Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors,integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf-/- mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.

Laminin-5, a major adhesive ligand for epithelial cells, undergoes processing of its ␥2 and ␣3 chains. This study investigated the mechanism of laminin-5 processing by keratinocytes. BI-1 (BMP-1 isoenzyme inhibitor-1), a selective inhibitor of a small group of astacin-like metalloproteinases, which includes bone morphogenetic protein 1 (BMP-1), mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mammalian Tolloid-like 2 (mTLL-2), inhibited the processing of laminin-5 ␥2 and ␣3 chains in keratinocyte cultures in a dose-dependent manner. In a proteinase survey, all BMP-1 isoenzymes processed human laminin-5 ␥2 and ␣3 chains to 105-and 165-kDa fragments, respectively. In contrast, MT1-MMP and MMP-2 did not cleave the ␥2 chain of human laminin-5 but processed the rat laminin ␥2 chain to an 80-kDa fragment. An immunoblot and quantitative PCR survey of the BMP-1 isoenzymes revealed expression of mTLD in primary keratinocyte cultures but little or no expression of BMP-1, mTLL-1, or mTLL-2. mTLD was shown to cleave the ␥2 chain at the same site as the previously identified BMP-1 cleavage site. In addition, mTLD/BMP-1 null mice were shown to have deficient laminin-5 processing. Together, these data identify laminin-5 as a substrate for mTLD, suggesting a role for laminin-5 processing by mTLD in the skin.Proteolysis of the extracellular matrix is emerging as a key mechanism in processes such as wound healing and tumor metastasis (1, 2). Although most studies have investigated the role of serine proteases and matrix metalloproteases, members of the astacin and ADAM (a disintegrin and metalloprotease) families have also been implicated in this process (1, 2). Laminin-5, the major component of epithelial basement membranes, is a heterotrimeric protein consisting of ␣3, ␤3, and ␥2 subunits (3, 4). Laminin-5 undergoes extracellular proteolysis of the ␣3 chain from a 200-to a 165-kDa form and of the ␥2 chain from a 155-to a 105-kDa form (5). Through its interaction with ␣ 3 ␤ 1 (6, 7), ␣ 6 ␤ 4 (8), and ␣ 2 ␤ 1 integrins (9), laminin-5 supports epithelial cell adhesion (3, 10), and migration (11, 12).Several proteases have been implicated in laminin-5 processing. Exogenous addition of matrix metalloprotease 2 (MMP-2) 1 cleaved the ␥2 subunit of rat laminin-5 (12). A subsequent study suggested that membrane type 1 matrix metalloprotease (MT1-MMP) may play a role in cleaving laminin-5 (13). Cleavage of laminin-5 by plasmin converted the ␣3 chain into the 165-kDa form observed in human breast and rat epithelial cells and capable of nucleating hemidesmosomes (14). Bone morphogenetic protein 1 (BMP-1) has also been implicated in laminin-5 proteolysis. N-terminal sequencing of the 105-kDa ␥2 chain obtained from human keratinocytes revealed a cleavage site that matched the minimal consensus sequence of this metalloprotease (15). In vitro studies demonstrated that BMP-1 cleaved the recombinant ␥2 short arm at the predicted site and that the enzyme cleaved both the ␣3 and ␥2 chains of whole laminin-5 to generate characterist...

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Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase, a hydrolytic, bacterial enzyme with an unusual substrate specificity for synthetic organophosphate triesters and phosphorofluoridates, which are common constituents of chemical warfare agents and agricultural pesticides. No natural substrate has been identified for phosphotriesterase, and it has been suggested that the enzyme may have evolved the ability to hydrolyze synthetic compounds in bacteria under selective pressure to meet nutritional needs. PHP, which has 28% sequence identity with phosphotriesterase, may belong to the family of proteins from which phosphotriesterase evolved. Here we report the cloning, expression, initial characterization, and high-resolution X-ray crystallographic structure of PHP. Biochemical analysis shows that PHP is monomeric and binds two zinc ions per monomer. Unlike phosphotriesterase, PHP does not catalyze the hydrolysis of nonspecific phosphotriesters. The structure, similar to that of phosphotriesterase, consists of a long, elliptical alpha/beta barrel and has a binuclear zinc center in a cleft at the carboxy end of the barrel at the location of the presumptive active site.

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