Jeffrey A. Engelman

Caveolins, Liquid-Ordered Domains, and Signal Transduction

Caveolae were originally identified as flask-shaped invaginations of the plasma membrane in endothelial and epithelial cells (14). Prior to the development of biochemical methods for their purification, caveolae were thought to principally mediate the transcellular movement of molecules (101, 145). Recently, the development of novel purification procedures has greatly expanded our knowledge regarding the putative functions of caveolae in vivo. In this review, we seek to update the working definition of caveolae, describe the functional roles of the caveolin gene family, and summarize the evidence that supports a role for caveolae as mediators of a number of cellular signaling processes.

Cell-type and Tissue-specific Expression of Caveolin-2 CAVEOLINS 1 AND 2 CO-LOCALIZE AND FORM A STABLE HETERO-OLIGOMERIC COMPLEX IN VIVO

Caveolae are microdomains of the plasma membrane that have been implicated in organizing and compartmentalizing signal transducing molecules. Caveolin, a 21–24-kDa integral membrane protein, is a principal structural component of caveolae membranein vivo. Recently, we and other laboratories have identified a family of caveolin-related proteins; caveolin has been re-termed caveolin-1. Here, we examine the cell-type and tissue-specific expression of caveolin-2. For this purpose, we generated a novel mono-specific monoclonal antibody probe that recognizes only caveolin-2, but not caveolins-1 and -3. A survey of cell and tissue types demonstrates that the caveolin-2 protein is most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts (L6, BC3H1, C2C12), fibroblasts, and 3T3-L1 cells differentiated to adipocytes. This pattern of caveolin-2 protein expression most closely resembles the cellular distribution of caveolin-1. In line with these observations, co-immunoprecipitation experiments with mono-specific antibodies directed against either caveolin-1 or caveolin-2 directly show that these molecules form a stable hetero-oligomeric complex. The in vivo relevance of this complex was further revealed by dual-labeling studies employing confocal laser scanning fluorescence microscopy. Our results indicate that caveolins 1 and 2 are strictly co-localized within the plasma membrane and other internal cellular membranes. Ultrastructurally, this pattern of caveolin-2 localization corresponds to caveolae membranes as seen by immunoelectron microscopy. Despite this strict co-localization, it appears that regulation of caveolin-2 expression occurs independently of the expression of either caveolin-1 or caveolin-3 as observed using two different model cell systems. Although caveolin-1 expression is down-regulated in response to oncogenic transformation of NIH 3T3 cells, caveolin-2 protein levels remain unchanged. Also, caveolin-2 protein levels remain unchanged during the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.

Targeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade

Caveolin‐1 is a principal component of caveolae membranes in vivo. Caveolin‐1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin‐1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether downregulation of caveolin‐1 is sufficient to mediate cell transformation or tumorigenicity. Here, we employ an antisense approach to derive stable NIH 3T3 cell lines that express dramatically reduced levels of caveolin‐1 but contain normal amounts of caveolin‐2. NIH 3T3 cells harboring antisense caveolin‐1 exhibit anchorage‐independent growth, form tumors in immunodeficient mice and show hyperactivation of the p42/44 MAP kinase cascade. Importantly, transformation induced by caveolin‐1 downregulation is reversed when caveolin‐1 protein levels are restored to normal by loss of the caveolin‐1 antisense vector. In addition, we show that in normal NIH 3T3 cells, caveolin‐1 expression levels are tightly regulated by specific growth factor stimuli and cell density. Our results suggest that upregulation of caveolin‐1 may be important in mediating contact inhibition and negatively regulating the activation state of the p42/44 MAP kinase cascade.

Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo: A role for the caveolin-scaffolding domain

The p42/44 mitogen‐activated protein (MAP)‐kinase cascade is a well‐established signal transduction pathway that is initiated at the cell surface and terminates within the nucleus. More specifically, receptor tyrosine kinases can indirectly activate Raf, which in turn leads to activation of MEK and ERK and ultimately phosphorylation of Elk, a nuclear transcription factor. Recent reports have suggested that some members of p42/44 MAP kinase cascade can be sequestered within plasmalemmal caveolae in vivo. For example, morphological studies have directly shown that ERK‐1/2 is concentrated in plasma membrane caveolae in vivo using immunoelectron microscopy. In addition, constitutive activation of the p42/44 MAP kinase cascade is sufficient to reversibly down‐regulate caveolin‐1 mRNA and protein expression. However, the functional relationship between the p42/44 MAP kinase cascade and caveolins remains unknown. Here, we examine the in vivo role of caveolins in regulating signaling along the MAP kinase cascade. We find that co‐expression with caveolin 1 dramatically inhibits signaling from EGF‐R, Raf, MEK‐1 and ERK‐2 to the nucleus. Using a variety of caveolin‐1 deletion mutants, we mapped this in vivo inhibitory activity to caveolin‐1 residues 32–95. Peptides derived from this region of caveolin 1 also inhibit the in vitro kinase activity of purified MEK‐1 and ERK‐2. Thus, we show here that caveolin‐1 expression can inhibit signal transduction from the p42/44 MAP kinase cascade both in vitro and in vivo. Taken together with previous data, our results also suggest that a novel form of reciprocal negative regulation exists between p42/44 MAP kinase activation and caveolin‐1 protein expression, i.e. up‐regulation of caveolin‐1 protein expression down‐modulates p42/44 MAP kinase activity (this report) and up‐regulation of p42/44 MAP kinase activity down‐regulates caveolin‐1 mRNA and protein expression.

