Samir K. Mandal

Modulation of Tissue Factor–Factor VIIa Signaling by Lipid Rafts and Caveolae

Objective—Coagulation factor VIIa (VIIa) binding to its cellular receptor, tissue factor (TF), not only initiates the coagulation cascade but also induces cell signaling by activating G-protein coupled protease-activated receptors. The objective of the present study is to investigate the role of lipid rafts and caveolae in modulating TF-VIIa signaling and coagulant functions. Methods and Results—TF-VIIa coagulant function was measured in factor X activation assay and the signaling function was evaluated in phosphoinositide hydrolysis and IL-8 gene induction. Buoyant density gradient centrifugation and immunofluorescence confocal microscopy were used to determine cellular localization of TF and protease-activated receptor 2. The data show that a substantial fraction of TF and protease-activated receptor 2 resides in lipid rafts/caveolae, and disruption of lipid rafts by cholesterol depletion or modification reduced TF-VIIa–induced cell signaling. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. Conclusion—Overall our data show that lipid raft/caveolae play a selective role in modulating the TF-VIIa signaling function without affecting the TF-VIIa coagulant activity.

A novel mechanism of plasmin-induced mitogenesis in fibroblasts.

Summary.  The plasminogen activator/plasmin system is believed to play an important role in diverse pathophysiological processes, including wound healing, vascular remodeling and pulmonary fibrosis. Our recent studies show that plasmin upregulates the expression of Cyr61, a growth factor‐like gene that has been implicated in cell proliferation and migration. In the present study, we investigated whether plasmin promotes fibroblast proliferation and, if so, determine the role of Cyr61 in the plasmin‐induced response. Human lung fibroblasts were exposed to varying concentrations of plasmin and DNA synthesis was monitored by measuring the incorporation of 3H‐thymidine into DNA. Plasmin increased DNA synthesis of fibroblasts in a dose‐dependent manner. Protease‐activated receptor‐1 (PAR‐1)‐specific antibodies, but not PAR‐2‐specific antibodies, reduced the plasmin‐induced DNA synthesis. Consistent with this, plasmin had no substantial effect on the DNA synthesis in PAR‐1‐deficient mouse fibroblasts. Plasmin activated both p38 and p44/42 MAPKs and specific inhibitors of these pathways inhibited the plasmin‐induced DNA synthesis. Plasmin‐induced increase in the DNA synthesis was completely abrogated by anti‐Cyr61 antibodies. Interestingly, thrombin, which is a potent inducer of Cyr61, had only a minimal effect on fibroblast proliferation. Additional experiments suggested that plasmin cleaved cell/extracellular matrix‐associated Cyr61 and the conditioned media from plasmin‐treated cells could support the cell proliferation. Overall, these data suggest that plasmin promotes fibroblast proliferation by a novel pathway, involving two independent steps. In the first step, plasmin induces Cyr61 expression via activation of PAR‐1, and in the second step, plasmin releases Cyr61 deposited in the extracellular matrix, thus making it accessible to act on cells.

Human placental protein/peptides stimulate melanin synthesis by enhancing tyrosinase gene expression

Placental protein/peptides as biological response modifier are well documented, but not much known about melanogenesis. We possibly for the first time, demonstrated melanogenesis in B16F10 mouse melanoma by a placental protein/peptide fraction (PPPF) prepared from a hydroalcoholic extract of fresh term human placenta. This study described the effect of PPPF on the induction of tyrosinase; the key enzyme of melanogenesis to investigate the basis of PPPF induced pigmentation in primary melanocyte and B16F10 melanoma. Tyrosinase induction by PPPF in B16F10 cells was found dose- and time dependent at the level of activity. Tyrosinase, at the level of transcription and protein expression when assessed by RT-PCR and Western blot analyses found to have considerable induction over untreated control. PPPF led to enhanced activation of tyrosinase promoter resulting higher transcription thus substantiating the role of PPPF as a stimulator of melanogenesis. Actinomycin D, the transcriptional inhibitor of protein synthesis, blocked the stimulatory action of PPPF since the induction of tyrosinase and melanin was markedly reduced in presence of this inhibitor. Thus the results suggested that PPPF mediated increase in tyrosinase expression occurred through transcriptional upregulation to stimulate melanogenesis in B16F10 cells and in primary melanocyte also. (Mol Cell Biochem xxx: 1–10, 2004)