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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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E2-induced lhcgr expression was dependent on cAMP-PKA without direct PKA activation in zebrafish follicle cells.(A–C) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM), forskolin (10 µM) and db-cAMP (1 mM) 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 = 3–6). (D) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM) and forskolin (10 µM) for 30 min before the end of the 24-h treatment period. Cells were lyzed in SDS sample buffer for Western blot analysis of phospho-CREB (p-CREB) and β-actin expression.
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pone-0062524-g002: E2-induced lhcgr expression was dependent on cAMP-PKA without direct PKA activation in zebrafish follicle cells.(A–C) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM), forskolin (10 µM) and db-cAMP (1 mM) 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 = 3–6). (D) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM) and forskolin (10 µM) for 30 min before the end of the 24-h treatment period. Cells were lyzed in SDS sample buffer for Western blot analysis of phospho-CREB (p-CREB) and β-actin expression.

Mentions: We then investigated if protein kinase A (PKA) played any role in cAMP regulation of lhcgr expression. Opposite to the effects of forskolin and db-cAMP, blocking PKA at 3 h with PKA inhibitor H89 slightly but not significantly increased the basal expression of lhcgr; however, it synergistically promoted E2-induced lhcgr expression from∼8-fold to∼24-fold compared to the control (Fig. 2A). In contrast to the enhancing effects of forskolin and db-cAMP at 24 h, H89 completely eradicated the stimulatory effect of E2 on lhcgr at 24 h (Fig. 2B).


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)

E2-induced lhcgr expression was dependent on cAMP-PKA without direct PKA activation in zebrafish follicle cells.(A–C) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM), forskolin (10 µM) and db-cAMP (1 mM) 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 = 3–6). (D) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM) and forskolin (10 µM) for 30 min before the end of the 24-h treatment period. Cells were lyzed in SDS sample buffer for Western blot analysis of phospho-CREB (p-CREB) and β-actin expression.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643932&req=5

pone-0062524-g002: E2-induced lhcgr expression was dependent on cAMP-PKA without direct PKA activation in zebrafish follicle cells.(A–C) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM), forskolin (10 µM) and db-cAMP (1 mM) 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 = 3–6). (D) The cells were pretreated with H89 (10 µM) for 15 min followed by treatment with E2 (50 nM) and forskolin (10 µM) for 30 min before the end of the 24-h treatment period. Cells were lyzed in SDS sample buffer for Western blot analysis of phospho-CREB (p-CREB) and β-actin expression.
Mentions: We then investigated if protein kinase A (PKA) played any role in cAMP regulation of lhcgr expression. Opposite to the effects of forskolin and db-cAMP, blocking PKA at 3 h with PKA inhibitor H89 slightly but not significantly increased the basal expression of lhcgr; however, it synergistically promoted E2-induced lhcgr expression from∼8-fold to∼24-fold compared to the control (Fig. 2A). In contrast to the enhancing effects of forskolin and db-cAMP at 24 h, H89 completely eradicated the stimulatory effect of E2 on lhcgr at 24 h (Fig. 2B).

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