Downregulation Of Caveolin-1 Research Articles

Identification of integrin β1 as a prognostic biomarker for human lung adenocarcinoma using 2D-LC-MS/MS combined with iTRAQ technology

To discover novel lung adenocarcinoma (AdC) biomarkers, isobaric tags for relative and absolute quantitation (iTRAQ)-tagging combined with 2D-LC-MS/MS analysis was used to identify differentially expressed plasma membrane proteins in lung AdC and paired paraneoplastic normal lung tissues (PNLTs) adjacent to tumors. In this study, significant caveolin-1 downregulation and integrin β1 upregulation was observed in primary lung AdC vs. PNLT. As there has been no report on the association of integrin β1 with lung AdC, immunohistochemical staining was performed to detect the expression of integrin β1 in an independent set of archival tissue specimens including 42 cases of PLNT, 46 cases of without lymph node metastasis primary AdC (non-LNM AdC) and 62 cases of LNM AdC; the correlation of their expression levels with clinicopathological characteristics and clinical outcomes were evaluated. Based on the data, upregulation of integrin β1 was significantly correlated with advanced clinical stage and lymph node metastasis. Integrin β1 overexpression was significantly associated with advanced clinical stage (P<0.05), lymph node metastasis (P<0.05), increased relapse rate (P<0.05) and decreased overall survival (P<0.05) in AdCs. Cox regression analysis indicated that integrin β1 overexpression is an independent prognostic factor. The data suggest that integrin β1 is a potential biomarker for LNM and prognosis of AdC and integrin β1 upregulation may play an important role in the pathogenesis of AdC.

Inhibition of nuclear factor-erythroid 2–related factor (Nrf2) by caveolin-1 promotes stress-induced premature senescence

Reactive oxygen species (ROS) can induce premature cellular senescence, which is believed to contribute to aging and age-related diseases. The nuclear erythroid 2 p45-related factor-2 (Nrf2) is a transcription factor that mediates cytoprotective responses against stress. We demonstrate that caveolin-1 is a direct binding partner of Nrf2, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain of Nrf2 (amino acids 281-289). Biochemical studies show that Nrf2 is concentrated into caveolar membranes in human and mouse fibroblasts, where it colocalizes with caveolin-1, under resting conditions. After oxidative stress, caveolin-1 limits the movement of Nrf2 from caveolar membranes to the nucleus. In contrast, Nrf2 is constitutively localized to the nucleus before and after oxidative stress in caveolin-1-null mouse embryonic fibroblasts (MEFs), which do not express caveolin-1. Functional studies demonstrate that caveolin-1 acts as an endogenous inhibitor of Nrf2, as shown by the enhanced up-regulation of NQO1, an Nrf2 target gene, in caveolin-1-null MEFs and the activation or inhibition of a luciferase construct carrying an antioxidant responsive element (ARE) after down-regulation of caveolin-1 by small interfering RNA or overexpression of caveolin-1, respectively. Expression of a mutant form of Nrf2 that cannot bind to caveolin-1 (Φ→A-Nrf2) hyperactivates ARE and inhibits oxidative stress-induced activation of the p53/p21(Waf1/Cip1) pathway and induction of premature senescence in fibroblasts. Finally, we show that overexpression of caveolin-1 in colon cancer cells inhibits oxidant-induced activation of Nrf2-dependent signaling, promotes premature senescence, and inhibits their transformed phenotype. Thus, by inhibiting Nrf2-mediated signaling, caveolin-1 links free radicals to the activation of the p53/senescence pathway.

Caveolin-1 Regulates Rac1 Activation and Rat Pulmonary Microvascular Endothelial Hyperpermeability Induced by TNF-α

A multiplicity of vital cellular and tissue level functions are controlled by caveolin-1 and it is considered to be an important candidate for targeted therapeutics. Rac1-cortactin signaling plays an important role in maintaining the functions of the endothelial barrier in microvascular endothelial cells. The activity of Rac1 has been shown to be regulated by caveolin-1. Therefore, the present study investigated the consequences of down-regulating caveolin-1 and the subsequent changes in activity of Rac1 and the endothelial barrier functions in primary rat pulmonary microvascular endothelial cells (RPMVECs). RPMVECs were transfected with a small hairpin RNA duplex to down-regulate caveolin-1 expression. This procedure significantly increased the activity of Rac1. Moreover, down-regulation of caveolin-1 attenuated TNF-α-induced decrease in TER, increase in the flux of FITC-BSA and the disappearance of cortactin from the cell periphery in RPMVEC. Rac1 inhibitors significantly abolished this barrier-protective effect induced by down-regulation of caveolin-1 in response to TNF-α in RPMVECs. In conclusion, our data suggest a mechanism for the regulation of Rac1 activity by caveolin-1, with consequences for activation of endothelial cells in response to TNF-α.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Fluorescence Studies of the Bradykinin 2 and µ-Opioid Receptors Suggest that Caveolae Localization of GPCRs is Mediated by their Attached G Proteins

