normal and OA human articular chondrocytes. FIG. 11B shows two putative sumoylation sites at proximity of a nuclear export sequence (SEQ ID NO: 68) found in human PHB-1 protein. FIG. 11C shows the two putative sumoylation sites at positions 202 (SEQ ID NO: 69) and 204 (SEQ ID NO: 70);

(13) FIG. 12 presents the nuclear (n) and cytoplasmic (c) protein expression of PHB-1 and Pan-SUMO in articular chondrocytes of OA patients (C73 and C74) and control patient (C75) analyzed by western blot. 3 markers are presented: Lamine (nucleus), GAPDH (cytoplasm) and ATP-Synthase (mitochondria);

(14) FIG. 13 graphically presents the repression of mir20a promoter by corepressors BCoR, PHB-1, PHB-2 and combinations thereof;

(15) FIG. 14 graphically presents the repression of human PITX1 gene promoter promoter by corepressors BCoR, PHB-1, PHB-2 and combinations thereof;

(16) FIGS. 15A-E show the sequence of a 10 kb pitx1 promoter region (SEQ ID NO: 28) and polymorphisms in that pitx1 promoter region between human subjects. The primers used to cover the different amplicons to cover the 10 kb regions are provided in Table 2 below;

(17) FIGS. 16A-B shows human PHB-1 mRNA nucleotide (obtained from gi|6031190|ref|NM_002634.2) (SEQ ID NO: 29) in FIG. 16A, and amino acid sequence (obtained from gi|4505773|ref|NP002625.1) (SEQ ID NO: 30) in FIG. 16B;

(18) FIGS. 17A-B shows human PHB-2 mRNA nucleotide (obtained from >gi|31543548|ref|NM_007273.3) (SEQ ID NO: 31) (FIG. 17A) and amino acid sequence (obtained from gi|6005854|ref|NP_009204.1)(SEQ ID NO: 32) (FIG. 17B); and

(19) FIGS. 18A-C show a sequence comprising 30 bp upstream and 30 bp downstream of the C to T mutation found in the human pitx1 promoter (SEQ ID NO: 1) (FIG. 18A); a nucleotide fragment showing restriction sites surrounding wild-type pitx1 E2F-like site (SEQ ID NO: 16) and (SEQ ID NO: 33) (FIG. 18B); and a nucleotide fragment showing restriction sites surrounding OA mutated human pitx1 E2F-like site (SEQ ID NO: 2) and (SEQ ID NO: 34) (FIG. 18C).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(20) The Applicant observed a correlation between accumulation of PHB-1, PHB-2 and BCoR in the nuclei of articular chondrocytes and OA.

(21) The present invention is illustrated in further details by the following non-limiting examples.

(22) Mice

(23) Pitx1+/ mice were prepared as previously reported (65).

(24) Human Specimens

(25) Human tissues were collected with the consent of patients. The Institutional Ethics Committee Board of Sainte Justine and Notre Dame Hospitals in Montreal, Canada approved the study protocol. All OA (n=58), RA (n=39) and control subjects (n=18) were evaluated by a certified rheumatologist based on the American College of Rheumatology Diagnostic Subcommittee for OA criteria (25). Of those, 93 patients (43 OA, 39 RA and 11 healthy controls) were used for the genetic study. Other patients (OA and controls) were used either for EMSA analysis, expression analysis or immunohistochemistry studies using cartilage sections.

(26) Articular Chondrocyte Cultures

(27) Cartilage was sectioned from the tibial plateaus, rinsed, finely chopped and cells released by enzymatic digestion performed as previously described (26).

(28) The cells were seeded in Falcon culture flasks at high density (10.sup.8 cells per 175 cm.sup.2 flask) and grown to confluence in Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Burlington, Ontario, Canada) containing 10% heated-inactivated foetal calf serum (FCS; Hyclone, Logan, Utah) and 1% penicillin/streptomycin (Gibco BRL). Only first passage cultured cells were used. Expression analysis was performed using RNA prepared from articular cartilage isolated from OA patients and age- and gender-matched control subjects.

(29) Total RNA Isolation and RT-PCR

(30) Total RNA was prepared by phenol/chloroform extraction. For RT-PCR, 2 g of total RNA was reversed transcribed using ThermoScript reverse transcriptase (Invitrogen), and the equivalent of 0.1 g of reverse-transcribed RNA used for PCR reactions. These were carried out in a final volume of 25 l containing 200 micromolar dNTPs, 1.5 mM MgCl2, 10 pM of each primer, and 1 U Pfx DNA-polymerase (Invitrogen). PCR reactions were performed using the following primers and conditions: human pitx1 (960-bp PCR product), forward primer 5-CCCACCTCCATGGACGCCTT-3 (SEQ ID NO: 35); reverse primer 5-GTCAGCTGTTGTACTGGCACGC-3 (SEQ ID NO: 36) (35 cycles: 94 C./45 seconds, 65 C./45 seconds, 68 C./1 minute), human -actin (233-bp PCR product), forward primer 5-GGAAATCGTGCGTGACAT-3 (SEQ ID NO: 37), reverse primer 5-TCATGATGGAGTTGAATGT AGTT-3 (SEQ ID NO: 38) (32 cycles: 94 C./1 minute, 55 C./1 minute, 72 C./1 minute), human PHB2 (730-bp PCR product), forward primer 5-GCCCAGAACTTGAAGGACTT-3 (SEQ ID NO: 39); reverse primer 5-TCTTGCTCAGTGCTTCTCCA-3 (SEQ ID NO: 40) (30 cycles: 94 C./45 seconds, 66 C./45 seconds, 72 C./1 minute), human PHB-1 (546-bp PCR product), forward primer 5-AGTATGTGTGGTTGGGGAAT-3 (SEQ ID NO: 41); reverse primer 5-GCTCGCTCTGTAAGGTCGTC-3 (SEQ ID NO: 42) (30 cycles: 94 C./45 seconds, 65 C./45 seconds, 72 C./1 minute), and human BCoR (640-bp PCR product), forward primer 5-*AAAGAGCCGGATCGCAGG-3 (SEQ ID NO: 43); reverse primer 5-CACCATTGATGTTGAGAGGGC-3 (SEQ ID NO: 44) (35 cycles: 94 C./45 seconds, 72 C./45 seconds, 72 C./1 minute). For quantitative and semi-quantitative analysis, all amplifications were normalized against that of the housekeeping gene -actin. PCR amplified product were separated on 1.5% agarose gel (1.0% for semi-quantitative analysis) and visualized by ethidium bromide staining.

