Faculty Bibliography

Mutations in human cartilage oligomeric matrix protein (COMP) have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia, however, the function of both wild-type and mutant COMP in the skeletogenesis remain unknown. In an effort to define the biological functions of COMP, a functional genetic screen based on the yeast two-hybrid system was performed. This led to the identification of Granulin-epithelin precursor (GEP), an autocrine growth factor, as a COMP-associated partner. COMP directly binds to GEP both in vitro and in vivo, as revealed by in vitro pull down and Co-immunoprecipitation assays. GEP selectively interacts with the EGF repeat domain of COMP but not with the COMP's other three functional domains. The Granulin A repeat unit of GEP is required and sufficient for association with COMP. COMP co-localizes with GEP predominantly in the pericellular matrix of transfected rat chondrosarcoma cell (RCS) and primary human chondrocytes. Staining of musculoskeletal tissues of day 19 mouse embryo with antibodies to GEP is restricted to chondrocytes and appears to be concentrated in areas where ossification will occur. Over expression of GEP stimulates the proliferation of chondrocytes and this stimulation is enhanced by COMP. In addition, COMP appears to be required for GEP-mediated chondrocyte proliferation, since chondrocyte proliferation induced by GEP is dramatically inhibited by an anti-COMP antiserum. These findings provide the first evidences linking the association of COMP and GEP, and identifying a previously unrecognized growth factor (i.e. GEP) in cartilage

Loss of articular cartilage due to extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme (s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the EGF-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis

Among 10 adult mouse tissues tested, the p204 protein levels were highest in heart and skeletal muscle. We described earlier that the MyoD-inducible p204 protein is required for the differentiation of cultured murine C2C12 skeletal muscle myoblasts to myotubes. Here we report that p204 was also required for the differentiation of cultured P19 murine embryonal carcinoma stem cells to beating cardiac myocytes. As shown by others this process can be triggered by dimethylsulfoxide (DMSO). We established that DMSO induced the formation of 204RNA and p204. Ectopic p204 could partially substitute for DMSO in inducing differentiation, whereas ectopic 204 antisense RNA inhibited the differentiation. Experiments with reporter constructs including regulatory regions from the Ifi204 gene (encoding p204) in P19 cells and in cultured newborn rat cardiac myocytes, as well as chromatin coimmunoprecipitations with transcription factors, revealed that p204 expression was synergistically transactivated by the cardiac Gata4, Nkx2.5 and Tbx5 transcription factors. Furthermore ectopic p204 triggered the expression of Gata4 and Nkx2.5 in P19 cells. p204 contains a Nuclear Export Signal (NES) and was partially translocated to the cytoplasm during the differentiation. p204 from which the NES was deleted was not translocated, and it did not induce differentiation. The various mechanisms by which p204 promoted the differentiation are reported in an accompanying article

We reported in an accompanying article that (i) the p204 protein is required for the differentiation of murine P19 embryonal carcinoma stem cells to beating cardiac myocytes, and (ii) the expression of p204 in the differentiating P19 cells is synergistically transactivated by the cardiac transcription factors Gata4, Nkx2.5 and Tbx5. Here we report that endogenous or ectopic Id (Inhibitor of differentiation) proteins inhibited the differentiation of P19 cells to myocytes. This was in consequence of the binding of Id1, Id2 or Id3 protein to the Gata4 and Nkx2.5 proteins and the resulting inhibition (i) of the binding of these transcription factors to each other and to DNA, as well as (ii) of their synergistic transactivation of the expression of various genes including atrial natriuretic factor and Ifi204 (encoding p204). p204 overcame this inhibition by Id proteins in consequence of (i) binding and sequestering Id proteins, (ii) accelerating their ubiquitination and degradation by proteasomes, and (iii) decreasing the level of Id proteins in the nucleus by increasing their translocation from the nucleus to the cytoplasm. (ii) and (iii) depended on the presence of the Nuclear Export Signal in p204. In the course of the differentiation Gata4, Nkx2.5 and p204 were components of a positive feedback loop. This arose in consequence of it that p204 overcame the inhibition of the synergistic activity of Gata4 and Nkx2.5 by the Id proteins