Caveolae are 50-100 nm plasma membrane invaginations that have several proposed functions. Some studies suggest that caveolae may influence cell signaling by sequestering certain signaling proteins such as G-protein coupled receptors. We have previously found that Caveolin-1, the main structural protein of caveolae specifically binds to Gαq and that their binding is strengthened upon Gαq activation. Here, we have used fluorescence methods to determine the effect of caveolae on the functional properties and localization of two G-protein coupled receptors: the bradykinin receptor type 2 (B2R), which is coupled to Gαq, and the µ opioid receptor (µOR), which is coupled to Gαi. While caveolae do not affect cAMP signals mediated by µOR, they prolong Ca2+ signals mediated by B2R. In A10 cells, down-regulation of Caveolin-1 ablates the prolonged calcium signal in accord with idea that caveolae binds to Gαq. Immunofluorescence and FRET studies show that a significant fraction of B2R resides at or close to caveolae domains while none or very little µOR resides in caveolae domains. FRET between B2R and caveolae is reduced by down-regulation of Gαq or by addition of a peptide that interferes with Gαq/Caveolin-1 interactions suggesting that Gαq promotes localization of B2R to caveolae domains. Fluorescence Correlation Spectroscopy studies on live cells show that the presence of caveolae changes the distribution of the apparent diffusion coefficients of B2R and Gαq but not µOR and Gαi. Our results suggest that Gαq can localize its associated receptors to caveolae domains to enhance their signals.

Cav1 Suppresses Tumor Growth and Metastasis in a Murine Model of Cutaneous SCC through Modulation of MAPK/AP-1 Activation

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Caveolin-1 is involved in radiation-induced ERBB2 nuclear transport in breast cancer cells

This study examined the radiation-induced ERBB2 nuclear transport in the BT474 breast cancer cell line and the relationship between caveolin-1 and radiation-induced ERBB2 nuclear transport. The BT474 cells were treated with herceptin (200 nmol/L), PP2 (a caveolin-1 inhibitor, 100 nmol/L) and irradiation combined or alone. Confocal microscopy was used to observe the nuclear import of ERBB2 and caveolin-1 after irradiation. Western blotting was employed to detect the expression of ERBB2, caveolin-1 and DNA-PKcs after irradiation, and immunoprecipitation to identify the ERBB2 and caveolin-1 complex before perinuclear ERBB2 localization. Confocal microscopy showed the transport of ERBB2 and caveolin-1 from the cell membrane to the nucleus 15 min after irradiation and the proteins accumulated at the perinuclear region within 45 min. Western blotting revealed that the expression levels of ERBB2, caveolin-1 and DNA-PKcs were increased after irradiation and reached a peak 45 min later. Both herceptin and PP2 treatments were found to decrease ERBB2 expression. An immune complex composed of ERBB2 and caveolin-1 was found in the herceptin group after irradiation. It was concluded that after irradiation, ERBB2 may be transported from the cell membrane to the nucleus and activate DNA-PKcs to trigger DNA double-strand break (DSB) repair; caveolin-1 may participate in this process. Treatments involving the downregulation of caveolin-1 may increase the radiosensitization of breast cancer cells.

