Expression Levels Of Caveolin-1 Research Articles

Caveolin‐1 expression regulates blood–retinal barrier permeability and retinal neovascularization in oxygen‐induced retinopathy

Caveolin-1 expression correlates with the permeability of endothelial barriers and angiogenesis. However, the role of caveolin-1 in retinal neovascularization remains unknown. We evaluated the effect of caveolin-1 on the blood-retina barrier and retinal neovascularization in a murine model of oxygen-induced retinopathy. Starting at postnatal day 7, mice were exposed to 75 ± 5% oxygen for 5 days and then returned to room air conditions to induce retinal neovascularization. Effects on blood-retina barrier were evaluated by Western blot analysis of extravasated albumin in the retina. Retinal neovascularization morphology was studied by fluorescence angiography and was quantified by counts of the endothelial nuclei that protruded into the vitreous cavity. Reverse transcription-polymerase chain reaction and Western blot analysis was used to examine retinal expression levels of caveolin-1. siRNA against caveolin-1 was injected intravitreally in the oxygen-induced retinopathy models. Effects on caveolin-1 mRNA and protein, and retinal neovascularization were assessed as described above. Caveolin-1 expression was found to increase during hypoxia and overexpression of caveolin-1 correlated with the appearance of extravascular albumin. Caveolin-1 siRNA reduced caveolin-1 mRNA and protein levels by 47.94% and 54.76%, respectively. Furthermore, caveolin-1 siRNA inhibition reduced retinal neovascularization by 51.3% and reduced albumin leakage by 56.32%. Caveolin-1 may play an important role in induction of retinal neovascularization. SiRNA against caveolin-1 can inhibit experimental retinal hyperpermeability and neovascularization. Therefore, the inhibition of caveolin-1 may be a powerful and novel therapeutic tool for the treatment of ischaemia-induced retinal diseases.

The Role of Caveolin-1 in Blood–Brain Barrier Disruption Induced by Focused Ultrasound Combined with Microbubbles

This research was designed to determine whether disrupting the blood-brain barrier (BBB) in rats by applying focused ultrasound (FUS) combined with microbubbles induced changes in the density of caveolae and/or the expression of the structural protein caveolin-1. To this end, two approaches were utilized. First, using enhanced magnetic resonance imaging, characteristics of BBB disruption induced by our specific FUS parameters and dose of microbubble were recorded, and the time after treatment when the BBB was the most permeable was determined. Second, rats were treated with FUS or microbubbles alone, both or neither, and a combination of Evans blue (EB) BBB permeability assays, streptavidin-peroxidase (SP) immunohistochemistry, western blot, and transmission electron microscopy (TEM) was employed to detect any changes in caveolae density and caveolin-1 expression at the previously determined time point when the BBB was the most permeable. The first set of studies revealed that our specific FUS parameters and dose of microbubbles were able to induce a transient, targeted, and reversible BBB opening in rats, and that the BBB was the most permeable 1 h after treatment with FUS and microbubbles. In the second set of experiments, the results of the SP immunohistochemistry, western blot, and TEM, taken together, revealed that caveolae and caveolin-1 were primarily localized in the brain microvascular endothelial cells of all of the rats regardless of treatment, and that caveolin-1 expression was highest in the rats treated with both FUS and microbubbles. In summary, treatment with FUS, in combination with a dose of microbubbles, can enhance BBB permeability through a caveolae-mediated transcellular approach by upregulating the expression level of caveolin-1 and, consequently, the amount of caveolae. This caveolin-1-mediated transcellular transport pathway may cooperate with other transport pathways to induce opening of the BBB. This research sheds light on the mechanism of a transient, targeted, and reversible opening of the BBB induced by FUS combined with microbubbles.

Transcriptional Repression of Caveolin-1 (CAV1) Gene Expression by GATA-6 in Bladder Smooth Muscle Hypertrophy in Mice and Human Beings

Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits down-regulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Silencing of GATA6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in bladder smooth muscle cells. Together, these data suggest that GATA-6 acts as a transcriptional repressor of CAV1 gene expression in PBOO-induced BSM hypertrophy in men and mice. GATA-6-induced transcriptional repression represents a new regulatory mechanism of CAV1 gene expression in pathologic BSM, and may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men.

