NF-κB and GATA-Binding Factor 6 Repress Transcription of Caveolins in Bladder Smooth Muscle Hypertrophy.

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.

Endocytosis and Degradation of Pegvisomant and a Potential New Mechanism That Inhibits the Nuclear Translocation of GHR.

CONTEXT: Pegvisomant, a growth hormone receptor (GHR) antagonist, is a well-known drug that was designed to treat acromegaly. However, recent studies have indicated that the GHR is a "moonlighting" protein that may exhibit dual functions based on its localization in the plasma membrane and nucleus. In light of this finding, we explored whether pegvisomant is a potential "moonlighting" GHR antagonist. In addition, the mechanisms of the endocytosis, postendocytic sorting, and degradation of pegvisomant are not fully understood. OBJECTIVE: This study investigated whether pegvisomant is a "moonlighting" antagonist and explored the mechanisms of the endocytosis, postendocytic sorting, and degradation of pegvisomant. METHODS: Indirect immunofluorescence and Western blot coupled with pharmacological inhibitors and gene silencing (small interfering RNA) were used to explore the mechanisms of the endocytosis, postendocytic sorting, and degradation of pegvisomant. Western blot, immunohistochemistry, and indirect immunofluorescence coupled with subcellular fractionation analysis were used to determine the effect of pegvisomant on GHR's nuclear localization in vitro and in vivo. RESULTS: Here, we show that the endocytosis of pegvisomant is mainly mediated though the clathrin pathway. Further study of the postendocytic sorting of pegvisomant shows that pegvisomant enters into different types of endosomes under GHR mediation. In addition, GHR is slightly downregulated by pegvisomant; further study indicates that proteasomes and lysosomes may cooperate to regulate pegvisomant/GHR degradation. Most importantly, we show that pegvisomant inhibits the nuclear localization of GHR. CONCLUSION: Our study showed that pegvisomant is a "moonlighting" antagonist. In addition, we revealed the mechanisms of the endocytosis, postendocytic sorting, and degradation of pegvisomant.

Inhibition of multiple myeloma­derived exosomes uptake suppresses the functional response in bone marrow stromal cell.

The communication between multiple myeloma (MM) cells and bone marrow stromal cells (BMSCs) serves a pivotal role in MM progression by supporting MM cell growth, proliferation and drug resistance. An exosomes­based endogenous transport system has been determined as a novel mechanism of this communication by revealing the capacity for exchange of functional components between cells. An exosomes transfer­mediated biological response in recipient cells is strongly determined by the detailed routes and mechanisms of exosomes internalization, which are diverse and can depend on surface molecules on the membrane of the vesicle and the recipient cell. Understanding the routes of exosomes uptake during MM cell­BMSC communication is of great importance for the development of blocking strategies beneficial for MM treatment. In the present study, fluorescently­labeled exosomes and pharmacological inhibitors, which are known to interfere with different internalization pathways, were used to characterize the cellular mechanisms involved in the uptake of MM cell­derived exosomes by BMSCs. MM cell­derived exosomes can promote BMSC viability and induce changes in multiple pro­survival and pro­proliferation pathways in BMSCs. As determined by flow cytometry and confocal microscopy, the uptake of MM cell­derived exosomes proceeded primarily through endocytosis, via special caveolin­dependent endocytosis, and partially through macropinocytosis and membrane fusion. Furthermore, treatment with endocytosis inhibitors suppressed the exosomes­induced changes in pathways in BMSCs. Collectively, these results indicate that endocytosis is the primary route of internalization of MM cell­derived exosomes by BMSCs and indicate that inhibition of exosomes uptake can interrupt the communication between MM cells and BMSCs and thus serve as a potential adjunctive strategy for MM treatment.

Intracellular Trafficking Pathways of Edwardsiella tarda: From Clathrin- and Caveolin-Mediated Endocytosis to Endosome and Lysosome.

Edwardsiella tarda is a Gram-negative bacterium that can infect a broad range of hosts including humans and fish. Accumulating evidences have indicated that E. tarda is able to survive and replicate in host phagocytes. However, the pathways involved in the intracellular infection of E. tarda are unclear. In this study, we examined the entry and endocytic trafficking of E. tarda in the mouse macrophage cell line RAW264.7. We found that E. tarda entered RAW264.7 and multiplied intracellularly in a robust manner. Cellular invasion of E. tarda was significantly impaired by inhibition of clathrin- and caveolin-mediated endocytic pathways and by inhibition of endosome acidification, but not by inhibition of macropinocytosis. Consistently, RAW264.7-infecting E. tarda was co-localized with clathrin, caveolin, and hallmarks of early and late endosomes, and intracellular E. tarda was found to exist in acid organelles. In addition, E. tarda in RAW264.7 was associated with actin and microtubule, and blocking of the functions of these cytoskeletons by inhibitors significantly decreased E. tarda infection. Furthermore, formaldehyde-killed E. tarda exhibited routes of cellular uptake and intracellular trafficking similar to that of live E. tarda. Together these results provide the first evidence that entry of live E. tarda into macrophages is probably a passive, virulence-independent process of phagocytosis effected by clathrin- and caveolin-mediated endocytosis and cytoskeletons, and that the intracellular traffic of E. tarda involves endosomes and endolysosomes.

Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells.

For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 µM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 µM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).

Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines.

Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types.

Nona-arginine facilitates delivery of quantum dots into cells via multiple pathways.

Semiconductor quantum dots (QDs) have recently been used to deliver and monitor biomolecules, such as drugs and proteins. However, QDs alone have a low efficiency of transport across the plasma membrane. In order to increase the efficiency, we used synthetic nona-arginine (SR9), a cell-penetrating peptide, to facilitate uptake. We found that SR9 increased the cellular uptake of QDs in a noncovalent binding manner between QDs and SR9. Further, we investigated mechanisms of QD/SR9 cellular internalization. Low temperature and metabolic inhibitors markedly inhibited the uptake of QD/SR9, indicating that internalization is an energy-dependent process. Results from both the pathway inhibitors and the RNA interference (RNAi) technique suggest that cellular uptake of QD/SR9 is predominantly a lipid raft-dependent process mediated by macropinocytosis. However, involvement of clathrin and caveolin-1 proteins in transducing QD/SR9 across the membrane cannot be completely ruled out.

On the mechanism and dynamics of uptake and permeation of polyether-copolyester dendrimers across an in vitro blood-brain barrier model.

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood-brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with P(app) of 19.7 +/- 1.9 x 10(-6) cm/s and 38.6 +/- 4.1 x 10(-6) cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11-14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. =

Caveolin-1 down-regulation activates estrogen receptor alpha expression and leads to 17beta-estradiol-stimulated mammary tumorigenesis.

Constitutive activation of estrogen receptor alpha (ER-alpha) expression is an early event in breast cancer tumorigenesis. However, the mechanism whereby ER-alpha is constitutively activated during transformation of normal mammary cells has not been well established. Previously, we reported that haploinsufficiency of caveolin-1, a major structural protein that forms caveolae, resulted in anchorage-independent growth of a normal mammary epithelial cell line, MCF10A. Here, we further demonstrated that ER-alpha but not ER-beta expression was constitutively activated in these caveolin-1 haploinsufficient cells. Transient treatment of MCF10A cells with beta-methyl-cyclodextrin, a chemical that can displace caveolin-1 from the plasma membrane, also stimulated ER-alpha expression. We further found that the 17beta-estradiol (E2) accelerated anchorage-independent growth of these cells in vitro and promoted their tumorigenesis in nude mice. These results suggest that dysregulation of caveolin-1 is one of the mechanisms by which ER-alpha expression is activated during initiation of breast tumorigenesis.

Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1.

BACKGROUND: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb uptake in pig renal proximal tubule cell line (LLC-PK1) cells; and ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells in a clathrin-dependent pathway. Our data also suggest a role of endocytosis in both ouabain-induced signal transduction and proximal tubule sodium handling. The present study addresses the molecular mechanisms involved in this process. METHODS: Studies were performed with cultured LLC-PK1 and a stable-expressed caveolin-1 knockdown LLC-PK1 cell line by SiRNA method. RESULTS: In wild-type LLC-PK1 cells, depletion of cholesterol by methyl beta-cyclodextrin reduced ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coated vesicles, as well as in endosomes. Depletion of cholesterol also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. In LLC-PK1 cells expressing mock-vehicle and caveolin-1 siRNA, depletion of caveolin-1 abolished ouabain-induced decrease in Rb uptake and decrease in the plasmalemmal Na/K-ATPase content. Depletion of caveolin-1 also significantly reduced the ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coat vesicles, as well as early and late endosomes. In addition, depletion of caveolin-1 also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. These data suggest that caveolae are involved in ouabain-induced endocytosis and signal transduction by initiating assembly of signaling cascades through the caveolar Na/K-ATPase and/or the interaction with clathrin-mediated endocytosis of the Na/K-ATPase.

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates.

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.

Promoter CpG methylation of caveolin-1 in sporadic colorectal cancer.

