Versatile Functions of Caveolin-1 in Aging-related Diseases / 전남의대학술지

Caveolin-1 (Cav-1) is a trans-membrane protein that is a major component of the caveolae structure on the plasma membrane. Cav-1 is involved in the regulation of various cellular processes, including cell growth, differentiation, endocytosis, and in particular it has been implied in cellular senescence. Here we review current knowledge about Cav-1 in cellular signaling and discuss the role of Cav-1 in aging-related diseases.

Versatile Functions of Caveolin-1 in Aging-related Diseases / 전남의대학술지

Caveolin-1 (Cav-1) is a trans-membrane protein that is a major component of the caveolae structure on the plasma membrane. Cav-1 is involved in the regulation of various cellular processes, including cell growth, differentiation, endocytosis, and in particular it has been implied in cellular senescence. Here we review current knowledge about Cav-1 in cellular signaling and discuss the role of Cav-1 in aging-related diseases.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Mechanism of cellular uptake and transport mediated by integrin receptor targeting trimethyl chitosan nanoparticles / 药学学报

This study investigated a nano drug delivery system built by one sort of modified trimethyl chitosan (TMC). The TMC was modified by cRGDyk, ligand of integrin receptor avβ3. Single factor screening was used to optimize the prescription in which the particle sizes of TMC nanoparticle (TMC NPs) and cRGDyk modified TMC nanoparticle (C-TMC NPs) were (240.3 ± 4.2) nm and (259.5 ± 3.3) nm. Electric potential of those two nanoparticles were (33.5 ± 0.8) mV and (25.7 ± 1.6) mV. Encapsulation efficiencies were (76.0 ± 2.2) % and (74.4 ± 2.0) %. Drug loading efficacies were (50.1 ± 2.1) % and (26.1 ± 1.0) %. Then the cellular uptake, uptake mechanism and transport efficacy of TMC NPs and C-TMC NPs were investigated using Caco-2 cell line. The uptake rate and accumulating drug transit dose of C-TMC NPs were 1.98 and 2.84 times higher than TMC NPs, separately. Mechanism investigations revealed that caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis were involved in the intercellular uptake of both TMC NPs and C-TMC NPs. What is more, free cRGDyk could remarkably inhibit the uptake of C-TMC NPs.

Clathrin and Lipid Raft-dependent Internalization of Porphyromonas gingivalis in Endothelial Cells

Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.

Exploration of conditions for releasing microvesicle from human bone marrow mesenchymal stem cells / 中国实验血液学杂志

The release of microvesicles(MV) is one of the critical mechanisms underlying the angiogenesis-promoting activity of mesenchymal stem cells(MSC). This study was aimed to explore the appropriate condition under which MSC releases MV. Bone marrow samples from 5 healthy adults were collected, and MSC were isolated, culture-expanded and identified. MSC at passage 5 were suspended in medium without or medium with 10% fetal(FCS) calf serum and seeded into culture dishes. The culture was separately maintained in hypoxia (1% oxygen) or normoxia (around 20% oxygen), and 20 dishes of cells (2×10(6)/dish) were used for each group. The supernatants were collected for MV harvesting. The cell number was counted with trypan blue exclusion test and the protein contents in the MV were determined. MV were identified by observation under an electron microscope. The surface markers on MV were analyzed by flow cytometry. MTT test was performed to observe the pro-proliferative activity of MV that were added into the culture of human umbilical cord vein endothelial cells at a concentration of 10 µg/ml. The results showed that the majority of MV released by MSC were with diameters of less than 100 nm, and MV took the featured membrane-like structure with a hypodense center. They expressed CD29, CD44, CD73 and CD105, while they were negative for CD31 and CD45. The increase multiples of the adherent trypan blue-resistant cells cultured in normoxia with serum, in normoxia without serum, in hypoxia with serum and hypoxia in the absence of serum were 4.05 ± 0.73, 1.77 ± 0.48, 5.80 ± 0.65 and 3.69 ± 0.85 respectively, and the estimated protein contents per 10(8) cells were 463.48 ± 138.74 µg, 1604.07 ± 445.28 µg, 2389.64 ± 476.75 µg and 3141.18 ± 353.01 µg. MTT test showed that MV collected from MSC in hypoxia seemed to promote the growth of endothelial cells more efficiently than those from cells in normoxia. It is concluded that hypoxia can enhance the release of microvesicles from MSC, and cultivation of MSC in hypoxia and medium without serum may provide an appropriate condition for MV harvesting.

