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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 could not directly activate PKC in zebrafish cultured follicle cells.The cells were treated with PKC activator, PMA (100 nM) or E2 (50 nM) for 20 min before the end of the 24-h treatment period. The treated cells were fractionated into cytosol (Cyto) and membrane (Mem) protein fractions followed by SDS-PAGE and Western blot analysis against phospho-PKCα/βII (p-PKCα/βII), p44/42 MAPK (cytosol marker) and pan-cadherin (membrane marker).
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pone-0062524-g006: E2 could not directly activate PKC in zebrafish cultured follicle cells.The cells were treated with PKC activator, PMA (100 nM) or E2 (50 nM) for 20 min before the end of the 24-h treatment period. The treated cells were fractionated into cytosol (Cyto) and membrane (Mem) protein fractions followed by SDS-PAGE and Western blot analysis against phospho-PKCα/βII (p-PKCα/βII), p44/42 MAPK (cytosol marker) and pan-cadherin (membrane marker).

Mentions: The strong dependence of E2-stimulated lhcgr expression on PKC pathway led us to speculate whether E2 could directly stimulate the PKC pathway to increase lhcgr expression in the zebrafish ovary. To explore this possibility, we examined membrane translocation of PKC after E2 treatment as PKC activation is associated with its translocation from the cytosol to the plasma membrane [20], [21]. As expected, PKC activator PMA induced a clear translocation of p-PKCα/βII from cytosol to plasma membrane. However, similar to the control, p-PKCα/βII remained in the cytosol fraction after E2 treatment while both PMA and E2 seemed to increase p-PKCα/βII abundance (Fig. 6).


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 could not directly activate PKC in zebrafish cultured follicle cells.The cells were treated with PKC activator, PMA (100 nM) or E2 (50 nM) for 20 min before the end of the 24-h treatment period. The treated cells were fractionated into cytosol (Cyto) and membrane (Mem) protein fractions followed by SDS-PAGE and Western blot analysis against phospho-PKCα/βII (p-PKCα/βII), p44/42 MAPK (cytosol marker) and pan-cadherin (membrane marker).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062524-g006: E2 could not directly activate PKC in zebrafish cultured follicle cells.The cells were treated with PKC activator, PMA (100 nM) or E2 (50 nM) for 20 min before the end of the 24-h treatment period. The treated cells were fractionated into cytosol (Cyto) and membrane (Mem) protein fractions followed by SDS-PAGE and Western blot analysis against phospho-PKCα/βII (p-PKCα/βII), p44/42 MAPK (cytosol marker) and pan-cadherin (membrane marker).
Mentions: The strong dependence of E2-stimulated lhcgr expression on PKC pathway led us to speculate whether E2 could directly stimulate the PKC pathway to increase lhcgr expression in the zebrafish ovary. To explore this possibility, we examined membrane translocation of PKC after E2 treatment as PKC activation is associated with its translocation from the cytosol to the plasma membrane [20], [21]. As expected, PKC activator PMA induced a clear translocation of p-PKCα/βII from cytosol to plasma membrane. However, similar to the control, p-PKCα/βII remained in the cytosol fraction after E2 treatment while both PMA and E2 seemed to increase p-PKCα/βII abundance (Fig. 6).

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