Evidence for rat organic anion transporter 3 association with caveolin-1 in rat kidney

The rat organic anion transporter 3 (rOAT3) has recently been identified as the third isoform of the OAT family. The mechanisms that regulate rOAT3's functions remain to be elucidated. rOAT3 contributes for moving a number of negatively charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role as a membrane transporter. To address the relationship of these two proteins, we investigated the protein-protein interaction between rOAT3 and Cav-1. The rOAT3 mRNA and protein expression were observed in the rat kidney, and the expressions of Cav-1 mRNA and protein were also detected in the kidney. Confocal microscopy of the immuno-cytochemistry experiments using primary cultured renal proximal tubular cells showed that rOAT3 and Cav-1 were co-localized at the plasma membrane. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions from the rat kidney and immuno-precipitation experimentation. When rOAT3's synthesized cRNA of rOAT3 along with the antisense oligo deoxynucleotide ofXenopusCav-1 were co-injected intoXenopusoocytes, the [(3)H] estrone sulfate uptake was significantly decreased. These findings suggest that rOAT3 and caveolin-1 share a cellular expression in the plasma membrane and Cav-1 up-regulates the organic anionic compound uptake via rOAT3 under normal physiological conditions.     

Caveolae-associated proteins in cardiomyocytes: caveolin-2 expression and interactions with caveolin-3

Caveolin-3 the muscle-specific caveolin isoform, acts like the more ubiquitously expressed caveolin-1 to sculpt caveolae, specialized membrane microdomains that serve as platforms to organize signal transduction pathways. Caveolin-2 is a structurally related isoform that alone does not drive caveolae biogenesis; rather, caveolin-2 cooperates with caveolin-1 to form caveolae in nonmuscle cells. Although caveolin-2 might be expected to interact in an fashion analogous to that of caveolin-3, it generally has not been detected in cardiomyocytes. This study shows that caveolin-2 and caveolin-3 are detected at low levels in ventricular myocardium and increase dramatically with age or when neonatal cardiomyocytes are placed in culture. In contrast, flotillins (caveolin functional homologs) are expressed at relatively constant levels in these preparations. In neonatal cardiac cultures, caveolin-2 and -3 expression is not influenced by thyroid hormone (a postnatal regulator of other cardiac gene products). The further evidence that caveolin-2 coimmunoprecipitates with caveolin-3 and floats with caveolin-3 by isopycnic centrifugation in cardiomyocyte cultures suggests that caveolin-2 may play a role in caveolae biogenesis and influence cardiac muscle physiology.     

大鼠脑内caveolin-1蛋白的表达及其在分辨学习中的作用

Caveolin-1（Cav—1）蛋白作为细胞质膜结构小窝（caveolae）的标志蛋白,在胆固醇运输、膜组装、信号转导和细胞转化过程中扮演重要的角色。为了探讨Cav-1蛋白在中枢神经系统可塑性及学习记忆中的作用,本文以Sprague—Dawley大鼠为实验对象,利用蛋白质免疫印迹杂交方法观察了Car-1蛋白在不同年龄大鼠脑内表达的特征,并研究了Y-迷宫训练前后Cav-1蛋白表达的变化。结果表明：（1）大鼠不同脑区Cav-1蛋白表达的年龄特征不同。海马内的表达属青年鼠最高,其次是老年鼠和幼年鼠;皮层内的表达属幼年鼠最高,其次是老年鼠,青年鼠最低;小脑内的表达无明显年龄差异。（2）Y-迷宫训练引起青年鼠海马和前额叶皮层内Cav-1蛋白的表达显著增加。结果提示,Cav-1蛋白与动物脑发育和学习记忆有密切关系,可能参与中枢可塑性的调节。     

Immunolocalization of caveolin-1 in rat and human mesothelium.

