Ramalinga Kuruba

Downregulation of Endothelin-1 by Farnesoid X Receptor in Vascular Endothelial Cells

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidney, adrenals, and intestine. FXR may play an important role in the pathogenesis of cardiovascular diseases via regulating the metabolism and transport of cholesterol. In this study, we report that FXR is also expressed in rat pulmonary artery endothelial cells (EC), a “nonclassical” bile acid target tissue. FXR is functional in EC, as demonstrated by induction of its target genes such as small heterodimer partner (SHP) after treatment with chenodeoxycholic acid, a FXR agonist. Interestingly, activation of FXR in EC led to downregulation of endothelin (ET)-1 expression. Reporter assays showed that activation of FXR inhibited transcriptional activation of the human ET-1 gene promoter and also repressed the activity of a heterologous promoter driven by activator protein (AP)-1 response elements. Electrophoretic mobility-shift and chromatin immunoprecipitation assays indicated that FXR reduced the binding activity of AP-1 transcriptional factors, suggesting that FXR may suppress ET-1 expression via negatively interfering with AP-1 signaling. These studies suggest that FXR may play a role in endothelial homeostasis and may serve as a novel molecular target for manipulating ET-1 expression in vascular EC.

Roles of microRNA-29a in the Antifibrotic Effect of Farnesoid X Receptor in Hepatic Stellate Cells

Liver fibrosis is a chronic disorder that is characterized by an alteration of the balance between fibrogenesis and fibrinolysis, which results in accumulation of excessive amounts of extracellular matrix (ECM) and distortion of the normal liver architecture. The activation and transformation of quiescent hepatic stellate cells (HSCs) into myofibroblast-like cells constitute a major mechanism for the increased production of ECM in the liver. The nuclear receptor farnesoid X receptor (FXR) shows potent antifibrotic activity in HSCs and protects animals in rodent models of liver fibrosis. However, the detailed mechanism remains incompletely understood. In this study, we report that treatment with 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]benzoic acid (GW4064), a synthetic FXR ligand, led to up-regulation of microRNA-29a (miR-29a) in HSCs isolated from wild-type mice, rats, and humans but not from FXR(−/−) mice. miR-29a seems to play an inhibitory role in the regulation of ECM production because of the following: 1) transfection of HSCs with miR-29a mimic resulted in drastic down-regulation of the mRNA expression of several genes that encode ECM proteins; and 2) miR-29a significantly inhibited the expression of a reporter expression plasmid that contains the 3′-untranslated region of the corresponding ECM genes. Our results suggest that miR-29a is a FXR target gene because miR-29a promoter activity was significantly increased by pharmacologic or genetic activation of FXR. Functional analysis of human miR-29a promoter identified an imperfect inverted repeat spaced by one nucleotide DNA motif, inverted repeat-1 (5′-AGGTCAcAGACCT-3′), as a likely FXR-responsive element that is involved in miR-29a regulation. Our study uncovers a new mechanism by which FXR negatively regulates the expression of ECM in HSCs.

A Functional Cross-Talk between Liver X Receptor-α and Constitutive Androstane Receptor Links Lipogenesis and Xenobiotic Responses

The liver X receptor (LXR) and constitutive androstane receptor (CAR) are two nuclear receptors postulated to have distinct functions. LXR is a sterol sensor that promotes lipogenesis, whereas CAR is a xenosensor that controls xenobiotic responses. Here, we show that LXRα and CAR are functionally related in vivo. Loss of CAR increased the expression of lipogenic LXR target genes, leading to increased hepatic triglyceride accumulation, whereas activation of CAR inhibited the expression of LXR target genes and LXR ligand-induced lipogenesis. On the other hand, a combined loss of LXR α and β increased the basal expression of xenobiotic CAR target genes, whereas activation of LXR inhibited the expression of CAR target genes and sensitized mice to xenobiotic toxicants. The mutual suppression between LXRα and CAR was also observed in cell culture and reporter gene assays. LXRα, like CAR, exhibited constitutive activity in the absence of an exogenously added ligand by recruiting nuclear receptor coactivators. Interestingly, although CAR competed with LXRα for coactivators, the constitutive activity and recruitment of coactivators was not required for CAR to suppress the activity of LXRα. In vivo chromatin immunoprecipitation assay showed that cotreatment of a CAR agonist compromised the LXR agonist responsive recruitment of LXRα to Srebp-1c, whereas an LXR agonist inhibited the CAR agonist-responsive recruitment of CAR to Cyp2b10. In conclusion, our results have revealed dual functions of LXRα and CAR in lipogenesis and xenobiotic responses, establishing a unique role of these two receptors in integrating xenobiotic and endobiotic homeostasis.

