Epigallocatechin-gallate stimulates NF-E2-related factor and heme oxygenase-1 via caveolin-1 displacement

Flavonoids, such as the tea catechin epigallocatechin-gallate (EGCG), can protect against atherosclerosis by decreasing vascular endothelial cell inflammation. Heme oxygenase-1 (HO-1) is an enzyme that plays an important role in vascular physiology, and its induction may provide protection against atherosclerosis. Heme oxygenase-1 can be compartmentalized in caveolae in endothelial cells. Caveolae are plasma microdomains important in vesicular transport and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that caveolae play a role in the uptake and transport of EGCG and mechanisms associated with the anti-inflammatory properties of this flavonoid. To test this hypothesis, we explored the effect of EGCG on the induction of NF-E2-related factor (Nrf2) and HO-1 in endothelial cells with or without functional caveolae. Treatment with EGCG activated Nrf2 and increased HO-1 expression and cellular production of bilirubin. In addition, EGCG rapidly accumulated in caveolae, which was associated with caveolin-1 displacement from the plasma membrane towards the cytosol. Similar to EGCG treatment, silencing of caveolin-1 by siRNA technique also resulted in up-regulation of Nrf2, HO-1 and bilirubin production. These data suggest that EGCG-induced caveolin-1 displacement may reduce endothelial inflammation.     

Caveolar Fatty Acids and Acylation of Caveolin-1

Purpose

Caveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.

Methods

Caveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.

Results

In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×10⁷ cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.

Conclusion

Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.     

Altered caveolin-3 expression disrupts PI(3) kinase signaling leading to death of cultured muscle cells

Caveolae and their coat proteins, caveolins, co-ordinate multiple signaling pathways. Caveolin-3 is a muscle-specific caveolin isoform that is deficient in limb girdle muscular dystrophy type 1 C (LGMD1C). Paradoxically, overexpression of this protein also causes muscle degeneration in vivo. We hypothesize that altered membrane expression of caveolin-3 in muscle cells causes a degenerative phenotype by disrupting the co-ordination of signaling pathways that are critical to the maintenance of cell survival. Here, we show for the first time that, in normal muscle cells subjected to oxidative stress, the phosphatidylinositol (3) kinase (PI(3) kinase)-associated proteins PDK1 and Akt associate with caveolae where they bind to caveolin-3, and that normal activation of this pathway promotes cell survival. Either increased or decreased expression of caveolin-3 at the membrane caused an increased susceptibility to oxidative stress, and myotube survival was markedly improved by PI(3) kinase inhibition. This occurred concomitantly with altered phosphorylation of the pro-apoptotic proteins GSK3beta and Bad, despite normal levels of Akt activation. Taken together, our results demonstrate that altered caveolin-3 expression can change the outcome of PI(3) kinase activation from cell survival to cell death. These findings indicate that normal expression and localization of caveolin-3 are required to appropriately co-ordinate PI(3) kinase/Akt-mediated cell survival signaling, and suggest that this pathway may be an effective therapeutic target for the treatment of muscular dystrophies associated with caveolin-3 mutations.     

Evidence for heme oxygenase-1 association with caveolin-1 and -2 in mouse mesangial cells

The interaction of heme oxygenase-1 (HO-1) and caveolin in the cultured mouse mesangial cells (MMC) was investigated. In normal MMCs, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Upon treating the MMCs either with cadmium (Cd) or spermine NONOate (SPER/NO), expression of HO-1 mRNA and protein was increased. Caveolae rich membranous fractions from the MMCs treated with Cd or SPER/NO contained both HO-1 and caveolin-1 or caveolin-2. The experiments of immuno-precipitation showed complex formation between the HO-1 and caveolin-1 or caveolin-2 in the Cd treated MMCs. Confocal microscopic results also support co-localization of HO-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with HO-1 in caveolae suggested that caveolin could also play an important role in regulating the function of HO-1.     

Interaction of caveolin-1 with Ku70 inhibits Bax-mediated apoptosis

Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101) to the caveolin-binding domain (CBD) of Ku70 (amino acids 471-478). Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 Φ→A), limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.     

The chaperone function of hsp70 is required for protection against stress-induced apoptosis

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.     

In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation

Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its "scaffolding domain" to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo, we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82-101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.     

Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

A high-cholesterol diet increases the association between caveolae and insulin receptors in rat liver

Caveolin-1, a component of caveolae, regulates signaling pathway compartmentalization by interacting with tyrosine (Tyr) kinase receptors and their substrates. Perturbations in caveolae lipid composition have been shown in vitro to displace proteins from lipid microdomains, thereby altering their functionality and subsequent downstream signaling. The role of caveolin-1 in insulin receptor (IR) signaling has been widely investigated in vitro mainly in 3T3-L1 adipocyte cells. However, in vivo experiments investigating this connection in liver tissue have not been carried out. The objective of the present study was to investigate the effects of a high-cholesterol diet on caveolin-1 expression and IR localization and activity in the rat liver. Compared with a standard diet, rats fed with diet rich in cholesterol significantly altered liver caveolae by increasing both caveolin-1 (66%, P < 0.05) and caveolin-2 (55%, P < 0.05) expression while caveolin-1 mRNA levels were reduced. Concomitantly, a 25% increase in localization of the caveolae-resident signaling protein IR was observed. The distribution of caveolar and noncaveolar phosphorylated IR was unaffected but insulin-induced IR activation was significantly enhanced following consumption of the high-cholesterol diet (120%, P < 0.001). However, the downstream molecules IRS-1 and Akt have shown impaired activity in cholesterol-fed rats suggesting insulin resistance condition. Insulin stimulation failed to induce Tyr phosphorylation of caveolin-1 in cholesterol-fed rats. These findings suggest a mechanism by which a high-cholesterol diet altered caveolin-1 expression in vivo accompanied by altered IR localization and activity.     

Modulation of the Stress-Induced Synthesis of hsp27 and αB-Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract: The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and αB-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β-adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB-crystallin is suppressed.     

Early effects of PP60v-src kinase activation on caveolae

Members of the nonreceptor tyrosine kinase family appear to be targeted to caveolae membrane. We have used a Rat-1 cell expressing a temperature sensitive pp60^v-src kinase to assess the initial changes that take place in caveolae after kinase activation. Within 24–48 h after cells were shifted to the permissive temperature, a set of caveolae-specific proteins became phosphorylated on tyrosine. During this period there was a decline in the caveolae marker protein, caveolin-1, a loss of invaginated caveolae, and a 70% decline in the sphingomyelin content of the cell. One of the phosphorylated proteins was caveolin-1 but it was associated in coimmunoprecipitation assays with both a 30 kDa and a 27 kDa tyrosine-phosphorylated protein. Finally, the cells changed from having a typical fibroblast morphology to a rounded shape lacking polarity. In light of the recent evidence that diverse signaling events originate from caveolae, pp60^v-src kinase appears to cause global changes to this membrane domain that might directly contribute to the transformed phenotype. J. Cell. Biochem. 71:524–535, 1998. © 1998 Wiley-Liss, Inc.     

Activation of Fas inhibits heat-induced activation of HSF1 and up-regulation of hsp70.

Activation of heat shock factor (HSF) 1-DNA binding and inducible heat shock protein (hsp) 70 (also called hsp72) expression enables cells to resist various forms of stress and survive. Fas, a membrane-bound protein, is a central proapoptotic factor; its activation leads to a cascade of events, resulting in programmed cell death. These two mechanisms with contradictory functions, promoting either cell survival or death, were examined for their potential to inhibit each other's activation. Induction of FAS-mediated signaling was followed by a rapid decrease in HSF1-DNA binding and inducible hsp70 expression. Inhibition of HSF1-DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS signaling. These effects of FAS activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1) inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase in apoptosis rates from 20% to 50% in response to heat stress. When analyzing the effects of HSF1/hsp70 activation on Fas-mediated apoptosis, protection from apoptosis was seen in cells with induced hsp70 protein levels, but not in cells that were just induced for HSF1-DNA binding. Thus, we conclude that inhibition of HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis, without the influence of inhibitory signals.     

Bipolar assembly of caveolae in retinal pigment epithelium

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function. caveolin; raft microdomains; membrane traffic; normal rat kidney     

Caveolin-1 inhibits TrkA-induced cell death by influencing on TrkA modification associated with tyrosine-490 phosphorylation

Caveolin-1, a main structural protein constituent of caveolae, plays an important role in the signal transduction, endocytosis, and cholesterol transport. In addition, caveolin-1 has conflictive role in the regulation of cell survival and death depending on intracellular signaling pathways. The receptor tyrosine kinase TrkA has been known to interact with caveolin-1, and exploits multiple functions such as cell survival, death and differentiation. In this report, we investigated how TrkA-induced cell death signaling is regulated by caveolin-1 in both TrkA and caveolin-1 overexpressing stable U2OS cells. Here we show that TrkA co-localizes with caveolin-1 mostly as a large aggresome around nucleus by confocal immunofluorescence microscopy. Interestingly, TrkA-mediated Bak cleavage was suppressed by caveolin-1, indicating an inhibition of TrkA-induced cell death signaling by caveolin-1. Moreover, caveolin-1 altered TrkA modification including tyrosine-490 phosphorylation and unidentified cleavage(s), resulting in the inhibition of TrkA-induced apoptotic cell death. Our results suggest that caveolin-1 could suppress TrkA-mediated pleiotypic effects by altering TrkA modification via functional interaction.     

