Role Of Cav-1 Research Articles

Caveolin-1 Deficiency Increases Cerebral Ischemic Injury

Caveolins (Cav), the principal structural proteins of the caveolar domains, have been implicated in the pathogenesis of ischemic injury. Indeed, changes in caveolin expression and localization have been reported in renal and myocardial ischemia. Genetic ablation of the Cav-1 gene in mice was further shown to increase the extent of ischemic injury in a model of hindlimb ischemia. However, the role of Cav-1 in the pathogenesis of cerebral ischemia remains unknown. Immunoblot and immunofluorescence analyses of rat brains subjected to middle cerebral artery occlusion revealed marked increases in endothelial Cav-1 and Cav-2 protein levels. To directly assess the functional role of caveolins in the pathogenesis of cerebral ischemic injury, we next investigated the effects of cerebral ischemia in caveolin knockout (KO) mice. Interestingly, Cav-1 KO mice showed a marked increase of cerebral volume of infarction, as compared with wild-type and Cav-2 KO mice. Immunofluorescence analyses showed an increased number of proliferating endothelial cells in wild-type ischemic brains, as compared with Cav-1 KO ischemic brains. Immunoblot analyses of wild-type ischemic brains showed an increase in endothelial nitric oxide synthase protein levels. Conversely, the protein levels of endothelial nitric oxide synthase remained unchanged in Cav-1 KO ischemic brains. TUNEL analysis also showed increased apoptotic cell death in Cav-1 KO ischemic brains, as compared with wild-type ischemic brains. Our findings indicate cerebral ischemia induces a marked increase in endothelial Cav-1 and Cav-2 protein levels. Importantly, genetic ablation of the Cav-1 gene in mice results in increased cerebral volume of infarction. Mechanistically, Cav-1 KO ischemic brains showed impaired angiogenesis and increased apoptotic cell death.

Dispensability and dynamics of caveolin-1 during liver regeneration and in isolated hepatic cells

Caveolae participate in several cellular processes such as vesicular transport, cholesterol homeostasis, regulation of signal transduction, integrin signaling, and cell growth. The expression and functional role of caveolin (Cav), the most abundant protein of caveolae, has been reported in liver and in different hepatocyte cell lines, in human cirrhotic liver, and in hepatocellular carcinomas. The role of Cav-1 in liver regeneration after partial hepatectomy (PH) has been investigated as a model of liver proliferation in vivo. Our results show that Cav-1 increases in liver after PH with a redistribution of the protein from the caveola-enriched domain to the noncaveolar fraction. Moreover, the Cav-1 located in the noncaveolar fraction is phosphorylated in tyrosine 14, even though the Cav-1 gene is dispensable for liver regeneration after PH, as deduced from data obtained with commercially available animals lacking this gene. In addition to this, the proinflammatory stimulation of hepatocytes induces Cav-1 translocation to a noncaveolar fraction and tyrosine 14 phosphorylation mainly through the activation of tyrosine kinases such as Src. These results support a dynamic role for Cav-1 in liver proliferation both in vivo after PH and in vitro in cultured hepatic cell lines, but with minimal implications for the liver regeneration process.

Ahnak, a new player in β-adrenergic regulation of the cardiac L-type Ca2+ channel

Ahnak, originally identified as a giant, tumour-related phosphoprotein, has emerged as an important signalling molecule in a wide range of physiological activities. In this article, current knowledge will be reviewed that places ahnak into the context of cardiac L-type Ca2+ channel function by its interaction with the beta2 subunit. Beginning with an overview on structural and functional properties of ahnak, basic features of beta subunits are highlighted. The review characterizes multiple ahnak/beta2 subunit binding sites and focuses on recent progress in understanding their functional role in Cav1.2 channel conductance (I(CaL)). Three main aspects of ahnak function in I(CaL) of cardiomyocytes emerge from available experimental data. First, ahnak acts as repressor towards I(CaL) by beta2 subunit sequestration. Second, PKA phosphorylation relieves the inhibition imposed by the C-terminal ahnak domain, ahnak-C1. Third, this action is mimicked by ahnak-derived fragments carrying a naturally occurring missense mutation Ile5236Thr. This paradigm introduces ahnak as a player in beta-adrenergic control of I(CaL) and sheds new light upon the molecular mechanism underlying this fundamental process of Cav1.2 channel physiology.