GENES ENCODING HUMAN CAVEOLIN-1 AND -2 ARE CO-LOCALIZED TO THE D7S522 LOCUS (7Q31.1), A KNOWN FRAGILE SITE (FRA7G) THAT IS FREQUENTLY DELETED IN HUMAN CANCERS

The (CA)n microsatellite repeat marker D7S522 is located on human chromosome 7q31.1 and is frequently deleted in a variety of human cancers, including squamous cell carcinomas of the head and neck, prostate cancers, renal cell carcinomas, ovarian adenocarcinomas, colon carcinomas, and breast cancers. In addition, D7S522 spans FRA7G, a known common fragile site on human chromosome 7. Based on these studies, it has been proposed that an as yet unidentified tumor suppressor gene (or genes) is contained within or located in close proximity to this locus. However, the identity of the candidate tumor suppressor gene at the D7S522 locus remains unknown. Here, we show that the human genes encoding caveolins 1 and 2 are contained within the same human genomic BAC clones and co‐localize to the q31.1‐q31.2 region of human chromosome 7, as seen by FISH analysis. In addition, we determined the intron‐exon boundaries of the human caveolin‐1 and ‐2 genes. The human caveolin‐1 gene contains three exons, while the human caveolin‐2 gene contains two exons. Interestingly, the boundary of the last exon of the human caveolin‐1 and caveolin‐2 genes are analogous, suggesting that they arose through gene duplication at this locus. (CA)n microsatellite repeat marker analysis of these caveolin genomic clones indicates they contain the marker D7S522 (located at 7q31.1), but not other microsatellite repeat markers tested. The close proximity of caveolins 1 and 2 to the D7S522 locus was independently confirmed by using a panel of MIT/Whitehead human STS markers that are known to map in the neighborhood of the D7S522 locus. As it has been previously shown that caveolin 1 possesses transformation suppressor activity (Koleske, A.J., Baltimore, D. and M.P. Lisanti (1995) Proc. Natl. Acad. Sci. USA 92, 1381–1385; Engelman, J.A. et al. (1997) J. Biol. Chem. 272, 16374–16381), we propose that the caveolin‐1 gene may represent the candidate tumor suppressor gene at the D7S522 locus on human chromosome 7q31.1.

Reciprocal Regulation of Neu Tyrosine Kinase Activity and Caveolin-1 Protein Expression in Vitro and in Vivo IMPLICATIONS FOR HUMAN BREAST CANCER

Neu (c-erbB2) is a proto-oncogene product that encodes an epidermal growth factor-like receptor tyrosine kinase. Amplification of wild-type c-Neuand mutational activation of Neu (Neu T) have been implicated in oncogenic transformation of cultured fibroblasts and mammary tumorigenesis in vivo. Here, we examine the relationship between Neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Recent studies have suggested that caveolins may function as negative regulators of signal transduction. Our current results show that mutational activation of c-Neu down-regulates caveolin-1 protein expression, but not caveolin-2, in cultured NIH 3T3 and Rat 1 cells. Conversely, recombinant overexpression of caveolin-1 blocks Neu-mediated signal transduction in vivo. These results suggest a reciprocal relationship between c-Neu tyrosine kinase activity and caveolin-1 protein expression. We next analyzed a variety of caveolin-1 deletion mutants to map this caveolin-1-dependent inhibitory activity to a given region of the caveolin-1 molecule. Results from this mutational analysis show that this functional in vivo inhibitory activity is contained within caveolin-1 residues 32–95. In accordance with thesein vivo studies, a 20-amino acid peptide derived from this region (the caveolin-1 scaffolding domain) was sufficient to inhibit Neu autophosphorylation in an in vitro kinase assay. To further confirm or refute the relevance of our findings in vivo, we next examined the expression levels of caveolin-1 in mammary tumors derived from c-Neu transgenic mice. Our results indicate that dramatic reduction of caveolin-1 expression occurs in mammary tumors derived from c-Neu-expressing transgenic mice and other transgenic mice expressing downstream effectors of Neu-mediated signal transduction, such as Src and Ras. Taken together, our data suggest that a novel form of reciprocal negative regulation exists between c-Neu and caveolin-1.

