Caveolin-1 and Caveolae Act as Regulators of Mitogenic Signaling in Vascular Smooth Muscle Cells
Smooth muscle cells (SMCs) build up the media of arterial walls and are normally highly differentiated, contractile cells that take part in the control of blood pressure and flow. In response to vascular injury (physical or chemical), they may become activated and gain the ability to migrate through the tissue (from the media to the intima), proliferate, and secrete extracellular matrix components. This change in differentiated properties (transition from a contractile to a synthetic phenotype) is a prerequisite for the participation of the SMCs in formation of intimal thickenings, eg, during atherosclerosis and restenosis.1,2⇓ Attention has lately also been paid to the possibility that progenitor cells circulating in the blood infiltrate the wounded intima and give rise to lesional SMCs.3 In both cases, a variety of extracellular matrix components, growth factors, and cytokines exert crucial regulatory roles.1,2⇓
See pages 1521 and 1528
Recently, a rapidly growing interest has further been focused on the function of caveolae and the protein caveolin in control of cell differentiation and proliferation, among others in the vascular system.4,5⇓ Caveolae are 50- to 80-nm, flask-shaped invaginations of the plasma membrane, enriched in cholesterol and sphingolipids. They contain the membrane protein caveolin (three isoforms described). Caveolin-1 and -2 are found in most cells, whereas caveolin-3 is muscle-specific (most strongly expressed in skeletal and heart muscle cells). The caveolin molecules form oligomers in the membrane and interact directly with cholesterol. Accordingly, both caveolin and cholesterol are required to give rise to the characteristic omega-like shape of caveolae. Caveolin also interacts with a multitude of signaling molecules (via a caveolin-binding motif in the latter).5 The receptors for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and major downstream components in their signaling chains, are just a few examples.6–10⇓⇓⇓⇓ Notably, the association with caveolin seems in most cases to be inhibitory and to maintain the signaling molecules in an inactive state.5 One possibility could be that the binding to caveolin serves to gather the components of a signal transduction system in a spatially defined cell compartment and to prevent inappropriate activation. Once the circumstances are suitable and the proper ligand is available, signaling is initiated. At the same time, the involved molecules dissociate from caveolin and leave caveolae (Figure). Partial evidence for such a mechanism has been gained in studies on PDGF and EGF receptor activation.6–10⇓⇓⇓⇓
Caveolae were originally described by Palade11 in electron microscopic (EM) analyses of endothelial cells in blood capillaries. In collaboration with the Simionescus,12,13⇓ he later made a series of experiments demonstrating the participation of caveolae in macromolecular transport across endothelial cells by transcytosis. In larger blood vessels, medial SMCs likewise display abundant caveolae.14 Other cell types containing plenty of these organelles are type I pneumocytes, adipocytes, and striated muscle cells (highly differentiated cells).1,2⇓ However, most studies on the function of caveolae have been made on cultivated fibroblasts and cell lines. During the last few years, gene targeting was used to create mice deficient in caveolin proteins. This made it possible to begin exploring the physiological roles of caveolins and caveolae also in vivo. Initial studies have revealed that caveolin-1–null mice show (1) lack of caveolae in the tissues examined, (2) down-regulation (degradation) of caveolin-2 but normal expression of caveolin-3, (3) vascular dysfunction with reduced endothelium (NO)-dependent relaxation, contractility, and myogenic tone, and (4) lung abnormalities with thickened alveolar septa.15,16⇓ It was further observed that caveolin-1/3 double-knockout mice lack caveolae both in muscle and nonmuscle cells and develop a severe cardiomyopathy with myocyte hypertrophy, inflammation, and interstitial fibrosis.17 Although the phenotypes of caveolin-deficient animals may appear less serious than expected, the findings stress the importance of caveolin proteins and caveolae in the cardiovascular system.
