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Cited by 4 publications
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Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes
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. nitric oxide; vasodilator-stimulated phosphoprotein; cGMP-dependent kinase; hepatic stellate cells NITRIC OXIDE (NO) generated by endothelial cells plays a major role in the control of vessel tone and vascular architecture (2, 10). In perivascular cells (pericytes), NO exerts its biological effects mainly by activating the cGMP-dependent protein kinase (PKG) via NO/cGMP/PKG paracrine signaling. Similar to pericytes in other vascular beds, liver-specific pericytes hepatic stellate cells (HSC) are also regulated by the NO/cGMP/PKG paracrine signaling. For instance, NO/cGMP/PKG signaling inhibits the contractility, migration, and survival of HSC in vitro (27,29,38). Indeed, impaired NO production or attenuated response of HSC to NO stimulation in animal models of liver cirrhosis suggest that a defective NO/cGMP/PKG signaling cascade is implicated in the pathophysiology of liver fibrosis and ensuing portal hypertension (3,12,15,17).Focal adhesions (FA) are dynamic protein complexes that connect extracellular matrix to cells and convey these external stimuli into intracellular signals, which is fundamental for cell adhesion, migration, and survival (5, 14, 31, 45). Although it is known that PKG signaling in smooth muscle cells mediates FA disassembly induced by counter-adhesive extracellular matrix proteins such as thrombospondin and tenascin (34), and that PKG inhibits integ...
Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes
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. nitric oxide; vasodilator-stimulated phosphoprotein; cGMP-dependent kinase; hepatic stellate cells NITRIC OXIDE (NO) generated by endothelial cells plays a major role in the control of vessel tone and vascular architecture (2, 10). In perivascular cells (pericytes), NO exerts its biological effects mainly by activating the cGMP-dependent protein kinase (PKG) via NO/cGMP/PKG paracrine signaling. Similar to pericytes in other vascular beds, liver-specific pericytes hepatic stellate cells (HSC) are also regulated by the NO/cGMP/PKG paracrine signaling. For instance, NO/cGMP/PKG signaling inhibits the contractility, migration, and survival of HSC in vitro (27,29,38). Indeed, impaired NO production or attenuated response of HSC to NO stimulation in animal models of liver cirrhosis suggest that a defective NO/cGMP/PKG signaling cascade is implicated in the pathophysiology of liver fibrosis and ensuing portal hypertension (3,12,15,17).Focal adhesions (FA) are dynamic protein complexes that connect extracellular matrix to cells and convey these external stimuli into intracellular signals, which is fundamental for cell adhesion, migration, and survival (5, 14, 31, 45). Although it is known that PKG signaling in smooth muscle cells mediates FA disassembly induced by counter-adhesive extracellular matrix proteins such as thrombospondin and tenascin (34), and that PKG inhibits integ...
Blood vessels form de novo through the tightly regulated programs of vasculogenesis and angiogenesis. Both processes are distinct but one of the steps they share is the formation of a central lumen, when groups of cells organized as vascular cords undergo complex changes to achieve a tube-like morphology. Recently, a protein termed epidermal growth factor-like domain 7 (EGFL7) was described as a novel endothelial cell-derived factor involved in the regulation of the spatial arrangement of cells during vascular tube assembly. With its impact on tubulogenesis and vessel shape EGFL7 joined the large family of molecules governing blood vessel formation. Only recently, the molecular mechanisms underlying EGFL7's effects have been started to be elucidated and shaping of the extracellular matrix (ECM) as well as Notch signaling might very well play a role in mediating its biological effects. Further, findings in knock-out animal models suggest miR-126, a miRNA located within the egfl7 gene, has a major role in vessel development by promoting VEGF signaling, angiogenesis and vascular integrity. This review summarizes our current knowledge on EGFL7 and miR-126 and we will discuss the implications of both bioactive molecules for the formation of blood vessels.
Tumor stroma importantly influences tumor growth and progression with pericytes representing an important stromal cell owing to their importance in angiogenesis and their transformation into highly motile tumor myofibroblasts.1 Focal adhesions (FAs) are specialized structures that connect extracellular environment to actin cytoskeleton thus facilitating the signal transduction and actin remodeling requisite for the process of pericyte migration during angiogenesis.2,3 Thus, mechanisms that govern FA assembly and pericyte motility may present potential targets for anti-cancer therapy but are not yet fully defined.vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that regulate actin cytoskeleton and cell migration. 4 It contains distinct subdomains that facilitate specific protein interactions and actin binding characteristics that culminate in an anticapping/branching function within the cytoskeleton.4,5 Although VASP resides within FAs, its precise role in FA dynamics has not been completely defined, probably due in part to cell-type specific VASP functions. 6 -8 Because the role of VASP in pericyte function and ensuing effects within the tumor microenvironment remain unexplored, we investigated the role of VASP in FA development in human hepatic stellate cells (HSC), liver pericytes that express a high level of VASP and which on transformation into myofibroblasts, develop a highly motile state that has been postulated to influence tumor growth. Our data reveal a requirement of VASP in FA dynamics and pericyte motility, which requires cooperation of each distinct VASP subdomain. Indeed, overexpression of the EVH2 domain alone results in a dominant negative function that inhibits FA assembly, cell motility, and angiogenesis. We also identify a role of Rac1 as a key binding partner of VASP that promotes its ability to regulate FA and cell motility. Perturbation of Ena/VASP function in tumor myofibroblast precursor cells signifi-
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