Caveolin-1 regulates neural differentiation of rat bone mesenchymal stem cells into neurons by modulating Notch signaling

Bone marrow mesenchymal stem cells (MSCs) are known to differentiate into neurons in vitro. However, the mechanism underlying MSC differentiation remains controversial. A recent analysis has shown that Notch signaling is involved in regulating the differentiation of MSCs. This study examines the potential mechanism of the differentiation of MSCs into neurons, and it considers the role of caveolin-1 in this process. We investigated neuron differentiation and Notch signaling by detecting the expression levels of microtubule-associated protein 2 (MAP-2), Neuron-specific Enolase (NSE), Notch-1, Notch intracellular domain (NICD) and hairy enhancer of split 5 (Hes5). We found that by down-regulating caveolin-1 during induction, MSCs were prone to neural differentiation and expressed high levels of neuronal markers. Meanwhile, the expression levels of Notch-1, NICD and Hes5 decreased. Our results indicate that down-regulation of caveolin-1 promotes the neuronal differentiation of MSCs by modulating the Notch signaling pathway.

Metronomic Ceramide Analogs Inhibit Angiogenesis in Pancreatic Cancer through Up-regulation of Caveolin-1 and Thrombospondin-1 and Down-regulation of Cyclin D1

AIMS: To evaluate the antitumor and antiangiogenic activity of metronomic ceramide analogs and their relevant molecular mechanisms. METHODS: Human endothelial cells [human dermal microvascular endothelial cells and human umbilical vascular endothelial cell (HUVEC)] and pancreatic cancer cells (Capan-1 and MIA PaCa-2) were treated with the ceramide analogs (C2, AL6, C6, and C8), at low concentrations for 144 hours to evaluate any antiproliferative and proapoptotic effects and inhibition of migration and to measure the expression of caveolin-1 (CAV-1) and thrombospondin-1 (TSP-1) mRNAs by real-time reverse transcription-polymerase chain reaction. Assessment of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt phosphorylation and of CAV-1 and cyclin D1 protein expression was performed by ELISA. Maximum tolerated dose (MTD) gemcitabine was compared against metronomic doses of the ceramide analogs by evaluating the inhibition of MIA PaCa-2 subcutaneous tumor growth in nude mice. RESULTS: Metronomic ceramide analogs preferentially inhibited cell proliferation and enhanced apoptosis in endothelial cells. Low concentrations of AL6 and C2 caused a significant inhibition of HUVEC migration. ERK1/2 and Akt phosphorylation were significantly decreased after metronomic ceramide analog treatment. Such treatment caused the overexpression of CAV-1 and TSP-1 mRNAs and proteins in endothelial cells, whereas cyclin D1 protein levels were reduced. The antiangiogenic and antitumor impact in vivo of metronomic C2 and AL6 regimens was similar to that caused by MTD gemcitabine. CONCLUSIONS: Metronomic C2 and AL6 analogs have antitumor and antiangiogenic activity, determining the up-regulation of CAV-1 and TSP-1 and the suppression of cyclin D1.

Hepatitis B virus X protein suppresses caveolin-1 expression in hepatocellular carcinoma by regulating DNA methylation

BackgroundTo understand the molecular mechanisms of caveolin-1 downregulation by hepatitis B virus X protein (HBx).MethodsThe DNA methylation status of the caveolin-1 promoter was examined by nested methylation-specific PCR of 33 hepatitis B virus (HBV)-infected hepatocellular carcinoma (HCC) samples. The SMMC-7721 hepatoma cell line was transfected with a recombinant HBx adenoviral vector, and the effects of HBx protein on caveolin-1 expression and promoter methylation were examined and confirmed by sequencing. A reporter gene containing the caveolin-1 promoter region was constructed, and the effects of HBx on the transcriptional activity of the promoter were also studied.ResultsMethylation of the caveolin-1 promoter was detected in 84.8% (28/33) of HBV-infected HCC samples. Expression of caveolin-1 was significantly downregulated (P = 0.022), and multiple CpG sites in the promoter region of caveolin-1 were methylated in SMMC-7721 cells after HBx transfection. Transfected HBx significantly suppressed caveolin-1 promoter activity (P = 0.001).ConclusionsHBx protein induces methylation of the caveolin-1 promoter region and suppresses its expression.

