Steven M. Ruben

Bone morphogenic protein

Pathogenesis of multiple myeloma is associated with an aberrant expression of pro-proliferative, pro-angiogenic and bone-metabolism-modifying factors by malignant plasma cells. Given the frequently long time span from diagnosis of early-stage plasma cell dyscrasias to overt myeloma and the mostly low proliferation rate of malignant plasma cells, we hypothesize these to similarly express a novel class of inhibitory factors of potential prognostic relevance. Bone morphogenic proteins (BMPs) represent possible candidates as they inhibit proliferation, stimulate bone formation and have an effect on the survival of cancer patients. We assessed the expression of BMPs and their receptors by Affymetrix DNA microarrays (n=779) including CD138-purified primary myeloma cell samples (n=635) of previously untreated patients. BMP6 is the only BMP expressed by malignant and normal plasma cells. Its expression is significantly lower in proliferating myeloma cells, myeloma cell lines or plasmablasts. BMP6 significantly inhibits the proliferation of myeloma cell lines, survival of primary myeloma cells and in vitro angiogenesis. A high BMP6 expression in primary myeloma cell samples delineates significantly superior overall survival for patients undergoing high-dose chemotherapy independent of conventional prognostic factors (International Staging System (ISS) stage, β2 microglobulin).

Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli.

During passage through the epididymis, sperm interact with secreted epididymal proteins that promote maturation, including the acquisition of motility and fertilization competence. Viewed previously as distinct from sperm maturation, host defence capabilities are now recognized functions of the human epididymis 2 (HE2) family of sperm-binding proteins. We analysed the potent dose and time-dependent bactericidal activity of recombinant HE2alpha, HE2beta1 and HE2beta2 and found that the full-length proteins (10 microg/ml or approximately 1 microM) caused more than a 50% decrease in Escherichia coli colony forming units within 15 min. By contrast, human beta-defensin-1, at a similar concentration, required more than 90 min to exhibit similar antibacterial activity. The epididymis-specific lipocalin, LCN6, failed to kill bacteria. Higher concentrations (25-100 microg/ml) of HE2 proteins and a longer duration of treatment resulted in near total inhibition of bacterial growth. The C-terminal peptides of HE2alpha, HEbeta1 and HEbeta2 proteins exhibited antibacterial activity similar to their full-length counterparts, indicating that the antibacterial activity of HE2 proteins resides in these C-terminal regions. Antibacterial activities of HE2 proteins and peptides were slightly inhibited by NaCl concentrations of up to 150 mM, while human beta-defensin-1 activity was nearly eliminated. Reduction and alkylation of disulphide bonds in HE2 proteins and their C-terminal peptides abolished their antibacterial activity. Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic E. coli membranes. A much longer exposure time was required for human beta-defensin-1-mediated permeabilization of membranes, suggesting a possible difference in mode of action compared with the HE2 antibacterial peptides.

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)