Effect of m-calpain in PKCα-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain.

There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate, PKC activity using an assay kit and assay of Na+/K+ATPase activity spectrophotometrically. We purified m-calpain and PKCα by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCα by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCα (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCα in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCα increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2+]i-mCalpain-PKCα signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation.

Cavins: new sights of caveolae-associated protein / 生物工程学报

Caveolae are specialized lipid rafts that form flask-shaped invaginations of the plasma membrane. Many researches show that caveolae are involved in cell signaling and transport. Caveolin-1 is the major coat protein essential for the formation of caveolae. Recently, several reports indicated that the other caveolae-associated proteins, Cavins, are required for caveola formation and organization. It's worth noting that Cavin-1 could cooperate with Caveolin-1 to accommodate the structural integrity and function of caveolae. Here, we reviewed that the relationship between Cavins and Caveolins and explore the role of them in regulating caveolae.

Papel de Caveolina-1 na produção de mediadores inflamatórios / Role of caveolin-1 in the production of inflammatory mediators

A caveolina-1 (Cav-1), uma proteína essencial para a formação de cavéolas, apresenta atividade na modulação da sinalização intracelular. Cav-1 é capaz de interagir com diversas proteínas através de seu domínio CSD (caveolin scaffolding domain) e, em geral, essa interação leva à inibição das proteínas associadas. O peptídeo CSD tem sido utilizado como um mimético de Cav-1 em relação à sua capacidade modulatória sobre a atividade de outras proteínas. Recentemente, tem sido mostrado que Cav-1 é capaz de modular a resposta inflamatória em diversos aspectos. Neste trabalho, examinamos o papel de Cav-1 na regulação da síntese de mediadores inflamatórios por macrófagos. O lipopolissacarídeo (LPS) de E.coli, um protótipo de estímulo inflamatório, foi capaz de induzir a expressão de Cav-1 e Cox-2 em macrófagos peritoneais in vitro. Estas proteínas são induzidas em um curso temporal semelhante, sendo detectadas por Western blot a partir de 3h com níveis de expressão crescentes até 18h. Por imunofluorescência, observamos que Cav-1 e Cox-2 apresentam um padrão de expressão mutualmente exclusivo em macrófagos estimulados com LPS. Mostramos por Western blot que a expressão de Cox-2 é induzida por LPS e que o tratamento com CSD leva à inibição da expressão de Cox-2, mas não de Cox-1. Observamos, também, a redução parcial dos níveis de PGE2 no sobrenadante de macrófagos estimulados com LPS e tratados com CSD. O tratamento com o peptídeo CSD também foi capaz de reduzir os níveis de IL-1beta, IL-6, e IL-12 induzidos por LPS. O LPS induz o aumento da expressão e fosforilação de STAT-1. A fosforilação de STAT-1 foi diminuída após o tratamento com CSD, indicando que Cav-1 modula negativamente a ativação de STAT-1. Estudos posteriores são necessários para complementar os dados obtidos até o momento para esclarecer os mecanismos de modulação da síntese de mediadores inflamatórios por Cav-1. Em conclusão, Cav-1 apresenta uma atividade inibitória sobre a expressão de Cox-2 e produção dos mediadores inflamatórios PGE2, IL1beta, IL-6, e IL-12 em macrófagos estimulados com LPS in vitro. O mecanismo de inibição possivelmente envolve inibição da ativação de STAT-1.