(31) Pitx1 Promoter Sequencing

(32) One hundred (100) ng of genomic DNA was mixed in a final volume of 25 l containing 200 micromolar dNTPs, 1.5 mM MgCl.sub.2, 10 pM of each primer (see Table 2 below for full list of primers used), and 1 U Pfx DNA-polymerase (Invitrogen). PCR reactions were performed using the following conditions (35 cycles: 94 C./30 seconds, 60 C./30 seconds, 68 C./1 minute 20 seconds) and primers:

(33) TABLE-US-00002 TABLE2 PITXPROMOTERPRIMERS PP1 forward 5-CTGTTTGCTCAAGACGCTGA-3 (962bp) primer (SEQIDNO:45) reverse 5-CTCGGCCTCACAAAAGAAAC-3 primer (SEQIDNO:46) PP2 forward 5-TGTCTGCATTCAGGCTGTTC-3 (966bp) primer (SEQIDNO:47) reverse 5-GATTCCCTCCTCGAGTCCTT-3 primer (SEQIDNO:48) PP3 forward 5-CAAGTGAGCTGGATGCTGAA-3 (1039bp) primer (SEQIDNO:49) reverse 5-AGGGAGTGTCCCTTCACAGA-3 primer (SEQIDNO:50) PP4 forward 5-GCTCAGCCATTCTCAGGAAC-3; (1085bp) primer (SEQIDNO:51) reverse 5-GCCATTGTCCCAGTCAAGAT-3 primer (SEQIDNO:52) PP5 forward 5-TCGCGTCAAGAGGGTATTTT-3 (1011bp) primer (SEQIDNO:53) reverse 5-TAGGACCCATGGCTCTACCC-3 primer (SEQIDNO:54) PP6 forward 5-CACGAGTCAGGTGGGAAACT-3 (1098bp) primer (SEQIDNO:55) reverse 5-GACGTCTGCTGCTTTTCTGC-3 primer (SEQIDNO:56) PP7 forward 5-AGGCACGGACTAGCAGGAC-3 (963bp) primer (SEQIDNO:57) reverse 5-ATGCGGACGAAGCCAGAG-3 primer (SEQIDNO:58) PP8 forward 5-TTAGCATTCAGCCCCTCTGT-3 (986bp) primer (SEQIDNO:59) reverse 5-TTCATGAGATGCAGTCAGCAG-3 primer (SEQIDNO:60) PP9 forward 5-ACAACTGGTAGGGGCAACAG-3 (951bp) primer (SEQIDNO:61) reverse 5-TGTGTGGCTTTGGCAAATAA-3 primer (SEQIDNO:62) PP10 forward 5-GCACTGTGCTCCAACTGTGT-3 (990bp) primer (SEQIDNO:63) reverse 5-GGGGGAGTGTTCTTTTCCTT-3 primer (SEQIDNO:64)
Immunohistochemistry Assays

(34) IHC assays for Pitx1 were performed using cartilage sections obtained from 8 OA patients (2 males and 6 females, 5910 years, meanSEM) undergoing total knee replacement, and from 8 donors without a known history of joint disorders. These tissues were obtained post-mortem from knee condyles within 24 h of death (2 males and six females aged 678 years, meanSEM). For IHC assay with PHB-1 antibody, cartilage was obtained from 3 OA patients (618 years, meanSEM) and 3 control patients (5728 years, meanSEM). Tissue specimens were embedded in paraffin, sectioned and examined by IHC for Pitx1 or PHB-1 (and counterstained with hematoxylin for PHB-1 experiments). OA severity was previously evaluated on adjacent sections using the Mankin's histological/histochemical scale (27).

(35) Nuclear Protein Extraction and EMSA

(36) For nuclear protein extraction, cells were rinced with cold 1PBS and scraped in a buffer containing 10 mM hepes (pH 7.9), 1.5 mM MgCl.sub.2, 10 mM KCl, 1% NP.sub.40, 0.5 mM DTT, 1 mM PMSF, 10 /ml of aprotinin, 10 g/ml of leupeptin and 10 g/ml of pepstatin. They were left on ice for 10-20 minute while mixed regularly using a vortex. After a 3 minute centrifugation at 3000 rpm, the pellet was resuspended in a buffer containing 20 mM hepes (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 2 mM PMSF, 10 g/ml of aprotinin, 10 Ug/ml of leupeptin and 10 Ug/ml of pepstatin. After a 20 minute incubation on ice and a 2 minute centrifugation at 12500 rpm, the supernatant was collected.

(37) For the analysis of E2F DNA complexes formed in the presence of OA articular chondrocytes, gel mobility shift reactions were performed essentially as previously described (28). Nuclear extracts (5 g of proteins) were incubated for 30 min at a temperature of between about 20-25 C. in 20 l of a solution containing 20 mM HEPES (pH 7.5), 40 mM KCl, 5% glycerol, 5 mM spermidine, and 100 ng of poly(dA-dT) and 4 g of anti-BCoR antibodies (Abcam, ab5276) were added to specific samples (with a + sign). The following probes were then added and incubated for another 30 min at room T: wild-type 5-CTGTGCCTCACTGGCGGCAGTCCTGCTCAA-3 (SEQ ID NO: 65); and mutant 5-CTGTGCCTCACTGGTGGCAGTCCTGCTCAA-3 (SEQ ID NO: 66). Samples were then separated on 7% native polyacrylamide gels (acrylamide:bis 29:1 in 0.5 Tris-borate EDTA). Gels were run at 180 V for 3 h with a recirculation pump for the buffer and a cooling tank.