A Novel Role for Caveolin-1 in B Lymphocyte Function and the Development of Thymus-Independent Immune Responses

Caveolin-1 (Cav-1) functions as a scaffold or platform for many molecules involved in signal transduction. However, the expression and function of Cav-1 in the immune system has been controversial. Here, we show that Cav-1 mRNA and protein is indeed expressed in murine B-lymphocytes in a regulated manner. Cav-1 deficient mice displayed reduced levels of antibody in their serum. In order to examine the role of Cav-1 in the development of immunoglobulin-mediated immune responses, we immunized wild-type and Cav-1 deficient mice with thymus-dependent and thymus independent antigens. Our results show that Cav-1 deficient mice have a normal response to thymus-dependent antigens, but have a reduced response to both type I and type II thymus independent antigens. However, lymphocyte populations in the spleen and peritoneum were not altered and no changes were observed in splenic architecture. Caveolin-1 deficient B-lymphocytes did not display altered proliferation in response to different stimuli. However, we found that Cav-1 deficient B cells have reduced IgG3 secretion in vitro in response to LPS. Finally, we also demonstrate that human plasma cells (mature B lymphocytes) express Cav-1 in vivo. Taken, together these results provide convincing evidence for expression of Cav-1 in activated B-lymphocytes and demonstrate a role for Cav-1 in the development of thymus-independent immune responses.

Caveolin-1 immuno-expression in human fetal tissues during mid and late gestation.

Caveolin-1 (Cav-1) is the main protein in caveolae, and serves as a scaffolding protein onto which many classes of signalling molecules are assembled. Through interaction with proto-oncogene products, Cav-1 may suppress cell proliferation; or when phosphorylated, may also stimulate cell growth. The aim of this study was to determine Cav-1 expression in human fetal tissues, tissues composed of cells undergoing growth and differentiation processes which require a nurturing environment provided by transmembrane vesicular transport. By using immunohistochemistry, Cav-1 was detected in several fetal tissues during mid- and late gestation (from 14 to 39 weeks). The protein was present in adipocytes, endothelial cells, smooth muscle fibers and in a number of sites with a pattern of distribution similar to that of the adult. Intriguingly, a positive immunoreaction for Cav-1 was also noticed in tissues, such as the urothelium, which normally do not express this protein in adulthood. This unexpected pattern of Cav-1 in human fetus may predict novel roles for Cav-1 during fetal development.

Caveolin-1 Functions as a Novel Cdc42 Guanine Nucleotide Dissociation Inhibitor in Pancreatic β-Cells

The cycling of the small Rho family GTPase Cdc42 is required for insulin granule exocytosis, although the regulatory proteins involved in Cdc42 cycling in pancreatic beta-cells are unknown. Here we demonstrate that the caveolar protein caveolin-1 (Cav-1) is a Cdc42-binding protein in beta-cells. Cav-1 associated with Cdc42-VAMP2-bound granules present near the plasma membrane under basal conditions. However, stimulation with glucose induced the dissociation of Cav-1 from Cdc42-VAMP2 complexes, coordinate with the timing of Cdc42 activation. Analyses of the Cav-1 scaffolding domain revealed a motif conserved in guanine nucleotide dissociation inhibitors (GDIs), which suggested a novel role for Cav-1 as a Cdc42 GDI in beta-cells. The novel role was further supported by: 1) in vitro binding analyses that demonstrated a direct interaction between Cav-1 and Cdc42; 2) GST-Cdc42 interaction assays showing preferential Cav-1 binding to GDP-Cdc42 over that of GTP-Cdc42; 3) Cav-1 depletion studies resulting in an inappropriate 40% induction of activated Cdc42 in the absence of stimuli and also a 40% increase in basal insulin release from both MIN6 cells and islets. Expression of wild-type Cav-1 in Cav-1-depleted cells restored basal level secretion to normal, whereas expression of a scaffolding domain mutant of Cav-1 failed to normalize secretion. Taken together, these data suggest that Cav-1 functions as a Cdc42 GDI in beta-cells, maintaining Cdc42 in an inactive state and regulating basal secretion in the absence of stimuli. Through its interaction with the Cdc42-VAMP2-bound insulin granule complex, Cav-1 may contribute to the specific targeting of granules to "active sites" of exocytosis organized by caveolae.

Caveolin-1 up-regulates CD147 glycosylation and the invasive capability of murine hepatocarcinoma cell lines

CD147 which is a regulator of matrix metalloproteinase (MMP) production on the surface of many malignant tumor cells, shows a highly specific association with caveolin-1 (Cav-1). As a result of heterogeneous N-glycosylation, CD147 exists in both highly glycosylated form, HG-CD147 (∼40–60 kDa) and lowly glycosylated form, LG-CD147 (∼32 kDa). This study investigated the possible role of Cav-1 in CD147 glycosylation in the HcaF, HcaP and Hepa1-6 mouse hepatocarcinoma cell lines, which have high, low and no metastatic potential in the lymph nodes, respectively, and in the normal mouse liver cell line IAR-20. Using an RNA interference (RNAi) strategy, we showed that the down-regulation of Cav-1 in Hca-F/RNAi cells could suppress the conversion of LG-CD147 to HG-CD147, down-regulate MMP-11 expression and decrease Hca-F/RNAi cell invasion. Conversely, a stable high expression of Cav-1 in Hepa1-6/Cav-1 cell could cause a specific increase of HG-CD147, up-regulate MMP-11 protein expression and enhance Hepa1-6/Cav-1 cell invasion. In conclusion, Cav-1 expression leads to an increased proportion of HG-CD147 relative to LG-CD147, increased production of MMP-11 and a higher invasive capability. Cav-1 is therefore proposed to act as both an oncogene and a tumor suppressor gene, and could represent a new potential target for gene therapy.

