David S. Park

Caveolin, Caveolae, and Endothelial Cell Function

Abstract—Caveolae are 50- to 100-nm cell-surface plasma membrane invaginations observed in terminally differentiated cells. They are particularly abundant in endothelial cells, where they are believed to play a major role in the regulation of endothelial vesicular trafficking and signal transduction. The use of caveolin-1–deficient mice has provided many new insights into the roles of caveolae and caveolin-1 in the regulation of endothelial cell function. These novel findings suggest an important role for caveolin-1 in the pathogenesis of cancer, atherosclerosis, and vascular disease.

Caveolin-1/3 Double-Knockout Mice Are Viable, but Lack Both Muscle and Non-Muscle Caveolae, and Develop a Severe Cardiomyopathic Phenotype

The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.

Genetic Ablation of Caveolin-1 Confers Protection Against Atherosclerosis

Objective—The development of atherosclerosis is a process characterized by the accumulation of lipids in the form of modified lipoproteins in the subendothelial space. This initiating step is followed by the subsequent recruitment and proliferation of other cell types, including monocytes/macrophages and smooth muscle cells. Here, we evaluate the potential role of caveolae membrane domains in the pathogenesis of atherosclerosis by using apolipoprotein E-deficient (ApoE−/−) mice as a model system. Methods and Results—Caveolin-1 (Cav-1) is a principal structural protein component of caveolae membrane domains. To directly assess the in vivo role of caveolae and Cav-1 in atherosclerosis, we interbred Cav-1−/− mice with ApoE−/− mice. Interestingly, loss of Cav-1 resulted in a dramatic >2-fold increase in non-HDL plasma cholesterol levels in the ApoE−/− background. However, despite this hypercholesterolemia, we found that loss of Cav-1 gene expression was clearly protective against the development of aortic atheromas, with up to an ≈70% reduction in atherosclerotic lesion area. Mechanistically, we demonstrated that loss of Cav-1 resulted in the dramatic downregulation of certain proatherogenic molecules, namely, CD36 and vascular cell adhesion molecule-1. Conclusions—Taken together, our results indicate that loss of Cav-1 can counteract the detrimental effects of atherogenic lipoproteins. Thus, Cav-1 is a novel target for drug development in the pharmacologic prevention of atheroma formation. Our current data also provide the first molecular genetic evidence to support the hypothesis that caveolar transcytosis of modified lipoproteins (from the blood to the sub-endothelial space) is a critical initiating step in atherosclerosis.

Caveolin-1 Mutations (P132L and Null) and the Pathogenesis of Breast Cancer: Caveolin-1 (P132L) Behaves in a Dominant-Negative Manner and Caveolin-1 (−/−) Null Mice Show Mammary Epithelial Cell Hyperplasia

Caveolin-1 (Cav-1) is the principal structural protein of caveolae membranes that are found in most cells types, including mammary epithelial cells. Recently, we mapped the human CAV1 gene to a suspected tumor suppressor locus (7q31.1/D7S522) that is deleted in a variety of human cancers, as well as mammary tumors. In addition, the CAV1 gene is mutated (P132L) in up to approximately 16% of human breast cancers. The mechanism by which deletion or mutation of the Cav-1 gene contributes to mammary tumorigenesis remains unknown. To understand the role of the Cav-1 (P132L) mutation in the pathogenesis of human breast cancers, we generated the same mutation in wild-type (WT) Cav-1 and studied its behavior in cultured cells. Interestingly, the P132L mutation leads to formation of misfolded Cav-1 oligomers that are retained within the Golgi complex and are not targeted to caveolae or the plasma membrane. To examine whether the Cav-1 (P132L) mutant behaves in a dominant-negative manner, we next co-transfected cells with Cav-1 (P132L) and WT Cav-1, and evaluated their caveolar targeting. Our results indicate that Cav-1 (P132L) behaves in a dominant-negative manner, causing the mislocalization and intracellular retention of WT Cav-1. Virtually identical results were obtained when Cav-1 (P132L) was stably expressed at physiological levels in a nontransformed human mammary epithelial cell line (hTERT-HME1). These data provide a molecular explanation for why only a single mutated CAV1 allele is found in patients with breast cancer. Thus, we next investigated if functional inactivation of Cav-1 gene expression leads to mammary tumorigenesis in vivo. For this purpose, we performed mammary gland analysis on Cav-1-deficient mice (-/-) that harbor a targeted disruption of the Cav-1 gene (a null mutation). Interestingly, we show that inactivation of Cav-1 gene expression leads to mammary epithelial cell hyperplasia, even in 6-week-old virgin female mice. These data clearly implicate loss of functional Cav-1 in the pathogenesis of mammary epithelial cell hyperplasia, and suggest that Cav-1-null mice represent a novel animal model to study premalignant mammary disease.

