The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro␣1(XXVII) chain has a domain structure similar to that of the type B clade chains (␣1(V), ␣3(V), ␣1(XI), and ␣2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro␣1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240 -110 kDa in tissue extracts and collagenous bands of 150 -140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro␣ chains.Fibril-forming or fibrillar collagens are one of the most ancient families of extracellular matrix molecules being found throughout the metazoan kingdom from the simplest (porifera (sponges)) to the most complex animals (vertebrates). Fibrillar collagens form major structural elements in extracellular matrices as diverse as the evolutionarily "primitive" mesoglea of cnidarians (1) to the highly specialized connective tissues of vertebrates (e.g. bone, cartilage, skin, and tendon) (2). Molecular features shared by all members of this family include a highly conserved C-terminal noncollagenous (NC1) domain and a long collagenous domain of ϳ1000 amino acid residues.Phylogenetic analyses have revealed that the previously known vertebrate fibrillar collagens fall into two related but distinct groups or clades (3, 4). The type A clade consists of the pro␣1(I), pro␣2(I), pro␣1(II), pro␣1(III), and pro␣2(V), whereas the type B clade contains the remaining chains encoding types V and XI collagens (with the exception of pro␣3(XI), which is derived from the COL2A1 gene). The division of the vertebrate fibrillar collagens into two clades is supported by two further observations. Firstly, the exon structures of the genes are virtually identical within a clade yet distinct between clades (5). Secondly, the members of each clade share homologous Nterminal noncollagenous domains (von Willebrand factor type C domain for type A and TSPN 1 for type B clade members) with the exception of the pro␣2(I) chain, where the N-terminal noncollagenous domain appears to have been deleted (6).We have recently described how the members of the two clades of vertebrate fibrillar collagens have apparently arisen early during vertebrate evolution from a single ...

HtrA1 (high-temperature requirement protein A1) is a secreted multidomain protein with proven serine protease activity and the ability to regulate TGF-beta (transforming growth factor-beta)/BMP (bone morphogenetic protein) signalling. There is increasing evidence that HtrA1 regulates several pathological processes, including tumour development, Alzheimer's disease, age-related macular degeneration and osteoarthritis, although the mechanism(s) by which it regulates these processes have not been fully elucidated. Using overexpression and knock-down strategies, we have evidence demonstrating that HtrA1 is also a key regulator of physiological and pathological matrix mineralization in vitro. We propose that HtrA1 regulates mineralization by inhibiting TGF-beta/BMP signalling and/or by cleaving specific matrix proteins, including decorin and MGP (matrix Gla protein). Taken together, these studies suggest that HtrA1 may be a novel therapeutic target for several diseases.

HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.Mammalian HtrA1 is a member of the family of HtrA (high temperature requirement) proteins that were originally identified in bacteria (1). These proteins are characterized by the presence of a trypsin-like serine protease domain and either one or two PDZ domains. However, in contrast to bacterial HtrA, mammalian HtrA1 is secreted, and it also contains an IGFBP 4 /mac25-like domain and a Kazal-type inhibitor domain at the N terminus (2, 3). Evidence is now accumulating to suggest that HtrA1 is involved in the development and progression of several pathologies. HtrA1 has been found to exhibit properties of a tumor suppressor protein, as its expression is downregulated in many cancer cell lines (2, 4), low levels of HtrA1 are detected in cancerous tissue compared with normal tissues (4, 5), and overexpression of this protein inhibits tumor cell growth in vivo and in vitro (4, 5). HtrA1 expression is also markedly up-regulated in several diseases, including rheumatoid arthritis and osteoarthritis (3, 6 -8), Alzheimer disease (9), and Duchenne muscular dystrophy (10). In addition, a single-nucleotide polymorphism in the gene encoding HtrA1 has been shown to increase susceptibility to age-related macular degeneration (...