Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase

Chen, Dong‐bao; Zangl, Amy L.; Zhao, Qin; Markley, John L.; Zheng, Jing; Bird, Ian M.; Magness, Ronald R.

doi:10.1016/s0303-7207(01)00403-8

Cited by 15 publications

(16 citation statements)

References 53 publications

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“…We were unable to detect caveolin 2 and caveolin 3 in uterine artery ex vivo by immunohistochemistry. The data agree with our previous report that these caveolins are undetectable in cultured uterine artery endothelial cells [26]. Upregulation of uterine artery endothelial NOS3 by estrogen and its combination with progesterone is not a surprise because these have been reported previously [13], and these treatment regimes are accompanied by the well-established chronic uterine vasodilatation [2,39].…”

Section: Reciprocal Regulation Of Uterine Artery Caveolinsupporting

confidence: 92%

“…Furthermore, upon estrogen or shear stress stimulation, increased NO production via NOS3 activation is associated with NOS3 dissociation from caveolin 1 [18,19], because binding to the latter tonically renders NOS3 less active or completely inactive [18,19]. We were the first to hypothesize downregulation of uterine artery caveolin 1 expression as an additional mechanism for estrogen-induced rise in uterine blood flow [26]. Thus, the observation that caveolin 1 is downregulated by estrogen in uterine artery highlights a novel additional mechanism for estrogen-induced uterine vasodilatation.…”

Section: Discussionmentioning

confidence: 92%

“…Western blot analysis of NOS3 and caveolin proteins was performed as previously described [26]. Briefly, the membranes were incubated with 20 mM Tris, pH7.5, 500 mM NaCl, and 0.1% Tween-20 (TBST) containing 5% skim milk to block nonspecific binding, followed by incubation with TBST containing 1% BSA anti-NOS3 monoclonal antibody (1:750, N30020) for 2h, or anti-caveolin 1 polyclonal antibody (1:20,000, N20) and b-actin monoclonal antibody (1:5000, Qiagen) for 1 h at room temperature.…”

Section: Western Blot Analysismentioning

confidence: 99%

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Global Protein Expression Profiling Underlines Reciprocal Regulation of Caveolin 1 and Endothelial Nitric Oxide Synthase Expression in Ovariectomized Sheep Uterine Artery by Estrogen/Progesterone Replacement Therapy1

Chen

Jia²,

Barker

et al. 2006

Biology of Reproduction

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Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17beta (E2, 5 microg/kg bolus + 6 microg kg(-1) day(-1)), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 microg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a approximately 133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a approximately 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.

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Section: Reciprocal Regulation Of Uterine Artery Caveolinsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 92%

Section: Western Blot Analysismentioning

confidence: 99%

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Global Protein Expression Profiling Underlines Reciprocal Regulation of Caveolin 1 and Endothelial Nitric Oxide Synthase Expression in Ovariectomized Sheep Uterine Artery by Estrogen/Progesterone Replacement Therapy1

Chen

Jia²,

Barker

et al. 2006

Biology of Reproduction

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“…S3A) or 12 and 48 h (data not shown). oFPAE cells expressed high levels of caveolin-1 protein (40,41). Consistently, VEGF and fibroblast growth factor 2 (FGF2) also did not alter oFPAE cell caveolin-1 protein expression up to 3 d in vitro (supplemental Fig.…”

Section: Caveolae Are Critical For Erk2/1 Activation By Vegfmentioning

confidence: 64%

Compartmentalizing VEGF-Induced ERK2/1 Signaling in Placental Artery Endothelial Cell Caveolae: A Paradoxical Role of Caveolin-1 in Placental Angiogenesis in Vitro

Liao

Feng

Zhang

et al. 2009

Molecular Endocrinology

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On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase Calpha, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

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“…22 The fact that caveolin genes exhibit significant evolutionary conservation suggests important biological functions for these proteins. [23][24][25][26][27] To investigate possible roles for the two Cav-1 isoforms in vertebrate development, zebrafish cav-1␣ and -1␤ cDNAs were isolated, the expression profiles of cav-1 mRNAs were defined, and Cav-1 proteins were functionally characterized using a targeted protein depletion approach. Our studies revealed unique developmental functions for zebrafish Cav-1␣ and -1␤ isoforms, including roles in actin cytoskeletal organization, which were rescued by expression of human Cav-1␣ and -1␤ isoforms.…”

mentioning

confidence: 99%

Caveolin-1α and −1β Perform Nonredundant Roles in Early Vertebrate Development

Fang

Solomon

Zhuang

et al. 2006

The American Journal of Pathology

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Caveolins are integral membrane proteins that localize predominantly to lipid rafts, where they oligomerize to form flask-shaped organelles termed caveolae and play important roles in membrane trafficking, signal transduction, and other cellular processes. To investigate potential roles for caveolin-1 (cav-1) in development, cav-1␣ and -1␤ cDNAs were functionally characterized in the zebrafish. Cav-1␣ and -1␤ mRNAs exhibited overlapping but distinct expression patterns throughout embryogenesis. Targeted depletion of either Cav-1 isoform, using antisense morpholino oligomers, resulted in a substantial loss of caveolae and dramatic neural, eye, and somite defects by 12 hours after fertilization, the time at which mRNA levels of both isoforms substantially increased in wildtype animals. Morphant phenotypes were rescued by injection of homotypic (cav-1␣/cav-1␣) but not heterotypic (cav-1␣/cav-1␤) zebrafish and human cav-1 cRNAs, revealing nonredundant and evolutionarily conserved functions for the individual Cav-1 isoforms. Mutation of a known Cav-1 phosphorylation site unique to Cav-1␣ (Y143 F) resulted in a failure to rescue the cav-1␣ morphant phenotype, verifying an essential role for Cav-1␣ specifically and implicating this residue in early developmental functions. Cav-1␣ and -1␤ morphants also exhibited disruption in the actin cytoskeleton. These results support important and previously unanticipated roles for the Caveolin-1 isoforms in vertebrate organogenesis. (Am J Pathol

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Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase

Cited by 15 publications

References 53 publications

Global Protein Expression Profiling Underlines Reciprocal Regulation of Caveolin 1 and Endothelial Nitric Oxide Synthase Expression in Ovariectomized Sheep Uterine Artery by Estrogen/Progesterone Replacement Therapy1

Global Protein Expression Profiling Underlines Reciprocal Regulation of Caveolin 1 and Endothelial Nitric Oxide Synthase Expression in Ovariectomized Sheep Uterine Artery by Estrogen/Progesterone Replacement Therapy1

Compartmentalizing VEGF-Induced ERK2/1 Signaling in Placental Artery Endothelial Cell Caveolae: A Paradoxical Role of Caveolin-1 in Placental Angiogenesis in Vitro

Caveolin-1α and −1β Perform Nonredundant Roles in Early Vertebrate Development

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