ADAMTS3 and ADAMTS14 Le Goff, Carine; Apte, Suneel S.
The ADAM Family of Proteases
Book Chapter
ADAMTS3 and ADAMTS14 belong to the procollagen aminopropeptidase subfamily of ADAMTS proteases that also includes ADAMTS2. These enzymes appear to have co-evolved with their substrates, the major ...fibrillar collagen types I, II and III by gene duplication from a primitive precursor. Mutations in ADAMTS2 cause an inherited connective tissue disorder, named dermatosparaxis or the Ehlers-Danlos syndrome type VIIC. Compensatory procollagen processing by ADAMTS3 and ADAMTS14 may explain why this disorder has its most severe manifestation in skin, whereas other collagen-containing tissues are relatively unaffected.
To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in ...chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.
OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1beta and/or TNFalpha. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1beta and/or TNFalpha. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1beta-stimulated OUMS-27 cells was investigated.
IL-1beta increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1beta stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1beta and TNFalpha had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1beta-stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up-regulation in OUMS-27 cells.
ADAMTS9 is an IL-1beta- and TNFalpha-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis.
The cell proliferation-associated nuclear antigen recognized by the Ki-67 monoclonal antibody was detected immunohistochemically in cells from skeletal tissues of humans, sheep and rabbits. In each ...case the distribution of Ki-67-positive cells was identical and consistent with the known distribution of dividing cells in growing long bones. The antigen recognized by Ki-67 is detectable only after fixation in cold acetone. In the 5-day-old rabbit, labelling with tritiated thymidine ( 3HTdR) in vivo, followed subsequently by a combination of Ki-67 immunohistochemistry and autoradiography for 3HTdR, demonstrated that, in addition to the expression of Ki-67 by the majority of 3HTdR labelled cells, other cells associated with proliferating populations also expressed the Ki-67 antigen. This suggests that the Ki-67 antibody identifies cycling cells in the skeleton in S-phase, as well as in other phases of the cell cycle. In Ki-67 reactive species, this method is a potentially useful tool for the study of cell proliferation in skeletal tissues in vivo and in vitro, having the advantage of not requiring prior labelling with DNA precursors.
Type X collagen, a homotrimer of alpha 1 (X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have ...previously characterized a genomic clone containing the major part of the mouse type X collagen gene (col10a1) and assigned the locus for col10a1 to mouse chromosome 10 (Apte et al., Eur. J. Biochem. 224: 217-224, 1992). In this paper, through additional characterization of cDNA and genomic clones, we describe the complete organization of the col10a1 gene. The col10a1 gene is 7.2 kb in size and, as in the chick, col10a1 gene transcripts are generated from only three exons. Exon 1 encodes only the 5' untranslated (5' UT) region of the mRNA and is separated from exon 2 by an intron 562 bp in length. Exon 2 (169 bp) encodes 15 bp of 5' UT message, the translation start plus 17-amino acid residues of the putative signal peptide, and 33 1/3 codons of the putative N-terminal non-collagenous (NC2) domain of type X collagen. Exon 3 is 2854 bp in size and encodes 4 2/3 codons of the NC2 domain, the entire collagenous domain of 463 residues (COL), the entire C-terminal non-collagenous (NC1) domain (161 amino acid residues) and the entire 965 bp 3' untranslated (3' UT) sequence of the mRNA. Two TATA boxes are present in tandem in the col10a1 promotor. Both TATA boxes are active in transcription, generating two populations of transcripts with different 5'-termini; the longer transcript is of low abundance and is detectable only by PCR in newborn mice. The col10a1 promotor contains a CCAAT box as well as other consensus sequence elements required for binding of potential transcription factors. Characterization of the col10a1 gene provides data essential for studies of the regulation of type X collagen expression during mammalian endochondral bone growth and development. Knowledge of the complete structure of the mouse type X collagen gene will also be useful for the investigation of type X collagen gene abnormalities in murine chondrodysplasias and for the generation of transgenic mice.
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