Flask-shaped vesicles have been described as caveolae in mesothelial cells in a number of animal species based on morphological criteria only. Using an antibody against caveolin-1, said to be a biochemical marker of caveolae, immunoelectron microscopy suggests that many but not all such vesicles in mesothelial cells are caveolae. Mesothelial cells from different anatomical sites showed obvious variations in both the population density and distribution of these flask-shaped vesicles and in their density of immunostaining. Lung and pericardial sac had the highest staining density. In some sites (e.g., lung, bladder, colon) caveolae were equally distributed between apical and basolateral surfaces, whereas in others (e.g., spleen, liver), they were predominantly apical. Additional immunopositive sites in the peritoneal membrane were identified, including the epineurium of peripheral nerves and the endothelium of lymphatic vessels. We further suggest that variations in the number of mesothelial cell caveolae and the density of their immunolabeling may have implications for our understanding of certain diseases such as malignant mesothelioma, especially in view of the recent hypothesis that it may be caused by SV40, a virus that appears to enter cells via caveolae.     

Real-time dynamic movement of caveolin-1 during smooth muscle contraction of human colon and aged rat colon transfected with caveolin-1 cDNA

Caveolin-1 (cav-1) plays a key role in PKC-α and RhoA signaling pathways during acetylcholine (ACh)-induced contraction of colonic smooth muscle cells (CSMC). Aged rat CSMC showed sluggish contractility, concomitant with reduced expression of cav-1 with an associated reduction in activation of PKC-α and RhoA signaling pathway. Real-time monitoring of live human CSMC transfected with yellow fluorescent protein-tagged wild-type caveolin 1 cDNA (YFP-wt-cav-1) cDNA in the present study suggests that cav-1 cycles within and along the membrane in a synchronized, highly organized cytoskeletal path. These studies provide, for the first time, the advantages of real-time monitoring of the dynamic movement of caveolin in living cells. Rapid movement of cav-1 in response to ACh suggests its dynamic role in CSMC contraction. Human CSMC transfected with YFP-ΔTFT-cav-1 dominant negative cDNA show fluorescence in the cytosol of the CSMC and no movement of fluorescent cav-1 in response to ACh mimicking the response shown by aged rat CSMC. Transfection of CSMC from aged rat with YFP-wt-cav-1 cDNA restored the physiological contractile response to ACh as well as the dynamic movement of cav-1 along the organized cytoskeletal path observed in normal adult CSMC. To study the force generation by CSMC, three-dimensional colonic rings were bioengineered. Colonic bioengineered rings from aged CSMC showed reduced force generation compared with colonic bioengineered rings from adult CSMC. Colonic bioengineered rings from aged CSMC transfected with wt-cav-1 cDNA showed force generation similar to colonic bioengineered rings from adult rat CSMC. The data suggest that contraction in CSMC is dependent on cav-1 reorganization dynamics, which restores the physiological contractile response in aged CSMC. We hypothesize that dynamic movement of cav-1 is essential for physiological contractile response of colonic smooth muscle.     

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.     

Immunolocalization of caveolin-1 and caveolin-3 in monkey skeletal,cardiac and uterine smooth muscles

Caveolin, a 20-24 kDa integral membrane protein, is a principal component of caveolar domains. Caveolin-1 is expressed predominantly in endothelial cells, fibroblasts, and adipocytes, while the expression of caveolin-3 is confined to muscle cells. However, their localization in various muscles has not been well documented. Using double-immunofluorescence labeling and confocal laser microscopy, we examined the localization of caveolins-1 and 3 in adult monkey skeletal, cardiac and uterine smooth muscles and the co-immunolocalization of these caveolins with dystrophin, which is a product of the Duchenne muscular dystrophy gene. In the skeletal muscle tissue, caveolin-3 was localized along the sarcolemma except for the transverse tubules, and co-immunolocalized with dystrophin, whereas caveolin-1 was absent except in the blood vessels of the muscle tissue. In cardiac muscle cells, caveolins-1 and -3 and dystrophin were co-immunolocalized on the sarcolemma and transverse tubules. In uterine smooth muscle cells, caveolin-1, but not caveolin-3, was co-immunolocalized with dystrophin on the sarcolemma.     