(38) In the presence of radiolabeled probe (14 ng/100,000 cpm) a cold competitor was added as indicated. For supershift analysis, the nuclear extracts were pre-incubated 30 min on ice then incubated in presence of 0.2 g of E2Fs antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif., USA; listed in Table 3 below) and incubated on ice for 1 h.

(39) TABLE-US-00003 TABLE 3 LIST OF ANTIBODIES USED Antibodies used for results presented in FIGS. 4A-D: E2F1 (KH95, sc-251 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA E2F3 (C-18, sc-878 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA E2F4 (A20, sc-1082 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA E2F5 (C-20, sc-1083) X Santa Cruz Biotechnology, Santa Cruz, CA, USA E2F6 (K-20, sc-8176 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA DP1 (K-20, sc-610 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA Sp1 (1C6, sc-420 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA Sp3 (D-20, sc-644 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA BCoR (ab5276) Abcam Inc., Camridge, MA, USA RXR (D-20, sc-553 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA Others not shown: E2F1 (H-137, sc-22820 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA E2F2 (L-20, sc-632 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA E2F3 (N-20, sc-879 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA E2F8 (H00079733-M01) Abnova Corporation, Taipei, Taiwan DP-2 (C-20, sc-829 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA Sp1 (H-225, sc-14027 X) Santa Cruz Biotechnology, Santa Cruz, CA, USA

(40) The sequences of the oligonucleotide probes were: E2F-like (wild-type) 5-CTGTGCCTCACTGGCGGCAGTCCTGCTCAA-3 (SEQ ID NO: 65); and E2F-like mutant (OA) site, 5-CTGTGCCTCACTGGTGGCAGTCCTGCTCAA-3 (SEQ ID NO: 66). Samples were then separated on 7% native polyacrylamide gels (acrylamide:bis 29:1 in 0.5 Tris-borate EDTA). Gels were run at 180 V for 3 h with a recirculation pump for the buffer and a cooling tank. For analysis of dissociation rate (off-rate), a 70-fold excess of unlabeled competitor oligonucleotide corresponding to the wild-type E2F site found in human pitx1 promoter was added after 30 min of incubation on ice with either labelled probe. Aliquots from the same binding mixture were taken at different times and frozen on dry ice to stop the reaction prior loading on gel.

(41) Nuclear Complexes Precipitation by DNA Pull Down

(42) Nuclear extracts (300 mg of protein) prepared from cultured human OA articular chondrocytes from knee joints were initially incubated in presence of 25 ml of streptavidin-magnetic beads slurry (BioClone Inc. San Diego, Calif., USA) in order to remove unspecific protein interactions. Following this, pre-cleared nuclear extracts were incubated with 5 mg of biotinylated ds probe (5-CTGTGCCTCACTGGTGGCAGTCCTG CTCAA-3 (SEQ ID NO: 66)) for 2 h at 4 C. with slow agitation in 1EMSA binding buffer. 50 ml of streptavidin-magnetic beads slurry was added and after 1 h of incubation at 4 C. the bound complexes were recovered using a magnetic stand and several washes according to the manufacturer's specifications. The complexes were then detached from the beads in a boiling mixture of NuPAGE 4LDS loading buffer (Invitrogen) and -mercaptoethanol. After centrifugation, the supernatant was loaded on a SDS-PAGE gel and visualized by Coomassie blue staining.

(43) Co-Immunoprecipitation

(44) Nuclear and cytoplasmic extracts obtained from chondrocytes of normal or OA patients were incubated overnight with goat anti-PHB-1 antibodies (Santa Cruz, sc-18196). Immunoprecipitated proteins were collected using protein A-sepharose beads. 50 g of proteins were loaded and run on SDS-PAGE. Proteins were then transferred to a nitrocellulose membrane at 100V for 70 minutes. For blotting, rabbit anti-PHB-1 antibodies (Santa Cruz, sc-28259) were used in a 15:10000 dilution.

(45) Transfection

(46) The Lipofectamine 2000 protocole from Invitrogen (www.invitrogen.com) was followed: All amounts and volumes are given on a per well basis. A DNA (g) to Lipofectamine 2000 (l) ratio of 1:2 to 1:3 was used. Cells were transfected at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity. One day before transfection, 0.5-210.sup.5 cells were plated in 500 l of growth medium without antibiotics so that cells were 90-95% confluent at the time of transfection. For each transfection sample, complexes were prepared as follows: a. DNA was diluted in 50 l of Opti-MEM I Reduced Serum Medium without serum and mixed gently. Lipofectamine 2000 was gently mixed before used, then the appropriate amount was diluted in 50 l of Opti-MEM I Medium and incubated for 5 minutes at room temperature. After the 5 minute incubation, the diluted DNA was combined with diluted Lipofectamine 2000 (total volume=100 l), mixed gently and incubated for 20 minutes at room temperature. The 100 l of complexes were added to each well containing cells and medium. The plate was mixed gently by rocking back and forth. The cells were incubated at 37 C. in a CO.sub.2 incubator for 18-48 hours prior to testing for transgene expression.

(47) Peptide Sequencing Analysis

(48) Bands containing proteins from the DNA pulldown assay and EMSA were cut, washed, sliced, dried and rehydrated in ammonium bicarbonate buffer. Proteins were digested with trypsin and extracted from the gel pieces using 50% acetonitrile and 0.1% formic acid. Samples were evaporated to dryness and resuspended in 3% acetonitrile-0.1% formic acid in a final volume of 20 l.