Differential Effects of Short Term Feeding of a Soy Protein Isolate Diet and Estrogen Treatment on Bone in the Pre-Pubertal Rat

BackgroundPrevious reports suggest that beneficial effects of soy on bone quality are due to the estrogenic actions of isoflavone phytochemicals associated with the protein. However, mechanistic studies comparing the effects of soy diet and estrogens on bone, particularly in rapidly growing animals are lacking.Methodology and Principal FindingsWe studied the effects of short term feeding of soy protein isolate (SPI) on bone in comparison to the effects of 17β-estradiol (E2) in pre-pubertal rats. Female rats were weaned to one of 4 treatments: 1) a control casein-based diet (CAS); 2) CAS with subcutaneous E2 (10 µg/kg/d) (CAS+E2); 3) a SPI-containing diet (SPI); or 4) SPI with subcutaneous E2 (SPI) or SPI with 10 µg/kg/d E2 (SPI+E2) for 14 days beginning on postnatal day 20. SPI increased while E2 decreased bone turnover compared to CAS. In contrast, both treatments decreased serum sclerostin levels. Microarray analysis of RNA isolated from bone revealed 652 genes regulated by SPI, 491 genes regulated by E2, and 266 genes regulated by both SPI diet and E2 compared to CAS. The expression of caveolin-1, a protein localized in the cell membrane, was down-regulated (p<0.05) in rats fed SPI, but not by E2 compared to rats fed casein. Down-regulation of caveolin-1 by SPI was associated with increased BMP2, Smad and Runx2 expression in bone and osteoblasts (p<0.05).Conclusions/SignificanceThese results suggest SPI and E2 have different effects on bone turnover prior to puberty. Approximately half of the genes are regulated in the same direction by E2 or SPI, but in combination, SPI blocks the estrogen effects and returns the profile towards control levels. In addition, there are E2 specific and SPI-specific gene changes related to regulation of bone formation.

Caveolin-1 knockdown is associated with the metastasis and proliferation of human lung cancer cell line NCI-H460

Caveolin-1 (CAV-1), one component of caveolae, involves in multiple cellular processes and signal transductions. We previously showed that the expression of CAV-1 gene in NCI-H446 cells inhibited cell proliferation and promoted cell metastasis. Here we explore the function of CAV-1 on tumor growth and metastasis by using NCI-H460 in vitro. First, we established NCI-H460 cell line, which CAV-1 was stably knockdown. Then we investigated the effects of CAV-1 on the morphology, proliferation, cell cycle and metastasis potential for NCI-H460 cell by crystal violet stains, CCK-8, colony formation, flow cytometry, scratch-wound assay and transwell assay. Western blot was used to examine the expression changes of cyclin D1, PCNA, E-cadherin and β-catenin. Our results showed stable knockdown of CAV-1 inhibited the proliferation of NCI-H460 cells. Cell cycle of the transfected cells was arrested in G1/S phase and the expressions of cyclin D1 and PCNA protein were downregulated. Downregulation of CAV-1 promoted the migration and invasion abilities of NCI-H460 cells in vitro. The expression of β-catenin increased and the level of E-cadherin decreased. In summary, our findings provide experimental evidence that CAV-1 may function as a proproliferative and antimetastatic gene in NCI-H460 cell line.

Caveolin‐1 orchestrates fibroblast growth factor 2 signaling control of angiogenesis in placental artery endothelial cell caveolae

Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-β-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis.

Altered Angiogenesis in Caveolin-1 Gene–Deficient Mice Is Restored by Ablation of Endothelial Nitric Oxide Synthase

Caveolin-1 is an essential structural protein of caveolae, specialized plasma membrane organelles highly abundant in endothelial cells, where they regulate multiple functions including angiogenesis. Caveolin-1 exerts a tonic inhibition of endothelial nitric oxide synthase (eNOS) activity. Accordingly, caveolin-1 gene-disrupted mice have enhanced eNOS activity as well as increased systemic nitric oxide (NO) levels. We hypothesized that excess eNOS activity, secondary to caveolin deficiency, would mediate the decreased angiogenesis observed in caveolin-1 gene-disrupted mice. We tested tumor angiogenesis in mice lacking either one or both proteins, using in vitro, ex vivo, and in vivo assays. We show that endothelial cell migration, tube formation, cell sprouting from aortic rings, tumor growth, and angiogenesis are all significantly impaired in both caveolin-1-null and eNOS-null mice. We further show that these parameters were either partially or fully restored in double knockout mice that lack both caveolin-1 and eNOS. Furthermore, the effects of genetic ablation of eNOS are mimicked by the administration of the NOS inhibitor N-nitro-L-arginine methyl ester hydrochloride (L-NAME), including the reversal of the caveolin-1-null mouse angiogenic phenotype. This study is the first to demonstrate the detrimental effects of unregulated eNOS activity on angiogenesis, and shows that impaired tumor angiogenesis in caveolin-1-null mice is, at least in part, the result of enhanced eNOS activity.