人Flt3配体在酵母中的表达及其生物学特性

Ｆｌｔ３配体（ＦＬ）是一种具有促进早期造血功能的细胞因子,能够促进造血干／祖细胞的增殖和分化。为了获得高效表达的人可溶性Ｆｌｔ３配体（ｒｈＦＬ）重组蛋白,采用酵母偏爱的密码子,人工合成ｒｈＦＬ基因,构建表达载体ｐＰＩＣＺａＡ－ＦＬ,电转化毕氏酵母９Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）,得到稳定分泌ｒｈＦＬ的基因工程菌株,在培养上清中,其表达量达３０ｍｇ／Ｌ。生物学活性检测提示,毕氏酵母系统表达的ｒｈＦ     

Expression of caveolin-1 is required for the transport of caveolin-2 to the plasma membrane. Retention of caveolin-2 at the level of the golgi complex.

Caveolins-1 and -2 are normally co-expressed, and they form a hetero-oligomeric complex in many cell types. These caveolin hetero-oligomers are thought to represent the assembly units that drive caveolae formation in vivo. However, the functional significance of the interaction between caveolins-1 and -2 remains unknown. Here, we show that caveolin-1 co-expression is required for the transport of caveolin-2 from the Golgi complex to the plasma membrane. We identified a human erythroleukemic cell line, K562, that expresses caveolin-2 but fails to express detectable levels of caveolin-1. This allowed us to stringently assess the effects of recombinant caveolin-1 expression on the behavior of endogenous caveolin-2. We show that expression of caveolin-1 in K562 cells is sufficient to reconstitute the de novo formation of caveolae in these cells. In addition, recombinant expression of caveolin-1 allows caveolin-2 to form high molecular mass oligomers that are targeted to caveolae-enriched membrane fractions. In striking contrast, in the absence of caveolin-1 expression, caveolin-2 forms low molecular mass oligomers that are retained at the level of the Golgi complex. Interestingly, we also show that expression of caveolin-1 in K562 cells dramatically up-regulates the expression of endogenous caveolin-2. Northern blot analysis reveals that caveolin-2 mRNA levels remain constant under these conditions, suggesting that the expression of caveolin-1 stabilizes the caveolin-2 protein. Conversely, transient expression of caveolin-2 in CHO cells is sufficient to up-regulate endogenous caveolin-1 expression. Thus, the formation of a hetero-oligomeric complex between caveolins-1 and -2 stabilizes the caveolin-2 protein product and allows caveolin-2 to be transported from the Golgi complex to the plasma membrane.     

Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU

We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological efficacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an effective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L efficiently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs. Intratumoral injection of Ad-mFlt3L followed by an intraperitoneal administration of 5-Fu significantly inhibited tumor growth and cured established tumors. Adenovirus mediated Flt3L gene therapy synergies with chemotherapeutic drug, 5-Fu, in elicitation of long-lasting antitumor immunity. The tumor specific immunity can be adoptively transferred into naïve animals successfully by transfusion of CD3⁺CD8⁺ T cells from the treated mice. The data suggests that adenovirus mediated Flt3L gene therapy in combination with 5-Fu chemotherapy may open a new avenue for development of anti-cancer chemogenetherapy.     

Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries

The role of caveolins, signature proteins of caveolae, in arterial Ca(2+) regulation is unknown. We investigated modulation of Ca(2+) homeostasis by caveolin-1 and caveolin-3 using smooth muscle cells from rat cerebral resistance arteries. Membrane current and Ca(2+) transients were simultaneously measured with voltage-clamped single cells. Membrane depolarization triggered Ca(2+) current and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). After repolarization, elevated [Ca(2+)](i) returned to the resting level. Ca(2+) removal rate was determined from the declining phase of the Ca(2+) transient. Application of caveolin-1 antibody or caveolin-1 scaffolding domain peptide, corresponding to amino acid residues 82-101 of caveolin-1, significantly slowed Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM, with little effect at a measured [Ca(2+)](i) of 600 nM. Application of caveolin-3 antibody or caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55-74 of caveolin-3, also significantly slowed Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM, with little effect at a measured [Ca(2+)](i) of 600 nM. Likewise, application of calmodulin inhibitory peptide, autocamtide-2-related inhibitory peptide, and cyclosporine A, inhibitors for calmodulin, Ca(2+)/calmodulin-dependent protein kinase II, and calcineurin, also significantly inhibited Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM but not at 600 nM. Application of cyclopiazonic acid, a sarcoplasmic reticulum Ca(2+) ATPase inhibitor, also significantly inhibited Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM but not at 600 nM. Our results suggest that caveolin-1 and caveolin-3 are important in Ca(2+) removal of resistance artery smooth muscle cells.     

Detection of caveolin-3/caveolin-1/P2X7R complexes in mice atrial cardiomyocytes in vivo and in vitro

Caveolae and caveolins, structural components of caveolae, are associated with specific ion channels in cardiac myocytes. We have previously shown that P2X purinoceptor 7 (P2X7R), a ligand-gated ion channel, is increased in atrial cardiomyocytes of caveolin-1 knockout mice; however, the specific biochemical relationship of P2X7R with caveolins in the heart is not clear. The aim of this work was to study the presence of the P2X7R in atrial cardiomyocytes and its biochemical relationship to caveolin-1 and caveolin-3. Caveolin isoforms and P2X7R were predominantly localized in buoyant membrane fractions (lipid rafts/caveolae) prepared from hearts using detergent-free sucrose gradient centrifugation. Caveolin-1 knockout mice showed normal distribution of caveolin-3 and P2X7R to buoyant membranes indicating the importance of caveolin-3 to formation of caveolae. Using clear native-PAGE, we showed that caveolin-1, -3 and P2X7R contribute to the same protein complex in the membranes of murine cardiomyocytes and in the immortal cardiomyocyte cell line HL-1. Western blot analysis revealed increased caveolin-1 and -3 proteins in tissue homogenates of P2X7R knockout mice. Finally, tissue homogenates of atrial tissues from caveolin-3 knockout mice showed elevated mRNA for P2X7R in atria. The colocalization of caveolins with P2X7R in a biochemical complex and compensated upregulation of P2X7R or caveolins in the absence of any component of the complex suggests P2X7R and caveolins may serve an important regulatory control point for disease pathology in the heart.     

A Novel Bicistronic High-Capacity Gutless Adenovirus Vector That Drives Constitutive Expression of Herpes Simplex Virus Type 1 Thymidine Kinase and Tet-Inducible Expression of Flt3L for Glioma Therapeutics