Caveolin-1 May Participate in the Pathogenesis of Bladder Pain Syndrome/ Interstitial Cystitis

Objectives: To determine whether caveolin-1 expression is associated with bladder pain syndrome/interstitial cystitis (BPS/IC) and to better understand the pathogenesis of BPS/IC. Methods: The study population was composed of 19 women with BPS/IC and 7 healthy women as controls. Midstream urine specimens were collected before cystoscopy and cold cup bladder biopsies were obtained from the trigone of the bladder. Caveolin-1 protein expression was determined by indirect immunofluorescence and Western blot analysis in cases and controls, using a rabbit polyclonal antibody against caveolin-1. χ² test and Student’s t test were used in the statistical analysis. Results: A statistical difference of caveolin-1 protein expression was observed between BPS/IC and healthy controls (p < 0.05, χ² test). Western blot analysis showed that the mean relative integrated density value of caveolin-1 in (BPS/IC) patients was significantly higher than that in the control group (p < 0.001, Student’s t test). Conclusions: The results of our study demonstrate that there is a relationship between the raised levels of caveolin-1 expression and BPS/IC. This preliminary study may provide a basis for further investigation of the role of caveolin-1 in the pathogenesis of BPS/IC.

Transcriptional Repression of Caveolin-1 gene Expression by Gata-6 in Bladder Smooth Muscle Hypertrophy in Mice and Humans

Caveolin-1 is a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Hypertrophied bladder smooth muscle (BSM) from the rabbit model of partial bladder outlet obstruction (PBOO) and men with benign prostatic hyperplasia (BPH)-induced PBOO shows a downregulation of Caveolin-1 and caveolar structural alteration. Here we report that caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. We characterized the proximal promoterof the human and mouse caveolin-1 gene and found that the transcriptionfactor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, we demonstrate that caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and humans. Importantly, silencing of GATA-6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in BSM cells. Together, our data suggest that GATA-6 acts as a transcriptional repressor of caveolin-1 gene expression in PBOO-induced BSM hypertrophy in men and mice. The GATA-6-induced transcriptional repression represents a new regulatory mechanism for caveolin-1 gene expression in pathologic BSM. Regulating the GATA-6 transcriptional regulation may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men. Supported by O’Brien Urology Center Grant P50 DK052620

Vascular Endothelial Growth Factor Increases Permeability of the Blood–Tumor Barrier via Caveolae-Mediated Transcellular Pathway

The first goal of this study was to determine the effect of vascular endothelial growth factor (VEGF) on permeability of the blood-tumor barrier (BTB). The second goal was to determine possible cellular mechanisms by which VEGF increases permeability of the BTB. In the rat C6 glioma model, the permeability of the BTB was significantly increased after VEGF injection at dose of 0.05ng/g and reached its peak at 45min. Meanwhile, we observed that the density of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) in the BTB increased dramatically by transmission electron microscopy. The immunohistochemistry and western blot analysis revealed that the expression level of caveolae structure proteins caveolin-1 and caveolin-2 in BMECs was increased after VEGF injection, peaked at 45min, and then decreased to the untreated level. The time peak of expression level of caveolin-1 and caveolin-2 was identical with the peak time of permeability of the BTB and the density of pinocytotic vesicles. All of these results strongly indicated that VEGF increased permeability of the BTB caused by enhancement of the density of pinocytotic vesicles, and the molecular mechanism might be associated with upregulated expression of caveolin-1 and caveolin-2.

Modulation of Insulin Sensitivity and Caveolin-1 Expression by Orchidectomy in a Nonobese Type 2 Diabetes Animal Model

