Publications

The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca2+]i, myosin activation, and migration were quantified using standard techniques. [Ca2+]i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca2+. Removal of extracellular Ca2+ did not alter endothelin-1-stimulated force development or [Ca2+]i. Ionomycin, a Ca2+ ionophore, triggered an increase in [Ca2+]i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca2+]i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca2+ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca2+]i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca2+]i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology.

The C-type natriuretic (CNP) peptide signals through the type B natriuretic peptide receptor (NPR-B) in vascular smooth muscle cells to activate the particulate guanylyl cyclase activity intrinsic to that receptor and raise cellular cyclic GMP levels. In the present study, we demonstrate that CNP down-regulates the expression of this receptor leading to a reduction in NPR-B activity. Pretreatment of rat aortic smooth muscle cells with CNP reduces NPR-B activity, NPR-B protein levels, NPR2 (NPR-B gene) mRNA levels, and NPR2 promoter activity. The decrease in NPR2 promoter activity is dependent on DNA sequence present between -441 and -134 relative to the transcription start site. The reduction in NPR2 gene expression appears to operate through generation of cyclic GMP. 8-Bromo cyclic GMP, a membrane-permeable cyclic GMP analog, reduced NPR2 mRNA levels and NPR2 promoter activity. Atrial natriuretic peptide, which signals through the type A natriuretic peptide receptor (NPR-A) to increase cyclic GMP levels in these cells, also reduced NPR-B mRNA levels and inhibited NPR-B promoter activity; however, this inhibition was not additive with that produced by CNP, implying that the two ligands traffic over a common signal transduction pathway. This report provides the first documentation that CNP is capable of autoregulating the expression of its cognate receptor.

Type I human T cell leukemia virus (HTLV-I) is etiologically linked with adult T cell leukemia, an aggressive and usually fatal expansion of activated CD4+ T lymphocytes that frequently traffic to skin. T cell transformation induced by HTLV-I involves the action of the 40-kDa viral Tax transactivator protein. Tax both stimulates the HTLV-I long terminal repeat and deregulates the expression of select cellular genes by altering the activity of specific host transcription factors, including cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor, NF-kappaB/Rel, and serum response factor. To study initiating events involved in HTLV-I Tax-induced T cell transformation, we generated "Tet-off" transgenic mice conditionally expressing in a lymphocyte-restricted manner (EmuSR alpha promoter-enhancer) either wild-type Tax or mutant forms of Tax that selectively compromise the NF-kappaB (M22) or CREB/activating transcription factor (M47) activation pathways. Wild-type Tax and M47 Tax-expressing mice, but not M22-Tax expressing mice, developed progressive alopecia, hyperkeratosis, and skin lesions containing profuse activated CD4 T cell infiltrates with evidence of deregulated inflammatory cytokine production. In addition, these animals displayed systemic lymphadenopathy and splenomegaly. These findings suggest that Tax-mediated activation of NF-kappaB plays a key role in the development of this aggressive skin disease that shares several features in common with the skin disease occurring during the preleukemic stage in HTLV-I-infected patients. Of note, this skin disease completely resolved when Tax transgene expression was suppressed by administration of doxycycline, emphasizing the key role played by this viral oncoprotein in the observed pathology.