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J. Biol. Chem., Vol. 283, Issue 9, 5928-5938, February 29, 2008

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HtrA1 Inhibits Mineral Deposition by Osteoblasts

REQUIREMENT FOR THE PROTEASE AND PDZ DOMAINS^*

Kristen D. Hadfield¹², Claire Farrington Rock¹, Colette A. Inkson, Sarah L. Dallas, Laure Sudre, Gillian A. Wallis, Raymond P. Boot-Handford, and Ann E. Canfield^¶3

From the Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences and ^¶Cardiovascular Research Group, School of Clinical and Laboratory Sciences, Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9PT, United Kingdom and the Department of Oral Biology, School of Dentistry, University of Missouri, Kansas City, Missouri 64108

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.

Received for publication, November 13, 2007

^* This work was supported by grants from the Arthritis Research Campaign and the Wellcome Trust (to A. E. C. and R. B. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Medical Genetics, Faculty of Medical & Human Sciences, University of Manchester, Manchester M13 0JH, UK.

³ To whom correspondence should be addressed: University of Manchester, Michael Smith Bldg., Oxford Road, Manchester, M13 9PT, UK. Tel.: 44-161-275-5066; Fax: 44-161-275-5082; E-mail: ann.canfield{at}manchester.ac.uk.

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