Vascular endothelial growth factor receptor-3 activity is modulated by its association with caveolin-1 on endothelial membrane

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.     

Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells

The P2X7 receptor has recently been described as a marker for lung alveolar epithelial type I cells. Here, we demonstrate both the expression of P2X7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X7 and the raft marker protein Caveolin-1; also, P2X7 protein was associated with caveolae. A marked reduction in P2X7 immunoreactivity was observed in lung sections prepared from Caveolin-1-knockout mice, indicating that Caveolin-1 expression was required for full expression of P2X7 protein. Indeed, suppression of Caveolin-1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X7 protein expression. Our data demonstrate a potential interaction between P2X7 protein and Caveolin-1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction.     

Lack of evidence for caveolin-1 and CD147 interaction before and after bleomycin-induced lung injury

Immunohistochemical and in vitro studies indicate that caveolin-1, which occurs abundantly in alveolar epithelial type I cells and microvascular endothelial cells of the lung, is selectively downregulated in the alveolar epithelium following exposure to bleomycin. Bleomycin is also known to enhance the expression levels of metalloproteinases and of the metalloproteinase inducer CD147/EMMPRIN in lung cells. Experimental in vitro data has showed that MMP-inducing activity of CD147 is under the control of caveolin-1. We studied the effects of bleomycin on the expression of caveolin-1, CD147 and metalloproteinases using an alveolar epithelial rat cell line R3/1 with properties of both alveolar type I and type II cells and explanted rat lung slices. In parallel, retrospective samples of bleomycin-induced fibrosis in rats and mice as well as samples of wild type and caveolin-1 knockout animals were included for immunohistochemical comparison with in vitro data. Here we report that treatment with bleomycin downregulates caveolin-1 and increases CD147 and MMP-2 and -9 expression/activity in R3/1 cells using RT-PCR, Western blot analysis, MMP-2 activity assay and immunocytochemistry. Immunofluorescence double labeling revealed that caveolin-1 and CD147 were not colocalized in vitro. The in vitro findings were confirmed through immunohistochemical studies of the proteins in paraffin embedded precision-cut rat lung slices and in fibrotic rat lung tissues. The caveolin-1-negative hyperplastic ATII cells exhibited enhanced immunoreactivity for CD147 and MMP-2. Caveolin-1-negative ATI cells of fibrotic samples were mostly CD147 negative. There were no differences in the pulmonary expression of CD147 between the normal and caveolin-1 deficient animals. The results demonstrate that bleomycin-induced lung injury is associated with an increase in CD147 expression and MMP activity, particularly in alveolar epithelial cells. In addition, our data exclude any functional interaction between CD147 and alveolar epithelial caveolin-1.     

Localization of caveolin-3 in the sinus endothelial cells of the rat spleen

The localization of caveolins in the sinus endothelial cells of the rat spleen has been demonstrated by confocal laser scanning and electron microscopy. Caveolin-3, a muscle-specific caveolin, was detected by Western blot analysis and immunofluorescence microscopy of isolated sinus endothelial cells and tissue cryosections of the spleen. During the immunofluorescence microscopy of isolated endothelial cells, both caveolin-3 and caveolin-1 were found. In tissue cryosections of the spleen, caveolin-3, as well as caveolin-1 and -2, was present in the contours and cytoplasm of the cells. Immunogold electron microscopy of tissue cryosections revealed caveolin-3, -1, and -2 to be present in caveolae in the apical, lateral, and basal plasma membranes and some vesicular profiles in the cytoplasm of sinus endothelial cells. Furthermore, caveolin-3 was colocalized with caveolin-1 in the same caveolae in the apical, lateral, and basal plasma membranes. Stress fibers and tubulovesicular structures were situated in the vicinity of caveolae labeled with anti-caveolin-3, anti-caveolin-1, and anti-caveolin-2 antibodies. It is speculated that caveolae in sinus endothelial cells play an important role in the constriction of stress fibers.     

Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures.

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-"like" phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.     

Loss of caveolin expression in type I pneumocytes as an indicator of subcellular alterations during lung fibrogenesis

 Caveolin is a major structural protein of caveolae, also known as plasmalemmal vesicles, which are particularly abundant in type I pneumocytes and capillary endothelial cells of lung parenchyma. Here we demonstrate that caveolin expression in the alveolar epithelium of rats and mini pigs is strikingly downregulated after irradiation-induced lung injury. Indirect immunoperoxidase staining with polyclonal anti-caveolin antibodies, confirmed by double fluorescence studies with type I cell-specific monoclonal anti-cytokeratin antibodies or lectins, revealed a dramatic loss of caveolin immunoreactivity in type I pneumocytes. In contrast, caveolin expression increased in endothelial cells. Immunoblotting of lung homogenates from normal and irradiated rats using specific anti-caveolin antibodies confirmed the presence of caveolin in normal tissue and its marked decrease of expression in fibrotic tissue. The loss of caveolin as an important structural protein of caveolae in alveolar epithelial cells may be an early indicator of serious type I cell injury during fibrogenesis. The increase of caveolin immunoreactivity in endothelia of blood vessels may indicate that different types of caveolae and/or different regulatory mechanisms of caveolin expression exist. Accepted: 28 May 1997     

The expression of scavenger receptor class B,type I (SR-BI) and caveolin-1 in parenchymal and nonparenchymal liver cells

The liver is the major site of cholesterol synthesis and metabolism, and the only substantive route for eliminating blood cholesterol. Scavenger receptor class B, type I (SR-BI) has been reported to be responsible for mediating the selective uptake of high-density lipoprotein cholesteryl esters (HDL-CE) in liver parenchymal cells (PC). We analysed the expression of SR-BI in isolated rat liver cells, and found the receptor to be highly expressed in liver PC at both the mRNA and protein levels. We also found SR-BI to be expressed in liver endothelial cells (LEC) and Kupffer cells (KC). SR-BI has not previously been reported to be present in LEC. CD36 mRNA was expressed in all three liver cell types. Since caveolin-1 appears to colocalize with SR-BI and CD36 in caveolae of several cell lines, the distribution and expression of caveolin-1 in the liver cells were investigated. Caveolin-1 was not detected in PC but was found in both LEC and KC. This led to the suggestion that caveolin-1 may be more important in the efflux of cholesterol than in the selective uptake of cholesterol in the liver.     

