Recent data have demonstrated that caveolin, a major structural protein of caveolae, negatively regulates signaling molecules localized to caveolae. The interaction of caveolin with several caveolae-associated signaling proteins is mediated by the binding of the scaffolding region of caveolin to a hydrophobic amino acid-containing region within the regulated proteins. The presence of a similar motif within the insulin receptor kinase prompted us to investigate the caveolar localization and regulation of the insulin receptor by caveolin. We found that overexpression of caveolin-3 augmented insulinstimulated phosphorylation of insulin receptor substrate-1 in 293T cells but not the phosphorylation of insulin receptor. Peptides corresponding to the scaffolding domain of caveolin potently stimulated insulin receptor kinase activity toward insulin receptor substrate-1 or a Src-derived peptide in vitro and in a caveolin subtype-dependent fashion. Peptides from caveolin-2 exhibited no effect, whereas caveolin-1 and -3 stimulated activity 10-and 17-fold, respectively. Peptides which increased insulin receptor kinase activity did so without affecting insulin receptor auto-phosphorylation. Furthermore, the insulin receptor bound to immobilized caveolin peptides, and this binding was inhibited in the presence of free caveolin-3 peptides. Thus, we have identified a novel mechanism by which the insulin receptor is bound and activated by specific caveolin subtypes. Furthermore, these data define a new role for caveolin as an activator of signaling.

Caveolar localization of protein kinase C and the regulation of caveolar function by protein kinase C are well known. This study was undertaken to examine whether caveolin subtypes interact with various protein kinase C isoenzymes using the caveolin scaffolding domain peptide. When protein kinase C-␣, -⑀, and -were overexpressed in COS cells followed by subcellular fractionation using the sucrose gradient method, all the isoenzymes (␣, ⑀, and ) were detected in the same fraction as caveolin. The scaffolding domain peptide of caveolin-1 and -3, but not -2, inhibited the kinase activity and autophosphorylation of protein kinase C-␣ and -, but not of protein kinase C-⑀, overexpressed in insect cells. Truncation mutation studies of the caveolin-1 and -3 peptides demonstrated that a minimum of 16 or 14 amino acid residues of the peptide were required for the inhibition or direct binding of protein kinase C. Thus, the caveolin peptide physically interacted with protein kinase C and regulated its function. Further, this regulation occurred in a protein kinase C isoenzyme-dependent manner. Our results may provide a new mechanism regarding the regulation of protein kinase C isoenzyme activity and the molecular interaction of protein kinase C with its putative binding proteins.Several studies from independent laboratories have demonstrated that multiple phorbol ester-sensitive, classic protein kinase C (PKC) 1 isoenzymes are accumulated in caveolae. Enrichment of PKC-␣ was detected by immunoelectron microscopy (1) as well as by the cell fractionation technique using buoyant density gradient centrifugation (2). PKC-␤ and -␥ were also detected in caveolae as separated by the silica coating method from lung endothelial cells (3). Whether all PKC isoenzymes, including nonclassic isoenzymes, are similarly accumulated in caveolae, however, remains uncharacterized. PKC also regulates the function and formation of caveolae. Caveolin, the major structural protein of caveolae, contains a conserved consensus phosphorylation site of PKC as well as of v-Src (4). Phorbol ester treatment of the cell inhibits caveolae-mediated internalization and markedly reduces the number of caveola (1). Further, activation of PKC-␣ by phorbol esters dislocates this isoenzyme from caveolae. Thus, PKC is not only present in caveolae, but interacts functionally with caveolae.It has been suggested that caveolin by itself regulates the function of certain molecules accumulated in caveolae. Caveolin may directly interact with G protein, Src kinase, and HaRas as has been demonstrated using a short stretch of membrane proximal regions of the cytosolic amino terminus caveolin domain (or the caveolin scaffolding domain) (5-8).Further, a small peptide derived from this domain bound G protein directly and regulated its function (9). The specificity of binding caveolin to target molecules has been confirmed, using a random peptide sequence library, by identifying a common amino acid sequence motif (XXXXX or XXXXXX; is an aromatic residue), which is contained in ma...

