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Evidence for gating roles of protein kinase A and protein kinase C in estradiol-induced luteinizing hormone receptor (lhcgr) expression in zebrafish ovarian follicle cells.

Liu KC, Ge W - PLoS ONE (2013)

Bottom Line: PKA inhibitor H89 showed reversed effects.In contrast, PKC pathway had consistent permissive effect on E2-induced lhcgr expression as evidenced by strong inhibition of E2 effect by PKC inhibitors GF109203X and Ro-31-8220 at both 3 and 24 h.One of the mechanisms by which PKA and PKC gated E2 effect might be through regulating nERs, particularly esr2a.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences and Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

ABSTRACT
Estradiol (E2) stimulates luteinizing hormone receptor (lhcgr) expression in zebrafish follicle cells via nuclear estrogen receptors (nERs) that are likely expressed on the membrane, and lhcgr responds to E2 in a biphasic manner during 24-h treatment. These observations raise an interesting question on the signaling mechanism underlying E2 regulation, in particular the biphasic response of lhcgr expression. In the present study, we demonstrated that E2 regulation of lhcgr was significantly influenced by the activity of cAMP-PKA pathway. Activation of cAMP-PKA pathway by forskolin or db-cAMP suppressed E2-stimulated lhcgr expression in short-term (3 h) but enhanced its effect in long-term (24 h), suggesting differential roles of PKA at these two phases of lhcgr response. PKA inhibitor H89 showed reversed effects. In contrast, PKC pathway had consistent permissive effect on E2-induced lhcgr expression as evidenced by strong inhibition of E2 effect by PKC inhibitors GF109203X and Ro-31-8220 at both 3 and 24 h. One of the mechanisms by which PKA and PKC gated E2 effect might be through regulating nERs, particularly esr2a. Despite the strong influence of PKA and PKC, our data did not suggest direct mediating roles for these two pathways in E2 stimulation of lhcgr expression; yet they likely play critical gating roles in E2 signal transduction. As a follow-up study to our previous report on E2 regulation of gonadotropin receptors in the zebrafish ovary, the present study provides further evidence for the involvement of classical intracellular signal transduction pathways in E2 stimulation of lhcgr expression in the follicle cells.

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Both basal and E2-induced lhcgr expression were highly dependent on PKC pathway.Effect of GF109203X (A and B) and Ro-31-8220 (C and D) on basal and E2-stimulated lhcgr expression at 3 h and 24 h of treatment in cultured zebrafish follicle cells. The cells were pretreated with GF109203X (10 µM) or Ro-31-8220 (1 µM) for 15 min followed by treatment with E2 (50 nM) for 3 h or 24 h before the end of the 24-h treatment period. The data were expressed as fold change compared to the control group after normalization to the expression of ef1a. Different letters in each data set indicated statistical significance (P<0.05; mean ± SEM, n = 3–4).
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pone-0062524-g005: Both basal and E2-induced lhcgr expression were highly dependent on PKC pathway.Effect of GF109203X (A and B) and Ro-31-8220 (C and D) on basal and E2-stimulated lhcgr expression at 3 h and 24 h of treatment in cultured zebrafish follicle cells. The cells were pretreated with GF109203X (10 µM) or Ro-31-8220 (1 µM) for 15 min followed by treatment with E2 (50 nM) for 3 h or 24 h before the end of the 24-h treatment period. The data were expressed as fold change compared to the control group after normalization to the expression of ef1a. Different letters in each data set indicated statistical significance (P<0.05; mean ± SEM, n = 3–4).

Mentions: Having shown the importance of cAMP-PKA pathway in E2-induced lhcgr expression, we turned our attention to protein kinase C (PKC), another signaling pathway that has been reported to play a role in E2 signaling [11], [12], [14], [19]. At 3-h of treatment, the PKC inhibitor GF109203X (added 15 min earlier) significantly suppressed E2-stimulated lhcgr expression from∼9-fold to∼3-fold (Fig. 5A) and it nearly abolished the effect of E2 at 24 h (Fig. 5B), which was in contrast to the biphasic effects of cAMP-PKA at the two time points. GF109203X also affected the basal lhcgr expression at both 3 and 24 h. It reduced the basal level albeit insignificantly at 3 h and the expression turned undetectable at 24 h (Fig. 5A and B). To further confirm the role of PKC, we tested another PKC inhibitor Ro-31-8220. Similarly, Ro-31-8220 reduced basal and E2-stimulated lhcgr expression at both 3-h and 24-h treatment; however, its potency was not as high as that of GF109203X (Fig. 5C and D).