In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, two articles highlight the significance of caveolin and caveolae in the control of mitogenic signaling in vascular SMCs.18,19⇓ In the first, Peterson et al18 use cultured human coronary vessel SMCs to study (1) how PDGF, a potent SMC mitogen and an important actor in atherogenesis,20,21⇓ affects caveolin-1 expression, and (2) how overexpression of caveolin-1 influences the response to PDGF.21 The results demonstrate that exposure of serum-starved SMCs to PDGF caused a dose-dependent reduction in caveolin-1 levels as determined by immunoblotting (60% reduction at 20 ng/mL PDGF). This effect became apparent 12 hours after addition of PDGF, a lag phase similar to that required for initiation of DNA synthesis.22 EM analysis further showed that the loss of caveolin-1 was accompanied by a decrease in the number of cell surface caveolae (75% reduction in 24 hours). An in vivo confirmation of these findings was obtained by immunocytochemical examination of balloon-injured rabbit iliac arteries. Thus, a diminished caveolin-1 staining was noted in the phenotypically modified SMCs of the growing neointima as compared with the cells of the media.
Interestingly, the decrease in caveolin-1 protein in the PDGF-treated cultures was paralleled by a distinct increase in caveolin-1 mRNA content as determined by real-time polymerase chain reaction (3-fold increase after 24 hours). Hence, the caveolin-1 downregulation in response to PDGF could not be ascribed to an inhibition of caveolin-1 gene transcription. On this basis, it was inferred that the PDGF-induced reduction in caveolin-1 was due to enhanced degradation, either via lysosomes or the ubiquitin-proteasome pathway. By using MG-132, a proteasome inhibitor, no support for the latter possibility was gained. In contrast, treatment of the cells with chloroquine, a weak base that accumulates in and raises the pH of acidic compartments such as the lysosomes, prevented the loss of caveolin-1. However, it remains to be clarified to what extent it was the digestion of caveolin-1 within the lysosomes or the transfer of the protein into these organelles that was inhibited. It is also not clear if a decrease in caveolin-1 synthesis may have contributed to the decline in the cellular levels of this protein.
In the second part of their investigation, Peterson et al18 used an adenoviral construct to study how caveolin-1 overexpression affected the SMCs. The results revealed that the DNA synthetic response to PDGF was completely blocked in the infected cells (as compared with vector controls). Similarly, the proliferation of caveolin-1 overexpressing cells kept in normal, growth medium was suppressed. The infected cells also showed a rounded-up appearance after PDGF exposure, and TUNEL staining indicated that 20% of them had become apoptotic after 24 hours. Additional evidence for activation of the apoptotic machinery (cleavage of caspase-9 and poly-ADP ribose polymerase) was obtained by immunoblot analyses. Notably, the phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2 was not cleary changed in the caveolin-1 overexpressing cells. On the other hand, no upregulation of cyclin D1 was seen. These findings suggest that the increase in caveolin-1 levels of the SMCs did not interfere with early events in PDGF signal transduction (Erk1/2 activation), but blocked entrance into the G1 phase of the cell cycle (cyclin D1 synthesis) and progress into S phase (DNA synthesis). Rather, the cells were shifted into an apoptotic pathway.
The study of Zeidan et al19 focuses on the role of cholesterol in supporting the integrity of caveolae and the growth response of vascular SMCs to stretch and stimulation with endothelin-1 (ET-1). They used an organ culture system in which strips of rat portal veins were depleted of cholesterol by treatment with methyl-β-cyclodextrin and then exposed to stretch and incubated in vitro for up to 24 hours. Initially, it was confirmed that 10 minutes of stretch induced tyrosine phosphorylation of several proteins in an isolated membrane fraction containing caveolin-1. Next, extraction of the vein strips with cyclodextrin was shown to bring about a noticeable decrease in the amount of SMC caveolae (>50% reduction by 5 mmol/L cyclodextrin for 3 hours). Culture of the strips under load (stretch) for 24 hours produced a distinct stimulation of protein and DNA synthesis (with a net increase in weight). These effects were significantly reduced after cholesterol depletion.
