Caveolins muscle their way into the regulation of cell differentiation, development, and function. Focus on "Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells."

Rennolds S Ostrom

Department of Pharmacology and the Vascular Biology Center of Excellence, University of Tennessee Health Science Center, Memphis, Tennessee

SINCE THEIR INITIAL CHARACTERIZATION in the early 1990s, the functions ascribed to caveolin proteins have steadily increased in complexity and sophistication. Caveolins were initially identified as the main component of the "coat" of caveolae, vesicles that were originally described in the 1950s (22). Caveolae were initially considered to be vesicular structures that mediated transcytosis of macromolecules; caveolins were thus viewed as structural proteins that aided formation of the vesicle. By the mid-1990s, caveolae and their "siblings," lipid rafts, rapidly became appreciated as "hot spots" for plasmalemmal signaling, with a newly recognized function as organizational or scaffolding proteins that attract and retain certain signaling moieties in efficient complexes. However, in recent years, evidence has steadily mounted to support the notion that caveolins are much more than simply structural components of vesicles or docking sites for signaling molecules. In fact, caveolins are now acknowledged to be critical regulators of several signaling pathways that control cell development, differentiation, and proliferation.

Caveolar vesicles, 50- to 100-nm flasklike indentations ("little caves"), can be morphologically identified in the plasma membrane of cells that express caveolin (1). Caveolae, which are expressed only by certain cells, have a lipid composition similar to that of lipid rafts (and can be considered subsets of lipid rafts), which are expressed by all mammalian cells (12). Three isoforms of caveolin have been identified, caveolin-1 (Cav-1), caveolin-2 (Cav-2), and caveolin-3 or M-caveolin (Cav-3) (10, 25, 32, 34). Cav-1 is the most widely expressed and well studied of the caveolins. Although structurally unique, Cav-3 is specific to striated muscle but is similar to Cav-1 in that both isoforms contain a scaffolding domain near their amino termini that can interact with a number of signaling proteins and both can induce caveolar biosynthesis. Cav-2 is the "black sheep" of the caveolin family because it appears to exist only as a heterooligomer with Cav-1 or Cav-3 and does not induce caveolar biosynthesis on its own (13, 20, 2325). The biology of caveolin proteins has been reviewed in detail elsewhere (15, 21, 35), as has the role of caveolae as microdomains that compartmentalize plasmalemmal signaling (8, 16, 17, 28).

As Cav-1 began to be intensively studied, it became clear that not only did it bind numerous signaling proteins via its amino-terminal scaffolding domain but also that this region could inhibit the activity of many of these binding partners (5, 27). Examples include endothelial nitric oxide synthase, epidermal growth factor receptor, Src tyrosine kinases, Ras, protein kinase C, G protein receptor kinase, and adenylyl cyclase. Recent data also demonstrate that Cav-1 is not restricted to the caveolar coat, but can be found in the cytosol and other cellular organelles in striated muscle and other cell types (4, 14). Such mechanistic studies helped provide a strong rationale for examining the physiology of caveolins using transgenic and knockout mice (21). Those more recent in vivo investigations have revealed even more complex and diverse roles for the caveolins than had been previously known. Cav-1 alters lipid homeostasis and regulates mitogenic pathways that influence cell proliferation, particularly in the lung and vasculature (6, 19). Cav-3 is central to normal skeletal muscle differentiation and function, with Cav-3 knockout mice displaying atypical muscle fibers and immature T tubules (7). Taken together, these studies have translated earlier molecular observations of caveolin function to illustrations of the impact Cav-1 and Cav-3 on cellular development, differentiation, and homeostasis.

Similar studies of Cav-2 were not expected to be as dramatic. Surprisingly, Cav-2-knockout animals display a profound pulmonary phenotype, suggesting a key role for this caveolin in lung cell mitogenesis (20). This finding, along with data showing that Cav-2 is a necessary complement to Cav-1 in the lung (13), may signal a resurgence in appreciation for this more poorly studied member of the caveolin family.

There is now even greater impetus for expending effort in studies of Cav-2. In this issue, Capozza and colleagues (Ref. 3, see p. C677 in this issue) report that Cav-1, Cav-2, and Cav-3 can interact to form oligomers in striated muscle, but in fibroblasts Cav-2 and Cav-3 do not interact. These studies build on reports by Rybin et al. (23), who examined cardiac myocytes and first demonstrated that Cav-2 and Cav-3 can interact, and Woodman et al. (36), who examined bladder smooth muscle and also observed oligomerization of all three caveolin isoforms. Together, these reports alter our views of the role of caveolin isoforms, particularly Cav-2, in muscle physiology. Capozza et al. (3) report that Cav-2 must be coexpressed with Cav-1 in fibroblasts to be localized in lipid rafts or caveolae or to be significantly expressed. These observations are consistent with previous reports from this group and others. Expression of Cav-3 did not rescue expression of Cav-2 and did not result in the formation of Cav-1/Cav-3 heterooligomers. These findings in fibroblasts presumably relate to a similar interaction of caveolins in other nonmuscle cells. However, in undifferentiated L6 myoblast cells that endogenously express both Cav-1 and Cav-2, transfection of Cav-3 leads to interaction of all three isoforms of caveolin. In fact, Cav-3 transfection increased the expression of both Cav-1 and Cav-2 in caveolae and created protein distributions in floating fractions (i.e., lipid rafts and caveolae) that are more reminiscent of a differentiated striated muscle cell (23). These observations are consistent with those of Rybin et al. and with reports that Cav-3 is not expressed in myoblastic precursor cells but is expressed in differentiated skeletal myocytes (23, 29). Because Cav-3 regulates muscle development and differentiation and since Cav-2 is coexpressed with Cav-3 in developing skeletal cardiac muscle (9), we must now consider the possibility that Cav-2 plays at least a modulatory role in muscle cell development and differentiation.

