RhoA, a ubiquitous intracellular GTPase, mediates cytoskeletal responses to extracellular signals. A 2.1 A resolution crystal structure of the human RhoA-GDP complex shows unique stereochemistry in the switch I region, which results in a novel mode of Mg2+ binding.
Like all Rho (Ras homology) GTPases, RhoA functions as a molecular switch in cell signaling, alternating between GTP- and GDP-bound states, with its biologically inactive GDP-bound form maintained as a cytosolic complex with RhoGDI (guanine nucleotide-exchange inhibitor). The crystal structures of RhoA-GDP and of the C-terminal immunoglobulin-like domain of RhoGDI (residues 67-203) are known, but the mechanism by which the two proteins interact is not known. The functional human RhoA-RhoGDI complex has been expressed in yeast and crystallized (P6(5)22, unit-cell parameters a = b = 139, c = 253 A, two complexes in the asymmetric unit). Although diffraction from these crystals extends to 3.5 A and is highly anisotropic, the experimentally phased (MAD plus MIR) electron-density map was adequate to reveal the mutual disposition of the two molecules. The result was validated by molecular-replacement calculations when data were corrected for anisotropy. Furthermore, the N-terminus of RhoGDI (the region involved in inhibition of nucleotide exchange) can be identified in the electron-density map: it is bound to the switch I and switch II regions of RhoA, occluding an epitope which binds Dbl-like nucleotide-exchange factors. The entrance of the hydrophobic pocket of RhoGDI is 25 A from the last residue in the RhoA model, with its C-terminus oriented to accommodate the geranylgeranyl group without conformational change in RhoA.
The role of nitric oxide (NO) in liver injury and fibrosis is unclear. The purpose of this study was to determine whether inducible NO synthase deficiency (iNOS ؊/؊ ) affects liver injury and fibrosis produced in mice by chronic carbon tetrachloride (CCl 4 ) administration. Wildtype (WT) or iNOS ؊/؊ mice were subjected to biweekly CCl 4 injections over 8 weeks, whereas controls were given isovolumetric injections of olive oil. Serum aminotransferases were lower after CCl 4 in the iNOS ؊/؊ than in the WT mice, which correlated with decreased necrosis on liver histology. There was increased apoptosis, a lower number of stellate cells, and a lesser degree of fibrosis after CCl 4 in the iNOS ؊/؊ as compared with the WT mice. ␣ 1 (I) collagen messenger RNA (mRNA) was markedly increased after CCl 4 in the WT and to a significantly lesser extent in the iNOS ؊/؊ mice. Liver matrix metalloproteinase-9 (MMP-9) mRNA and MMP-2 mRNA were increased more in the WT than in the iNOS ؊/؊ mice after CCl 4 . Also tissue inhibitor metalloproteinase 1 (TIMP-1) mRNA was increased to a much greater extent in the WT than in the iNOS ؊/؊ mice after CCl 4 (P < 0.05). However, MMP-9 and TIMP-1 protein, determined by western blot, were similarly increased after CCl 4 in both groups of mice. Conclusion: NO protects against CCl 4 -induced apoptosis. In the absence of iNOS, there is decreased necrosis, increased apoptosis, and reduced liver fibrosis. (HEPATOLOGY 2008;47:2051-2058 N itric oxide (NO) is a free radical that has a broad range of functions. It is synthesized from L-arginine by three known NO synthase isoforms: neuronal NO synthase, inducible NO synthase (iNOS-2), and endothelial NO synthase. NO has many roles ranging from neurotransmission, prevention of blood clotting, and regulation of blood pressure (mainly through the constitutive expression of neuronal NO synthase or endothelial NO synthase). In the liver, NO production by endothelial NO synthase has a protective role by maintaining perfusion and preventing platelet aggregation, whereas the exact role of iNOS remains unclear. There are differing results on whether NO production by iNOS is hepatoprotective or detrimental, depending on the level, intensity, and duration of an insult. In response to inflammation, iNOS is up-regulated in hepatocytes and macrophages. 1 In the presence of iNOS inhibitors, apoptotic cell death is increased, suggesting that NO has antiapoptotic properties. 2 Interactions between NO and reactive oxygen species (ROS)
Background Vitamin D deficiency is common in chronic liver disease particularly in those with severe liver fibrosis. Aims: To determine the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the human α1(I) collagen promoter and collagen formation by human stellate LX-2 cells and the mechanism of the effect of the vitamin D receptor (VDR) on the promoter. Methods Type I collagen was assessed by measurements of collagen mRNA and collagen protein and by transfection experiments. Binding of VDR to the α1(I) collagen promoter was determined by EMSA and ChIP assays. Results 1,25-(OH)2D3 decreased human α1(I) collagen mRNA and protein and the secretion of type I collagen by stellate cells after exposure to TGFβ1. Furthermore, 1,25-(OH)2D3 inhibited TGFβ1–induced activation of the α1(I) collagen promoter in transfected LX-2 cells. The effect of 1, 25-(OH)2D3 is mediated by the VDR, which binds at a proximal Sp1 site and also at a newly identified distal site on the collagen promoter. A VDR expression vector reduced the activities of the collagen promoter in transfected LX-2 cells. Conclusions 1,25-(OH)2D3 inhibits type I collagen formation in human stellate cells. The effect of 1,25-(OH)2D3 is mediated by its receptor which binds at a proximal Sp1.1 site and at a newly identified distal site on the collagen promoter. Correction of vitamin D deficiency in patients with chronic liver disease is a potential therapy to inhibit progression of fibrosis.
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