Fig 1: T-regulated MIGA2 expression dependent on androgen receptor in a concentration dependent manner in KGN cells. (A) Relative mRNA expression of AR after treatments of T at indicated concentrations or combined treatment of Flu (5 µM). (B, C) Relative mRNA expression of MIGA1 (B) and MIGA2 (C) after treatments of indicated concentrations of T or combined treatment of Flu. (D, E) LHCGR protein expression after the indicated concentrations of T or combined treatment of Flu. (F-I) LHCGR protein expression levels after the indicated treatment of gradient concentration of T or DHT (F, G), or treatments of T or DHT combined with FSK/PMA for 24 hours (H, I). (J-N) Representative western blots of MIGA2, pAMPK (T172), AMPK, PRKACA, and PRKACB after treatments of T at indicated concentrations or combined treatment of Flu (5 µM) (L). Quantification of relative protein expression levels of MIGA2 (J) pAMPK (K), PRKACA (M), and PRKACB (N). Data presented as mean ± SD. *, P < .05, **, P < .01. Abbreviations: AMPK, AMP-activated protein kinase; AR, androgen receptor; DHT, dihydrotestosterone; Flu, flutamide; FSK, forskolin; LHCGR, LH/choriogonadotropin receptor; MIGA1, mitoguardin 1; MIGA2, mitoguardin 2; pAMPK, phosphorylation of AMPK; PMA, phorbol 12-myristate 13-acetate; PRKACA, PKA catalytic subunit a; PRKACB, PKA catalytic subunit ß; T, testosterone.
Fig 2: Proposed schematic representation of testosterone regulation through MIGA2 during steroidogenesis in ovarian granulosa cells. Testosterone may promote mitochondrial fusion mediated by MIGA1/2 via AR or LHCGR partially. LH or hCG binds to LHCGR and activates PKA through Gs-mediated activation of Ac and the increase of cAMP. Likewise, FSK activates PKA and PMA activates PKC. Increase of cAMP regulates MIGA2 expression through PKA signaling in a time-dependent manner. MIGA2 increases TOMM20, interacts with StAR, and induces StAR localization on mitochondria in luteal cells. Testosterone or FSK/PMA increases the expression and interactions of MIGA2 and StAR. However, MIGA2 overexpression increases PRKACA and pAMPK (T172) levels but inhibits StAR protein expression. The pAMPK (T172) is speculated to be increased by PRKACA subunit but inhibited by PRKACB subunit and has been suggested to play a role in inhibiting progesterone synthesis. The key steroidogenic genes of CYP11A1, HSD3B2, and CYP11A1 were all increased by either testosterone or MIGA2 overexpression. As a result, MIGA2 decreases progesterone levels but increases estradiol levels by promoting the aromatization of testosterone. In addition, estradiol regulates MIGA2 expression in turn. However, whether MIGA2 regulates steroidogenesis via AMPK in granulosa cells remains to be clarified. Ac, Abbreviations: Ac, adenylyl cyclase; AR, androgen receptor; BMI, body mass index; FSK, forskolin; Gs, stimulatory G proteins that activate Ac; HA-PCOS, 75 hyperandrogenic PCOS; hCG, human chorionic gonadotropin; LHCGR, LH/choriogonadotropin receptor; MIGA1, mitoguardin 1; MIGA2, mitoguardin 2; NA-PCOS, 54 normo-androgenic PCOS; PCOS, polycystic ovary syndrome; PKA, cAMP-activated protein kinase A; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; StAR, steroidogenic acute regulatory protein; TOMM20, translocase of outer mitochondrial membrane 20.
Fig 3: PKA and AMPK signalings are involved in testosterone regulation on MIGA2 and StAR. (A) Representative western blots of PRKACA, PRKACB, pAMPK (T172), AMPK, and ATP5A after MIGA1 or MIGA2 overexpression and treatments with FSK/PMA for 24 hours or not. (B) Quantification of the relative protein expression levels of PRKACA, PRKACB, and ATP5A to ß-ACTIN in (B) compared to the control. (C) Quantification of phosphorylation of pAMPK (T172)/AMPK of (C). (D) ATP levels after MIGA1 or MIGA2 overexpression and treatments with FSK/PMA for 24 hours or not. (E) Representative western blots of MIGA2, StAR, PRKACA, PRKACB, pAMPK (T172), and AMPK after FSK/PMA or H-89 treatments for 2 or 24 hours. (F) Quantification of the relative protein expression levels of (E) to GAPDH compared to the control. (G) Representative western blots of MIGA2, StAR, PRKACA, PRKACB, pAMPK (T172), and AMPK after T, DHT, or H-89 treatments for 24 hours. (H) Quantification of relative protein expression levels of (G) to GAPDH compared to the control. (I, J) Representative western blots images of pAMPK (T172) and AMPK after E2 treatment for 24 hours at the concentration of 1, 10, 100 nM. (J) The quantification of relative protein expression levels of pAMPK (T172) to GAPDH compared to the control. Data presented as mean ± SD. *, P < .05, **, P < .01. Abbreviations: AMPK, AMP-activated protein kinase; DHT, dihydrotestosterone; E2, estradiol; FSK, forskolin; MIGA1, mitoguardin 1; MIGA2, mitoguardin 2; pAMPK, phosphorylation of AMPK; PKA, cAMP-activated protein kinase A; PMA, phorbol 12-myristate 13-acetate; PRKACA, PKA catalytic subunit a; PRKACB, PKA catalytic subunit ß; StAR, steroidogenic acute regulatory protein; T, testosterone.
Supplier Page from Proteintech Group Inc for PRKACB antibody