Caveolin-1 Expression Enhances Endothelial Capillary Tubule Formation

The level of caveolin-1 expression closely correlates with the oncogenic transformation of NIH 3T3 cells, the proliferation of human cancer cells, and the differentiation of adipocytes and muscle cells. However, the role of caveolin-1 in endothelial cell proliferation and differentiation remains unknown. Here, we have shown that angiogenic growth factors that stimulate endothelial cell proliferation lead to dramatic reductions in caveolin-1 expression. In addition, using an in vitroMatrigel assay system, we studied the potential role of caveolin-1 in capillary-like tubule formation (i.e. endothelial cell differentiation) using human microvascular endothelial cells (HMEC-1). We showed that the level of endogenous caveolin-1 expression increased in a time-dependent manner when endothelial cells underwent differentiation and that the maximum level of caveolin-1 expression occurred just prior to the formation of capillary-like tubules. Interestingly, overexpression of caveolin-1, via an adenoviral gene delivery system, clearly accelerated endothelial cell differentiation/tubule formation and led to a dramatic ∼3-fold increase in the number of capillary-like tubular structures. Conversely, down-regulation of caveolin-1 expression, via an antisense adenoviral approach, reduced the number of capillary-like tubules formed by >10-fold. Consistent with the unique function of caveolin-1 in interacting with key signaling molecules, delivery of the caveolin-1 scaffolding domain into the cytoplasm of living endothelial cells was also sufficient to enhance capillary-like tubule formation. Taken together, these results clearly demonstrate that caveolin-1 and the caveolin-1 scaffolding domain play an important positive role in the regulation of endothelial cell differentiation, a prerequisite step in the process of angiogenesis.

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

Glomerular Endothelial Fenestrae In Vivo Are Not Formed from Caveolae

Previous reports indicate that endothelial fenestrae in vitro can form by fusion of caveolae or caveolae-like vesicles. The principal aim of this study was to determine whether formation of glomerular endothelial cell fenestrae in vivo similarly involves caveolae and caveolin-1. Whereas caveolin-1 immunofluorescence was found around the circumference of human and mouse glomerular capillary loops, it co-localized only partially with the endothelium-specific lectin Ulex Europaeus I in human glomeruli, leaving portions of the endothelium devoid of caveolin-1. Immunogold electron microscopy, used to definitively localize caveolin-1 in glomeruli, showed that caveolin-1 was completely excluded from the fenestrated portion of the endothelium. Moreover, in caveolin-1-deficient mice, which cannot form caveolae, the ultrastructure of glomerular endothelial fenestrae appeared entirely normal. Interestingly, strong caveolin-1 immunogold labeling was observed in podocytes, where some caveolin-1 localized to filtration slits. Caveolin-1 co-immunoprecipitated with the podocyte slit diaphragm proteins nephrin and CD2AP, and dual immunofluorescence confirmed co-localization of caveolin-1 and nephrin. Nevertheless, in caveolin-1-deficient mice, podocyte ultrastructure appeared normal, and the podocyte proteins synaptopodin, nephrin, and podocin were expressed normally. In addition, blood urea nitrogen concentrations and urinary protein excretion in these mice were similar to those in wild-type mice. Thus, unlike caveolae formation, glomerular endothelial cell fenestrae formation in vivo does not require caveolin-1, ruling out the previous hypothesis that endothelial fenestrae represent fused caveolae, at least for glomerular endothelial cells. Localization of caveolin-1 to podocytes and their filtration slits is consistent with the view that the filtration slit plasma membrane represents a type of lipid raft microdomain.

Prolactin Negatively Regulates Caveolin-1 Gene Expression in the Mammary Gland during Lactation, via a Ras-dependent Mechanism

Caveolin-1 is a 22-kDa integral membrane protein that has been suggested to function as a negative regulator of mitogen-stimulated proliferation in a variety of cell types, including mammary epithelial cells. Because much of our insight into caveolin-1 function has come from the study of human breast tumor-derived cell lines in culture, the normal physiological regulators of caveolin-1 expression in the mammary gland remain unknown. Here, we examine caveolin-1 expression in mice at different stages of mammary gland development. We show that caveolin-1 expression is significantly down-regulated during late pregnancy and lactation. Upon weaning, mammary gland expression of caveolin-1 rapidly returns to non-pregnant “steady-state” levels. Injection of virgin mice with a battery of hormones normally up-regulated during lactation demonstrates that prolactin is the main mediator of caveolin-1 down-regulation. Virtually identical results were obtained with human mammary epithelial cells (hTERT-HME1) in culture. In addition, we demonstrate that prolactin-mediated down-regulation of caveolin-1 expression occurs at the level of transcriptional control and via a Ras-dependent mechanism. Interestingly, in the mammary gland, both mammary epithelial cells and the surrounding mammary adipocytes show prolactin-mediated down-regulation of caveolin-1. This hormone-dependent regulation of caveolin-1 expression is specific to the mammary fat pad. Finally, we employed HC11 cells, a well-established model of mammary epithelial cell differentiation, to study the possible functional effects of caveolin-1 expression. In the presence of lactogenic hormones, recombinant expression of caveolin-1 in HC11 cells dramatically suppresses the induction of the promoter activity and the synthesis of β-casein, an established reporter of lactogenic differentiation and milk production. These findings may explain why caveolin-1 levels are normally down-regulated during lactation. This report is the first demonstration that caveolin-1 levels are down-regulated during a normal physiological event in vivo, i.e. lactation, because previous reports have only documented that down-regulation of caveolin-1 occurs during cell transformation and tumorigenesis.