To look into possible underlying mechanisms, the phosphorylation of the MAP kinases Erk1/2 in response to stretch was examined. Cholesterol depletion inhibited stretch-induced Erk1/2 phosphorylation by ≈70%. For the main part, this effect could be prevented if the cholesterol-depleted vessels were exposed to cholesterol-loaded cyclodextrin before stretching to reconstitute membrane cholesterol and caveolae. It was also reported that pretreatment with cyclodextrin hindered Erk1/2 phosphorylation in response to endothelin-1 (ET-1), a possible endogenous mediator of the stretch effect. Moreover, the effect of stretch on Erk1/2 activation was repressed by an ET-1 type A receptor blocker (RF139317). Taken together, these findings suggest that stretch-induced growth signaling in vascular SMCs is dependent on cholesterol-rich plasma membrane domains such as caveolae. They further imply that ET-1 of endogenous origin and ET-1 type A receptors are involved.
Summing up, the two studies referred to above attract attention to the fundamental role of caveolin proteins and caveolae in the regulation of SMC growth (Figure). The findings add to earlier observations indicating that caveolae are less abundant in synthetic than in contractile SMCs23 and in proliferating than in nonproliferating cells.5,24⇓ Nevertheless, the analysis of caveolin and caveolae functions in SMC physiology and pathophysiology has just started. Future work will hopefully open up novel avenues for the understanding of the biology of these cells and provide ideas for development of improved therapies of vascular disease. Issues that remain to be clarified include the precise mechanism for how the cellular level of caveolin-1 is downregulated after vascular injury and growth stimulation (eg, by peptide mitogens and/or stretch). Is there a change in protein synthesis? Is caveolin-1 transferred to lysosomes for degradation together with growth factor receptors and other signaling components and, if so, via what route (direct from caveolae or via clathrin-coated pits – see the Figure)? Can down-regulation of caveolin-1 be prevented by pharmaceutical means in order to inhibit loss of caveolae and activation of SMC migration and proliferation?
It should finally be stressed that the role of caveolins and caveolae in the biology of SMCs and other cell types is certainly not restricted to mitogenic signaling and cell growth. Another major function in which these proteins and plasma membrane structures are involved is cellular cholesterol homeostasis.5,25⇓ Caveolin gene transcription and caveolae numbers are upregulated by free cholesterol derived from LDL. Moreover, caveolin is believed to take part in transport of newly synthesized cholesterol as well as free cholesterol released during lysosomal degradation of endocytized LDL from the endoplasmic reticulum to the plasma membrane. There are also results indicating that excess cholesterol may be released from the membrane in caveolae to extracellular particles of HDL. Supposedly, such a reverse cholesterol transport may explain why the caveolae-rich SMCs of the arterial media seldom show signs of abnormal cholesterol accumulation.25,26⇓
The work in the author’s laboratory is supported by the Swedish Research Council, the Swedish Heart Lung Foundation, and the King Gustaf V 80th Birthday Fund.
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- ↵Mineo C, Gill GN, Anderson RGW. Regulated migration of epidermal growth factor receptor from caveolae. J Biol Chem. 1999; 274: 30636–30643.
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- ↵Palade GE, Simionescu M, Simionescu N. Structural aspects of the permeability of the microvascular endothelium. Acta Physiol Scand Suppl. 1979; 463: 11–32.
- ↵Simionescu M, Simionescu N. Ultrastructure of the microvascular wall: functional correlations. In: Renkin EM, Michel CC, eds. Handbook of Physiology, the Cardiovascular System, Microcirculation.vol IV. Bethesda, Md: American Physiological Society; 1984: 41–101.
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- ↵Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, Schedl A, Haller H, Kurzchalia TV. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science. 2001; 293: 2449–2452.
- ↵Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, Marks CB, Macaluso F, Russell RG, Li M, Pestell RG, Di Vizio D, Hou H Jr, Kneitz B, Lagaud G, Christ GJ, Edelmann W, Lisanti MP. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J Biol Chem. 2001; 276: 38121–38138.
- ↵Park DS, Woodman SE, Schubert W, Cohen AW, Frank PG, Chandra M, Shirani J, Razani B, Tang B, Jelicks LA, Factor SM, Weiss LM, Tanowitz HB, Lisanti MP. Caveolin-1/3 double-knockout mice are viable, but lack both muscle and non-muscle caveolae, and develop a severe cardiomyopathic phenotype. Am J Pathol. 2002; 160: 2207–2217.
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