Glycosphingolipid Modification: Structural Diversity, Functional and Mechanistic Integration of Diabetes

Glycosphingolipids (GSLs) are present in all mammalian cell plasma membranes and intracellular membrane structures. They are especially concentrated in plasma membrane lipid domains that are specialized for cell signaling. Plasma membranes have typical structures called rafts and caveola domain structures, with large amounts of sphingolipids, cholesterol, and sphingomyelin. GSLs are usually observed in many organs ubiquitously. However, GSLs, including over 400 derivatives, participate in diverse cellular functions. Several studies indicate that GSLs might have an effect on signal transduction related to insulin receptors and epidermal growth factor receptors. GSLs may modulate immune responses by transmitting signals from the exterior to the interior of the cell. Guillain-Barre syndrome is one of the autoimmune disorders characterized by symmetrical weakness in the muscles of the legs. The targets of the immune response are thought to be gangliosides, which are one group of GSLs. Other GSLs may serve as second messengers in several signaling pathways that are important to cell survival or programmed cell death. In the search for clear evidence that GSLs may play critical roles in various biological functions, many researchers have made genetically engineered mice. Before the era of gene manipulation, spontaneous animal models or chemical-induced disease models were used.

Cholesterol, a Major Component of Caveolae, Down-regulates Matrix Metalloproteinase-1 Expression through ERK/JNK Pathway in Cultured Human Dermal Fibroblasts

BACKGROUND: Cholesterol is a major component of specialized membrane microdomains known as lipid rafts or caveolae, which modulate the fluidity of biological membranes. Membrane cholesterol therefore plays an important role in cell signaling and vesicular transport. OBJECTIVE: In this study, we investigated the effects of cholesterol on matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. METHODS: MMP-1 mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. AP-1 DNA binding activity was detected by electrophoretic mobility shift assays. The amount of cholesterol was analyzed by cholesterol assay kit. RESULTS: We observed that MMP-1 mRNA and protein expression was dose-dependently decreased by cholesterol treatment. In contrast, cholesterol depletion by a cholesterol depletion agent, methyl-beta-cyclodextrin (M beta CD) in human dermal fibroblasts, increased MMP-1 mRNA and protein expression in a dose-dependent manner. Also, we investigated the regulatory mechanism of M beta CD-induced MMP-1 expression: cholesterol depletion by M beta CD, activated ERK1/2 and JNK, but not p38 MAPK cascade, and it also significantly increased c-Jun phosphorylation, c-Fos expression and activator protein-1 binding activity. Furthermore, the inhibition of ERK or JNK with specific chemical inhibitors prevented M beta CD-induced MMP-1 expression, which indicates that ERK and JNK play an important role in cholesterol depletion-mediated MMP-1 induction. In addition, M beta CD-induced phosphorylation of ERK and JNK and MMP-1 expression were suppressed by cholesterol repletion. CONCLUSION: Our results suggest that cholesterol regulates MMP-1 expression through the control of ERK and JNK activity in human dermal fibroblasts.

A second protein marker of caveolae: caveolin-2 / 中国医学科学杂志(英文版)

Caveolin-2, a protein about 20 kD, is a major component of the inner surface of caveolae, small invaginations of the plasma membrane. Similar with caveolin-1 and caveolin-3, it serves as a protein marker of caveolae. Caveolin-1 and -2 are located next to each other at 7q31.1 on human chromosome, the proteins encoded are co-localized and form a stable hetero-oligomeric complex, distributing similarly in tissue and cultured cells. Caveolin-3 is located on different chromosomes but confirmed to interact with caveolin-2. Caveolin-2 is similar to caveolin-1 in many respects but differs from the latter in functional domains, especially in G-protein binding domain and caveolin scaffolding domain. The mRNAs of both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during differentiation of 3T3-L1 cells to adipocytes. Caveolin-2-deficient mice demonstrate clear pulmonary defects, with little or no change in caveolin-1 expression and caveolae formation, suggesting that caveolin-2 plays a selective role in lung functions. Caveolin-2 is also involved in lipid metabolism and human cancers.