Upregulation of scavenger receptor class B type I expression by activation of FXR in hepatocyte

OBJECTIVE The farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been proposed to play an important role in the pathogenesis of cardiovascular diseases by regulating the metabolism and transport of cholesterol and triglyceride. Scavenger receptor class B type I (SR-BI), a high-density lipoprotein receptor, plays an important role in decreasing lipid metabolism-associated cardiovascular diseases by regulating reverse cholesterol transport. Recent studies have shown that SR-BI expression is upregulated by several nuclear receptors. However, the role of FXR in the regulation of SR-BI expression is not well known. In the present study, we investigate the regulation of SR-BI by FXR in hepatocyte and the corresponding mechanism. METHODS AND RESULTS Treatment of human hepatoma cell line HepG2 with FXR ligands resulted in upregulation of SR-BI at the levels of both mRNA and protein. Reporter assays showed that activation of FXR significantly enhanced the SR-BI promoter activity. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that FXR induced SR-BI expression by binding to a novel FXR element (FXRE), a directed repeat DNA motif, DR8 (-703 AGGCCAcgttctagAGCTCA -684). The in vivo experiment demonstrated that gavaging mice with a natural ligand of FXR increased SR-BI expression in liver tissues. CONCLUSIONS FXR can directly upregulate SR-BI expression in hepatocyte, and DR8 is a likely novel FXRE that is involved in SR-BI regulation. FXR may serve as a novel molecular target for manipulating SR-BI expression in hepatocyte.

Noble metal-free bifunctional oxygen evolution and oxygen reduction acidic media electro-catalysts

Identification of low cost, highly active, durable completely noble metal-free electro-catalyst for oxygen reduction reaction (ORR) in proton exchange membrane (PEM) fuel cells, oxygen evolution reaction (OER) in PEM based water electrolysis and metal air batteries remains one of the major unfulfilled scientific and technological challenges of PEM based acid mediated electro-catalysts. In contrast, several non-noble metals based electro-catalysts have been identified for alkaline and neutral medium water electrolysis and fuel cells. Herein we report for the very first time, F doped Cu1.5Mn1.5O4, identified by exploiting theoretical first principles calculations for ORR and OER in PEM based systems. The identified novel noble metal-free electro-catalyst showed similar onset potential (1.43 V for OER and 1 V for ORR vs RHE) to that of IrO2 and Pt/C, respectively. The system also displayed excellent electrochemical activity comparable to IrO2 for OER and Pt/C for ORR, respectively, along with remarkable long term stability for 6000 cycles in acidic media validating theory, while also displaying superior methanol tolerance and yielding recommended power densities in full cell configurations.

Inhibition of endothelin-1-mediated contraction of hepatic stellate cells by FXR ligand.

Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen and the enhanced contractile response to vasoactive mediators such as endothelin-1 (ET-1). The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidneys, adrenals, and intestine. FXR is also expressed in HSCs and activation of FXR in HSCs is associated with significant decreases in collagen production. However, little is known about the roles of FXR in the regulation of contraction of HSCs. We report in this study that treatment of quiescent HSCs with GW4064, a synthetic FXR agonist, significantly inhibited the HSC transdifferentiation, which was associated with an inhibition of the upregulation of ET-1 expression. These GW4064-treated cells also showed reduced contractile response to ET-1 in comparison to HSCs without GW4064 treatment. We have further shown that GW4064 treatment inhibited the ET-1-mediated contraction in fully activated HSCs. To elucidate the potential mechanism we showed that GW4064 inhibited ET-1-mediated activation of Rho/ROCK pathway in activated HSCs. Our studies unveiled a new mechanism that might contribute to the anti-cirrhotic effects of FXR ligands.