Previously, we found that male JYD mice developed type 2 diabetes but female mice did not, and that decreased expression levels of caveolin-1 were correlated with the development of a diabetic phenotype in these mice. Therefore, we hypothesized that sex hormones affect the expression of caveolin-1 and contribute to the development of insulin resistance and hyperglycemia in JYD mice. We used glucose and insulin tolerance tests to examine insulin sensitivity in male, female and orchidectomized male JYD mice. Glucose uptake was analyzed by using (18)F-fluorodeoxyglucose positron emission tomography. We also examined insulin-signaling molecules and caveolin proteins in various tissues in these mice by Western blotting. In addition, we examined changes of caveolin-1 expression in L6 skeletal muscle cells treated with 17-β estradiol or dihydroxytestosterone. We found that glucose and insulin tolerance were impaired and hyperglycemia developed in male, but not female, JYD mice. Expression of insulin-signaling molecules such as insulin receptor, protein kinase B, and glucose transporter-4 were decreased in male JYD mice compared with female mice. Orchidectomized JYD male mice showed improved glucose and insulin tolerance with a concomitant increase in the expression of insulin-signaling molecules and caveolin-1 in adipose tissue and skeletal muscle. Moreover, 17-β-estradiol treatment increased the expression of caveolin-1 in differentiated skeletal muscle cells. We conclude that sex hormones modulate the expression of caveolin-1 and insulin-signaling molecules, subsequently affecting insulin sensitivity and the development of type 2 diabetes in JYD mice.

RNA interference-directed caveolin-1 knockdown sensitizes SN12CPM6 cells to doxorubicin-induced apoptosis and reduces lung metastasis

Human renal cell carcinoma (HRCC) is characterized by a high level of resistance to all treatment modalities. Therefore, the investigation of global gene expression in HRCC might help understand its biologic behavior and develop treatment strategies. Using cDNA microarray analysis, we initially compared gene expression profiles between HRCCs and adjacent normal tissues, and found that 87 were up-regulated and 127 genes were down-regulated. Next, a subset of genes, twofold differentially expressed, were validated by Northern blotting. Unexpectedly, caveolin-1, a gene reported to be a tumor suppressor gene, was found to be up-regulated in HRCC tissues. Expression level of caveolin-1 in SN12CPM6 (high metastatic clone) was higher than in SN12C (low metastatic clone), and SN12CPM6 was more resistant to doxorubicin (DXR) than SN12C. Caveolin-1 gene was slightly induced in surviving SN12C cells after DXR treatment. Furthermore, SN12CPM6-siCav1 cells, which were transfected with siRNA of cavelon-1 gene, were more sensitive to DXR, compared to SN12CPM6, but reduction of caveolin-1 gene expression did not affect tumor formation in subcapsule of kidney and lung metastasis. On the other hand, induction of caveolin-1 gene affected the production of lung metastasis under anti-cancer drug treatment: the incidence of pulmonary metastasis was significantly lower in SCID mice injected with SN12CPM6-siCav1 cells, and the number of pulmonary nodules decreased significantly (p = 0.0004). The above results together suggest that caveolin-1 may confer a growth advantage to cancer cells during DXR chemotherapy and surviving HRCC cells eventually might develop lung metastasis.

Caveolin-1 P132L Mutation in Human Cancers: 1 CAVeat to be Voiced

Caveolae are 50–100 nm flask-shaped invaginations of the plasma membrane that function as membrane organizing centers and play important roles in intracellular trafficking of cellular components (eg, lipid and cholesterol) and signal transduction.1 The CAV1 gene maps to 7q31.1 and encodes the ubiquitously expressed caveolin-1, which is an essential component of caveolae. Caveolin-1 is believed to regulate the activity of a plethora of signaling molecules highly represented in caveolae, such as H-ras, c-src, and src-like kinases; endothelial nitric oxide synthase; G-proteins and membrane-associated receptors coupled to G-proteins; or tyrosine kinase receptors including epidermal growth factor receptor.1 In normal breast tissue, caveolin-1 is expressed at high levels in adipocytes, endothelial cells, fibroblasts, and myoepithelial cells of the breast.1 Initial studies have suggested that caveolin-1 would have properties consistent with those of a tumor suppressor protein.2,3,4 A dominant negative mutation of the CAV1 gene was reported to be found in up to 20% of estrogen receptor (ER)-positive breast cancers.2,4 Surprisingly, however, CAV1 knock-out mice failed to develop any tumor.1,3 Furthermore, the presence of CAV1 gene mutations could not be independently validated.5,6,7,8,9 Concurrent with the controversies about the role of CAV1 as a tumor suppressor gene, evidence of caveolin-1 as a potential oncogene has been mounting.7,8,9,10,11,12,13,14,15 In fact, multiple independent groups have reported overexpression of caveolin-1 in aggressive subtypes of cancer,10,11,13,14,15 and even CAV1 gene amplification has been documented,14,16 although this has been shown to be a rare phenomenon. Furthermore, expression of caveolin-1 in stromal cells of breast cancer has also been shown to be associated with the outcome of breast cancer patients.17,18 Despite the interest in the tumor suppressive and oncogenic functions of caveolin-1, numerous issues remain unanswered. For instance, caveolin-1 expression in normal luminal epithelial cells of normal breast lobules and ducts remains a matter of controversy, as does the existence and prevalence of the CAV1 P132L dominant-negative mutation. In this issue of The Journal of Molecular Diagnostics, Koike et al19 have addressed the question of the prevalence of the CAV1 P132L mutation in human cancer. Using three different methods for mutation detection (ie, reverse-transcriptase direct sequencing, DNA-based direct sequencing, and cycleave PCR assay), the authors failed to detect the CAV1 P132L mutation in a large series of 409 human tumors, including 139 breast cancers [114 estrogen receptor (ER)-positive and 25 ER-negative tumors] and 270 cancers from other anatomical sites (ie, gastrointestinal tract, lung, ovary, and pancreas). Taken together, the findings reported by Koike et al19 and those by others5,6,7,8,9 call into question the presence of the CAV1 P132L mutation in breast cancers and other cancer types and heat up the debate about the roles of caveolin-1 in human cancer.

