Coexpression Of Caveolin-1 Research Articles

Coexpression of CAV-1, AT1-R and FOXM1 in prostate and breast cancer and normal cell lines and their influence on metastatic properties.

The aim of this study was to evaluate the coexpression of caveolin-1 (CAV-1), angiotensin II type 1 receptor (AT1-R) and forkhead box Ml (FOXM1) in prostate and breast cancer cell lines, in comparison with normal cell lines. CAV-1, AT1-R and FOXM1 expression was determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis in the prostate cancer cell lines PC3, DU145 and LNCaP; prostate normal cell line PNT1A; breast cancer cell lines MCF-7 and MDA-MB-231; and the normal breast cell line 184A1. A correlation between the expression levels of the investigated genes and their metastatic properties was determined by the Spearman's rank test (P<0.05) and Aspin-Welsch t-test, respectively. In prostate cell lines, a significant correlation was noted between CAV-1 and AT1-R expression and between FOXM1 and CAV-1 expression. A correlation between the expression levels of the investigated genes and their metastatic potential was also observed, with relatively high expression of all the investigated genes in the normal prostate cell line PNT1A. In comparison to prostate cancer cell lines, an adverse dependency between CAV-1, AT1-R, FOXM1 expression and metastatic potential was observed in the breast cancer cell lines. Relatively high expression of all tested genes was observed in the normal breast cell line 184A1, which was decreasing respectively with increasing metastatic potential of breast cancer cell lines. The results obtained here indicate that CAV-1, FOXM1 and AT1-R may be potential markers of tumorigenesis in certain types of cancer in vitro.

Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.

Up-regulation of Na+-coupled glucose transporter SGLT1 by caveolin-1

The Na+-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (Ig), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal Ig without significantly modifying the glucose concentration required to trigger half maximal Ig (KM). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5μM) resulted in a decline of Ig, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.

E‐cadherin determines Caveolin‐1 tumor suppression or metastasis enhancing function in melanoma cells

The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells.

MicroRNA 802 stimulates ROMK channels by suppressing caveolin-1.

Dietary potassium stimulates the surface expression of ROMK channels in the aldosterone-sensitive distal nephron, but the mechanism by which this occurs is incompletely understood. Here, a high-potassium diet increased the transcription of microRNA (miR) 802 in the cortical collecting duct in mice. In addition, high-potassium intake decreased the expression of caveolin-1, whose 3' untranslated region contains the seed sequence of miR-802. In vitro, expression of miR-802 suppressed the expression of caveolin-1, and conversely, downregulation of endogenous miR-802 increased the expression of caveolin-1. Sucrose-gradient centrifugation suggested that caveolin-1 closely associated with ROMK channels, and immunoprecipitation showed that caveolin-1 interacted with the N terminus of ROMK. Expression of caveolin-1 varied inversely with the expression of ROMK1 in the plasma membrane, and caveolin-1 inhibited ROMK1 channel activity. Removal of the clathrin-dependent endocytosis motif from ROMK1 failed to abolish the effect of caveolin-1 on ROMK1 channel activity. Last, expression of miR-802 increased ROMK1 channel activity, an effect blocked by coexpression of caveolin-1. Taken together, miR-802 mediates the stimulatory effect of a high-potassium diet on ROMK channel activity by suppressing caveolin-1 expression, which leads to increased surface expression of ROMK channels in the distal nephron.

Caveolin-1 inhibits membrane-type 1 matrix metalloproteinase activity

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent proteinase found in cholesterol-rich lipid rafts on the plasma membrane. MT1-MMP hydrolyzes extracellular matrix (ECM) proteins, activates pro-matrix metalloproteinase-2 (proMMP-2) and plays an important role in ECM remodeling, cancer cell migration and metastasis. The role of caveolin-1, an integral protein of caveolae, in the activation of MT1-MMP remains largely unknown. Here, we show that the expression of caveolin-1 attenuates the activation of proMMP-2, reduces proteolytic cleavage of ECM and inhibits cell migration. We utilized the cytoplasmic tail domain deletion (DeltaCT) or the E240A mutant of MT1-MMP. Co-expression of caveolin-1 with the wild-type or the DeltaCT MT1-MMP decreased the proMMP-2 activation and inhibited collagen degradation and cell migration. Caveolin-1 had no effect on the catalytically inert E240A MT1-MMP. Our findings suggest that caveolin-1 is essential in the down-regulation of MT1-MMP activity by promoting internalization from the cell surface.

Isoforms of caveolin-1 and caveolar structure.

The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.

Caveolin-1 Potentiates Estrogen Receptor α (ERα) Signaling: CAVEOLIN-1 DRIVES LIGAND-INDEPENDENT NUCLEAR TRANSLOCATION AND ACTIVATION OF ERα

Estrogen receptor alpha (ERalpha) is a soluble protein that mediates the effects of the gonadal estrogens such as 17beta-estradiol. Upon ligand binding, a cytoplasmic pool of ERalpha translocates to the nucleus, where it acts as a transcription factor, driving the expression of genes that contain estrogen-response elements. The activity of ERalpha is regulated by a number of proteins, including cytosolic chaperones and nuclear cofactors. Here, we show that caveolin-1 potentiates ERalpha-mediated signal transduction. Coexpression of caveolin-1 and ERalpha resulted in ligand-independent translocation of ERalpha to the nucleus as shown by both cell fractionation and immunofluorescence microscopic studies. Similarly, caveolin-1 augmented both ligand-independent and ligand-dependent ERalpha signaling as measured using a estrogen-response element-based luciferase reporter assay. Caveolin-1-mediated activation of ERalpha was sensitive to a well known ER antagonist, 4-hydroxytamoxifen. However, much higher concentrations of tamoxifen were required to mediate inhibition in the presence of caveolin-1. Interestingly, caveolin-1 expression also synergized with a constitutively active, ligand-independent ERalpha mutant, dramatically illustrating the potent stimulatory effect of caveolin-1 in this receptor system. Taken together, our results identify caveolin-1 as a new positive regulator of ERalpha signal transduction.