BACKGROUND: Caveolin-1 has been shown to be down-regulated in human colon cancer and involved in colon tumorigenesis. We investigated the mechanism. MATERIALS AND METHODS: Cancerous and nearby non-cancerous tissues of 185 sporadic colorectal cancer samples were enrolled in this study. Methylation-specific PCR was performed to explore the mechanism of regulation of caveolin-1 gene expression. RESULTS: Aberrant promoter methylation in the caveolin-1 gene was 3.8% and 5.9% for cancerous and nearby non-cancerous tissues, respectively. All the cancerous and non-cancerous tissue contained unmethylated promoters in the caveolin-1 gene. The methylation status of caveolin-1 had no clear relationship with age, cell grade, location of tumor or lymph node metastasis. However, female gender showed statistically significant difference (p=0.045). The immunohistochemistry study demonstrated that expression of caveolin-1 correlated with aberrant promoter methylation status in sporadic colorectal cancer tissues. CONCLUSION: Our findings suggested that aberrant promoter methylation of the caveolin-1 gene may occur at the precancerous stage, regulated by gender-related factors and is associated with gene silencing of caveolin-1 in the development of colorectal cancer.

Daidzein and 17 beta-estradiol enhance nitric oxide synthase activity associated with an increase in calmodulin and a decrease in caveolin-1.

Isoflavones, such as daidzein, are proposed to possess vasculoprotective properties, perhaps through a mechanism similar to estrogen. Our experiments aimed to test the hypothesis that daidzein and 17 beta-estradiol enhance endothelium-dependent relaxation through an increase in NO synthesis due to an increase in activity or expression of endothelial nitric oxide synthase (eNOS). Male rats were treated with daidzein (0.2 mg/kg per day sc), 17 beta-estradiol (0.1 mg/kg per day sc), or vehicle for 7 days and reactivity of isolated aortic rings was then determined. ACh-induced relaxation was significantly enhanced in aortic rings from rats treated with daidzein or 17 beta-estradiol but the relaxant responses to the endothelium-independent dilators sodium nitroprusside or isoprenaline were not different. Nitrite production and the level of cGMP were significantly greater in aortae from daidzein and 17 beta-estradiol compared with vehicle-treated rats. Daidzein and 17 beta-estradiol did not alter eNOS protein in endothelium-intact aortae but reduced expression of caveolin-1 and increased expression of calmodulin, changes that would account for an increase in eNOS activity. There were no differences between groups in the expression of calmodulin and caveolin-1 in arteries when the endothelium was removed. Daidzein or 17 beta-estradiol treatment selectively enhances endothelium-dependent relaxation in male rats through an increase in eNOS activity. The increase in eNOS activity is associated with a decreased expression of caveolin-1 and an increased expression of calmodulin in endothelial cells.

Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death.

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.

Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids.

Peptide nucleic acids (PNAs) are a potentially powerful approach for the recognition of cellular mRNA and the inhibition of gene expression. Despite their promise, the rules for using antisense PNAs have remained obscure, and antisense PNAs have been used sparingly in research. Here we investigate the ability of PNAs to be effective antisense agents inside mammalian cells, to inhibit expression of human caveolin-1 (hCav-1), and to discriminate between its alpha and beta isoforms. Many human genes are expressed as isoforms. Isoforms may play different roles within a cell or within different tissues, and defining these roles is a challenge for functional genomics and drug discovery. PNAs targeted to the translation start codons for the alpha and beta isoforms inhibit expression of hCav-1. Inhibition is dependent on PNA length. The potency and duration of inhibition by PNAs are similar to inhibition of gene expression by short interferring RNA (siRNA). Expression of the alpha isoform can be blocked selectively by a PNA. Cell proliferation is halted by inhibition of expression of both hCav-1 isoforms, but not by inhibition of the alpha hCav-1 isoform alone. Efficient antisense inhibition and selective modulation of isoform expression suggest that PNAs are versatile tools for controlling gene expression and dissecting the roles of closely related protein variants. Potent inhibition by PNAs may supply a "knock down" technology that can complement and "cross-check" siRNA and other approaches to antisense gene inhibition that rely on oligomers with phosphate or phosphorothioate backbone linkages.

RNA interference in mammalian cells by chemically-modified RNA.

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.

Secreted caveolin-1 stimulates cell survival/clonal growth and contributes to metastasis in androgen-insensitive prostate cancer.