(49) Mass Spectroscopy Analysis

(50) Samples (10 l volume) were analyzed using a LC-MS/MS system consisting of Agilent 1100 Series nanoflow liquid chromatography system and 1100 Series LC MSD SL ion trap mass spectrometer (Agilent Technologies, Palo Alto, Calif., USA). Peptides were enriched on a Zorbax 300SB-C18 trap column (5 m, 50.3 mm) and separated by reversed phase on a Zorbax 300SB-C18 analytical column (3.5 m, 1500.075 mm, Agilent) with a gradient of 5-90% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. The column eluent was sprayed directly into the mass spectrometer. Spectra were interpreted using Spectrum Mill software (Agilent) and NCBI NR mammalian database.

(51) Extraction Parameters with Spectrum Mill Software

(52) Cysteine modification: carbamidomethylation; sequence tag length >1; mass range of precursor ions: 600.0 to 4000.0 Da; scan time range: 0 to 300 minutes; scans were merged for same precursor m/z: +/15 seconds; +/1.4 m/z.

(53) Chromatin Immunoprecipitation Assays

(54) ChIP experiment was carried out with the ChIP-IT kit (Active Motif, Carlsbad, Calif., USA) ChIP-ITT kit. Briefly, cells were cross-linked with 1% formaldehyde and collected in cell scraping solution. Nuclear extraction was then performed using lysis buffer and total DNA was sheared to an approximate length of 0.3-1.5 kb (Branson Sonifier 450). Chromatin was pre-cleared with Protein G beads and separated as follows: total input, negative control without antibody, and ChIP with either anti-PHB-1 (Santa Cruz), anti-BCoR (Abcam) or anti-E2F1 antibodies (Santa Cruz). After overnight incubation with the appropriate antibody, Protein G beads were added and several washes were carefully done. Chromatin was eluted from the beads using ChIP elution buffer. Cross-linking reversal was done by heating samples at 65 C. for 4 h, and DNA was purified using columns provided in the kit. PCR analysis was performed using the following primers and conditions: human pitx1 promoter harbouring the E2F site (372 bp PCR product), forward primer 5-GCTCAGCCATTCTCAGGAAC-3 (SEQ ID NO: 51); reverse primer 5-CCACCTACCTCTTTCTGCCT-3 (SEQ ID NO: 67) (35 cycles: 94 C./45 seconds, 68 C./45 seconds, 68 C./1 minute).

(55) Statistical Analysis

(56) The 95% confidence intervals for sensitivity, specificity, positive predictive value and negative predictive value presented in Table 4 were calculated according to Deeks and Altman (29). The association between the presence of mutation and diagnosis was assessed using the Fisher's Exact Test (two-tailed test). A p value <0.05 was considered statistically significant.

Example 1

Comparison of Pitx1 Expression in Articular Chondrocytes of OA Subjects with that of in Articular Chondrocytes Matched Controls

(57) To determine whether pitx1 plays a role in the genetic control of OA onset, an expression analysis of pitx1 gene using RNA prepared from articular chondrocyte cultures derived from knee cartilage of OA patients (n=7) and age- and gender-matched control subjects (n=4) was performed. Pitx1 expression was detected only in articular chondrocytes derived from matched controls, while in OA articular chondrocytes, Pitx1 expression was abrogated or barely detectable by RT-PCR (FIG. 2A). Analysis of Pitx1 protein levels and distribution in human knee joint sections showed Pitx1 proteins only in control cartilages (n=8), while Pitx1 proteins were hardly detected in OA cartilage sections (n=8) (FIG. 2B-D).

Example 2

Identification of Pitx1 Promoter Mutation

(58) To examine the mechanisms turning off pitx1 gene expression in OA patients, the 5 regulatory region of human pitx1 gene was examined for specific mutations leading to a progressive loss of Pitx1 expression during adulthood. Sequencing analysis of genomic DNA obtained from OA, rheumatoid arthritis (RA) and matched control subjects revealed, along a 10 kb promoter region of human pitx1 gene, a single homozygous mutation (3727 C.fwdarw.T) (position corresponds to distance from transcription point) affecting only OA patients (11/43) with a high frequency (25%) while none of the RA patients (0/29) and matched control subjects (0/11) had the homozygous mutation. The specificity, the positive predictive values and negative predictive values of the mutation were calculated for each group as reported in Table 4 below. A statistically significant association between the mutation and diagnosis was calculated (two-tailed test) by comparing OA versus RA patients (p=0.002) or by combining RA and control subjects (p<0.001). Heterozygous mutation was present in OA patients (3/43), RA subjects (6/29) and control subjects (3/11) although the association between the heterozygous mutation and diagnosis was not statistically significant (p=0.14 for OA versus RA and p=0.09 for OA versus control).

(59) TABLE-US-00004 TABLE 4 SENSITIVITY, SPECIFICITY, POSITIVE PREDICTIVE VALUE* (PPV) AND NEGATIVE PREDICTIVE VALUE** (NPV) OF PITX1 mutation (3727 C.fwdarw.T) for OA Mutation Sensitivity Specificity PPV NPV Homozygous 25.6 (13.8-39.5) 100.0 (71.5-100.0) 100.0 (71.5-100.0) 25.6 (13.8-39.5) OA-Control 100.0 (88.1-100.0) 100.0 (71.5-100.0) 47.5 (35.2-60.0) OA-RA 100.0 (97.6-100.0) 100.0 (71.5-100.0) 55.6 (44.0-66.8) OA-(Control & RA) Heterozygous 7.0 (1.4-16.4) 72.7 (39.0-94.0) OA-Control 79.3 (60.3-92.0) OA-RA 77.5 (63.4-88.9) OA-(Control & RA)

