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Routray C.,GI Research Unit and Cancer Cell Biology Program | Liu C.,GI Research Unit and Cancer Cell Biology Program | Yaqoob U.,GI Research Unit and Cancer Cell Biology Program | Billadeau D.D.,Mayo Medical School | And 4 more authors.
American Journal of Physiology - Cell Physiology

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMPdependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1. © 2011 the American Physiological Society. Source

Tu K.,GI Research Unit and Cancer Cell Biology Program | Tu K.,Xian Jiaotong University | Li J.,GI Research Unit and Cancer Cell Biology Program | Li J.,University of Minnesota | And 12 more authors.

Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-β) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-β-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-β-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-β receptor II (TβRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TβRII to the plasma membrane, thereby desensitizing HSCs to TGF-β1 stimulation. Conclusions: Our study demonstrates a requirement of VASP for TGF-β-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TβRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver. © 2014 by the American Association for the Study of Liver Diseases. Source

Liu C.,GI Research Unit and Cancer Cell Biology Program | Li J.,GI Research Unit and Cancer Cell Biology Program | Li J.,University of Minnesota | Li J.,Chongqing Medical University | And 9 more authors.
American Journal of Physiology - Gastrointestinal and Liver Physiology

Platelet-derived growth factor (PDGF) and transforming growth factor-ß(TGF-ß) signaling are required for hepatic stellate cell (HSC) activation under pathological conditions such as liver metastatic tumor growth. These two signaling pathways are functionally divergent; PDGF signaling promotes proliferation and migration of HSCs, and TGF-ß induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-ß-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-ß activation of HSCs has not been investigated. Here we report that PDGF receptor-α (PDGFR α) is required for TGF-ß signaling of cultured human HSCs although HSCs express both PDGF α and α receptors. PDGFR α knockdown inhibits TGF- _-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with noncanonical TGF-ß signaling. PDGFR α knockdown suppresses TGF-ß receptor I (T_RI) but increases T_RII gene transcription. At the protein level, PDGFR α is recruited to T_RI/T_RII complexes by TGF-ß stimulation. PDGFR α knockdown blocks TGF-ß-mediated internalization of T_RII and induces accumulation of T_RII at the plasma membrane, thereby inhibiting TGF-ß phosphorylation of SMAD2. Functionally, knockdown of PDGFR α reduces paracrine effects of HSCs on colorectal cancer cell proliferation and migration in vitro. In mice and patients, colorectal cancer cell invasion of the liver induces upregulation of PDGFR α of HSCs. In summary, our finding that PDGFR α knockdown inhibits SMAD-dependent TGF-ß signaling by repressing T_RI transcriptionally and blocking endocytosis of TGF-ß receptors highlights a convergence of PDGF and TGF-ß signaling for HSC activation and PDGFR α as a therapeutic target for liver metastasis and other settings of HSC activation. © 2014 the American Physiological Society. Source

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