HtrA1 inhibits mineral deposition by osteoblasts - Requirement for the protease and PDZ domains
HtrA1 inhibits mineral deposition by osteoblasts - Requirement for the protease and PDZ domains
复制标题
DOI：
10.1074/jbc.m709299200
复制
发表时间：
2008-02-29
影响因子：
4.8
通讯作者：
Canfield, Ann E.
中科院分区：
文献类型：
--
作者：
Hadfield, Kristen D.;Rock, Claire Farrington;Canfield, Ann E.
HtrA 1是一种具有丝氨酸蛋白酶活性的分泌型多结构域蛋白。鉴于越来越多的证据表明这种蛋白质参与骨骼发育和病理学的调节，我们研究了HtrA 1在成骨细胞矿化中的作用，并确定了这种活性所必需的结构域。我们证明了在矿化出现之前，分化的2 T3成骨细胞中HtrA 1表达增加。HtrA 1随后在完全矿化的培养物中下调。使用三种互补的方法研究了HtrA 1在基质钙化中的功能作用。首先，我们将全长HtrA 1表达质粒转染到2 T3细胞中，结果表明HtrA 1的过表达延迟了矿化，减少了Cbfa 1和I型胶原mRNA的表达，并阻止了BMP-2诱导的矿化。第二，使用短干扰RNA敲低HtrA 1表达诱导2 T3细胞的矿物质沉积。第三，通过表达一系列重组HtrA 1蛋白，我们证明了蛋白酶结构域和PDZ结构域是HtrA 1对成骨细胞矿化的抑制作用所必需的。最后，我们测试了HtrA 1是否切割已知调节成骨细胞分化、矿化和/或BMP-2活性的特异性基质蛋白。全长重组HtrAl切割重组核心蛋白聚糖、纤连蛋白和基质Gla蛋白。蛋白酶结构域和PDZ结构域都是切割基质Gla蛋白所必需的，而PDZ结构域不是切割核心蛋白聚糖或纤连蛋白所必需的。I型胶原不被重组HtrA 1裂解。这些结果表明，HtrA 1可能通过抑制BMP-2信号传导，调节成骨细胞基因表达，和/或通过降解特定的基质蛋白来调节基质钙化。
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.