Polyhydroxylalkyleneamines: A class of hydrophilic cationic polymer-based gene transfer agents

A new series of cationic polymers, poly[N,N-bis-(2-hydroxylpropyl) alkylalcoholamine-co-ethylenediamine] were synthesized by directly cross-linking several dichloro alkylating agents with ethylenediamine and its derivatives. Co-polymerization with cystamine introduces biodegradable disulfide bonds to the polymer backbone. When tested on COS-1 cells, PHAs showed reduced cytotoxicity, broad polymer to DNA ratios, and enhanced transfection activity that was 2-9-fold better than that of polyethylenimine. Comparison studies also revealed several chemical and physical parameters related to the biological activities of these polymers. The length of the side chain groups affects both transfection activity and toxicity of the polymers; a side chain group of moderate size appeared to be optimal for both high transfection activity and low toxicity. Introduction of biodegradable disulfide bonds to the polymer backbone further enhanced transfection activity and reduced toxicity of the polymer. Fractionated PHAs with molecular weight of > or =5000 Da were equally effective but smaller polymers were ineffective in transfection. This flexible synthesis route enables the determination of key structural and physical parameters related to polymer activity and could aid further improvement of polymer-based transfection agents.

Targeted Delivery of Nucleic Acid-Based Therapeutics to the Pulmonary Circulation

Targeted delivery of functional nucleic acids (genes and oligonucleotides) to pulmonary endothelium may become a novel therapy for the treatment of various types of lung diseases. It may also provide a new research tool to study the functions and regulation of novel genes in pulmonary endothelium. Its success is largely dependent on the development of a vehicle that is capable of efficient pulmonary delivery with minimal toxicity. This review summarizes the recent progress that has been made in our laboratory along these research directions. Factors that affect pulmonary nucleic acids delivery are also discussed.

Upregulation of decorin by FXR in vascular smooth muscle cells

Decorin is a member of the family of small leucine-rich proteoglycans that are present in blood vessels and synthesized by vascular smooth muscle cells (VSMCs). Decorin plays complex roles in both normal vascular physiology and the pathogenesis of various types of vascular disorders. However, the mechanisms of regulation of decorin expression in vasculature are not clearly understood. Particularly little information is available about a role of nuclear receptors in the regulation of decorin expression. In the present study, we report that activation of vascular FXR by a specific ligand resulted in upregulation of decorin at the levels of both mRNA and protein. FXR appears to induce decorin expression at a transcriptional level because (1) upregulation of decorin mRNA expression was abolished by the treatment of a transcription inhibitor, actinomycin D; and (2) decorin promoter activity was significantly increased by activation of FXR. Functional analysis of human decorin promoter identified an imperfect inverted repeat DNA motif, IR8 (-2313TGGTCAtagtgtcaTGACCT-2294), as a likely FXR-responsive element that is involved in decorin regulation.

Chronic Activation of FXR in Transgenic Mice Caused Perinatal Toxicity and Sensitized Mice to Cholesterol Toxicity

The nuclear receptor farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4, or NR1H4) is highly expressed in the liver and intestine. Previous reports have suggested beneficial functions of FXR in the homeostasis of bile acids, lipids, and glucose, as well as in promoting liver regeneration and inhibiting carcinogenesis. To investigate the effect of chronic FXR activation in vivo, we generated transgenic mice that conditionally and tissue specifically express the activated form of FXR in the liver and intestine. Unexpectedly, the transgenic mice showed several intriguing phenotypes, including partial neonatal lethality, growth retardation, and spontaneous liver toxicity. The transgenic mice also displayed heightened sensitivity to a high-cholesterol diet-induced hepatotoxicity but resistance to the gallstone formation. The phenotypes were transgene specific, because they were abolished upon treatment with doxycycline to silence the transgene expression. The perinatal toxicity, which can be rescued by a maternal vitamin supplement, may have resulted from vitamin deficiency due to low biliary bile acid output as a consequence of inhibition of bile acid formation. Our results also suggested that the fibroblast growth factor-inducible immediate-early response protein 14 (Fn14), a member of the proinflammatory TNF family, is a FXR-responsive gene. However, the contribution of Fn14 induction in the perinatal toxic phenotype of the transgenic mice remains to be defined. Because FXR is being explored as a therapeutic target, our results suggested that a chronic activation of this nuclear receptor may have an unintended side effect especially during the perinatal stage.