Regulation of cAMP-mediated Signal Transduction via Interaction of Caveolins with the Catalytic Subunit of Protein Kinase A

cAMP-dependent processes are essential for cell growth, differentiation, and homeostasis. The classic components of this system include the serpentine receptors, heterotrimeric G-proteins, adenylyl cyclase, protein kinase A (PKA), and numerous downstream target substrates. Evidence is accumulating that some members of this cascade are concentrated within membrane microdomains, termed caveolae and caveolae-related domains. In addition, the caveolin-1 protein has been shown to interact with some of these components, and this interaction inhibits their enzymatic activity. However, the functional effects of caveolins on cAMP-mediated signaling at the most pivotal step, PKA activation, remain unknown. Here, we show that caveolin-1 can dramatically inhibit cAMP-dependent signaling in vivo. We provide evidence for a direct interaction between caveolin-1 and the catalytic subunit of PKA both in vitro and in vivo. Caveolin-1 binding appears to be mediated both by the caveolin scaffolding domain (residues 82-101) and a portion of the C-terminal domain (residues 135-156). Further functional analysis indicates that caveolin-based peptides derived from these binding regions can inhibit the catalytic activity of purified PKA in vitro. Mutational analysis of the caveolin scaffolding domain reveals that a series of aromatic residues within the caveolin scaffolding domain are critical for mediating inhibition of PKA. In addition, co-expression of caveolin-1 and PKA in cultured cells results in their co-localization as seen by immunofluorescence microscopy. In cells co-expressing caveolin-1 and PKA, PKA assumed a punctate distribution that coincided with the distribution of caveolin-1. In contrast, in cells expressing PKA alone, PKA was localized throughout the cytoplasm and yielded a diffuse staining pattern. Taken together, our results suggest that the direct inhibition of PKA by caveolin-1 is an important and previously unrecognized mechanism for modulating cAMP-mediated signaling.

Caveolin-2 Localizes to the Golgi Complex but Redistributes to Plasma Membrane, Caveolae, and Rafts when Co-expressed with Caveolin-1

We have characterized comparatively the subcellular distributions of caveolins-1 and -2, their interactions and their roles in caveolar formation in polarized epithelial cells. In Fischer rat thyroid (FRT) cells, which express low levels of caveolin-2 and no caveolin-1, caveolin-2 localizes exclusively to the Golgi complex but is partially redistributed to the plasma membrane upon co-expression of caveolin-1 by transfection or by adenovirus-mediated transduction. In Madin-Darby canine kidney (MDCK) cells, which constitutively express both caveolin-1 and -2, caveolin-2 localized to both the Golgi complex and to the plasma membrane, where it co-distributed with caveolin-1 in flat patches and in caveolae. In FRT cells, endogenous or overexpressed caveolin-2 did not associate with low density Triton insoluble membranes that floated in sucrose density gradients but was recruited to these membranes when co-expressed together with caveolin-1. In MDCK cells, both caveolin-1 and caveolin-2 associated with low density Triton-insoluble membranes. In FRT cells, transfection of caveolin-1 promoted the assembly of plasma membrane caveolae that localized preferentially (over 99%) to the basolateral surface, like constitutive caveolae of MDCK cells. In contrast, as expected from its intracellular distribution, endogenous or overexpressed caveolin-2 did not promote the assembly of caveolae; rather, it appeared to promote the assembly of intracellular vesicles in the peri-Golgi area. The data reported here demonstrate that caveolin-1 and -2 have different and complementary subcellular localizations and functional properties in polarized epithelial cells and suggest that the two proteins co-operate to carry out specific as yet unknown tasks between the Golgi complex and the cell surface.

Points: Dual posts in the NHS and the Third World

Dietary potassium stimulates the surface expression of ROMK channels in the aldosterone-sensitive distal nephron, but the mechanism by which this occurs is incompletely understood. Here, a high-potassium diet increased the transcription of microRNA (miR) 802 in the cortical collecting duct in mice. In addition, high-potassium intake decreased the expression of caveolin-1, whose 3′ untranslated region contains the seed sequence of miR-802. <i>In vitro</i>, expression of miR-802 suppressed the expression of caveolin-1, and conversely, downregulation of endogenous miR-802 increased the expression of caveolin-1. Sucrose-gradient centrifugation suggested that caveolin-1 closely associated with ROMK channels, and immunoprecipitation showed that caveolin-1 interacted with the N terminus of ROMK. Expression of caveolin-1 varied inversely with the expression of ROMK1 in the plasma membrane, and caveolin-1 inhibited ROMK1 channel activity. Removal of the clathrin-dependent endocytosis motif from ROMK1 failed to abolish the effect of caveolin-1 on ROMK1 channel activity. Last, expression of miR-802 increased ROMK1 channel activity, an effect blocked by coexpression of caveolin-1. Taken together, miR-802 mediates the stimulatory effect of a high-potassium diet on ROMK channel activity by suppressing caveolin-1 expression, which leads to increased surface expression of ROMK channels in the distal nephron.