(60) Promoter sequence analysis performed with the ALGGEN PROMO search program (using TRANSFAC version 8.3; (35, 36) revealed that the homozygous mutation was localized within the core of an E2F-like site (10, 11). The sequence encompassing the E2F-like site found upstream of the human pitx1 gene is more distal although it shows an overlap with several functional E2F sites and also with an adjacent region termed cyclin homology region (CHR) found in promoter of genes involved in the regulation of cell cycle 12 (FIG. 3). The transcriptional relationship between pitx1 and E2Fs is further strengthened by the works of Muller et al. showing that E2F1, E2F2 and E2F3 up regulate Pitx1 expression by several fold in osteosarcoma cell line U2OS (13). To date, eight different mammalian E2Fs have been cloned; each of them can heterodimerize with either DP1 or DP2/3 proteins and bind with similar affinity to the same collection of target sites (14). These factors could be broadly divided into two classes: activators (E2F1-3) and repressors (E2F4-8) of transcription (15), which could explain why E2F sites on a promoter do not always indicate that it is induced by E2F but, on the contrary, that it can be repressed through those sites as well (16-18).

Example 3

Determination of Functional Consequences of Mutation in the E2F-like Site on Complex Binding

(61) To determine the functional consequences of the homozygous mutation found in OA patients, it was investigated whether E2Fs were able to bind this E2F-like site using nuclear extracts prepared with OA articular chondrocytes as described above. EMSA analysis using both radiolabeled E2F-like sites (wild-type FIG. 4A versus mutant FIG. 4B) showed no supershift of the bound complex with any antibodies against E2Fs, or their dimerization partners DP-1 or DP-2 (E2F2, E2F8 and DP2 data not shown). The Sp1 and Sp3 transcription factors were also analysed since they bind GC-rich regions such as the E2F-like site found in the human Pitx1 promoter. Unfortunately, there was no supershift with either anti-Sp1 or anti-Sp3 antibodies. Addition of BCoR antibodies generated the binding of an additional lower complex bound in presence of either probes although the binding was increased with the mutant one as is mostly apparent by comparing lanes 4 to 6. Functional analysis by EMSA revealed a slower dissociation rate when the complex was bound to the mutant E2F probe indicating that C.fwdarw.T mutation increased the stability of the bound complex.

Example 4

Identification of BCoR as a Member of the Pitx1 Repressor Complex

(62) In order to characterize the nature of the complex identified in Example 3, bands corresponding to the bound complex were cut and extracted from an EMSA wet gel to perform a peptide sequencing analysis combined with tandem mass spectrometry.

(63) This experiment led to the identification of peptides corresponding to BCoR, a known co-repressor (see FIG. 7). The presence of BCoR in the bound complex was confirmed also by the detection of an additional lower band in EMSA when BCoR antibodies were added (see Example 3 and FIG. 4A-B).

Example 5

Identification of PHB-1 and PHB-2 as Members of the Pitx1 Repressor Complex

(64) A DNA-pull down method as described above was used with a biotinylated double-stranded oligonucleotide harboring the mutant E2F-like site to allow the identification of peptides corresponding to prohibitin (PHB-1) and prohibitone (PHB-2) (FIG. 7). To confirm the presence of PHB-1 in the bound complex, an immuno-depletion of PHB-1 in nuclear extracts prepared from OA articular chondrocytes was performed and no binding was observed in EMSA with PHB-1 depleted nuclear extracts using either radiolabeled probes (FIG. 8).

Example 6

Identification of the PHB-1 and PHB-2 Nucleus Localization in OA and Normal Articular Chondrocytes

(65) The cellular localization of PHB-1 was compared in normal and OA articular chondrocytes by IHC assays using anti-PHB-1 antibodies. As may be seen in FIG. 6A-C, an increased nuclear localization of PHB-1 was observed in OA cartilages when compared to age-matched control cartilages. See also FIG. 10 for a larger magnification using different anti-PHB-1 antibodies. The cellular localization of PHB-2 is also compared in normal and OA articular chondrocytes by IHC assays using anti-PHB-2 antibodies.

Example 7

Co-immunoprecipitation Assay of PHB-1, PHB-2 and E2F1

(66) Co-immunoprecipitation assay of PHB-1, PHB-2 and E2F1 were performed as described above. Physical interactions between PHB-1, PHB-2 and E2F1 were detected in nuclear extracts prepared from human OA articular chondrocytes while controls did not show any interaction with PHB-2 (FIG. 5A).

Example 8

Chip Assay to Demonstrate the Functionality of the EF2 Site in OA Chondrocytes and Co-occupancy of PHB-1 and BCoR

(67) Chromatin immunoprecipitation (ChIP) assays were performed as described above with primary OA articular chondrocytes cultures to examine the co-occupancy of PHB-1, BCoR and E2F1 on the E2F-responsive element identified in human pitx1 promoter. PHB-1 and BCoR co-localised in vivo only in OA chondrocytes confirming also the functionality and specificity of this E2F site although E2F1 was not detected (FIG. 5B). The interaction is however not dependent on the 3727 C.fwdarw.T mutation since wild-type also interacts with the repressor complex.

Example 9

Identification of the 3727 C to T Homozygous Mutation in a Subject Sample

(68) Genomic DNA is isolated (directly using available commercial kits or after extraction of lymphocytes) from the subject saliva or blood. The human pitx1 promoter region harbouring the 3727 C.fwdarw.T homozygous mutation is then amplified by PCR using specific primers. Amplified products are identified by direct sequencing using genomic DNA extracted from lymphocytes. They may also be digested with either restriction enzyme Acil or Fnu4H to detect the presence of the mutation. Because replacement of C by T in OA patients abrogates Acil and Fnu4HI restriction sites normally present in the wild-type sequence, the presence of the mutation is assessed by absence of relevant restriction fragments.