Reactive Nitrogen Species:Dual Roles for Blood Brain Barrier Disruption and Brain Repairs in Cerebral Ischemia and Reperfusion Injury

Free radicals play an important role in cerebral ischemia-reperfusion injury. Cerebral ischemia and reperfusion insults induce the production and accumulation of free radicals and trigger numerous molecular cascades, leading to increased blood-brain barrier (BBB) permeability, brain edema, hemorrhage and inflammation, and cell death. As an important component of free radicals, reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO ), is critical player in the process of cerebral ischemia-reperfusion injury. Activation of matrix metalloproteinases (MMPs) mediated by RNS is one of the key steps in BBB opening. As proteolytic zinc-containing enzymes, MMPs induce the degradation of the extracellular matrix around cerebral blood vessels and neurons. Both NO and ONOO can activate MMPs and degrade tight junctions (TJs), leading to BBB breakdown in cerebral ischemia-reperfusion injury. On the other hand, recent progress indicates that RNS also participate in the regulations of spontaneous neurogenesis and brain repair in post-ischemic brains. It is of interesting why and how such small and short lifetime species have the complex biological activites on mediating BBB disruption as well as regulating neurogenesis. Caveolin-1, a membrane integral protein located at caveolae, appears to be a key player in the process. Caveolin-1 can protect BBB integrity through inhibiting MMPs activation, but inhibit neural differentiation of neural progenitor/stem cells through regulating multiple cell growth signaling pathways. NO could down-regulate caveolin-1 whereas the down-regulation of caveolin-1 subsequently induce the increase of NOS activity and produce more NO in the ischemic brains. The interaction of RNS and caveolin-1 forms a positive feedback loop through MMP activations, which provides amplified impacts on BBB dysfunction during cerebral ischemia-reperfusion injury. RNS production also potentially regulates neurogenesis through interacting with caveolin-1 and other cell signaling mechanisms. Herewith, we reviewed the recent progress in the roles of RNS on BBB permeability and neurogenesis. In conclusion, RNS can be a cytotoxic factor as well as neurogenesis signaling. The neurotoxicity or neurogenesis properties of RNS depend on the amount of RNS production in neurons, endothelial cells and their microenvironment during cerebral ischemia-reperfusion injury.

Effects of RNA interference-induced Caveolin-1 downregulation on lung edema of rats

Objective To explore the effects of down-regulation of Caveolin-1 gene expression on the permeability of lung capillaries. Methods Small interfering RNA (siRNA) was used to knock-down Caveolin-1 expression in rat lungs. A single-stranded siRNA construct was designed, and another sequence was used as a control construct. Pulmonary injection of siRNA against Caveolin-1 mRNA incorporated in liposomes with three dosage gradients of 0. 4, 0. 8 and 1.2 mg/kg. The protein expression of Caveolin-1 was detected by Western blotting and immunohistochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the expression of caveolin-1 expression. The expression of Aquaporin (AQP)-1protein was detected by using Western blotting. Chest fluoroscopy showed the degree of lung edema. 1% of Evans blue was injected through vena caudalis with a dosage of 1.0 mL/kg, and then lobus medius pulmonis was dissected and immersed in formamide. Results At 72nd h in experimental group, the gene silencing of Caveolin-1 siRNA was about 85%. There was slight difference in the Caveolin-1 expression in the control group with the three dosage gradients. Caveolin-1 siRNA had little influence over the AQP-1 protein level. Under the chest fluoroscopy, density of pulmonary lobes was increased. In comparison with the control group, the absorbance values of formamide in experimental group were significantly increased (P ＜0. 01 ). Conclusion Caveolin-1 may play an important role in the process of lung edema, which was AQP-1 independent. Key words: Lung; Caveolin-1; Permeability; Aquaporin

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.

