Regulation of caveolin-1 expression and phosphorylation by VEGF in ovine amnion cells

Abstract

Vascular endothelial growth factor (VEGF) has been implicated in the regulation of vesicular transport of amniotic fluid via caveolae across the amnion. This study tested the hypothesis that VEGF regulates caveolar function by stimulating caveolin-1 expression and phosphorylation in ovine amniotic epithelial cells (oAECs). Using primary cultures of oAECs, caveolin-1 was identified by immunofluorescent staining. Caveolin-1 messenger RNA (mRNA) abundance was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and protein by Western blotting. The effects of VEGF( 165) on caveolin-1 expression and phosphorylation were determined. Caveolin-1 immunoreactivity was detected in oAECs. In response to 10 ng/mL VEGF( 165), caveolin-1 mRNA levels increased whereas the protein levels were unaffected. Furthermore, VEGF stimulated caveolin-1 phosphorylation, an effect abrogated by the inhibition of c-Src protein kinase. These data suggest that VEGF upregulates caveolin-1 activity through c-Src signaling pathways. Our observations support the hypothesis that VEGF regulates amniotic fluid transport across the amnion by stimulating caveolin-1 activity to mediate caveolar function in amnion cells.

Figure 1

Immunofluorescent labeling of ovine amniotic epithelial cells for caveolin-1, VEGFR-1, and VEGFR-2 proteins. Ovine amnion cells fixed on coverslips were reacted with specific antibodies and visualized under the florescence microscope. The images were captured and digitized. A. oAECs treated with anti-caveolin-1 polyclonal antibody at 1:200 followed by secondary antibody conjugated to Cy3. B. High magnification of oAEC immunostained for caveolin-1. C. oAECs immunostained for VEGFR-1 with anti-Flt-1 polyclonal antibody followed by secondary antibody conjugated to Alexa Flour 488. D. oAECs immunostained for VEGFR-2 with anti-Flk-1/KDR monoclonal antibody followed by secondary antibody conjugated to Alexa Flour 488. VEGFR indicates vascular endothelial growth factor receptor; oAECs, ovine amniotic epithelial cells.

Figure 2

Vascular endothelial growth factor (VEGF) regulation of caveolin-1 expression in oAEC. The cells were treated with VEGF₁₆₅ at 1 to 25 ng/mL for 6 to 24 hours, and caveolin-1 mRNA and proteins levels were analyzed. A. Caveolin-1 mRNA levels as determined by semi-quantitative RT-PCR and expressed as the ratio of caveolin-1 abundance to the internal reference L-19 at 12 hours of VEGF treatment (F = 3.5, P < .02, 2-factor analysis of variance); *P < .05; **P < .01. Top panel, representative agarose gel stained with ethidium bromide for the caveolin-1 and L-19 PCR products. B. Western immunoblot determination of caveolin-1 protein levels referenced to the respective α-actin levels at 12 hours of VEGF treatment. (F = 1.66, P = nonsignificant, 2-factor analysis of variance). Results are mean ± SE, n = 4 experiments. M indicates, 100 bp ladder; mRNA, messenger RNA; SE, standard error; cav-1, caveolin-1.

Figure 3

Agonist activation of caveolin-1 phosphorylation in oAECs. Results are expressed as caveolin-1 phoshorylation (IP/total) and presented as the increase from the control level (F = 4.70, P < .02, 2-factor analysis of variance). Top panel, SDS-PAGE analysis of phosphorylated caveolin-1 protein and total caveolin-1 protein. Data are mean ± SE, n = 6 experiments. *P < .05; **P < .01. H₂O₂ indicates hydrogen peroxide; VEGF, VEGF₁₆₅; PMA, phorbol 12-myristate 13-acetate; oAECs, ovine amniotic epithelial cells; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Cav-1, caveolin-1.

Figure 4

Effect of c-Src inhibition on VEGF165-induced caveolin-1 phosphorylation. Levels of caveolin-1 phosphorylation (IP/total) are presented as the increase from control level (F = 5.39, P < .01, 2-factor analysis of variance). Data are mean ± SE, n = 3 experiments. *P < .05. PP2 indicates pyrazolopyrimidine 2; VEGF, vascular endothelial growth factor.