HtrA1 Inhibits Mineral Deposition by Osteoblasts

Abstract

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 (...

LRP1 Protects the Vasculature by Regulating Levels of Connective Tissue Growth Factor and HtrA1

“…This enzyme, high-temperature requirement factor A1 (HtrA1) is a secreted serine protease that degrades several matrix components including decorin, fibronectin, aggrecan, type II collagen 30,31 and impairs elastogenesis by cleaving fibulin 5 30 . Interestingly, while HtrA1 protein levels were increased in smLRP1-/- aortic extracts, mRNA levels for htra1 were not altered in these mice (Fig 4b).…”

Role of HTRA1 in bone formation and regeneration: In vitro and in vivo evaluation

“…However, it wasn’t until several years later that evidence emerged of a possible functional role for HTRA1 in bone formation. Studies performed using mouse-derived osteoblasts demonstrated that despite its upregulation in response to BMP-2, HTRA1 acted as a negative regulator of bone formation [29, 30]. Moreover, the observation that Htra1 -deficient mice have a moderately improved bone phenotype [31], suggested that HTRA1’s influence on bone may go beyond simply affecting mineral formation in vitro .…”

“…In support of this, HTRA1 protein has been detected in developing bones in vivo , as well as in fracture callus [9, 15, 19]. However, in contrast to these findings, several studies now exist in which HTRA1 has been demonstrated to impart a negative influence over osteogenesis [29, 30]. Although the cause of these conflicting results remains unclear, it is important to note that inherent differences exist between the cell culture systems used in each of these studies, and may therefore indicate that cell specific effects of HTRA1 need to be taken into account.…”

The Autolysis of Human HtrA1 Is Governed by the Redox State of Its N-Terminal Domain

“…29 The N- and C-terminal domains of HtrA1 comprise an IGFBP-like/Kazal-like tandem domain motif (termed the N-domain) and a PDZ domain, respectively. Both seem largely dispensable for catalytic function, 30 and whereas the PDZ domain facilitates protein–protein interactions by tethering of the HtrA1 protease to specific sites in the ECM or in the cell, 4,26,31 the role of the N-domain remains enigmatic. The domain contains a total of 16 cysteines, all forming part of an intensive disulfide bond network (Figure 1).…”