RhoA and DIAPH1 mediate adrenocorticotropin-stimulated cortisol biosynthesis by regulating mitochondrial trafficking

Abstract

Steroid hormones are formed by the successive action of enzymes that are localized in mitochondria and the endoplasmic reticulum (ER). Compartmentalization of these enzymes in different subcellular organelles dictates the need for efficient transfer of intermediary metabolites between the mitochondrion and ER; however, the molecular determinants that regulate interorganelle substrate exchange are unknown. The objective of this study was to define the molecular mechanism by which adrenocorticotropin (ACTH) signaling regulates communication between mitochondria and the ER during steroidogenesis. Using live cell video confocal microscopy, we found that ACTH and dibutyryl cAMP rapidly increased the rate of mitochondrial movement. Inhibiting tubulin polymerization prevented both basal and ACTH/cAMP-stimulated mitochondrial trafficking and decreased cortisol secretion. This decrease in cortisol secretion evoked by microtubule inhibition was paralleled by an increase in dehydroepiandrosterone production. In contrast, treatment with paclitaxel to stabilize microtubules or latrunculin B to inhibit actin polymerization and disrupt microfilament organization increased both mitochondrial trafficking and cortisol biosynthesis. ACTH-stimulated mitochondrial movement was dependent on RhoA and the RhoA effector, diaphanous-related homolog 1 (DIAPH1). ACTH signaling temporally increased the cellular concentrations of GTP-bound and Ser-188 phosphorylated RhoA, which promoted interaction with DIAPH1. Expression of a dominant-negative RhoA mutant or silencing DIAPH1 impaired mitochondrial trafficking and cortisol biosynthesis and concomitantly increased the secretion of adrenal androgens. We conclude that ACTH regulates cortisol production by facilitating interorganelle substrate transfer via a process that is mediated by RhoA and DIAPH1, which act to coordinate the dynamic trafficking of mitochondria.

Figure 1

ACTH/cAMP stimulate mitochondrial movement. A, H295R human adrenocortical cells were plated on coverslips and mitochondria labeled with MitoTracker Red (Invitrogen). Mitochondrial movement was observed by time-lapsed fluorescence video microscopy in control, ACTH- (5 nm), and Bt₂cAMP (0.4 mm)-treated cells. Imaging was carried out on at least five separate occasions and the rates of at least 20 mitochondria quantified on each occasion. Error bars, sd. *, Statistically significant, P < 0.05. B, Cells were treated for 5–30 min with 5 nm ACTH and the levels of intracellular cAMP quantified by ELISA. cAMP concentrations are normalized to total cellular protein content and data graphed represent the mean ± sem of three experiments, each carried out in triplicate. *, Statistical significance, P < 0.05. C, Cells were plated onto glass coverslips, labeled with MitoTracker Red, and treated with 10 μm H89 and/or 5 nm ACTH. Imaging was performed on three separate cell preparations and the rates of approximately 25 mitochondria quantified on each occasion. *, Statistically significant difference compared with untreated control, P < 0.001.

Figure 2

A, Video microscopy was carried out on cells MitoTracker-stained cells (Invitrogen) that were treated with 0.4 mm Bt₂cAMP in the presence and absence of 1 μm colchicine (colc.), 1 μm latrunculin B (LaB), 1 μm nocodazole (noc.), or 2 μm paclitaxel (paclitax.). Imaging was carried out on three cell preparations and the rates of approximately 20 mitochondria quantified each time. Error bars, sd. *, Treatments that are statistically different from the control group, P < 0.001; ^, statistically significant difference (P < 0.001) when compared with Bt₂cAMP-treated group. B, The amount of cortisol and DHEA secreted into the media was determined by ELISA and normalized to total cellular protein. Data represent the average of three experiments, each carried out in duplicate. * and ^, Statistically significant difference compared with untreated controls and Bt₂cAMP-treated cells, respectively (P < 0.001).