Caveolin-1 Promotes Tumor Progression in an Autochthonous Mouse Model of Prostate Cancer: Genetic Ablation of Cav-1 Delays Advanced Prostate Tumor Development in Tramp Mice

Caveolin-1 (Cav-1) is the primary structural component of caveolae and is implicated in the processes of vesicular transport, cholesterol balance, transformation, and tumorigenesis. Despite an abundance of data suggesting that Cav-1 has transformation suppressor properties both in vitro and in vivo, Cav-1 is expressed at increased levels in human prostate cancer. To investigate the role of Cav-1 in prostate cancer onset and progression, we interbred Cav-1(-/-) null mice with a TRAMP (transgenic adenocarcinoma of mouse prostate) model that spontaneously develops advanced prostate cancer and metastatic disease. We found that, although the loss of Cav-1 did not affect the appearance of minimally invasive prostate cancer, its absence significantly impeded progression to highly invasive and metastatic disease. Inactivation of one (+/-) or both (-/-) alleles of Cav-1 resulted in significant reductions in prostate tumor burden, as well as decreases in regional lymph node metastases. Moreover, further examination revealed decreased metastasis to distant organs, such as the lungs, in TRAMP/Cav-1(-/-) mice. Utilizing prostate carcinoma cell lines (C1, C2, and C3) derived from TRAMP tumors, we also showed a positive correlation between Cav-1 expression and the ability of these cells to form tumors in vivo. Furthermore, down-regulation of Cav-1 expression in these cells, using a small interfering RNA approach, significantly reduced their tumorigenic and metastatic potential. Mechanistically, we showed that loss or down-regulation of Cav-1 expression results in increased apoptosis, with increased prostate apoptosis response factor-4 and PTEN levels in Cav-1(-/-) null prostate tumors. Our current findings provide the first in vivo molecular genetic evidence that Cav-1 does indeed function as a tumor promoter during prostate carcinogenesis, rather than as a tumor suppressor.

Insights into voltage-gated calcium channel regulation from the structure of the CaV1.2 IQ domain–Ca2+/calmodulin complex

Changes in activity-dependent calcium flux through voltage-gated calcium channels (Ca(V)s) drive two self-regulatory calcium-dependent feedback processes that require interaction between Ca(2+)/calmodulin (Ca(2+)/CaM) and a Ca(V) channel consensus isoleucine-glutamine (IQ) motif: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Here, we report the high-resolution structure of the Ca(2+)/CaM-Ca(V)1.2 IQ domain complex. The IQ domain engages hydrophobic pockets in the N-terminal and C-terminal Ca(2+)/CaM lobes through sets of conserved 'aromatic anchors.' Ca(2+)/N lobe adopts two conformations that suggest inherent conformational plasticity at the Ca(2+)/N lobe-IQ domain interface. Titration calorimetry experiments reveal competition between the lobes for IQ domain sites. Electrophysiological examination of Ca(2+)/N lobe aromatic anchors uncovers their role in Ca(V)1.2 CDF. Together, our data suggest that Ca(V) subtype differences in CDI and CDF are tuned by changes in IQ domain anchoring positions and establish a framework for understanding CaM lobe-specific regulation of Ca(V)s.

Loss of Caveolin-1 Causes the Hyper-Proliferation of Intestinal Crypt Stem Cells, with Increased Sensitivity to Whole Body ?-Radiation

Caveolin-1 (Cav-1) is a protein marker for caveolae organelles, and acts as a scaffolding protein to negatively regulate the activity of signaling molecules by binding to and releasing them in a timely fashion. We have previously shown that loss of Cav-1 promotes the proliferation of mouse embryo fibroblasts (MEFs) in vitro. Here, to investigate the in vivo relevance of these findings, we evaluated the turnover rates of small intestine crypt stem cells from WT and Cav-1 deficient mice. Interestingly, we show that Cav-1 null crypt stem cells display higher proliferation rates, as judged by BrdU and PCNA staining. In addition, we show that Wnt/?-catenin signaling, which normally controls intestinal stem cell self-renewal, is up-regulated in Cav-1 deficient crypt stem cells. Because the small intestine constitutes one of the main targets of radiation, we next evaluated the role of Cav-1 in radiation-induced damage. Interestingly, after exposure to 15 Gy of ?-radiation, Cav-1 deficient mice displayed a decreased survival rate, as compared to WT mice. Our results show that after radiation treatment, Cav-1 null crypt stem cells of the small intestine exhibit far more apoptosis and accelerated proliferation, leading to a faster depletion of crypts and villi. As a consequence, six days after radiation treatment, Cav-1 -/- mice lost all their crypt and villus structures, while WT mice still showed some crypts and intact villi. In summary, we show that ablation of Cav-1 gene expression induces an abnormal amplification of crypt stem cells, resulting in increased susceptibility to ?-radiation. Thus, our studies provide the first evidence that Cav-1 normally regulates the proliferation of intestinal stem cells in vivo.

