Gregg A. Hastings

Cathepsin K, but Not Cathepsins B, L, or S, Is Abundantly Expressed in Human Osteoclasts

Random high throughput sequencing of a human osteoclast cDNA library was employed to identify novel osteoclast-expressed genes. Of the 5475 ESTs obtained, approximately 4% encoded cathepsin K, a novel cysteine protease homologous to cathepsins S and L; ESTs for other cathepsins were rare. In addition, ESTs for cathepsin K were absent or at low frequency in cDNA libraries from numerous other tissues and cells. In situ hybridization in osteoclastoma and osteophyte confirmed that cathepsin K mRNA was highly expressed selectively in osteoclasts; cathepsins S, L, and B were not detectable. Cathepsin K was not detected by in situ hybridization in a panel of other tissues. Western blot of human osteoclastoma or fetal rat humerus demonstrated bands of 38 and 27 kDa, consistent with sizes predicted for pro- and mature cathepsin K. Immunolocalization in osteoclastoma and osteophyte showed intense punctate staining of cathepsin K exclusively in osteoclasts, with a polar distribution that was more intense at the bone surface. The abundant expression of cathepsin K selectively in osteoclasts strongly suggests that it plays a specialized role in bone resorption. Furthermore, the data suggest that random sequencing of ESTs from cDNA libraries is a valuable approach for identifying novel cell-selective genes.

METH-1, a Human Ortholog of ADAMTS-1, and METH-2 Are Members of a New Family of Proteins with Angio-inhibitory Activity

We have studied two related proteins that contain a repeated amino acid motif homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (TSP1). Complete sequence analysis revealed no other similarities with TSP1, but identified unique signal sequences, as well as metalloprotease and disintegrin-like domains in the NH2 termini. We named these proteins METH-1 and METH-2 due to the novel combination of metalloprotease andthrombospondin domains. Overall amino acid sequence identity between METH-1 and METH-2 is 51.7%, yet transcript distribution revealed non-overlapping patterns of expression in tissues and cultured cell lines. To characterize these proteins functionally, we isolated full-length cDNAs, produced recombinant protein, and generated antisera to the recombinant proteins. Both METH-1 and METH-2 represent single copy genes, which encode secreted and proteolytically processed proteins. METH proteins suppressed fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited vascular endothelial growth factor-induced angiogenesis in the chorioallantoic membrane assay. Suppression of vessel growth in both assays was considerably greater than that mediated by either thrombospondin-1 or endostatin on a molar basis. Consistent with an endothelial specific response, METH-1 and METH-2 were shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth. We propose that METH-1 and METH-2 represent a new family of proteins with metalloprotease, disintegrin, and thrombospondin domains. The distinct distribution of each gene product suggests that each has evolved distinct regulatory mechanisms that potentially allow for fine control of activity during distinct physiological and pathological states.

A novel regulatory function of proteolytically cleaved VEGF-2 for vascular endothelial and smooth muscle cells.

By high throughput sequencing, we have identified a cDNA encoding a polypeptide related to vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF) in the VEGF/PDGF gene family. It is designated vascular endothelial growth factor 2 (VEGF‐2). Similar to VEGF, expression of VEGF‐2 mRNA is abundant in vascular smooth muscle cells and several highly vascularized tissues. VEGF‐2 protein is expressed as a secreted 52 kDa precursor as well as the 30 kDa ammo‐terminal and 27 kDa carboxy‐terminal cleavage products. The latter two cleavage products are linked via a disulfide bridge (or bridges) and can be copurified. Using copurified 30 and 27 kDa proteins, the effect of VEGF‐2 on growth of several cell types, including vascular endothelial and smooth muscle cells, was determined. Our results demonstrate that VEGF‐2 protein stimulates the growth of human vascular endothelial cells but inhibits growth of human aortic smooth muscle cells induced by platelet‐derived growth factor. These studies establish VEGF‐2 as a novel regulator for growth of vascular endothelial and smooth muscle cells.—Hu, J.‐S., Hastings, G. A., Cherry, S., Gentz, R., Ruben, S., Coleman, T. A. A novel regulatory function of proteolytically cleaved VEGF‐2 for vascular endothelial and smooth muscle cells. FASEB J. 11, 498–504 (1997)

Identification and characterization of three cDNAs that encode putative novel hyaluronan-binding proteins, including an endothelial cell-specific hyaluronan receptor.

The glycosaminoglycan hyaluronan (HA) and HA-binding proteins (HABPs) serve important structural and regulatory functions during development and in maintaining adult tissue homeostasis. Here we have identified and partially characterized the sequence and expression pattern of three putative novel HABPs. DNA sequence analysis revealed that two of the novel HABPs, WF-HABP and BM-HABP, form a unique HA-binding subfamily, whereas the third protein, OE-HABP, is more closely related to the LINK subfamily of HABPs. Northern blotting experiments revealed that the expression of BM-HABP was highly restricted, with substantial expression detected only in human fetal liver. In contrast, WF-HABP and OE-HABP mRNAs were detected in a number of tissues, with particularly prominent expression in highly vascularized tissues such as the heart, placenta, and lung. Additional studies showed that OE-HABP was expressed by cultured human endothelial cells, smooth muscle cells, and differentiated monocytes. However, only endothelial cells expressed WF-HABP mRNA, and its expression was regulated by growth state, being most prominent in quiescent endothelial cells. We further characterized the expression of WF-HABP in vivo and found that its expression colocalized with CD31-positive cells and was prominently expressed in microvessels in the human aorta and in atherectomy samples. Our data suggest that WF-HABP is an endothelial cell-specific HA receptor and that it may serve a unique function in these cells. The WF-HABP gene was localized to chromosome 3p21.31 and the OE-HABP gene to 15q25.2-25.3.