Figure 3

RhoA mediates ACTH-stimulated mitochondrial trafficking and cortisol production. A, Cells were treated with 0.4 mm Bt₂cAMP (or 50 nm ACTH, not shown) for time periods ranging from 5 to 30 min and the amount of active Rho determined by rhoteklin binding as shown in the representative blot. B, Densities obtained from Rho activation Western blots were graphed as fold change over control group mean and represent the mean ± sem of three experiments, each carried out in triplicate. *, Statistically different from untreated 0-min control group mean, P < 0.05. C, Time-lapsed video imaging was performed on cells transfected with wild-type or dominant-negative (T19N) GFP-tagged RhoA and stained with MitoTracker Red (Invitrogen). The rate of mitochondrial movement in control and Bt₂cAMP-treated cells and the average rates of movement graphed. Rates were quantified only for cells expressing wild-type or mutant GFP-tagged RhoA. Data represent the mean ± sd of three separate experiments, each quantifying the rate of mitochondrial movement in at least 20 transfected cells. *, Statistically different from wild-type control group mean, P < 0.05; ^, statistically different from Bt₂cAMP-treated wild-type cells, P < 0.001. D, Cells were transfected with wild-type or mutant RhoA and then treated with 0.4 mm Bt₂cAMP. The amount of cortisol and DHEA secreted into the cell culture media was quantified by ELISA and normalized to total cellular protein content. Data represent the mean ± sd of three experiments, each carried out in triplicate. * and ^, Statistical significant differences (P < 0.01) from wild-type cortisol or DHEA control and wild-type Bt₂cAMP-stimulated cortisol or DHEA samples, respectively.

Figure 4

ACTH signaling promotes the interaction of RhoA and DIAPH1 and RhoA phosphorylation. A, Cells were treated with 50 nm ACTH or 0.4 mm Bt₂cAMP for 30 min and isolated lysates separated by SDS-PAGE. Western blotting was carried out using an antiphospho Ser-188 RhoA antibody (top panel) and total RhoA (bottom panel). B, Graphical representation of phospho-RhoA amounts quantified by densitometric scanning. Data represent the mean ± sem of three experiments, each carried out in triplicate. *, Statistically different from control group mean, P < 0.05. C, Lysates isolated from cells treated for 15, 30, or 60 min with 0.4 mm Bt₂cAMP were precleared and immunoprecipitated (IP) with anti-DIAPH1 antibody. The immobilized proteins resolved by SDS-PAGE and Western blotting (WB). Membranes were probed with antiphospho Ser-188 RhoA antibody (top panel) or anti-DIAPH1 (bottom panel) and imaged using ECF and fluorometric scanning. D, Cells were treated for 30 min with 0.4 mm Bt₂cAMP and 10 μm H89 and lysates subjected to immunoprecipitation (anti-DIAPH1 antibody), SDS-PAGE and Western blotting (antiphospho Ser-188 RhoA antibody for output and anti-DIAPH1 for input).

Figure 5

DIAPH1 regulates mitochondrial movement and steroid hormone biosynthesis. A, MitoTracker-labeled (Invitrogen) wild-type or mutant GFP-tagged, DIAPH1-expressing cells were treated with Bt₂cAMP and the movement of mitochondria assessed by time-lapsed video microscopy. Frames of 0-min time point and 5-min Bt₂cAMP-treated cells expressing wild-type or mutant DIAPH1 correspond to movies provided in supplemental data. B, H295R cells were plated onto coverslips and transfected with pEGFP-DIAPH1. Forty-eight hours after transfection, cells were labeled with MitoTracker Red and treated with 1 μm colchicine (colc.), 1 μm latrunculin B (LaB), 1 μm nocodazole (noc.), or 0.4 mm Bt₂cAMP. The rate of mitochondrial movement was quantified and the average rates from three experiments graphed. *, Statistically different from wild-type (WT) control group mean, P < 0.05; ^, statistically different from mutant (mut) control cells, P < 0.001. C, H295R cells were transfected with pEGFP-DIAPH1 and then and treated with 1 μm colchicine (colc.), 1 μm latrunculin B (LaB), 1 μm nocodazole (noc.), or 0.4 mm Bt₂cAMP for 48 h. Cortisol and DHEA secreted into the media were quantified by ELISA and normalized to the total cellular protein content in each well. The data graphed represent the mean ± sem of two experiments, each performed in triplicate. D, Cells were transfected with scrambled siRNA or siRNA oligonucleotides directed against DIAPH1 and the expression of DIAPH1 determined Western blotting using an anti-DIAPH1 antibody. E, The amount of cortisol and DHEA secreted into the cell culture media was quantified in cells transfected with DIAPH1 siRNA oligonucleotides. The amount of steroid hormone was normalized to total protein content and the data graphed represent the average of two separate experiments, each performed in triplicate. * and ^, Statistical difference from untransfected control (P < 0.05) and Bt₂cAMP-treated (P < 0.01) groups for each steroid, respectively.

Figure 6

Model for ACTH regulation of microtubule-dependent, interorganelle substrate delivery. ACTH increases intracellular cAMP. PKA stimulates RhoA activation and phosphorylation and promotes RhoA interaction with DIAPH1. In response to ACTH, DIAPH1 mediates microtubule-dependent mitochondrial repositioning. ER, Endoplasmic reticulum.