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The Journal of the American Academy of Orthopaedic Surgeons 10/2010; 18(10):638-41. · 2.66 Impact Factor
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ABSTRACT: Matrix metalloproteinases (MMP)-13 activity is necessary for normal skeletal development and plays a central role in cartilage degeneration associated with osteoarthritis (OA). The studies we described here examine the interactions of the hemopexin domain of MMP-13 with proteins secreted by human chondrocytes in culture. The hemopexin domain of the MMPs and many other proteins in which this structure is found mediates protein function by forming the primary site of interaction with other proteins. We have modified a tandem affinity expression tag (hTAP) to enable efficient expression of the tagged bait protein. In this case the MMP-13 C-terminal domain (CTD) comprises hinge and hemopexin domain, and we immobilized the fusion construct on a column of agarose bound immunoglobin G. The MMP-13 CTD affinity column so generated enabled the efficient and gentle isolation of interacting proteins from the culture medium of human articular chondrocytes. TIMP1 and alpha2-macroglobulin previously shown to interact with MMP-13 as well as several proteins, fibronectin, type VI collagen and xylosyltransferase 1 and several proteoglycans, decorin, syndecan 4 and serglycin not previously recognized as interacting with MMP-13 were identified by mass spectrometry. The interaction between isolated proteins and MMP-13 CTD was verified by yeast two hybrid analysis. We also demonstrated serglycin expression by chondrocytes for the first time and its co localization with MMP-13 in a cytoplasmic granular morphology. The consequence of these interactions remains to be demonstrated, however; binding to MMP-13 suggests a role in the regulation of cartilage degradation.
Connective tissue research 06/2010; 51(3):230-9. · 1.55 Impact Factor
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The Journal of the American Academy of Orthopaedic Surgeons 01/2010; 18(1):59-62. · 2.66 Impact Factor
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ABSTRACT: Transforming and insulin-like growth factors are important in regulating bone mass. Thus, one would anticipate correlations between matrix concentrations of growth factors and functional properties of bone. We therefore investigated the relationships of (1) TGF-beta2 and (2) IGF-I matrix concentrations with the trabecular microstructure, stress distribution, and mechanical properties of tibial cancellous bone from six male human cadavers. Trabecular stress amplification (VMExp/sigma(app)) and variability (VMCOV) were calculated using microcomputed tomography (muCT)-based finite element simulations. Bone volume fraction (BV/TV), surface/volume ratio (BS/BV), trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), connectivity (Eu.N), and anisotropy (DA) were measured using 3-D morphometry. Bone stiffness and strength were measured by mechanical testing. Matrix concentrations of TGF-beta2 and IGF-I were measured by ELISA. We found higher matrix concentrations of TGF-beta2 were associated with higher Tb.Sp and VMExp/sigma(app) for pooled data and within subjects. Similarly, a higher matrix concentration of IGF-I was associated with lower stiffness, strength, BV/TV and Tb.Th and with higher BS/BV, Tb.Sp, VMExp/sigma(app) and VMCOV for pooled data and within subjects. IGF-I and Tb.N were negatively associated within subjects. It appears variations of the stress distribution in cancellous bone correlate with the variation of the concentrations of TGF-beta2 and IGF-I in bone matrix: increased local matrix concentrations of growth factors are associated with poor biomechanical and architectural properties of tibial cancellous bone.
Clinical Orthopaedics and Related Research 06/2009; 467(12):3079-86. · 2.53 Impact Factor
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ABSTRACT: Like other members of the transforming growth factor beta (TGF-beta) family of growth factors, the biological activity of TGF-beta2 is believed to be regulated by the formation and dissociation of multiprotein complexes. To isolate the molecular complex formed by TGF-beta2 secreted by hypertrophic chondrocytes we have used expression of TGF-beta2 fused with the humanized, tandem affinity purification (hTAP) tag and mass spectrometry for the identification of interacting proteins. The hTAP synthetic gene was assembled by systematically replacing the rare codons of the original TAP tag with codons most preferred in highly expressed human genes to circumvent the poor translation efficiency of the original TAP tag in animal cells. TGF-beta2 was shown to interact with Type X collagen and this interaction confirmed using V5 tagged TGF-beta2. Functional interaction was suggested by the inhibition of TGF-beta2 activity by type X collagen in culture and the influence of a mutation in type X collagen on the distribution of TGF-beta2 in growth cartilage.
Journal of Chromatography B 11/2008; 875(2):493-501. · 2.89 Impact Factor
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ABSTRACT: Membrane-type 1 matrix metalloproteinase (MT1-MMP) is the most ubiquitous and widely studied of the membrane-type metalloproteinases (MT-MMPs). It was thus surprising to find no published data on chicken MT1-MMP. We report here the characterization of the chicken gene. Its low sequence identity with the MT1-MMP genes of other species, high GC content, and divergent catalytic domain explains the absence of data and our difficulties in characterizing the gene. The absence of structural features in the chicken gene that have been suggested to be critical for the activation of MMP-2 by MT1-MMP; for the effect of MT1-MMP on cell migration and for the recycling of MT1-MMP suggest these features are either not essential or that MT1-MMP does not perform these functions in chickens. Comparison of the expression of chicken MT1-MMP with MT3-MMP and with MMP-2 and MMP-13 has confirmed the previously recognized co-expression of MT1-MMP with MMP-2 and MMP-13 in fibrous and vascular tissues, particularly those surrounding the developing long bones in other species. By contrast, MT3-MMP expression differs markedly from that of MT1-MMP and of both MMP-2 and MMP-13. MT3-MMP is expressed by chondrocytes of the developing articular surface. Similar expression patterns of this group of MT-MMPs and MMPs have been observed in mouse embryos and suggest distinct and specific functions for MT1-MMP and MT3-MMP in skeletal development.
Cell and Tissue Research 08/2008; 333(1):81-90. · 3.11 Impact Factor
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Matrix Biology. 01/2008; 27:60-60.
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The Journal of the American Academy of Orthopaedic Surgeons 08/2006; 14(7):445-6. · 2.66 Impact Factor
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