Evidence for gating roles of protein kinase A and protein kinase C in estradiol-induced luteinizing hormone receptor (lhcgr) expression in zebrafish ovarian follicle cells.

Liu KC, Ge W - PLoS ONE (2013)

Both basal and E2-induced lhcgr expression were highly dependent on PKC pathway.Effect of GF109203X (A and B) and Ro-31-8220 (C and D) on basal and E2-stimulated lhcgr expression at 3 h and 24 h of treatment in cultured zebrafish follicle cells. The cells were pretreated with GF109203X (10 µM) or Ro-31-8220 (1 µM) for 15 min followed by treatment with E2 (50 nM) for 3 h or 24 h before the end of the 24-h treatment period. The data were expressed as fold change compared to the control group after normalization to the expression of ef1a. Different letters in each data set indicated statistical significance (P<0.05; mean ± SEM, n = 3–4).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643932&req=5

pone-0062524-g005: Both basal and E2-induced lhcgr expression were highly dependent on PKC pathway.Effect of GF109203X (A and B) and Ro-31-8220 (C and D) on basal and E2-stimulated lhcgr expression at 3 h and 24 h of treatment in cultured zebrafish follicle cells. The cells were pretreated with GF109203X (10 µM) or Ro-31-8220 (1 µM) for 15 min followed by treatment with E2 (50 nM) for 3 h or 24 h before the end of the 24-h treatment period. The data were expressed as fold change compared to the control group after normalization to the expression of ef1a. Different letters in each data set indicated statistical significance (P<0.05; mean ± SEM, n = 3–4).
Mentions: Having shown the importance of cAMP-PKA pathway in E2-induced lhcgr expression, we turned our attention to protein kinase C (PKC), another signaling pathway that has been reported to play a role in E2 signaling [11], [12], [14], [19]. At 3-h of treatment, the PKC inhibitor GF109203X (added 15 min earlier) significantly suppressed E2-stimulated lhcgr expression from∼9-fold to∼3-fold (Fig. 5A) and it nearly abolished the effect of E2 at 24 h (Fig. 5B), which was in contrast to the biphasic effects of cAMP-PKA at the two time points. GF109203X also affected the basal lhcgr expression at both 3 and 24 h. It reduced the basal level albeit insignificantly at 3 h and the expression turned undetectable at 24 h (Fig. 5A and B). To further confirm the role of PKC, we tested another PKC inhibitor Ro-31-8220. Similarly, Ro-31-8220 reduced basal and E2-stimulated lhcgr expression at both 3-h and 24-h treatment; however, its potency was not as high as that of GF109203X (Fig. 5C and D).

Bottom Line: PKA inhibitor H89 showed reversed effects.In contrast, PKC pathway had consistent permissive effect on E2-induced lhcgr expression as evidenced by strong inhibition of E2 effect by PKC inhibitors GF109203X and Ro-31-8220 at both 3 and 24 h.One of the mechanisms by which PKA and PKC gated E2 effect might be through regulating nERs, particularly esr2a.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences and Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.

ABSTRACT
Estradiol (E2) stimulates luteinizing hormone receptor (lhcgr) expression in zebrafish follicle cells via nuclear estrogen receptors (nERs) that are likely expressed on the membrane, and lhcgr responds to E2 in a biphasic manner during 24-h treatment. These observations raise an interesting question on the signaling mechanism underlying E2 regulation, in particular the biphasic response of lhcgr expression. In the present study, we demonstrated that E2 regulation of lhcgr was significantly influenced by the activity of cAMP-PKA pathway. Activation of cAMP-PKA pathway by forskolin or db-cAMP suppressed E2-stimulated lhcgr expression in short-term (3 h) but enhanced its effect in long-term (24 h), suggesting differential roles of PKA at these two phases of lhcgr response. PKA inhibitor H89 showed reversed effects. In contrast, PKC pathway had consistent permissive effect on E2-induced lhcgr expression as evidenced by strong inhibition of E2 effect by PKC inhibitors GF109203X and Ro-31-8220 at both 3 and 24 h. One of the mechanisms by which PKA and PKC gated E2 effect might be through regulating nERs, particularly esr2a. Despite the strong influence of PKA and PKC, our data did not suggest direct mediating roles for these two pathways in E2 stimulation of lhcgr expression; yet they likely play critical gating roles in E2 signal transduction. As a follow-up study to our previous report on E2 regulation of gonadotropin receptors in the zebrafish ovary, the present study provides further evidence for the involvement of classical intracellular signal transduction pathways in E2 stimulation of lhcgr expression in the follicle cells.

Show MeSH
Related in: MedlinePlus