Downregulation of Caveolin-1 in a Murine Model of Acute Allergic Airway Disease

Airway remodeling refers to the structural changes in the airways of asthma. Caveolin-1 reduces cell growth and negatively regulates smooth muscle cell proliferation. The aim was to investigate lung caveolin-1 status in a murine model of acute allergic airway disease. Six- to eight-week-old female BALB/c mice were sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) and aluminium hydroxide on Days 0 and 14, challenged with aerosolized saline or OVA (1%) on Days 21-25, 28-32, and 35. The mice were killed 1 day after the last OVA/saline challenge. Serum OVA-specific immunoglobulin E (IgE) was measured by enzyme-linked immunosorbent assay. Peribronchial inflammation was quantified by morphometric analysis. Lung caveolin-1 and Type I collagen mRNA expression was determined by real-time reverse-transcription polymerase chain reaction. Total lung collagen was measured using Sircol Assay Kit. Serum OVA-specific IgE levels were significantly elevated in OVA-challenged mice when compared with saline-challenged mice. Percentage of inflammatory cells in the bronchoalveolar lavage was significantly higher in the OVA-challenged animals. The animals' lungs that were sensitized and challenged with OVA contained large numbers of inflammatory cells concentrated near the airways and in the perivascular areas. The thickness of the bronchial epithelial layer and smooth muscle layer and the numbers of total inflammatory cells and eosinophils significantly increased in OVA-challenged mice. Caveolin-1 mRNA expression significantly decreased and Type I collagen mRNA expression significantly increased in the lung tissue of OVA-challenged mice. These results suggest that caveolin-1 seems to be involved in the pathogenesis of airway remodeling of acute allergic airway disease.

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

Abstract P4-06-19: Role of Caveolin-1 and PDGFR in Inflammatory Breast Cancer

Abstract Inflammatory breast cancer is a highly metastatic form of locally advanced breast cancer. Its uniqueness lies in the fact that it is found to be highly metastatic from the inception. Given the clinical differences between IBC and non-IBC, it is hypothesized that IBC has a characteristic molecular profile. Microarray data reveals interesting differences in the genetic makeup of these two cancer types. Caveolin-1, 2 proteins typically have much reduced expression in other breast cancers; however, this is not the case in inflammatory breast cancer. Caveolins, primarily caveolin-1 and 2 are over-expressed in IBC cells SUM 149. Caveolin-1 is membrane-associated protein located in lipid rafts called ‘caveolae'. Its unique scaffolding domain is known to concentrate several signaling molecules including PDGFR, Rho GTPases etc. leading to unique signaling cascades. Due to this characteristic feature, caveolin-1 has become one of the potential regulators of many cellular signaling and trafficking pathways that control cell growth and migration. Previously, our laboratory demonstrated that RhoC GTPase is responsible for the invasive phenotype of IBC. However, the mechanism of RhoC activation has not been elucidated yet. Preliminary data from Translational cancer research group Antwerp, Belgium shows over-expression of PDGFR alfa at mRNA level in IBC patient samples. Therefore, primary objectives of our study are to determine whether the highly invasive phenotype of IBC is due to increased PDGFR signaling through Akt1 leading to increased RhoC activation and what role does caveolin-1 play in regulating the interaction of PDGFRA and RhoC GTPase in Inflammatory breast cancer. We first determined the down-regulation of caveolin-1 at the mRNA level using varying doses of caveolin-1-specific siRNA in the inflammatory breast cancer cell line, SUM 149. QPCR showed a maximum down regulation of 50% at the mRNA level 72 h post siRNA treatment; bringing caveolin-1 mRNA levels close to the levels in normal human mammary epithelial cells. Next we quantitatively analyzed the effect of caveolin-1 down-regulation on SUM 149 cell invasiveness through a Matrigel coated filter. After 72 h caveolin-1 siRNA treatment, the percent invasion of SUM149 IBC cells was significantly reduced to 10% as compared to the control SUM 149 cells. We also determined that siRNA treatment does not affect cell viability or proliferation. This data supports our hypothesis that over-expression of caveolin-1 is involved in the invasive phenotype of IBC and is possibly due to interactions with RhoC GTPase. We are particularly interested in PDGFR that is found to be associated with caveolin-1 at plasma membrane. So far, we have found mRNA and protein expression of PDGFR in SUM 149 cells. However, instead of doublet at 150 and 180kDa in control NIH3T3 cells, SUM 149 cells produce only single band at 140-150kDa. Data from our lab shows that SUM 149 cells express high levels of active Akt1 and selective downregulation of Akt1 significantly reduces SUM 149 cells invasion. These results support our hypothesis that high levels of PDGFR in SUM 149 cells act through Akt1 causing RhoC activation at caveolin-1 and thus regulate invasive and metastatic behavior of IBC. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-19.