Molecular Genetics of the Caveolin Gene Family: Implications for Human Cancers, Diabetes, Alzheimer Disease, and Muscular Dystrophy

Because of space limitations, we were unable to cite many important primary references. This work was supported by National Institutes of Health FIRST Award GM-50443 (to M.P.L.) and by grants from the Charles E. Culpeper Foundation (to M.P.L.), the G. Harold and Leila Y. Mathers Charitable Foundation (to M.P.L and P.E.S.), and the Sidney Kimmel Foundation for Cancer Research (to M.P.L.). P.E.S. was supported by a grant from the Pfizer Corporation, a pilot grant from the Diabetes Research and Research Training Center of the Albert Einstein College of Medicine, and a research grant from the American Diabetes Association. T.O. is supported by National Institutes of Health FIRST Award MH-56036 and the Prentiss Foundation. J.A.E. was supported by National Institutes of Health Medical Scientist Training Program grant T32-GM07288. C.M. was supported by Italian Telethon grant 1111. R.G.P. was supported in part by National Institutes of Health grants R29CA70897, RO1 CA75503, and P50-HL56399 and is a recipient of the Ira T. Hirschl award and an award from the Susan G. Komen Breast Cancer Foundation.

Crowded little caves: Structure and function of caveolae

Caveolae are small vesicular invaginations of the cell membrane. It is within this organelle that cells perform transcytosis, potocytosis and signal transduction. These little caves are composed of a mixture of lipids and proteins unlike those found in the plasma membrane proper. The chief structural proteins of caveolae are caveolins. To date, three caveolins (Cav-1, -2 and -3) with unique tissue distributions have been identified. Caveolins form a scaffold onto which many signalling molecules can assemble, to generate pre-assembled signalling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signalling molecules.

p42/44 MAP Kinase-dependent and -independent Signaling Pathways Regulate Caveolin-1 Gene Expression ACTIVATION OF RAS-MAP KINASE AND PROTEIN KINASE A SIGNALING CASCADES TRANSCRIPTIONALLY DOWN-REGULATES CAVEOLIN-1 PROMOTER ACTIVITY

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are down-regulated in NIH 3T3 cells in response to transformation by activated oncogenes, such as H-Ras(G12V) and v-Abl. The mechanisms governing this down-regulation event remain unknown. Here, we show that caveolin-1 gene expression is directly regulated by activation of the Ras-p42/44 MAP kinase cascade. Down regulation of caveolin-1 protein expression by Ras is independent of (i) the type of activating mutation (G12V versus Q61L) and (ii) the form of activated Ras transfected (H-Ras versus K-Rasversus N-Ras). Treatment of Ras or Raf-transformed NIH 3T3 cells with a well characterized MEK inhibitor (PD 98059) restores caveolin-1 protein expression. In contrast, treatment of v-Src and v-Abl transformed NIH 3T3 cells with PD 98059 does not restore caveolin-1 expression. Thus, there must be at least two pathways for down-regulating caveolin-1 expression: one that is p42/44 MAP kinase-dependent and another that is p42/44 MAP kinase-independent. We focused our efforts on the p42/44 MAP kinase-dependent pathway. The activity of a panel of caveolin-1 promoter constructs was evaluated using transient expression in H-Ras(G12V) transformed NIH 3T3 cells. We show that caveolin-1 promoter activity is up-regulated ∼5-fold by inhibition of the p42/44 MAP kinase cascade. Using electrophoretic mobility shift assays we provide evidence that the caveolin-1 promoter (from −156 to −561) is differentially bound by transcription factors in normal and H-Ras(G12V)-transformed cells. We also show that activation of protein kinase A (PKA) signaling is sufficient to down-regulate caveolin-1 protein expression and promoter activity. Thus, we have identified two signaling pathways (Ras-p42/44 MAP kinase and PKA) that transcriptionally down-regulate caveolin-1 gene expression.

Sequence and detailed organization of the human caveolin-1 and -2 genes located near the D7S522 locus (7q31.1). Methylation of a CpG island in the 5' promoter region of the caveolin-1 gene in human breast cancer cell lines.

The CA microsatellite repeat marker, D7S522, is located at the center of a ∼1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (∼100–200 kb) containing D7S522 and the human genes encoding caveolins 1 and 2. Here, we present the detailed organization of the caveolin locus and its relationship to D7S522, as deduced using a shot‐gun sequencing approach. We derived two adjacent contigs for a total coverage of ∼250 kb. Analysis of these contigs reveals that D7S522 is located ∼67 kb upstream of the caveolin‐2 gene and that the caveolin‐2 gene is located ∼19 kb upstream of the caveolin‐1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the caveolin genes; these include the intron‐exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the caveolin genes. The first and second exons of both caveolin genes are embedded within CpG islands. These results suggest that regulation of caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5′ promoter region of the caveolin‐1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T‐47D) that fail to express the caveolin‐1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non‐methylated and these cells express high levels of the caveolin‐1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the caveolin‐1/‐2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the caveolin genes in a variety of human tumors.