Overexpression of caveolin-1 in adult T-cell leukemia

AbstractCaveolin-1 is implicated in the regulation of signal pathways. Adult T-cell leukemia (ATL) is a T-cell malignancy causatively associated with human T-cell leukemia virus type 1 (HTLV-1). To determine the role of caveolin-1 in leukemogenesis, we examined caveolin-1 expression levels in HTLV-1–infected T-cell lines and ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Caveolin-1–positive ATL cells were detected in ATL lymph nodes and skin lesions, and caveolin-1 was also detected in the plasma of patients with ATL. Infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 expression. The viral protein Tax transcriptionally activated caveolin-1 gene through nuclear factor-κB and cAMP response element binding protein signal pathways. HTLV-1–infected T-cell lines, and ATL cells are known to be resistant to transforming growth factor β (TGF-β)–induced growth inhibition. Caveolin-1 was colocalized with TGF-β type I receptor in HTLV-1–infected T-cell lines and suppressed TGF-β signaling. Caveolin-1 knockdown in an HTLV-1–infected T-cell line exhibited susceptibility to TGF-β. Thus, we describe a new function for Tax, repression of TGF-β signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

The mechanism of polyplex internalization into cells: testing the GM1/caveolin-1 lipid raft mediated endocytosis pathway.

The GM1/caveolin-1 lipid raft mediated endocytosis mechanism was explored for generation 5 and 7 poly(amidoamine) dendrimer polyplexes employing the Cos-7, 293A, C6, HeLa, KB, and HepG2 cell lines. Expression levels of GM1 and caveolin-1 were measured using dot blot and Western blot, respectively. The level of GM1 in the cell plasma membrane was adjusted by incubation with exogenous GM1 or ganglioside inhibitor PPMP, and the level of CAV-1 was adjusted by upregulation with the adenovirus vector expressed caveolin-1 (AdCav-1). Cholera toxin B subunit was employed as a positive control for uptake in all cases. No evidence was found for a GM1/caveolin-1 lipid raft mediated endocytosis mechanism for the generation 5 and 7 poly(amidoamine) dendrimer polyplexes.

Transforming growth factor-β1 downregulates caveolin-1 expression and enhances sphingosine 1-phosphate signaling in cultured vascular endothelial cells