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, affecting ∼18,000 new patients every year; its prognosis remains poor despite standard treatment with surgery, radiotherapy, and chemotherapy (temozolomide) (36-38, 44; J. C. Buckner, presented at the ASCO Annual Meeting, 2009). Complete resection is mostly impossible due to the highly infiltrative nature of this disease. Residual GBM cells remaining within the nonneoplastic brain parenchyma eventually lead to tumor recurrence that is resistant to conventional chemotherapy and radiotherapy, ultimately leading to the patient''s death (44). Several dendritic cell vaccination strategies aiming to stimulate the patient''s immune system to seek out and destroy residual brain tumor cells are currently under preclinical and clinical development and constitute a promising adjuvant treatment for GBM (18, 24, 42, 43, 45).We have developed a novel immunotherapeutic approach for GBM using first-generation adenoviral vectors (Ads) to deliver a combination of therapeutic transgenes into the tumor mass (2, 7, 9, 12, 14, 17, 48) which is slated to begin phase I clinical testing this year. In our strategy, rather than vaccinating against tumor antigens, we aim to reconstruct an immune circuit that is absent from the normal brain. Our gene therapy strategy consists of the conditionally cytotoxic herpes simplex virus type 1 thymidine kinase (TK) (2, 12), which kills proliferating tumor cells in the presence of the prodrug ganciclovir (GCV), used in combination with human soluble Fms-like tyrosine kinase 3 ligand (Flt3L), which recruits bone marrow-derived dendritic cells (DCs) to the normal brain or brain tumor microenvironment in mice (11, 12) and rats (1, 13). We previously showed that codelivery of Ad-Flt3L and Ad-TK into the brain tumor milieu induces an anti-GBM-specific immune response (9, 12, 17, 27, 48), leading to long-term survival of rats bearing intracranial CNS1, 9L, and F98 tumors (2, 17, 27) and mice bearing intracranial GL26, GL261, and B16-F10 tumors (12, 48). In addition, the combination of Ad-Flt3L plus Ad-TK induces GBM-specific immunological memory that improves survival in intracranial multifocal and recurrent models of GBM in both rats and mice (9, 12, 17, 20, 22, 27).First-generation adenoviral vectors have been used in clinical trials to deliver HSV1-TK (31). Ad-TK delivered into the margins of the tumor cavity after surgical resection of GBM was well tolerated in over 70 patients in six early clinical trials (19, 34). Publication of final results from a large, multicenter phase III trial involving 251 patients is awaited; preliminary analysis indicates small but statistically significant benefits from adding gene therapy to standard care. Final analysis will have to wait for the detailed publications (3, 16).To provide stable, long-term therapeutic transgene expression, “gutless,” high-capacity adenoviral (HC-Ad) vectors have been engineered in which all viral encoding genes have been eliminated and replaced with inert, noncoding stuffer DNA sequences (26, 30). In the presence of an anti-adenoviral immune response, transgene expression in the brain from first-generation Ads is reduced, while expression from HC-Ad vectors remains stable for at least 1 year (4, 25, 28). Therefore, we engineered two HC-Ad vectors to either constitutively express TK (HC-Ad-TK) (21, 27) or express Flt3L under the control of the tightly regulatable mCMV-TetOn expression system (HC-Ad-TetOn-Flt3L) (6, 8, 27, 47). Intratumoral administration of HC-Ad-TK and HC-Ad-TetOn-Flt3L led to high levels of therapeutic efficacy, and the therapy was well tolerated, with no overt toxicity and no spread of vector genomes outside the injected brain hemisphere in treated Lewis rats bearing syngeneic intracranial brain tumors (27).To reduce vector dose and facilitate GMP manufacturing of the clinical product, we engineered a novel, bicistronic HC-Ad vector that encodes, for the first time, both constitutively expressed HSV1-TK and inducible Flt3L from a single HC-Ad vector genome. Herein we demonstrate therapeutic efficacy of the bicistronic HC-Ad in naïve rats bearing intracranial tumors and also in tumor-bearing rats which had been exposed to adenoviruses and therefore exhibited anti-Ad immunity. The safety profile of this approach was assessed by evaluating the biodistribution of HC-Ad vector genomes, systemic and neurological toxicity, and behavioral abnormalities over the course of 1 year posttreatment. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.     

PHLCX Nflag3/小窝蛋白-1重组质粒的构建及融合基因在293T细胞中的表达

构建重组真核表达质粒PHLCX Nflag3／小窝蛋白-1,并在293T细胞中表达．用PCR的方法扩增cDNA文库中的人小窝蛋白-1基因,连接在真核表达栽体PHLCX Nflag3的短肽标签flag的下游,用限制性酶切和泖l序的方法鉴定;将重组质粒以脂质体法转染293T细胞,Western blotting法检测蛋白质的表达．结果显示,双酶切出现两个片段,分别与空栽体和人小窝蛋白-1的cDNA分子质量大小相符,测序结果符合人小窝蛋白-1的cDNA序列;Western blotting显示构建的新栽体能够在293T细胞中表达小窝蛋白-1／flag融合蛋白,表明已成功构建了能在293T细胞中高效表达小窝蛋白-1／flag融合蛋白的真核表达栽体PLHCX Nflag3／小窝蛋白-1．     

Colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts.