Modulation of the caveolin-3 localization to caveolae and STAT3 to mitochondria by catecholamine-induced cardiac hypertrophy in H9c2 cardiomyoblasts

We investigated the effect of phenylephrine (PE)- and isoproterenol (ISO)-induced cardiac hypertrophy on subcellular localization and expression of caveolin-3 and STAT3 in H9c2 cardiomyoblast cells. Caveolin-3 localization to plasma membrane was attenuated and localization of caveolin-3 to caveolae in the plasma membrane was 24.3% reduced by the catecholamine-induced hypertrophy. STAT3 and phospho-STAT3 were up-regulated but verapamil and cyclosporin A synergistically decreased the STAT3 and phospho-STAT3 levels in PE- and ISO-induced hypertrophic cells. Both expression and activation of STAT3 were increased in the nucleus by the hypertrophy. Immunofluorescence analysis revealed that the catecholamine-induced hypertrophy promoted nuclear localization of pY705-STAT3. Of interest, phosphorylation of pS727-STAT3 in mitochondria was significantly reduced by catecholamine-induced hypertrophy. In addition, mitochondrial complexes II and III were greatly down-regulated in the hypertrophic cells. Our data suggest that the alterations in nuclear and mitochondrial activation of STAT3 and caveolae localization of caveolin-3 are related to the development of the catecholamine-induced cardiac hypertrophy.

Interleukin-1beta Decreases Caveolin-1 Expression in Human Nasal Epithelium / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Caveolin-1 (Cav-1) is the structural protein that is necessary for the formation of caveolae membrane domains. It is known as an inhibitor of various signaling pathways and associated with several diseases such as cancer, atherosclerosis, restrictive lung disease and obesity. However, studies for Cav-1 in nose has been hardly performed. The objectives of our study were to detect Cav-1 expression in human nasal epithelium and to investigate the change of Cav-1 expression in the inflammation of nasal epithelium. SUBJECTS AND METHOD: We obtained nasal polyp specimens from three patients undergoing endoscopic sinus surgery. Cells from specimens were cultured using the air-liquid interface technique and IL-1beta was treated. The expression of Cav-1 mRNA and protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis, respectively. RESULTS: Both RT-PCR and Western blot analysis demonstrated the presence of Cav-1 mRNA and protein in human nasal epithe-lium. Furthermore, the expression of both Cav-1 mRNA and protein was decreased by IL-1beta stimulation. CONCLUSION: Cav-1 was expressed in human nasal epithelial cells. It is assumed that Cav-1 may play a role in nasal inflammatory disease. However, further studies to confirm the interaction between Cav-1 and signaling molecules in the nasal inflammatory process should be followed.

Association of rOAT1 with Caveolin-1 in Rat Kidney / 대한신장학회지

PURPOSE: A family of organic anion transporters (OAT) has been identified, and several isoforms have been reported. The regulatory mechanisms of OATs functions, however, still remain to be elucidated. The rat OAT1 contributes to move a number of negatively-charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. To address this issue, we investigated the protein-protein interaction between rOAT1 and Cav-1. METHODS: The expressions of rOAT1 and Cav-1 (mRNA and protein) were evaluated using RT-PCR and Western blot analysis. The localization of rOAT1 and Cav-1 was determined in the caveolae-rich membrane fraction isolated by sucrose density gradient ultra-centrifugation. For the direct binding between the rOAT1 and Cav-1 proteins, the immuno-precipitation method and confocal microscopy were employed. To perform functional analysis, a Xenopus oocytes expression system with the antisense oligonucleotides (ODN) technique was used. RESULTS: The expressions of rOAT1 and Cav-1 were detected in the kidney. The caveolae-rich membranous fractions from the kidney contained both rOAT1 and Cav-1 in the same fractions. The immuno-precipitation experiments showed the formation of a complex between the rOAT1 and Cav-1. The confocal microscopy using primary cultured renal proximal epithelial cells also supported the co-localization of rOAT1 and Cav-1 at the plasma membrane. The uptake function of rOAT1, as assessed by using a Xenopus oocytes expression system, was inhibited by the Xenopus Cav-1 antisense ODN. CONCLUSION: rOAT1 co-localizes with caveolin-1 in the caveolae, and caveolin-1 plays an important role in regulating the function of rOAT1.