In vascular endothelial cells, specialized microdomains of plasma membrane termed caveolae modulate various receptor signal transduction pathways regulated by caveolin-1, a resident protein of caveolae. We examined whether transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine, alters expression levels of caveolin-1 and influences heterologous receptor signaling. Treatment of cultured bovine aortic endothelial cells (BAEC) with TGF-beta1 induces marked decreases in caveolin-1 expression in a time- and dose-dependent fashion at both levels of protein and mRNA. A pharmacological inhibitor of activin receptor-like kinase 5 (ALK-5) counteracts caveolin-1 downregulation by TGF-beta1, indicating the involvement of ALK-5 receptor subtype for TGF-beta1. Sphingosine 1-phosphate (S1P) is a serum-borne angiogenic lipid growth factor that exerts a wide variety of biological actions. S1P modulates G protein-coupled S1P receptors, activating downstream molecules kinases AMP-activated protein kinase (AMPK), and Akt as well as a small G protein Rac1, ultimately to promote migration. Because S1P receptor signaling is associated with caveolae/caveolin-1, we examined whether pretreatment with TGF-beta1 enhances effects of S1P on BAEC. Whereas S1P alone evokes robust BAEC responses to S1P, pretreatment with TGF-beta1 leads to even higher magnitudes of S1P-elicited signaling responses and cell migration. Conversely, genetic knockdown of caveolin-1 using small interfering RNA mimics TGF-beta1-induced promotion of BAEC responses to S1P. Collectively, these data demonstrate that TGF-beta1 downregulates caveolin-1 of cultured endothelial cells, involving ALK-5 receptor subtype. Because downregulation of caveolin-1 by TGF-beta1 promotes subsequent heterologous receptor signaling by S1P, these results may also identify novel point of cross-talk between cytokines and sphingolipids within endothelial signal transduction machineries.

Bleomycin and its Role in Inducing Apoptosis and Senescence in Lung Cells - Modulating Effects of Caveolin-1

Bleomycin, a widely used anti-tumor agent, is well-known to cause single- and double-strand breaks in cellular DNA in vivo and in vitro leading finally to genomic instability of damaged cells. Bleomycin causes an increase of reactive oxygen species resulting in oxidative stress and pulmonary fibrosis. Further, bleomycin induces apoptosis and senescence in epithelial and non-epithelial cells of the lung. Caveolin-1 is a scaffold protein of caveolae, which are particularly abundant in alveolar epithelial type I cells, in endothelial and smooth muscle cells, and in fibroblasts of lung tissue. Caveolin-1 directly interacts with signaling molecules and effects diverse signaling pathways regulating cell proliferation, apoptosis, differentiation and growth. In this review we discuss aspects of bleomycin resistance. We summarize recent data about the effects of bleomycin in terms of lung cell biology and emphasize that bleomycin-induced injury of lung cells is accompanied by altered expression levels of caveolin-1. Caveolin-1 is involved in bleomycin-induced apoptosis and senescence of normal and lung cancer cells. Investigating the role of caveolin-1 may provide new tools for therapeutic interventions in lung disease and for the understanding of tumor biology.

Caveolin‐1 and Rac regulate endothelial capillary‐like tubular formation and fenestral contraction in sinusoidal endothelial cells

Rho guanidine triphosphatases (GTPases) are major regulators of cell migration. We investigated the cytoskeleton and Rho GTPases during cell migration and morphogenesis processes in isolated rat liver sinusoidal endothelial cells (LSECs) cultured on Matrigel while stimulated by the vascular endothelial growth factor (VEGF). To obtain primary monolayers, LSECs were cultured on Matrigel for 5-17 h with or without VEGF. Sinusoidal endothelial fenestrae (SEF) morphology was observed using scanning electron microscopy and transmission electron microscopy. RhoA, Rac1 and phosphorylated myosin light-chain kinase, Rho-binding domain of Rhotekin and the p21-binding domain of p21-activated protein kinase were analysed using Western blotting. The LSECs showed cellular protrusions and or cords of aligned cells resembling primitive capillary-like structures, with SEF contraction. Time course analyses of Rac1 activation matched specific morphological changes. Rac1 activity increased progressively to 17 h in cells cultured without VEGF, but markedly increased at 7 h in the presence of VEGF. RhoA activity was slightly elevated at 5 h. The levels of endogenous caveolin-1 (CAV-1) expression increased in a time-dependent manner, reaching a peak at 7 h. CAV-1 expression occurred immediately before the formation of the capillary-like tube. Moreover, treatment with VEGF regulated CAV-1 expression in LSECs. Spatial activation of Rac1 is involved in the formation of a capillary-like tubular network accompanying SEF contraction in LSECs, implying that endothelial migration and adhesion are necessary for LSECs tubular formation in the liver. CAV-1 might play an important positive role in the regulation of LSEC tubular formation.