Multiple domains in caveolin-1 control its intracellular traffic

Caveolin-1 is an integral membrane protein of caveolae that is thought to play an important role in both the traffic of cholesterol to caveolae and modulating the activity of multiple signaling molecules at this site. The molecule is synthesized in the endoplasmic reticulum, transported to the cell surface, and undergoes a poorly understood recycling itinerary. We have used mutagenesis to determine the parts of the molecule that control traffic of caveolin-1 from its site of synthesis to the cell surface. We identified four regions of the molecule that appear to influence caveolin-1 traffic. A region between amino acids 66 and 70, which is in the most conserved region of the molecule, is necessary for exit from the endoplasmic reticulum. The region between amino acids 71 and 80 controls incorporation of caveolin-1 oligomers into detergent-resistant regions of the Golgi apparatus. Amino acids 91-100 and 134-154 both control oligomerization and exit from the Golgi apparatus. Removal of other portions of the molecule has no effect on targeting of newly synthesized caveolin-1 to caveolae. The results suggest that movement of caveolin-1 among various endomembrane compartments is controlled at multiple steps.     

Functional interaction of caveolin-1 with Bruton's tyrosine kinase and Bmx.

Bruton's tyrosine kinase (Btk), a member of the Tec family of protein-tyrosine kinases, has been shown to be crucial for B cell development, differentiation, and signaling. Mutations in the Btk gene lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Using a co-transfection approach, we present evidence here that Btk interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. In addition, we found that native Bmx, another member of the Tec family kinases, is associated with endogenous caveolin-1 in primary human umbilical vein endothelial cells. Second, in transient transfection assays, expression of caveolin-1 leads to a substantial reduction in the in vivo tyrosine phosphorylation of both Btk and its constitutively active form, E41K. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82--101) functionally suppressed the autokinase activity of purified recombinant Btk protein. Third, we demonstrate that mouse splenic B-lymphocytes express substantial amounts of caveolin-1. Interestingly, caveolin-1 was found to be constitutively phosphorylated on tyrosine 14 in these cells. The expression of caveolin-1 in B-lymphocytes and its interaction with Btk may have implications not only for B cell activation and signaling, but also for antigen presentation.     

Analysis of the variation in the hsp70-1 and hsp90alpha mRNA expression in human myocardial tissue that has undergone surgical stress

In the present work we reported a semiquantitative detection of messenger ribonucleic acids (mRNAs) encoding the human heat shock proteins Hsp70-1, the stress inducible member of the HSP70 family, and hsp90alpha, the inducible member of the HSP90 family. We investigated the change in the expression of these mRNAs in tissue samples taken from the right atrium of 48 pediatric patients, soon after the ischemic period during surgery to correct congenital heart diseases, in which a crystalloid cold cardioplegic solution was used. No significant variations were found for either hsp70-1 or hsp90alpha expressions. Moreover, we searched for an association between the hsp70-1 promoter region polymorphism and the expression of the hsp70-1 in a smaller group of these patients (n = 27). The -110AA genotype was on average significantly associated with a decrease in the hsp70-1 mRNA level (P < 0.05), whereas the other genotypes -110AC or -110CC did not seem to be associated with the hsp70-1 expression level. The lack of any observed increase in the hsp70-1 expression level may be due to the high basal level of the Hsp70 protein in the tissues examined.     

A new N-terminal recognition domain in caveolin-1 interacts with sterol carrier protein-2 (SCP-2)

Although plasma membrane domains, such as caveolae, provide an organizing principle for signaling pathways and cholesterol homeostasis in the cell, relatively little is known regarding specific mechanisms, whereby intracellular lipid-binding proteins are targeted to caveolae. Therefore, the interaction between caveolin-1 and sterol carrier protein-2 (SCP-2), a protein that binds and transfers both cholesterol and signaling lipids (e.g., phosphatidylinositides and sphingolipids), was examined by yeast two-hybrid, in vitro binding and fluorescence resonance energy transfer (FRET) analyses. Results of the in vivo and in vitro assays identified for the first time the N-terminal amino acids (aa) 1-32 amphipathic alpha helix of SCP-2 functionally interacted with caveolin-1. This interaction was independent of the classic caveolin-1 scaffolding domain, in which many signaling proteins interact. Instead, SCP-2 bound caveolin-1 through a new domain identified in the N-terminal domain of caveolin-1 between aa 34-40. Modeling studies suggested that electrostatic interactions between the SCP-2 N-terminal aa 1-32 amphipathic alpha-helical domain (cationic, positively charged face) and the caveolin-1 N-terminal aa 33-59 alpha helix (anionic, negatively charged face) may significantly contribute to this interaction. These findings provide new insights on how SCP-2 enhances cholesterol retention within the cell as well as regulates the distribution of signaling lipids, such as phosphoinositides and sphingolipids, at plasma membrane caveolae.