Reduction of blood pressure elevation by losartan in spontaneously hypertensive rats through suppression of LARG expression in vascular smooth muscle cells.

BACKGROUND/PURPOSE: This study sought to elucidate the mechanism by which losartan inhibits blood pressure (BP) elevation in spontaneously hypertensive rats (SHRs). METHODS: Four-week-old Wistar-Kyoto (WKY) rats and SHRs were either treated with losartan (20 mg/kg/day) for 8 weeks or served as untreated controls. BP was measured by the tail-cuff method. At 12 weeks, isometric contraction of the aortic rings of the rats was evaluated with a force transducer and recorder. The mRNA and protein levels of the target Rho guanine nucleotide exchange factors (RhoGEFs), and the extent of myosin phosphatase target subunit 1 (MYPT-1) phosphorylation in the aorta, were determined using quantitative real-time polymerase chain reaction (qPCR) assay and Western blot analysis. RESULTS: The BP of the four-week-old SHRs did not differ from that of the age-matched WKY rats, whereas the BP of the twelve-week-old control group SHRs was higher than that of the control group WKY rats. Losartan treatment, however, inhibited BP elevation in both rat strains, doing so to a greater extent in the treatment group SHRs. The contractile force in response to angiotensin II of the aortic rings from the SHRs treated with losartan was significantly lower than that of the aortic rings from the non-treated SHRs. The protein expression of leukemia-associated RhoGEF (LARG) was significantly higher in the non-treated SHRs compared to the non-treated WKY rats. CONCLUSION: The study results showed that the reduction of BP elevation by losartan in SHRs occurs through the suppression of LARG expression and MYPT-1 phosphorylation in vascular smooth muscle cells.

Peptidomimetic inhibitors of APC-Asef interaction block colorectal cancer migration.

The binding of adenomatous polyposis coli (APC) to its receptor Asef relieves the negative intramolecular regulation of Asef and leads to aberrant cell migration in human colorectal cancer. Because of its crucial role in metastatic dissemination, the interaction between APC and Asef is an attractive target for anti-colorectal-cancer therapy. We rationally designed a series of peptidomimetics that act as potent inhibitors of the APC interface. Crystal structures and biochemical and cellular assays showed that the peptidomimetics in the APC pocket inhibited the migration of colorectal cells by disrupting APC-Asef interaction. By using the peptidomimetic inhibitor as a chemical probe, we found that CDC42 was the downstream GTPase involved in APC-stimulated Asef activation in colorectal cancer cells. Our work demonstrates the feasibility of exploiting APC-Asef interaction to regulate the migration of colorectal cancer cells, and provides what to our knowledge is the first class of protein-protein interaction inhibitors available for the development of cancer therapeutics targeting APC-Asef signaling.

XPLN is modulated by HDAC inhibitors and negatively regulates SPARC expression by targeting mTORC2 in human lung fibroblasts.

Pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unclear. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that participates in the assembly and turnover of the extracellular matrix, whose expression is regulated by transforming growth factor (TGF)-ß1 through activation of mammalian target of rapamycin complex 2 (mTORC2). Exchange factor found in platelets, leukemic, and neuronal tissues (XPLN) is an endogenous inhibitor of mTORC2. However, whether XPLN modulates SPARC expression remains unknown. Herein, we investigated the regulatory mechanisms of XPLN in human lung fibroblasts. Effect of XPLN on mTORC2 activity was evaluated by silencing XPLN in human foetal lung fibroblasts (HFL-1 cells), using small interfering RNA. SPARC expression was quantified by quantitative real-time RT-PCR and western blotting. Fibroblasts were treated with TGF-ß1, histone deacetylase (HDAC) inhibitors, entinostat, or vorinostat, to assess their effects on XPLN expression. Moreover, the effect of mTORC1 inhibition on SPARC and XPLN was examined. XPLN depletion stimulated SPARC expression and Akt phosphorylation on Ser473. TGF-ß1 treatment down-regulated XPLN via Smad 2/3. XPLN mRNA expression was up-regulated upon treatment with HDAC inhibitors in a concentration-dependent manner, and TGF-ß1-induced SPARC expression was reversed by entinostat treatment. mTORC1 inhibition by rapamycin and Raptor depletion stimulated SPARC expression. In conclusion, this is the first study describing the involvement of XPLN in the regulation of SPARC. These findings may help uncover the regulatory mechanisms of the mTORC2-SPARC axis. The up-regulation of XPLN by HDAC inhibitors may be a novel therapeutic approach in patients with IPF.

Angiotensin II activates the RhoA exchange factor Arhgef1 in humans.

Although a causative role for RhoA-Rho kinase has been recognized in the development of human hypertension, the molecular mechanism(s) and the RhoA guanine exchange factor(s) responsible for the overactivation of RhoA remain unknown. Arhgef1 was identified as a RhoA guanine exchange factor involved in angiotensin II (Ang II)-mediated regulation of vascular tone and hypertension in mice. The aim of this study was to determine whether Arhgef1 is activated and involved in the activation of RhoA-Rho kinase signaling by Ang II in humans. In vitro stimulation of human coronary artery smooth muscle cells and human peripheral blood mononuclear cells by Ang II (0.1 µmol/L) induced activation of Arhgef1 attested by its increased tyrosine phosphorylation. Silencing of Arhgef1 expression by siRNA inhibited Ang II-induced activation of RhoA-Rho kinase signaling. In normotensive subjects, activation of the renin-angiotensin system by a low-salt diet for 7 days increased RhoA-Rho kinase signaling and stimulated Arhgef1 activity in peripheral blood mononuclear cells. In conclusion, our results strongly suggest that Arhgef1 mediates Ang II-induced RhoA activation in humans. Moreover, they show that measurement of RhoA guanine exchange factor activity in peripheral blood mononuclear cells might be a useful method to evaluate RhoA guanine exchange factor activity in humans.

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1.

The RhoGEF GEF-H1 can be sequestered in an inactive state on polymerized microtubules by the dynein motor light-chain Tctex-1. Phosphorylation of GEF-H1 Ser885 by PKA or PAK kinases creates an inhibitory 14-3-3-binding site. Here we show a new mode of GEF-H1 activation in response to the G-protein-coupled receptor (GPCR) ligands lysophosphatidic acid (LPA) or thrombin that is independent of microtubule depolymerization. LPA/thrombin stimulates disassembly of the GEF-H1:dynein multi-protein complex through the concerted action of Gα and Gßγ. Gα binds directly to GEF-H1 and displaces it from Tctex-1, while Gßγ binds to Tctex-1 and disrupts its interaction with the dynein intermediate chain, resulting in the release of GEF-H1. Full activation of GEF-H1 requires dephosphorylation of Ser885 by PP2A, which is induced by thrombin. The coordinated displacement of GEF-H1 from microtubules by G-proteins and its dephosphorylation by PP2A demonstrate a multistep GEF-H1 activation and present a unique mechanism coupling GPCR signalling to Rho activation.