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.     

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.     

Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.

The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time- and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium.     

Caveolae: biochemical analysis

Caveolae appear in a multitude of processes encompassing growth regulation and trafficking. We demonstrate the abundant presence of ESA/reggie-1/flotillin-2, ATP synthase beta subunit and annexin V in endothelial caveolae by immunopurification of caveolae from vascular endothelial membrane. Five proteins are abundant in a caveolin-1 protein complex, analyzed by sucrose gradient velocity sedimentation following octyl-beta-D-glucopyranoside extraction. Caveolin-1 alpha interacts with caveolin-1beta, caveolin-2, actin, the microsomal form of NADH cytochrome B5 reductase and ESA/reggie-1/flotillin-2 as shown by co-immunoprecipitation. We propose the concept that ATP biosynthesis in caveolae regulates mechanosignaling and is induced by membrane depolarization and a proton gradient. Pressure stimuli and metabolic changes may trigger gene regulation in endothelial cells, involving a nuclear conformer of caveolin-1, shown here with an epitope-specific caveolin-1 antibody, and immediate response of ion channel activity, regulated by ESA/reggie-1/flotillin-2.     

小窝蛋白-1在免疫调节中的作用

小窝（caveolae）是一类特殊的膜脂筏,富含鞘磷脂和胆固醇。小窝蛋白-1（caveolin-1）是小窝的标志蛋白质,分子量约22 kD。后者不但直接参与小窝结构的形成、膜泡运输、胆固醇稳态维持,还通过其脚手架结构域（caveolin scaffolding domain,CSD）与众多信号分子相互作用调控细胞的生长、发育和分化,最终影响机体的生理和病理过程。近年发现,小窝蛋白-1和胞膜窖不但存在于内皮细胞、脂肪细胞、血管平滑肌细胞和纤维细胞中,还广泛表达于免疫细胞中,参与调节免疫细胞活化引起的炎症应答反应。本文结合最新的研究进展和前期结果,简要综述小窝蛋白-1在巨噬细胞、T细胞、B细胞以及中性粒细胞等免疫细胞内的调节作用,以及在细菌感染如绿脓杆菌、沙门氏菌和克雷伯杆菌的炎症中的信号转导研究进展。     

Caveolin-1 and caveolae in atherosclerosis: differential roles in fatty streak formation and neointimal hyperplasia

PURPOSE OF REVIEW: Caveolae are 50-100 nm cell surface plasma membrane invaginations observed in terminally differentiated cells. They are characterized by the presence of the protein marker caveolin-1. Caveolae and caveolin-1 are present in almost every cell type that has been implicated in the development of an atheroma. These include endothelial cells, macrophages, and smooth muscle cells. Caveolae and caveolin-1 are involved in regulating several signal transduction pathways and processes that play an important role in atherosclerosis. RECENT FINDINGS: Several recent studies using genetically engineered mice (Cav-1 (-/-) null animals) have now clearly demonstrated a role for caveolin-1 and caveolae in the development of atherosclerosis. In fact, they suggest a rather complex one, either proatherogenic or antiatherogenic, depending on the cell type examined. For example, in endothelial cells, caveolin-1 and caveolae may play a proatherogenic role by promoting the transcytosis of LDL-cholesterol particles from the blood to the sub-endothelial space. In contrast, in smooth muscle cells, the ability of caveolin-1 to negatively regulate cell proliferation (neointimal hyperplasia) may have an antiatherogenic effect. SUMMARY: Caveolin-1 and caveolae play an important role in several steps involved in the initiation of an atheroma. Development of new drugs that regulate caveolin-1 expression may be important in the prevention or treatment of atherosclerotic vascular disease.     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.     

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.     

Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae

Caveolin-2 is a member of the caveolin gene family with no known function. Although caveolin-2 is coexpressed and heterooligomerizes with caveolin-1 in many cell types (most notably adipocytes and endothelial cells), caveolin-2 has traditionally been considered the dispensable structural partner of the widely studied caveolin-1. We now directly address the functional significance of caveolin-2 by genetically targeting the caveolin-2 locus (Cav-2) in mice. In the absence of caveolin-2 protein expression, caveolae still form and caveolin-1 maintains its localization in plasma membrane caveolae, although in certain tissues caveolin-1 is partially destabilized and shows modestly diminished protein levels. Despite an intact caveolar membrane system, the Cav-2-null lung parenchyma shows hypercellularity, with thickened alveolar septa and an increase in the number of endothelial cells. As a result of these pathological changes, these Cav-2-null mice are markedly exercise intolerant. Interestingly, these Cav-2-null phenotypes are identical to the ones we and others have recently reported for Cav-1-null mice. As caveolin-2 expression is also severely reduced in Cav-1-null mice, we conclude that caveolin-2 deficiency is the clear culprit in this lung disorder. Our analysis of several different phenotypes observed in caveolin-1-deficient mice (i.e., abnormal vascular responses and altered lipid homeostasis) reveals that Cav-2-null mice do not show any of these other phenotypes, indicating a selective role for caveolin-2 in lung function. Taken together, our data show for the first time a specific role for caveolin-2 in mammalian physiology independent of caveolin-1.