Results from our previous study suggest that cyclooxygenase-2 (COX-2) induced by phorbol 12-myristate 13-acetate (PMA) may be localized to caveolae-like structures (Liou, J.-Y., Shyue, S.-K., Tsai, M.-J., Chung, C.-L., Chu, K.-Y., and Wu, K. K. (2000) J. Biol. Chem. 275, 15314-15320). In this study, we determined subcellular localization of COX-2 and caveolin-1 by confocal microscopy. COX-2 in human foreskin fibroblasts stimulated by PMA (100 nm) or interleukin-1beta (1 ng/ml) for 6 h was localized to plasma membrane in addition to endoplasmic reticulum and nuclear envelope. Caveolin-1 was localized to plasma membrane, and image overlay showed colocalization of COX-2 with caveolin-1. This was confirmed by the presence of COX-2 and caveolin-1 in the detergent-insoluble membrane fraction of cells stimulated by PMA. Immunoprecipitation showed complex formation of COX-2 with caveolin-1 in a time-dependent manner. A larger quantity of COX-2 was complexed with caveolin-1 in PMA-treated than in interleukin-1beta-treated cells. Purified COX-2 complexed with glutathione S-transferase-fused caveolin-1, which was not inhibited by the scaffolding domain peptide. Caveolin-1-bound COX-2 was catalytically active, and its activity was not inhibited by the scaffolding domain peptide. These results suggest that COX-2 induced by PMA and interleukin-1beta is colocalized with caveolin-1 in the segregated caveolae compartment. Because caveolae are rich in signaling molecules, this COX-2 compartment may play an important role in diverse pathophysiological processes.     

Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts

Previous studies have shown that estrogen modulation of endothelial nitric oxide (NO) synthase (eNOS) may confer protection against heart disease. Here, we demonstrate an association between reductions in baroreflex-mediated bradycardia and in cardiac NOS activity in ovariectomized (Ovx) rats compared with controls. The latter resulted, at least in part, from a reduction in cardiac eNOS protein. eNOS-derived NO and its biological effects are determined by the levels of eNOS protein and by eNOS catalytic activity; the latter is regulated partly through the dynamic interaction with an inhibitory protein (caveolin) and a stimulatory protein (calmodulin). The association of eNOS immunoprecipitated with caveolin-3 and calmodulin was examined. Caveolin-3 and calmodulin binding with eNOS was increased and decreased, respectively, in Ovx rats. 17 beta-Estradiol replacement restored, to within normal levels, the baroreflex-mediated bradycardic responses along with eNOS activity, eNOS expression, and the association of eNOS with caveolin-3 and calmodulin. Our findings may help to elucidate the molecular mechanism underlying the favorable effects of estrogen on cardiac responses to baroreflex activation.     

Flt3 ligand induces tyrosine phosphorylation of gab1 and gab2 and their association with shp-2, grb2, and PI3 kinase

The receptor tyrosine kinase Flt3 has been shown to play an important role in proliferation, differentiation, and survival of hematopoietic stem and progenitor cells. Although some postreceptor signaling events of Flt3 have been characterized, the involvement of Gab family proteins in Flt3 signaling is not known. In this study, we show that both Gab1 and Gab2 are rapidly tyrosine phosphorylated after Flt3 ligand stimulation of Flt3 ligand-responsive cells. They interact with tyrosine-phosphorylated Shp-2, p85, Grb2, and Shc. The results suggest that Gab proteins are engaged in Flt3 signaling to mediate downstream activation of Shp-2 and PI3 kinase pathways and possibly the Ras/Raf/MAPK pathway.