title: HtrA1 inhibits mineral deposition by osteoblasts: requirement for the protease and PDZ domains.
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.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
authors.forename: Kristen D
authors.initials: KD
authors.lastname: Hadfield
authors.forename: Claire Farrington
authors.initials: CF
authors.lastname: Rock
authors.forename: Colette A
authors.initials: CA
authors.lastname: Inkson
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Dallas
authors.forename: Laure
authors.initials: L
authors.lastname: Sudre
authors.forename: Gillian A
authors.initials: GA
authors.lastname: Wallis
authors.forename: Raymond P
authors.initials: RP
authors.lastname: Boot-Handford
authors.forename: Ann E
authors.initials: AE
authors.lastname: Canfield
mesh_terms: D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002113:Calcification, Physiologic / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D016326:Extracellular Matrix Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; D012697:Serine Endopeptidases