Caveolin-1 is an integral protein of caveolae, known to play important roles in signal transduction and lipid transport. We demonstrate that caveolin-1 expression is significantly increased in primary and metastatic human prostate cancer after androgen ablation therapy. We also show that caveolin-1 is secreted by androgen-insensitive prostate cancer cells, and that this secretion is regulated by steroid hormones. Significantly, caveolin-1 was detected in the MDL(3) fraction of serum specimens from patients with advanced prostate cancer and to a lesser extent in normal subjects. Conditioned media from high passage caveolin-1 secreting, androgen-insensitive, LNCaP cells stimulated increased viability and clonal growth of low passage, caveolin-1-negative, androgen-sensitive, LNCaP cells in vitro, and this effect was blocked by treating the media with caveolin-1 antibody. i.p. injections of caveolin-1 antibody suppressed the orthotopic growth and spontaneous metastasis of highly metastatic, androgen-insensitive caveolin-1-secreting mouse prostate cancer. Overall, our results establish caveolin-1 as an autocrine/paracrine factor that is associated with androgen-insensitive prostate cancer. We demonstrate the potential for caveolin-1 as a therapeutic target for this important malignancy.

Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia-reperfusion via nitric oxide mechanism.

Caveolin-1 is a protein constituent of cell membranes. The caveolin-1 scaffolding region (residues 82-101) is a known inhibitor of protein kinase C. Inhibition of protein kinase C results in maintained nitric oxide (NO) release from the endothelium, which attenuates cardiac dysfunction after ischemia-reperfusion (I/R). Therefore, we hypothesized that the caveolin-1 scaffolding region of the molecule, termed caveolin-1 peptide, might attenuate postischemia polymorphonuclear neutrophil (PMN)-induced cardiac dysfunction. We examined the effects of caveolin-1 peptide in isolated ischemic (20 min) and reperfused (45 min) rat hearts reperfused with PMNs. Caveolin-1 peptide (165 or 330 microg) given intravenously 1 h before I/R significantly attenuated postischemic PMN-induced cardiac dysfunction, as exemplified by left ventricular developed pressure (LVDP) (P < 0.01) and the maximal rate of developed pressure (+dP/dt(max)) (P < 0.01), compared with I/R hearts obtained from rats given 0.9% NaCl. In addition, caveolin-1 peptide significantly reduced cardiac PMN infiltration from 195 +/- 5 PMNs/mm2 in untreated hearts to 103 +/- 5 and 60 +/- 5 PMNs/mm2 in hearts from 165 and 330 microg caveolin-1 peptide-treated rats, respectively (P < 0.01). PMN adherence to the rat coronary vasculature was also significantly reduced in rats given either 165 or 330 microg caveolin-1 peptide compared with rats given 0.9% NaCl (P < 0.01). Moreover, caveolin-1 peptide-treated rat aortas exhibited a 2.2-fold greater basal release of NO than vehicle-treated aortas (P < 0.01), and this was inhibited by NG-nitro-L-arginine methyl ester. These results provide evidence that caveolin-1 peptide significantly attenuated PMN-induced post-I/R cardiac contractile dysfunction in the isolated perfused rat heart, probably via enhanced release of endothelium-derived NO.

Evidence that Myc isoforms transcriptionally repress caveolin-1 gene expression via an INR-dependent mechanism.

The c-Myc oncoprotein contributes to oncogenesis by activating and repressing a repertoire of genes involved in cellular proliferation, metabolism, and apoptosis. Increasing evidence suggests that the repressor function of c-Myc is critical for transformation. Therefore, identifying and characterizing Myc-repressed genes is imperative to understanding the mechanisms of Myc-induced tumorigenesis. Here, we employ NIH 3T3 cell lines harboring c-Myc-ER or N-Myc-ER to dissect the relationship between Myc activation and caveolin-1 expression. In this well-established inducible system, treatment with estrogen like molecules, such as tamoxifen, leads to activation of Myc, but in a tightly controlled fashion. Using this approach, we show that Myc activation induces the repression of caveolin-1 expression at the transcriptional level. We also provide two independent lines of evidence suggesting that caveolin-1 is a direct target of Myc: (i) the effect of Myc activation on caveolin-1 expression is independent of new protein synthesis, as revealed through the use of cycloheximide; and (ii) Myc-mediated repression of the caveolin-1 promoter is dependent on an intact INR sequence. Moreover, we show that expression of caveolin-1, via an adenoviral vector approach, can suppress cell transformation that is mediated by Myc activation. In support of these observations, treatment with an adenoviral vector harboring anti-sense caveolin-1 specifically potentiates transformation induced by Myc activation. Taken together, our results indicate that caveolin-1 is a direct target of Myc repression, and they also provide evidence for an additional mechanism by which Myc repression can elicit a malignant phenotype.