HB-EGF induces mitochondrial dysfunction via estrogen hypersecretion in granulosa cells dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway

Abstract

Polycystic ovarian syndrome (PCOS) is one of the most prevalent endocrinopathies and the leading cause of anovulatory infertility, but its pathogenesis remains elusive. Although HB-EGF is involved in ovarian cancer progression, there is still no clarity about its relevance with PCOS. The present study exhibited that abundant HB-EGF was noted in follicular fluid from PCOS women, where it might induce the granulosa cells (GCs) production of more estrogen via the elevation of CYP19A1 expression after binding to EGFR. Furthermore, HB-EGF transduced intracellular downstream cAMP-PKA signaling to promote the phosphorylation of JNK and ERK whose blockage impeded the induction of HB-EGF on estrogen secretion. Meanwhile, HB-EGF enhanced the accumulation of intracellular Ca²⁺ whose chelation by BAPTA-AM abrogated the stimulation of HB-EGF on FOXO1 along with an obvious diminishment for estrogen production. cAMP-PKA-JNK/ERK-Ca²⁺ pathway played an important role in the crosstalk between HB-EGF and FOXO1. Treatment of GCs with HB-EGF resulted in mitochondrial dysfunction as evinced by the reduction of ATP content, mtDNA copy number and mitochondrial membrane potential. Additionally, HB-EGF facilitated the opening of mitochondrial permeability transition pore via targeting BAX and raised the release of cytochrome C from mitochondria into the cytosol to trigger the apoptosis of GCs, but this effectiveness was counteracted by estrogen receptor antagonist. Collectively, HB-EGF might induce mitochondrial dysfunction and GCs apoptosis through advancing estrogen hypersecretion dependent on cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway and act as a promising therapeutic target for PCOS.

Keywords: HB-EGF; cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway; estrogen; granulosa cell; mitochondrial dysfunction.

Figure 1

HB-EGF induces the hypersecretion of estrogen and GCs apoptosis via EGFR. A, HB-EGF content in follicular fluid between PCOS and non-PCOS patients. B, Effect of HB-EGF on estrogen secretion in the absence or presence of EGFR inhibitor PF299804. PF, PF299804. C, Regulation of HB-EGF on CYP19A1 expression with/without PF299804. N = 6. D, Effect of HB-EGF on GCs apoptosis in the existence or not of PF299804. N = 3. E and F, Real-time PCR and western blot analyses of CASP3 and BAX expression after treatment with rHB-EGF in the absence or presence of PF299804. N = 3. G, Effect of HB-EGF on CASP3 activity with/without PF299804. N = 5. * P < 0.05 versus control, # P < 0.05 versus rHB-EGF treatment.

Figure 2

cAMP-PKA signaling mediates the effects of HB-EGF on estrogen secretion and GCs apoptosis. A, HB-EGF induced the accumulation of intracellular cAMP via EGFR. B and C, cAMP-PKA signaling mediated the effect of HB-EGF on estrogen secretion (N = 9) and CYP19A1 expression (N = 4). D, cAMP-PKA signaling mediated the effect of HB-EGF on GCs apoptosis. N = 3. E-G, Regulation of HB-EGF on the expression or activity of CASP3 and BAX was mediated by cAMP-PKA signaling. N = 3. ^* P < 0.05 versus control, ^# P < 0.05 versus rHB-EGF treatment, ^& P < 0.05 versus rHB-EGF plus PF299804 treatment.

Figure 3

HB-EGF induces estrogen secretion and GCs apoptosis via cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway. A, HB-EGF enhanced the expression of JNK and ERK via cAMP-PKA signaling. N = 3. B and C, HB-EGF induced estrogen secretion (N = 9) and CYP19A1 expression (N = 4) via JNK/ERK-Ca²⁺-FOXO1 pathway. SP, SP600125; GDC, GDC-0994; BAPTA, BAPTA-AM; AS, AS1842856. D, HB-EGF induced GCs apoptosis via JNK/ERK-Ca²⁺-FOXO1 pathway. N = 3. E-G, HB-EGF raised the expression or activity of CASP3 and BAX through JNK/ERK-Ca²⁺-FOXO1 pathway. N = 3. H-J, HB-EGF enhanced intracellular Ca²⁺ content via cAMP-PKA-ERK/JNK pathway. N = 3. K, Luciferase activity was assessed after GCs were transfected with pGL6-CYP19A1 plasmid and then treated with rHB-EGF in the absence or presence of FOXO1 inhibitor AS1842856. N = 6. L-N, HB-EGF increased FOXO1 expression via cAMP-PKA- JNK/ERK-Ca²⁺ pathway. N = 3.

Figure 4

HB-EGF causes the aberration of ATP level, mtDNA copy number and MMP via cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway. A and B, HB-EGF brought about the reduction of ATP content (N = 6) and mtDNA copy number (N = 5) via cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway. C and D, HB-EGF attenuated the MMP via cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway by flow cytometry analysis or visualization in fluorescence microscope. N = 3. Scale bar, 20 µm.

Figure 5

HB-EGF induces mPTP opening and cytochrome C release via cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway. A-C, Flow cytometry analysis evidenced that HB-EGF induced the opening of mPTP via cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway. N = 3. D, Visualization of mPTP after treatment with rHB-EGF in the absence or presence of different inhibitor or 8-Br-cAMP. N = 3. E, HB-EGF induced the release of cytochrome C via cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway. N = 3.

Figure 6

HB-EGF induces GCs apoptosis through promoting mPTP opening and enhancing estrogen secretion. A and B, mPTP opening inhibitor ER-000444793 hampered the induction of HB-EGF on cytochrome C release and GCs apoptosis. N = 3. C-E, Blockage of mPTP opening weakened the induction of HB-EGF on cleaved CASP3 expression and activity, while did not alter change its regulation on BAX. N = 4. F, ER antagonist ICI 182780 impeded GCs apoptosis by HB-EGF. N = 3. ICI, ICI 182780. G-I, ER antagonist ICI 182780 attenuated the facilitation of HB-EGF on the expression or activity of CASP3 and BAX. N = 3.

Figure 7

HB-EGF impairs mitochondrial function through enhancing estrogen secretion. A and B, Estrogen receptor antagonist ICI 182780 prevented the impairment of HB-EGF on ATP level and mtDNA copy number. N = 5. C and D, ICI 182780 resisted the regulation of HB-EGF on MMP. N = 3. E and F, ICI 182780 counteracted the induction of HB-EGF on mPTP opening. N = 3. G, ICI 182780 impeded the induction of HB-EGF on cytochrome C release. N = 3.

Figure 8

Schematic depiction of HB-EGF regulation to GCs. Elevated HB-EGF was noted in follicular fluid of PCOS patients, where it might induce the GCs production of more estrogen through cAMP-PKA-JNK/ERK-Ca²⁺-FOXO1 pathway after binding to EGFR and brought about mitochondrial dysfunction, resulting in the release of cytochrome C from mitochondria into the cytosol to trigger GCs apoptosis.