Expression of caveolin-1 gene in hepatitis B-related hepatocellular carcinoma and the clinical significance thereof

To study the correlation between the expression of caveolin-1 in hepatitis B-related hepatocellular carcinoma (HCC) and the relationship thereof with the invasive and metastatic ability of HCC. Real-time PCR and immunohistochemistry were used to detect the expression of caveolin-1 in 123 specimens of HCC tissues obtained during operation. Human HCC cells of the lines MHCC-97L and MHCC-97H were cultured, and Western-blotting was used to detect the caveolin-1 expression in these cells. The expression level of caveolin-1 in the HCC tissues was significantly lower than that of the adjacent noncancerous tissues (P = 0.026). The caveolin-1 mRNA expression in the HBV-related HCC cells was negatively correlated with the tumor size, expression of AFP, major venous invasion, being single or multiple, pTNM staging, and HBx expression level, factors closed to the prognosis of HCC. The caveolin-1 protein expression of the MHCC-97H cells was significantly lower than that of the MHCC-97L cells. Caveolin-1 expression is associated with the invasive and metastatic ability of HCC, low expression of caveolin-1 shows poor prognosis of HCC, and caveolin-1 is probably down-regulated by HBx protein.

Caveolin‐1 promotes the transformation and anti‐apoptotic ability of mouse hepatoma cells

Caveolin-1 is a major structural protein of caveolae and plays important roles in signal transduction, cellular transformation and tumor metastasis. Our previous study demonstrated that caveolin-1 expression level was positively correlated with the invasive ability of mouse hepatoma Hepa1-6 and Hca-F cells. However, the role of caveolin-1 in cellular transformation and apoptosis remains undetermined. We found that exogenous expression of caveolin-1 in Hepa1-6 cells enhanced cell transformation capability both in vitro and in vivo and prevented actinomycin D-induced apoptosis via the activation of survivin-mediated survival pathway. Conversely, downregulation of caveolin-1 in Hca-F cells significantly attenuated cell transformation ability in vitro and in vivo and increased cell sensitivity to actinomycin D by inhibiting survivin-mediated survival pathway. These results indicate that caveolin-1 could play an active role in mediating the transformation and survival of mouse hepatoma cells and might be a potential target for gene and antitumor drugs therapy.

Enhanced expression of caveolin-1 in articular chondrocytes in osteoarthritis

To study the expression level of caveolin-1 in articular chondrocytes in patients with osteoarthritis (OA), and whether catabolic factor IL-1beta stimulate caveolin-1 expression in articular chondrocytes. In human OA cartilage, caveolin-1 mRNA was investigated by quantitative real-time RT-PCR while caveolin-1 protein was detected by immunohistologic analysis in 8 cases. OA model was prepared by unilateral anterior cruciate ligament and medial collateral ligament transection in 20 rats. In cultured OA chondrocytes, caveolin-1 mRNA expression was studied by RT-PCR and quantitative real-time RT-PCR, and caveolin-1 protein was analyzed by Western blotting. In 6 of 8 OA cases, human OA articular cartilage, higher expression levels of both caveolin-1 mRNA and caveolin-1 protein were found in advanced degenerated cartilage than less degenerated cartilage of the same joints. In rat OA model, upregulated caveolin-1 expression was found, which was associated with the degree of cartilage destruction. IL-1beta (10 ng/ml) upregulated caveolin-1 mRNA/protein expression in cultured OA chondrocytes for at least 48 hours. Enhanced expression level of caveolin-1 is associated with cartilage degeneration in OA. IL-1beta stimulates caveolin-1 expression in articular chondrocytes, which may be responsible for OA development.