Example 10

Assay to Identify Blood and Synovial Concentration Levels of PHB-1, PHB-2 or Native Peptide Agonists to Cell Surface PHB-1 in Subjects

(69) PHB-1 and PHB-2 are also found in the bloodstream (Mishra et al. 2006). Diet-induced obesity was successfully reversed in mice through subcutaneous injection of a chimeric proapoptotic peptide that binds to PHB-1 at the cell surface of white fat vasculature (57). The peptide cut off the blood supply to fat tissue leading to its reabsorption. Most of the OA patients are obese. Dosage of PHB-1 in biological fluids like plasma/serum, urine or synovial fluids are performed using an ELISA method (see for instance 58).

Example 11

Assay to Identify RNA Levels of PHB-1, PHB-2 and BCoR Between Normal and OA Human Articular Chondrocytes

(70) Expression analysis was performed as described above by semi-quantitative RT-PCR of PHB-2, PHB-1 and BCoR in human articular chondrocytes (normal vs. OA). RNA was extracted from 4 normal patients and 10 OA patients. No significant change was observed between normal and OA articular chondrocytes as may be seen in FIG. 9.

Example 12

Detection of PHB-1 Immunoreactive Bands in Nuclear and Cytoplasm of Human Articular Chondrocyte Fractions from OA Subjects

(71) Co-immunoprecipitation of PHB-1 was performed as described above.

(72) The detection using an anti-PHB-1 antibody revealed (FIG. 11A) a major PHB-1 immunoreactive band of 32 kDa, which corresponds to the molecular weight of the PHB-1 alone. PHB-1 immunoreactive bands of higher molecular weight suggest post-translational modifications of PHB-1 modifying its weight.

(73) On FIG. 11A, a higher molecular weight bands pattern is detected mostly in the nuclear fraction of OA patients. This pattern is not found in the nuclear fraction of the control subject.

Example 13

Comparison of Sumoylation of PHB-1 Between Normal and OA Human Articular Chondrocytes

(74) Sumoylation is the binding of one or more small proteins of 12 kDa, designated SUMO, to another protein. SUMO proteins will lead to a laddering profile of the protein to which they are binding in a western blot. The hypothesis that the laddering of PHB-1 immunoreactive bands observed in FIG. 11A, was caused by sumoylation was thus tested.

(75) SUMOsp (SUMOsp: a web server for sumoylation site prediction. Yu Xue, Fengfeng Zhou, Chuanhai Fu, Ying Xu, and Xuebiao Yao. Nucl Acids Res 34: W254-W257, 2006.) predicted that PHB-1 contains two putative sumoylation sites at proximity of a nuclear export sequence (NES), namely at positions 204 and 240. FIG. 11B presents the position of these putative sites and the sequence of the NES (SEQ ID NO: 68). The table in FIG. 11C presents the sequences of these sites, namely 204 (SEQ ID NO: 69) and 240 (SEQ ID NO: 70).

(76) Total proteins of nuclear and cytoplasmic fractions from articular chondrocytes of OA patients (C73 and C74) and age-matched control patient (C75) were then analyzed by western blot against PHB-1 and Pan-SUMO. They were loaded and run on SDS-PAGE. Proteins were then transferred to a PVDF membrane at 100V for 70 minutes. The 3 markers used, namely lamine (nucleus), GAPDH (cytoplasm) and ATP-Synthase (mitochondria), show that the samples have been separated efficiently into a nuclear fraction (X-N) and a cytoplasmic fraction (Cyto). These antibodies were used for blotting: anti-PHB-1 (Santa Cruz, sc-28259), Pan SUMO antibodies (ABGENT, AP1299a), anti-Lamin A/C (Cell Signaling, #2032), anti-GAPDH (Santa Cruz, sc-20357) and anti-F1-ATPase (Santa Cruz, sc-16689).

(77) Pan-SUMO immunoreactive bands in FIG. 12 reveal proteins (different proteins or a single protein) modified by the binding of one or more SUMO proteins, as reflected by bands with increasing molecular weights.

(78) FIG. 12 shows that the two OA subjects have the same sumoylation pattern in the nuclear fraction, and that this pattern is absent from the control subject. Arrows (except for the 32 kDa bands) point to bands that are immunoreactive to both PHB-1 and PAN SUMO, and are detected in the nuclear fraction of OA subjects but not in the control nuclear fraction. These results suggest that sumoylation contributes to PHB-1 nuclear localization in OA patients.

(79) Clinically, this suggests that increased sumoylation is the primary event leading to PHB-1 nuclear accumulation and repression of Pitx1 in primary OA. The biological consequences of sumoylation include the increase of protein stability, increase targeting of proteins (including transcription factors) from the cytoplasm to the nucleus, regulates transcriptional activities of proteins, mediates the binding of the protein to other proteins and increases the repressor activity of certain transcription factors. Recent studies linked sumoylation of several proteins to important diseases (neurodegenerative diseases, acute promyelocytic leukemia, type I diabetes and other disorders). The regulation of these postranslational modifications may provide new targets for therapeutic intervention in several human diseases.

(80) PHB-1 is a molecule having a molecular weight of less than 50 kDa and can passively penetrate the cell nucleus. In normal subjects, PHB-1 is quickly exported out of the cell nucleus through the exportin system by the recognition of a nucleus export signal (NES). Without being bound by such hypothesis, the proximity of the 2 sumoylation sites to the NES, could contribute to the nuclear retention of PHB-1 in OA subjects by hiding the NES and thereby prevent the recognition PHB-1 by exportin.

Example 14

Determination of Repression of mir20a Promoter by Co-Repressors PHB-1, PHB-2, BCoR and Combinations Thereof

(81) MG-63 cells were transfected as described above with Lipofectamine 2000 during 6 hours. After the transfection, fresh media was added to cells during 24 hours and 10.sup.7 M 4-OH-Tamoxifen was added to cells during 24 hours.

