Requirement of Caveolae Microdomains in Extracellular Signal‐Regulated Kinase and Focal Adhesion Kinase Activation Induced by Endothelin‐1 in Primary Astrocytes

Abstract

Endothelin-1 (ET-1) mitogenic activity in astrocytes is mediated by the activation of the extracellular signal-regulated kinase (ERK) pathway together with the Rho-dependent activation of the focal adhesion kinase (FAK) pathway. To clarify the mechanisms responsible for the coordinate activation of both pathways in the ET-1 signal propagation, the involvement of caveolae microdomains, suggested to play a role in signal transduction, was evaluated. In this study, it is reported that caveolae of primary astrocytes are enriched in endothelin receptor (ET B -R). Furthermore, signaling molecules such as the adaptor proteins Shc and Grb2, and the small G protein Rho, also reside within these microdomains. Selective disassembly of caveolae by filipin III impairs the ET-1-induced tyrosine phosphorylation of proteins including ERK and FAK. In agreement with these observations, astrocytes pretreated with filipin III also failed to form stress fibers and focal adhesions and did not undergo the associated morphological changes in response to ET-1. This study reveals that structural integrity of caveolae is necessary for the adhesion-dependent mitogenic signals induced by ET-1 in astrocytes, through compartmentation of ET B -R with the upstream signaling molecules of the ERK and FAK pathways. Key Words: Endothelin-1-Caveolae -Primary astrocyte -Extracellular signal-regulated kinase -Focal adhesion kinase -Cytoskeleton.

Cross-Linking of Brain Endothelial Intercellular Adhesion Molecule (ICAM)-1 Induces Association of ICAM-1 With Detergent-Insoluble Cytoskeletal Fraction

“…Caveolae are low density membrane microdomains that have been implicated in initiating the signaling events induced by growth factors, such as epidermal growth factor and endothelin-1, and receptors for these ligands are enriched within caveolae. 17 As expected, caveolin-1, a characteristic caveolar marker protein in the brain, 18 appeared to be retained in the Triton X-100 -insoluble fraction under basal conditions and after ICAM-1 crosslinking ( Figure 5D). Under basal ( Figure 5A) and ICAM-1 cross-linking conditions (data not shown), ICAM-1 and caveolin-1 showed no significant colocalization in GP8/3.9 cells, although data from sucrose density gradient fractionation of GP8/3.9 cell lysates showed that caveolin-1 and ICAM-1 cofractionated ( Figure 5C).…”

Caveolin-1 Knockdown by Small Interfering RNA Suppresses Responses to the Chemokine Monocyte Chemoattractant Protein-1 by Human Astrocytes

“…This observation of possible caveolae involvement differs from those noted by others, who described chemokine-induced receptor internalization to be achieved by the more commonly acknowledged clathrincoated pit pathway (33)(34)(35). Insofar as caveolae and their main structural protein, caveolin-1, both of which are expressed by astrocytes (36,37), have been implicated in regulating signal transduction events as well as subserving endocytosis and transcytosis (38), caveolin-1-bearing membrane domains may be critical in mediating one or more of the responses of astrocytes to stimulation by MCP-1.…”

Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1

“…It may be relevant in regard to endothelin-induced contraction that both ET A and ET B receptors have been proposed to depend on caveolae (3,35). The present results showed that impaired contractility in response to ET-1 in the ileum was not associated with reduced ET-1 receptor expression, nor was it due to reduced Ca 2ϩ sensitization.…”