Focal Adhesions in (Myo)fibroblasts Scaffold Adenylyl Cyclase with Phosphorylated Caveolin

Abstract

Fibroblast-myofibroblast transformation, a critical event for enhanced extracellular matrix deposition, involves formation of an actin stress fiber contractile apparatus that radiates from focal adhesions (FA) in the plasma membrane. Activation of adenylyl cyclase (AC, i.e. increases in cAMP) negatively regulates such transformation. Caveolae and their resident protein caveolins scaffold signaling molecules, including AC isoforms, whereas phosphorylated caveolin-1 (phospho-cav-1) may localize at FA. Here, we used adult rat cardiac fibroblasts to examine distribution and expression of AC, phospho-cav-1, and FA proteins to define mechanisms that link increases in cAMP to caveolin-1 phosphorylation, actin/FA assembly, and fibroblast-myofibroblast transformation. Sucrose density gradient centrifugation, immunoblot, and immunohistochemical analysis revealed that, unlike cav-1, phospho-cav-1 enriches in membrane fractions that express FA proteins and localize at the ends of actin stress fibers. We detected AC in both cav-1 and phospho-cav-1 immunoprecipitates, but FA kinase (FAK), phospho-FAK (FAK Tyr-397), paxillin, and vinculin were detected only in phospho-cav-1 immunoprecipitates. Treatment with the AC activator forskolin or a cAMP analog increased cav-1 phosphorylation but decreased FAK Tyr-397 phosphorylation in a cAMP-dependent protein kinase-dependent manner. These events preceded actin cytoskeletal disruption, an effect that was blocked by small interfering RNA knock-down of cav-1. Inhibition of protein tyrosine phosphatase 1B abrogated cAMP-mediated disruption of actin cytoskeleton, cav-1 phosphorylation, and FAK Tyr-397 dephosphorylation. The data thus define a novel organization of signaling molecules that regulate fibroblasts: scaffolding of AC by phospho-cav-1 at FA sites in a caveolaefree microdomain along with components that mediate inhibition of actin/FA assembly and fibroblast-myofibroblast transformation via increases in cAMP.Fibroblast-myofibroblast transformation is a key event in the deleterious remodeling that results in exaggerated production of connective tissue following injury of the lung, liver, kidneys, skin, and heart (1-4). Relatively little is known regarding the precise cellular mechanisms that lead to and regulate myofibroblast formation, although a primary component is the formation of a contractile apparatus composed of ␣-smooth muscle actin-containing microfilaments: i.e. stress fibers that anchor and radiate from FA 2 along the plasma membrane (5). Recent evidence suggests that FA serve as organizing centers for regulatory and structural proteins so as to facilitate rapid, precise control of cell proliferation, differentiation, and function (6, 7). Among these are anchoring proteins, such as vinculin, paxillin, talin, and ␣-actinin, which link the actin cytoskeleton to transmembrane integrin receptors at FA (8). Formation of focal contacts involves tyrosine phosphorylation of the non-receptor protein tyrosine kinase, FAK (9). In response to growth factor stimulation and integrin ...

Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion

“…pY14Cav1 has been localized to the major sites of tyrosine-kinase signaling, FAs, where it generates a docking site for SH2-domain containing proteins, such as Grb7 (42), as well as for the COOH terminal Src kinase Csk that is able to down-regulate Src activity via phosphorylation (43). Cav1 also functions as a membrane adaptor that, when phosphorylated upon integrin ligation in FAs, promotes cell signaling and actin reorganization (20,21,(44)(45)(46)(47). The ability of Cav1 to regulate domain formation in FAs (22) is consistent with the ability of pY14Cav1 to stabilize FAK within FAs, limiting exchange of FAK between FAs and cytosol (23).…”

The Prostanoid EP4 Receptor and Its Signaling Pathway

“…In contrast to Epac, PKA, the other downstream effector, inhibited cardiac fetal gene expression and cardiomyocyte hypertrophy through activation of histone deacetylase 5 (Ha et al, 2010). In the heart, AC5 and AC6 are major AC isoforms (Ishikawa et al, 2005;Swaney et al, 2006;Gao et al, 2011). However, studies using genetically engineered mice have shown that neither AC5 nor AC6 promotes cardiomyocyte hypertrophy (Ishikawa et al, 2005;Gao et al, 2011;Sugano et al, 2011).…”

Mechanically induced osteogenic differentiation – the role of RhoA, ROCKII and cytoskeletal dynamics

“…Furthermore, chemically activating RhoA is sufficient to induce osteogenic differentiation and acts synergistically with oscillatory fluid flow in Runx2 upregulation. There are multiple potential mechanisms whereby actin fibril tension may play a role in intracellular signal transduction, including connections to caveolae (Stahlhut and van Deurs, 2000;Hjalm et al, 2001;Swaney et al, 2006) and focal adhesions (Petit and Thiery, 2000;Rajfur et al, 2002;Humphries et al, 2007;Wehrle-Haller, 2007), as well as adherens junctions (Wheelock and Knudsen, 1991;Aberle et al, 1996;Ganz et al, 2006;Liedert et al, 2006) and primary cilia (Alenghat et al, 2004;Oishi et al, 2006;Montalbetti et al, 2007). Distinguishing between these mechanisms as well as the family of signaling proteins responsible for RhoA activation will provide novel insight into future therapeutics for tissue engineering.…”