(82) The luciferase assay was then performed. Results are presented in FIG. 13. Results are expressed by the fold induction of luciferase signal of reporter construct compared to that of a control. The control (CTRL) luciferase activity was that measured in osteoblastic MG-63 cells co-transfected with mir20A reporter promoter, known to be regulated by E2Fs, from Sylvestre Y et al. 2007, pBAPE-ER and pLPC NEP-Flag vectors. The E2F1 was the condition where cells were co-transfected with E2F1-ER over-expressing vector. All luciferase signal was normalized by a beta-gal reporter vector. The X-axis represents the names of each repressor co-overexpressed in the conditions tested. This transient transfection assay shows that BCoR alone or in combinations with PHB-1 (designated PHB in FIG. 13) and/or PHB-2 is sufficient to repress the induction of an E2F reporter construct like mir20a even in the presence of the mir20a agonist E2F1.

Example 15

Determination of Repression of PITX1 Promoter by Corepressors PHB-1, PHB-2, BCoR and Combinations Thereof

(83) MG-63 cells were transfected as described above with a vector containing 3000 or 2000 bp of PITX1 gene promoter upstream of a firefly luciferase gene. Cells were synchronised 16 h by serum starvation. Fresh serum was added during 48 h and the luciferase assay was performed. The luciferase signal was normalised against beta-gal. Results are expressed by the fold induction of luciferase signal compared to a luciferase signal from an empty vector. Results are presented in FIG. 14. These results show the repression driven when BCoR, PHB-1 and PHB2 are combined together using a fragment of human pitx1 promoter (fragment 3000 bp). This demonstrates the repressor activity of this complex. Without being bound by this hypothesis, it is suggested that more than one E2F-like sites may be present on the PITX1 gene.

Example 16

Analysis of Prohibitin-CRM-1 Interaction in Normal and OA Articular Chondrocytes

(84) PHB-1 physical interaction with CRM-1 is measured by co-IP assays as described above, while Western blot with anti-CRM-1 antibodies (Santa Cruz) are performed with nuclear extracts prepared with normal and OA articular chondrocytes to assess changes in CRM-1 levels in normal versus OA cartilage cells. In parallel, functionality of CRM-1 is tested by an immunostaining method. Cells are either untreated or treated with 30 M camptothecin (Sigma) for 4 h, a topoisomerase I inhibitor that stimulates the nuclear export of PHB-1 through a CRM-1-dependent mechanism. Subcellular localisation of p53 is used as internal control since p53 normally co-localises with PHB-1 and migrate to the cytoplasm upon camptothecin treatment. Cells are fixed in 4% PFA for 5 min and blocked in 5% BSA in PBS buffer at RT for 1 h, followed by primary antibody incubations overnight at 4 C. After washing, secondary antibody incubation is performed with goat anti anti-mouse IgG Alexa Fluor-488 (green) and goat anti-rabbit IgG Alexa Fluor-546 (red) for 30 min at room temperature. Cells are visualised with a Zeiss LSM 510 confocal microscope. Finally, the contribution of TGF- signalling to PHB-1 nuclear export is investigated since it was reported that TGF-1 mediates the nuclear export of PHB-1 in a prostate cancer cell line.

Example 17

OA Genetic Association Studies

(85) Genotyping of the (3727 C.fwdarw.T) found in the promoter of human pitx1 gene is performed in a large cohort that currently comprises 1,400 cases (510 males and 890 females) and 750 controls (350 females and 400 males) well characterised. The cases have each undergone elective joint-replacement surgery (hip or knee) due to severe, end-stage primary OA. The controls have no symptoms of OA or of any other joint or musculoskeletal disease. The cases and controls are all aged 45 or over, they are unrelated to each other and are of UK Caucasian origin. The DNA from the cases and the controls are arrayed on to 96-well microtitre plates at concentrations of 100 ng/l and 10 g/l. Results are analysed in function of differences in genotype or allele frequencies between OA cases and controls. The cases are stratified by sex, age, by joint replaced or by sex combined with joint replaced or age combined with joint replaced.

(86) Genetic association, and Hardy-Weinberg equilibrium for the distribution of genotypes, are tested by X.sup.2 analysis with Yates's correction. Odds ratios are calculated with 95 percent confidence intervals. The pair wise linkage disequilibrium coefficient (r2).sup.Z is calculated using the GOLD program (59). Haplotype frequencies between variants showing evidence of linkage disequilibrium at r2>0.2 is estimated using the EH-PLUS program (60). Haplotype frequency differences are then compared using X.sup.2.

Example 13

Characterisation of Nuclear Factors Known to Interact with PHB-1 and/or PHB-2

(87) Additional partners of PHB-1 and PHB-2 are expected to exist since there are no indications in the literature that either molecule can bind directly to the DNA to exert their transcriptional repression. It was shown that prohibitin recruits Brg-1/Brm to E2F-responsive promoters, and that this recruitment is required for the repression of E2F-mediated transcription by PHB-1. Although PHB-1 associates with, and recruits, Brg-1 and Brm independently of pRb, prohibitin/Brg-1/Brm-mediated transcriptional repression requires pRb. PHB-1 and PHB-2 mediated transcriptional repression required also histone-deacetylase activity (HDAC1), but unlike pRb, additional co-repressors like N-CoR are also involved (24,41). In addition, PHB-2 also associates with the class II histone deacetylase HDAC5. Finally, it was reported that PHB-2 specifically interacts with the chicken ovalbumin upstream binding transcription factors I and II (COUP-TFI and COUP-TF-II). The nuclear receptor chicken ovalbumin upstream binding transcription factor I was found to cooperate with PHB-2 and histone deacetylases in the repression of target genes. PHB-1 and PHB-2 thus appear to repress E2F-mediated transcription utilising different molecular mediators and facilitate channelling of specific signalling pathways to the cell cycle machinery.