Abstract-Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27 Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with ␤-cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. ␣ V ␤ 3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of ␣ V ␤ 3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt. (Circ Res. 2005;96:635-642.) Key Words: remodeling Ⅲ muscle, smooth Ⅲ signal transduction Ⅲ stress Ⅲ vasculature I t is currently recognized that the machinery governing the cell cycle regulates multiple cellular functions in the cardiovascular system, thereby maintaining the homeostasis of the vasculature and allowing its adaptation to acute and chronic changes. Besides organizing cellular proliferation, the cell cycle is involved in migration, apoptosis, and hypertrophy. 1 One of the major constituents of the blood vessel wall responsible for the maintenance of vessel structures and functions are vascular smooth muscle cells (VSMCs). In the vasculature, VSMCs are constantly exposed to alternating mechanical forces. Under normal tensile stress, VSMCs are relatively insensitive to mitogens. During altered mechanical stress (eg, high blood pressure), however, VSMCs upregulate protein synthesis in response to growth factors, dedifferentiate, and increase their proliferative rate, resulting in medial hypertrophy and intimal hyperplasia. 2 Whereas the commonly accepted "response to injury" hypothesis suggests that growth factors are locally released, thereby initiating cell cycle entry and progression of vascular cells, the signaling pathways arising solely from mechanical force have just partially c...

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BackgroundAtrial fibrillation is associated with higher mortality. Identification of causes of death and contemporary risk factors for all‐cause mortality may guide interventions.Methods and ResultsIn the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF) study, patients with nonvalvular atrial fibrillation were randomized to rivaroxaban or dose‐adjusted warfarin. Cox proportional hazards regression with backward elimination identified factors at randomization that were independently associated with all‐cause mortality in the 14 171 participants in the intention‐to‐treat population. The median age was 73 years, and the mean CHADS 2 score was 3.5. Over 1.9 years of median follow‐up, 1214 (8.6%) patients died. Kaplan–Meier mortality rates were 4.2% at 1 year and 8.9% at 2 years. The majority of classified deaths (1081) were cardiovascular (72%), whereas only 6% were nonhemorrhagic stroke or systemic embolism. No significant difference in all‐cause mortality was observed between the rivaroxaban and warfarin arms (P=0.15). Heart failure (hazard ratio 1.51, 95% CI 1.33–1.70, P<0.0001) and age ≥75 years (hazard ratio 1.69, 95% CI 1.51–1.90, P<0.0001) were associated with higher all‐cause mortality. Multiple additional characteristics were independently associated with higher mortality, with decreasing creatinine clearance, chronic obstructive pulmonary disease, male sex, peripheral vascular disease, and diabetes being among the most strongly associated (model C‐index 0.677).ConclusionsIn a large population of patients anticoagulated for nonvalvular atrial fibrillation, ≈7 in 10 deaths were cardiovascular, whereas <1 in 10 deaths were caused by nonhemorrhagic stroke or systemic embolism. Optimal prevention and treatment of heart failure, renal impairment, chronic obstructive pulmonary disease, and diabetes may improve survival.Clinical Trial Registration URL: https://www.clinicaltrials.gov/. Unique identifier: NCT00403767.

The identification of various signaling molecules found within caveolae and their functional interaction with the integral membrane protein caveolin, a major structural component of caveolae, suggests that these membrane microdomains participate in transmembrane signaling. Several lines of evidence indicate that caveolin may act as a scaffolding protein by direct interaction with and modulation of the activity of multiple signaling molecules. The compartmentation of various signaling molecules in caveolae and their direct and functional interaction with caveolin provides a paradigm by which these membrane microdomains are involved in regulating signal transduction pathways. By dysregulation of these signal transduction pathways caveolins may be involved in the pathogenesis of various diseases. This review focuses on the implications as well as controversies of the contribution of caveolae and caveolins for several human diseases and the potential implications to therapeutic strategies.

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The rapid amplification of ␤-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein ␣-and ␤␥-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of ␤-adrenergic receptors with caveolin under resting conditions. ␤1-and ␤2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. ␤1-and ␤2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gs␣-and ␤␥-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of ␤-adrenergic receptors with caveolin, indicating a nonrandom distribution of ␤-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, ␤-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize ␤-adrenergic receptors.