Caveolae are an essential component of the pathway for endothelial cell signaling associated with abrupt reduction of shear stress

Abrupt cessation of flow representing the acute loss of shear stress (simulated ischemia) to flow-adapted pulmonary microvascular endothelial cells (PMVEC) leads to reactive oxygen species (ROS) generation that signals for EC proliferation. We evaluated the role of caveolin-1 on this cellular response with mouse PMVEC that were preconditioned for 72 h to laminar flow at 5 dyn/cm2 followed by stop of flow (“ischemia”). Preconditioning resulted in a 2.7-fold increase in cellular expression of KATP (KIR 6.2) channels but no change in expression level of caveolin-1, gp91phox, or MAP kinases. The initial response to ischemia in wild type cells was cell membrane depolarization that was abolished by gene targeting of KIR 6.2. The subsequent response was increased ROS production associated with activation of NADPH oxidase (NOX2) and then phosphorylation of MAP kinases (Erk, JNK). After 24 h of ischemia in wild type cells, the cell proliferation index increased 2.5 fold and the % of cells in S+G2/M phases increased 6-fold. This signaling cascade (cell membrane depolarization, ROS production, MAP kinase activation and cell proliferation) was abrogated in caveolin-1 null PMVEC or by treatment of wild type cells with filipin. These studies indicate that caveolin-1 functions as a shear sensor in flow-adapted EC resulting in ROS-mediated cell signaling and endothelial cell proliferation following the abrupt reduction in flow.

Curcumin inhibits cellular cholesterol accumulation by regulating SREBP-1/caveolin-1 signaling pathway in vascular smooth muscle cells

To investigate the protective effect and the possible mechanism of curcumin on anti-atherosclerosis. Morphological changes of atherosclerotic lesions taken from apoE knockout (apoE-/-) mice were determined by hematoxylin- eosin staining. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC. The protein expression of caveolin-1 was quantified by Western blotting. Translocation and the expression of sterol response element-binding protein-1 (SREBP-1) were indirectly detected by an immunofluorescence analysis. The administration of 20 mg. kg(-1 ). d(-1 )curcumin to apoE-/- mice for 4 months induced a 50% reduction of atherosclerotic lesions and yielded a 5- fold increase in the caveolin-1 expression level as compared to the model group. Rat vascular smooth muscle cells (VSMC) pretreated with 50 mg/L ox-lipid density lipoprotein(ox-LDL) for 48 h increased cellular lipid contents, and stimulated SREBP-1 translocation, but decreased the caveolin-1 expression level. Lipid-loaded cells exposed to curcumin at various concentrations (12.5, 25, and 50 micromol/L) for different durations (0, 6, 12, 24, and 48 h) significantly diminished the number and area of cellular lipid droplets, total cholesterol, cholesterol ester, and free cholesterol accompanying the elevation of the caveolin-1 expression level (approximately 3-fold); the translocation of SREBP-1 from the cytoplasm to the nucleus was inhibited compared with the models. Lipid-loaded VSMC exposed to N-acetyl- Leu-Leu-norleucinal, a SREBP-1 protease inhibitor, showed increased nuclear translocation of SREBP-1, reduced caveolin-1 expression level, and upregulated cellular lipid levels. Curcumin inhibits ox-LDL-induced cholesterol accumulation in cultured VSMC through increasing the caveolin-1 expression via the inhibition of nuclear translocation of SREBP-1.

Compartment‐dependent modulation of Ras ubiquitination

Ras GTPases are key regulators of cell growth, differentiation and transformation. Ubiquitination of Ras modulates its intracellular trafficking and signaling output. Yet, detailed knowledge about the in vivo dynamics of this process is limited. Here, we took advantage of the Bioluminescence Resonance Energy Transfer (BRET) technology to monitor HRas ubiquitination in living cells and to investigate its relationship to membrane compartmentalization. We demonstrate that HRas ubiquitination is dynamically altered upon growth factor stimulation. The growth factor‐dependent modulation of HRas ubiquitination is temporally coupled to changes in HRas microdomain localization as determined by BRET analysis of HRas/Caveolin1 interactions. Significantly, an HRas chimera that is unable to interact with Caveolin1 is refractory to ubiquitination, indicating that HRas ubiquitination requires an interaction with Caveolin1. Consistent with this observation, disruption of lipid microdomains by membrane cholesterol depletion inhibits HRas ubiquitination. Furthermore, Caveolin1 expression levels are tightly correlated with HRas ubiquitination. Taken together, our data demonstrate that HRas ubiquitination is dynamically regulated and requires the integrity of caveolae and their scaffolding protein Caveolin1.Supported by Human Frontier Science Program (S.T.) and NIH grant CA055360 (D.B.‐S.)