Degradative fragments of cartilage oligomeric matrix protein( )(COMP) have been observed in arthritic patients. The physiological( )enzyme(s) that degrade COMP, however, remain unknown. We performed( )a yeast two-hybrid screen (Y2H) to search for proteins that( )associate with COMP to identify an interaction partner that( )might degrade it. One screen using the epidermal growth factor( )(EGF) domain of COMP as bait led to the discovery of ADAMTS-7.( )Rat ADAMTS-7 is composed of 1595 amino acids, and this protein( )exhibits higher expression in the musculoskeletal tissues. COMP( )binds directly to ADAMTS-7 in vitro and in native articular( )cartilage. ADAMTS-7 selectively interacts with the EGF repeat( )domain but not with the other three functional domains of COMP,( )whereas the four C-terminal TSP motifs of ADAMTS-7 are required( )and sufficient for association with COMP. The recombinant catalytic( )domain and intact ADAMTS-7 are capable of digesting COMP in( )vitro. The enzymatic activity of ADAMTS-7 requires the presence( )of Zn(2+) and appropriate pH (7.5-9.5), and the concentration( )of ADAMTS-7 in cartilage and synovium of patients with rheumatoid( )arthritis is significantly increased as compared to normal cartilage( )and synovium. ADAMTS-7 is the first metalloproteinase found( )to bind directly to and degrade COMP.--Liu, C., Kong, W., Ilalov,( )K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., Di( )Cesare, P. E. ADAMTS-7: a metalloproteinase that directly binds( )to and degrades cartilage oligomeric matrix protein.( )

A novel gene therapy approach for treating damaged cartilage is proposed that involves placing endotoxin-free cDNA containing the gene for bone morphogenetic protein-2 (BMP-2) in type I collagen sponges and then transferring the naked plasmid DNA construct to the injury site. A full-thickness cartilaginous defect in rabbits implanted with plasmid containing a marker gene (beta-galactosidase) showed expressed protein as detected by immunostaining. At 1 week postimplantation, mesenchymal cells subjacent to the defect had incorporated the implanted naked plasmid DNA and, once transfected, served as local bioreactors, transiently producing the gene product. Plasmids containing the gene for BMP-2 implanted in collagen sponges in cartilage lesions stimulated hyalinelike articular cartilage repair at 12 weeks postimplantation, nearly equivalent in quality to that induced by collagen sponges with recombinant BMP-2 protein. Our approach circumvents the risks of inflammation and immunogenic response associated with the use of viral vectors. Naked plasmid DNA as a vehicle for transferring therapeutic genes has been shown to be effective in a therapeutic model within rabbit articular cartilage and appears to be safe and cost effective.

BACKGROUND: The erythrocyte sedimentation rate, the C-reactive protein serum level, and the white blood-cell count are routinely used to diagnose periprosthetic infection. In the present study, the diagnostic accuracy of the interleukin-6 serum level was compared with the accuracy of these standard tests for the evaluation of a group of patients who had had a total hip or total knee arthroplasty and were undergoing a reoperation for the treatment of an infection or another implant-related problem. METHODS: A prospective, case-control study of fifty-eight patients who had had a total hip or knee replacement and were undergoing a reoperation because of an infection (seventeen patients) or another implant-related problem (forty-one patients) was conducted. The serum levels of interleukin-6 and C-reactive protein, the erythrocyte sedimentation rate, and the white blood-cell count were measured. The definitive diagnosis of an infection was determined on the basis of positive histopathological evidence of infection and growth of bacteria on culture of intraoperative specimens. Two-sample Wilcoxon rank-sum (Mann-Whitney) tests were used to determine the presence of a significant difference between patients with and without infection with regard to each laboratory value studied. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of each text were also calculated. RESULTS: The serum interleukin-6 level, erythrocyte sedimentation rate, and C-reactive protein level were significantly higher in patients who had an infection than in those who did not, both when all patients were considered together and when the total hip arthroplasty and total knee arthroplasty groups were considered separately. With the numbers available, there was no significant difference with regard to the white blood-cell count between patients with and without infection. With a normal serum interleukin-6 level defined as <10 pg/mL, the serum interleukin-6 test had a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 1.0, 0.95, 0.89, 1.0, and 97%, respectively. CONCLUSIONS: An elevated serum interleukin-6 level correlated positively with the presence of periprosthetic infection in patients undergoing a reoperation at the site of a total hip or knee arthroplasty. The serum interleukin-6 level is valuable for the diagnosis of periprosthetic infection in patients who have had a total hip or total knee arthroplasty. LEVEL OF EVIDENCE: Diagnostic Level IV. See Instructions to Authors for a complete description of levels of evidence

The voltage-gated sodium channel Na(v)1.8 produces a tetrodotoxin-resistant current and plays a key role in nociception. Annexin II/p11 binds to Na(v)1.8 and facilitates insertion of the channel within the cell membrane. However, the mechanisms responsible for removal of specific channels from the cell membrane have not been studied. We have identified a novel protein, clathrin-associated protein-1A (CAP-1A), which contains distinct domains that bind Na(v)1.8 and clathrin. CAP-1A is abundantly expressed in DRG neurons and colocalizes with Na(v)1.8 and can form a multiprotein complex with Na(v)1.8 and clathrin. Coexpression of CAP-1A and Na(v)1.8 in DRG neurons reduces Na(v)1.8 current density by approximately 50% without affecting the endogenous or recombinant tetrodotoxin-sensitive currents. This effect of CAP-1A is blocked by bafilomycin A1 treatment of transfected DRG neurons. CAP-1A thus is the first example of an adapter protein that links clathrin and a sodium channel and may regulate Na(v)1.8 channel density at the cell surface