(88) Whether pitx1 repression is mediated by recruitment of Brg-1/Brm to this E2F site is investigated. The contribution of specific histone deacetylases (HDACs) is also investigated. To test whether Brg-1, Brm and prohibitin physically interact in OA nuclear extracts, co-immunoprecipitation studies are carried out using nuclear extracts from OA articular chondrocytes, which contain PHB-1 and PHB-2 endogenously. Immunoprecipitation are then performed with anti-cmyc (negative control), anti-Brg-1 or anti-Brm antibodies, and the precipitated proteins are then immunoblotted for PHB-1 or PHB-2. Normal articular chondrocytes obtained from control subjects or purchased (PromoCell) are tested in parallel as negative controls. Positive controls are generated with the use of anti-E2F1, anti-E2F2 and anti-E2F3 antibodies since E2F1 and E2F3 mRNA were detected in normal and OA articular chondrocytes.

(89) The same approach is used to determine whether additional known partners of PHB-1 and PHB-2 are present in the repressor complex detected in OA patients (the candidate proteins are indicated in Table 1 above). Notably, the presence of pRb or of one of its family members (p107 and p130) are investigated since prohibitin/Brg-1/Brm-mediated transcriptional repression was shown to require pRb. To assess whether PHB-1 co-localises in vivo with Brg-1 and Brm to the E2F-responsive element found in the human pitx1 promoter, chromatin immunoprecipitation (ChIP) assays, using anti-Brg-1 and anti-Brm antibodies are performed. ChIP assays are performed with OA articular chondrocytes derived from at least ten distinct OA patients according to the manufacturer's specifications (ActiveMotif, Carlsbad Calif., USA). Articular chondrocyte cultures are obtained from distinct OA patients in order to investigate whether these factors and repressing mechanism are predominantly found only in a specific subset of OA patients or they are conserved among all primary OA patients (excluding secondary knee joint OA). Presence of the mutation is determined by direct sequencing of the promoter region harbouring the E2F-like site for each OA and control subjects to assess whether the mutation seen in human pitx1 promoter has effects in the recruitment of different nuclear interacting partners. Whether pRb or one of its family members (p107 and p-130) is also recruited in vivo with Brg-1 and Brm is investigated. Control experiments is carried out in parallel with normal human articular chondrocytes using anti-E2F1, anti E2F-2 and anti-E2F3 antibodies as positive controls to demonstrate that this E2F site is also subjected to a positive transcriptional regulation by E2Fs in normal cartilage cells. Negative controls will be provided by anti-PHB-1 and anti-PHB-2 antibodies.

Example 18

Determination of Mechanisms Modulating Prohibitins Interactions with Brg-1/Brm in OA

(90) Several reports showed that JNK1 promotes the association of PHB-1 with Brg-1 or Brm on E2F-responsive promoters and represses the transcriptional activity of E2F. The finding that JNK1 phosphorylates PHB-1 (in vitro) and regulates prohibitin/Brg1/Brm associations in vivo now adds a testable potential mechanistic link to this pathway. Furthermore, PHB-1 interacts with MLK2, a binding partner of, activator of, and substrate for JNK1. To determine whether JNK1 could affect the physical association between PHB-1 and Brg-1/Brm, co-IP methods are used with normal and OA articular chondrocyte cultures untreated or treated with 100 M of SP600125, a specific inhibitor of JNK1 for two hours (Stressgene Bioreagents, Ann Arbor, Mich., USA). The effect of JNK1 on the recruitment of PHB-1, PHB-2, Brg-1 and Brm to the endogenous and mutant E2F site in the human pitx1 promoter is then tested by ChIP assays.

(91) Akt subcellular localisation is compared in normal and OA chondrocytes by IHC methods as well as by Western blot analysis with corresponding cytoplasmic and nuclear extracts. Detection of Akt and phospho-Akt (pAkt) is preformed with specific antibodies (Santa Cruz). In the event that cytoplasmic Akt is predominantly detected in OA cells, Akt phosphorylation is stimulated by adding physiological doses of 17--estradiol (10.sup.10M, Sigma) since estrogenic stimulation was reported to promote accumulation of activated Akt in the nucleus (61).

(92) Additional PHB-1 and PHB-2 interacting partners such as HDAC1 and HDAC5 are tested while others are determined by DNA-pull down method. The analysis of the proteins complexed is carried-out by peptide sequencing coupled to mass spectrometry as described above. The role of estrogens in Akt activation and nuclear localisation is of interest in OA pathogenesis because postmenopausal women are more affected by OA than males after 65 years.

Example 19

Determination of Post-translational Modifications Masking/interfering with NES of PHB-1

(93) There is evidence that PHB-1 and PHB-2 can undergo post-translational modification such as phosphorylation and ubiquitination, but the role of post-translational modifications in modulating the various functions of the prohibitins has yet to be determined. In-silico analysis revealed that PHB-1 NES can be phosphorylated by tyrosine kinases MTOR, PAK, EGFR and JAK. The effect of individual kinases is tested by adding specific kinase inhibitors to the cells prior to analysing PHB-1 interaction with CRM-1 as described above.

(94) Whether the NES of PHB-1 is a substrate for an unknown kinase or any other enzymatic action reducing its interaction with CRM-1 is also tested. A peptide corresponding to the NES of PHB-1 is synthesized and conjugated to a carrier peptide, Penetratin (Q-Biogene), for delivery into the cells (cytoplasm and nucleus). The effect of delivering this peptide on the nuclear export of PHB-1 is assessed by double immunofluorescence experiments. Conformation changes in PHB-1-PHB-2 heterodimers or homodimers are also examined by cross-linkage analyses and Western blot.

(95) Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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