As do many pioneering studies, the report by Capozza et al. also raises several questions. Foremost among these questions is which molecular entities do striated muscle cells possess to allow coassembly of Cav-2 and Cav-3? Capozza et al. raise two possibilities: 1) muscle-specific expression of an accessory protein or 2) muscle-specific expression of a chaperone that directs the Cav-2/Cav-3 assembly. While either idea is possible, the former is intriguing because it implies a higher order of complex assembly involving other proteins. Considering the intricate architecture of a striated muscle cell, it would be tempting to hypothesize that caveolar complexes in these cells contain muscle-specific proteins that modify other components in the complexes. A related question is how Cav-2/Cav-3 heterooligomers alter muscle cell signaling and/or function. Mutations in Cav-3 have been associated with various forms of muscular dystrophies (11). If Cav-2 can modify the function of Cav-3, then one would predict that Cav-2 would play a role in regulation of muscle development or function. Capozza et al. cite as-yet unpublished observations from the Cav-2-knockout mice as an indication that this may indeed be true.

An additional question is how these findings in striated muscle translate to smooth muscle physiology. Arterial, ileal, uterine, and stomach smooth muscle cells express all three caveolin isoforms (18, 26, 29, 31), whereas many other smooth muscle cell types express only Cav-1 and Cav-2 (24). Uterine smooth muscle appears to express heterooligomers of Cav-1, Cav-2, and Cav-3, but knockout of Cav-1 expression leads to a sizeable reduction in Cav-2 expression (31, 36). Thus Cav-3 may not be able to rescue Cav-2 expression, meaning that smooth muscle may have limited ability to form Cav-2/Cav-3 heterooligomers. While this observation from one smooth muscle cell type may not apply to other smooth muscle-containing tissues, there does not appear to be an obligatory role for Cav-2 in normal smooth muscle cell development or function based on the reported phenotype of Cav-2-null mice (20). The answer may come from determining whether Cav-2 expression modifies the signaling attributes of coexpressed Cav-1 and/or Cav-3. In other words, does coexpression of Cav-2, which does not appear to inhibit mitogenic signaling, dilute the inhibitory effects of Cav-1 and/or Cav-3 on these signaling pathways? As yet, this idea has not been directly tested.

There does not appear to be a consistent role for Cav-3 in smooth muscle function based upon its intermittent expression across a variety of smooth muscle-containing tissues, but recent work by Woodman et al. (36) is revealing. These investigators have shown that expression of Cav-3 in smooth muscle suppresses Cav-1-mediated caveolar biogenesis. Because caveolae are more prevalent in contractile vascular smooth muscle cells than in the secretory phenotype (33), Cav-3 expression may confer a less contractile phenotype. Does the balance between Cav-1 and Cav-3 expression play a role in smooth muscle cell differentiation from a contractile to a secretory phenotype or alter the regulation of smooth muscle contraction or proliferation (2, 30)? Cav-1-knockout animals display decreased muscarinic receptor-mediated smooth muscle contractions, illustrating a physiological role of this caveolin isoform (36). In contrast, the contractile phenotype of smooth muscle cells from Cav-2- and Cav-3-knockout animals have not been reported.

In conclusion, it is increasingly evident that all caveolins (and probably caveolae as well) are not the same and that different oligomeric combinations of caveolins can impart different characteristics to cells in which they are expressed. It remains to be seen whether different oligomeric assemblies of caveolin isoforms can exist in the same cell. It is also increasingly clear that caveolins are important determinants of cell physiology. The understanding of caveolins has evolved from simple markers of caveolae to recognition of their role as scaffolding proteins and regulators of cell signaling. Our understanding of the role of these proteins is now expanding with the realization that they affect not only muscle cell development and differentiation but also function. These seemingly simple, svelte proteins are thus turning out to have significant power and influence: certainly more than originally proposed or perhaps even imagined.

FOOTNOTES


Address for reprint requests and other correspondence: R. S Ostrom, Dept. of Pharmacology, Univ. of Tennessee Health Science Center, 874 Union Ave., Crowe 115, Memphis, TN 38163 (E-mail: rostrom{at}utmem.edu)

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