Mecanismos envolvidos na arritmogênese em cardiomiócitos e desenvolvimento de modelo in vitro de fibrose e hipertrofia cardíaca induzidos por Trypanosoma cruzi / Mechanisms involved in arrythmogenesis into cardiomyocytes and development of in vitro model of cardiac hypertrophy and fibrosis induced by Trypanosoma cruzi

A infecção pelo protozoário Trypanosoma cruzi causa a doença de Chagas que entre as suas manifestações, apresenta uma severa forma cardíaca que pode gerar arritmias e morte súbita. No primeiro bloco desta dissertação nós avaliamos o efeito da infecção por T. cruzi no acoplamento celular e no cálcio intracelular de monocamadas de cardiomiócitos. Nós verificamos que durante o processo de invasão da célula hospedeira, o T. cruzi gera aumento dos níveis basais de cálcio e arritmias dos sinais cíclicos deste íon. Como já descrito por nosso grupo, este aumento é transmitido às células adjacentes. Neste trabalho buscamos verificar a expressão de conexina43, principal formadora de junções comunicantes no tecido cardíaco responsável pela passagem de íons e a sincronização da contratilidade. Nós verificamos que há um aumento significativo na expressão desta proteína nos tempos de invasão por t. cruzi (1hpi). Após 72 horas de infecção dos cardiomiócitos por T. cruzi nós observamos alterações na distribuição de cavéolas e diminuição na expressão de Cx43 e caveolina-3. Estudos in vivo demonstraram que a expressão de Cx43 também foi reduzida durante infecção aguda dos camundongos por T. cruzi. Estes resultados indicaram alteração em mecanismos envolvidos na manutenção da homeostase do cálcio intracelular e comunicação celular, esclarecendo alguns aspectos relacionados à aritmogênese causada pela infecção de cardiomiócitos por T. cruzi. No segundo bloco, utilizamos um modelo de interação do T. cruzi com um microtecido cardíaco in vitro, desenvolvido por nós. Os microtecidos apresentam estruturas similares às do tecido cardíaco in vivo e quando infectados por T. cruzi, observamos hipertrofia e fibrose, com aumento de moléculas de matriz extracelular tais como fibronectina, laminina e colágeno IV. Este modelo mimetiza in vitro importantes aspectos observados durante a doença de Chagas mais fidedignamente do que os modelos em monocamadas. O modelo de esferóides ...anti-T. cruzi.

Evidence for Cyclooxygenase-2 Association with Caveolin-3 in Primary Cultured Rat Chondrocytes

The purpose of this study was to demonstrate the cellular localization of cyclooxygenase-2 (COX-2) and caveolin-3 (Cav-3) in primarily cultured rat chondrocytes. In normal rat chondrocytes, we observed relatively high levels of Cav-3 and a very low level of COX-2 mRNA and protein. Upon treating the chondrocytes with 5 microM of CdCl2 (Cd) for 6 hr, the expressions of COX-2 mRNA and protein were increased with the decreased Cav-3 mRNA and protein expressions. The detergent insoluble caveolae-rich membranous fractions that were isolated from the rat chondrocytes and treated with Cd contained the both proteins of both COX-2 and Cav-3 in a same fraction. The immuno-precipitation experiments showed complex formation between the COX-2 and Cav-3 in the rat chondrocytes. Purified COX-2 with glutathione S-transferase-fused COX-2 also showed complex formation with Cav-3. Confocal and electron microscopy also demonstrated the co-localization of COX-2 and Cav-3 in the plasma membrane. The results from our current study show that COX-2 and Cav-3 are co-localized in the caveolae of the plasma membrane, and they form a protein-protein complex. The co-localization of COX-2 with Cav-3 in the caveolae suggests that the caveolins might play an important role for regulating the function of COX-2.