Association of aCAV-1 haplotype to familial aggressive prostate cancer

Multiple lines of evidence have implicated the CAV-1 gene in prostate cancer progression. CAV-1 is located within the prostate cancer aggressiveness locus at 7q31-33, and was identified as being overexpressed in prostate tumors. Mutation screening was performed as well as a case-control study to examine if polymorphisms in CAV-1 are associated with prostate cancer aggressiveness in a German population. We sequenced the CAV-1 promoter region and its open reading frame in prostate cancer families with linkage to chromosome 7q31-33. Additionally, 105 unrelated familial prostate cancer probands, 190 sporadic cases, and 191 controls were genotyped at four intronic single nucleotide polymorphisms. Resulting haplotypes were tested for association using age at diagnosis, tumor grade, TNM stage, and follow up information to stratify for aggressive disease. No mutation was found in the CAV-1 coding region or in the promoter. One of the 11 observed haplotypes showed an increased frequency in cases with high tumor stage (P = 0.03). This is the first report providing evidence for CAV-1 being involved in predisposition to aggressive prostate cancer. The association of a potential risk haplotype agrees well with a role of CAV-1 in tumor progression but needs further confirmation.

Different roles for caveolin-1 in the development of non-small cell lung cancer versus small cell lung cancer.

Caveolin-1 (CAV1), an essential structural constituent of caveolae that plays an important role in cellular processes such as transport and signaling, has been implicated in the development of human cancers. However, it is unclear whether CAV1 is acting like an oncogene or tumor suppressor gene. We found that CAV1 expression was reduced or absent in 95% of small cell lung cancers (SCLCs; n = 21 lines), whereas it was retained in 76% of non-small cell lung cancers (NSCLCs; n = 25 lines) compared with normal human lung epithelial cultures, where it was abundantly expressed. CAV1 expression was tightly linked to the ability to grow attached to the plastic cell culture surface, whereas CAV1-nonexpressing lung cancers of both SCLC and NSCLC type grew as suspension cultures. In addition, attached lung cancer cultures expressed phosphorylated focal adhesion kinase, whereas suspension cultures did not. Lack of CAV1 expression was tightly associated with CAV1 promoter methylation (P < 0.0001) such that CAV1 methylation was found in 93% of SCLCs (n = 15) and 9% of NSCLCs (n = 11), whereas 5-aza-2'deoxycytidine treatment restored CAV1 expression in SCLCs. Exogenous CAV1 expression in SCLCs significantly inhibited soft-agar colony formation but did not lead to attachment. By contrast, CAV1 knockdown in NSCLCs mediated by small interfering RNA against CAV1 led to inhibition of cellular proliferation and soft-agar and liquid colony formation. Importantly, CAV1 knockdown led to reduced phospho-focal adhesion kinase and RalA, but not RalB, levels in NSCLC cells. These results suggest different roles for CAV1 in SCLC, where CAV1 acts like a tumor suppressor gene, and NSCLC, where it appears required for survival and growth.

A role for Ca v1.3 in rat intestinal calcium absorption

Active Ca 2+ absorption through epithelial Ca 2+ channels TRPV5/6 in duodenum is activated by hyperpolarisation. However, when diet and Ca 2+ are plentiful, digestion products cause depolarisation. We therefore used homology-based PCR from a rat jejunal mucosal cDNA preparation to reveal the presence of the neuroendocrine L-type isoform Ca v1.3α 1. Immunocytochemical labelling and immunoblotting localised Ca v1.3α 1 protein in apical membrane from proximal jejunum to mid ileum. Perfusion studies in vivo with 1.25 mM luminal Ca 2+ revealed L-type channel activity. Inhibition of glucose absorption with phloridzin strongly inhibited 45Ca 2+ absorption; absorption was inhibited by nifedipine and Mg 2+ and activated by Bay K 8644, none of which affect TRPV5/6. At 10 mM Ca 2+, nifedipine inhibited 45Ca 2+ absorption with a time course similar to that at 1.25 mM Ca 2+: absorption was therefore channel-mediated rather than paracellular. We suggest that in times of dietary sufficiency, Ca v1.3 may mediate a significant route of Ca 2+ absorption into the body.