Introduction of Organic Anion Transporters (SLC22A) and a Regulatory Mechanism by Caveolins

The kidney is an important organ for controlling the volume of body fluids, electrolytic balance and excretion/reabsorption of endogenous and exogenous compounds. Among these renal functions, excretion/reabsorption of endogenous and exogenous substance is very important for the maintenance of physiological homeostasis in the body. Recently discovered organic anion transporters (OAT or SLC22A) have important roles for renal functions. It is well known as drug transporter. Several isoforms belong to SLC22A family. They showed different transport substrate spectrums and different localizations within the kidney. Their gene expressions are changed by some stimulus. The functional transport properties are regulated by protein kinase C. In addition, the function of organic anion transporters are also regulated by protein-protein interaction, such as caveolin which is compositional protein of caveolae structure. In this review, we will give an introduction of organic anion transporters and its regulatory mechanisms.

p38 Kinase Regulates Nitric Oxide-induced Dedifferentiation and Cyclooxygenase-2 Expression of Articular Chondrocytes

BACKGROUND: Caveolin, a family of integral membrane proteins are a principal component of caveolae membranes. In this study, we investigated the effect of p38 kinase on differentiation and on inflammatory responses in sodium nitroprusside (SNP)- treated chondrocytes. METHODS: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. SNP was used as a nitric oxide (NO) donor. In this experiments measuring SNP dose response, primary chondrocytes were treated with various concentrations of SNP for 24 h. The time course of the SNP response was determined by incubating cells with 1 mM SNP for the indicated time period (0~24 h). The cyclooxygenase-2 (COX-2) and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin E2 (PGE2) assay was used to measure the COX-2 activity. The tyrosine phosphorylation of caveolin-1 was determined by immunoblot analysis and immunostaining. RESULTS: SNP treatment stimulated tyrosine phosphorylation of caveolin-1 and activation of p38 kinase. SNP additionally caused dedifferentiation and inflammatory response. We showed previously that SNP treatment stimulated activation of p38 kinase and ERK-1/-2. Inhibition of p38 kinase with SB203580 reduced caveolin-1 tyrosine phosphorylation and COX-2 expression but enhanced dedifferentiation, whereas inhibition of ERK with PD98059 did not affect caveolin-1 tyrosine phosphorylation levels, suggesting that ERK at least is not related to dedifferentiation and COX-2 expression through caveolin-1 tyrosine phosphorylation. CONCLUSION: Our results indicate that SNP in articular chondrocytes stimulates dedifferentiation and inflammatory response via p38 kinase signaling in association with caveolin-1 phosphorylation.

Ectopic Expression of Caveolin-1 Induces COX-2 Expression in Rabbit Articular Chondrocytes via MAP Kinase Pathway

BACKGROUND: Caveolin-1 is a principal component of caveolae membranes in vivo. Although expression of caveolae structure and expression of caveolin family, caveolin-1, -2 and -3, was known in chondrocytes, the functional role of caveolae and caveolins in chondrocytes remains unknown. In this study, we investigated the role of caveolin-1 in articular chondrocytes. METHODS: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. Caveolin-1 cDNA was transfected to articular chondrocytes using LipofectaminePLUS. The cyclooxygenase-2 (COX-2) expression levels were determined by immunoblot analysis, immunostaining, immunohistochemistry, and prostaglandin E2 (PGE2) assay was used to measure the COX-2 activity. RESULTS: Ectopic expression of caveolin-1 induced COX-2 expression and activity, as indicated by immunoblot analysis and PGE2 assay. And also, overexpression of caveolin-1 stimulated activation of p38 kinase and ERK-1/ -2. Inhibition of p38 kinase and ERK-1/-2 with SB203580 and PD98059, respectively, led to a dose-dependent decrease COX-2 expression and PGE2 production in caveolin-1-transfected cells. CONCLUSION: Taken together, our data suggest that ectopic expression of caveolin-1 contributes to the expression and activity of COX-2 in articular chondrocytes through MAP kinase pathway.