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. 2010 Jun 30;151(9):4313–4323. doi: 10.1210/en.2010-0044

RhoA and DIAPH1 Mediate Adrenocorticotropin-Stimulated Cortisol Biosynthesis by Regulating Mitochondrial Trafficking

Donghui Li 1, Marion B Sewer 1
PMCID: PMC2940507  PMID: 20591975

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.

These studies define how the cytoskeleton, RhoA, and DIAPH1 control cortisol and DHEA production in the adrenal cortex.

Glucocorticoids are essential regulators of varied physiological processes including the inflammatory response, gluconeogenesis, and vascular tone. These molecules are synthesized in the adrenal cortex from cholesterol via a series of reactions that are catalyzed by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases (1,2,3,4). Steroidogenesis is tightly regulated by the peptide hormone ACTH, which acts by varied, temporally distinct mechanisms to increase the transcription of genes required for steroidogenesis, facilitate the uptake and intracellular delivery of free cholesterol, promote electron transfer, and modulate the activity of steroidogenic enzymes (5,6,7).

Steroid hormone biosynthesis in the adrenal cortex takes place in two organelles, the mitochondrion and the endoplasmic reticulum. Both the first step and last reactions in cortisol production occur in mitochondria, with intermediary reactions taking place in the ER. Although mechanisms that control the transcription and enzymatic activity of steroidogenic genes are extensively characterized, the factors that regulate interorganelle substrate delivery are not well understood.

Studies using chemical inhibitors to modulate the polymerization of cytoskeletal proteins have implicated both microtubules and microfilaments as key regulators of steroid hormone biosynthesis (8,9,10,11,12,13,14,15,16). A relationship between steroidogenesis and the cytoskeleton, in which microtubule depolymerization agents have been found to impair steroid hormone production, has also been identified (10,11,17,18,19,20,21). Moreover, microscopic studies in Y1 mouse adrenal cells has found that lipid droplets move along microtubule tracts (22). Collectively these data point to a key role for the cytoskeletal architecture in steroid hormone production. In contrast to studies carried out in neurons, in which mitochondria have also been visualized in association with microfilaments (23), microtubules (24,25), and intermediate filaments (26,27) and in which motor proteins, such as kinesins, facilitate the movement of these organelles along cytoskeletal fibers (28,29,30,31,32), the precise molecular mechanism by which cytoskeletal proteins control steroidogenesis is unclear. Furthermore, the specific protein(s) that couple steroid hormone output to microfilaments and/or microtubules are unknown.

Rho GTPases are a family of proteins that use the hydrolysis of GTP to transduce a wide variety of extracellular and intracellular signals, including cell migration, cytokinesis, vesicular trafficking, and phagocytosis (33,34,35,36). Signal transduction cascades stimulate the activation of Rho proteins by facilitating the exchange of GDP for GTP. In the GTP-bound form, Rho proteins act to relay extracellular signals to intracellular effectors. Diaphanous-related homolog (DIAPH)-1 is a member of the diaphanous-related formin family of Rho effector proteins that coordinate cell motility and migration by regulating the organization of microtubules and microfilaments (37). DIAPH proteins contain an N-terminal Rho-binding domain, two formin-homology regions, and a C-terminal diaphanous-autoregulatory domain (DAD) that stabilizes the proteins in an inactive conformation in the absence of Rho binding. Binding of active Rho stimulates dissociation of intermolecular interactions between the N-terminal region and the DAD, thereby activating the protein (38,39,40,41,42). Thus, truncation mutants that lack the DAD or Rho-binding domain are constitutively active (40,41,43,44). Activation of signaling pathways promote the release of the intermolecular autoinhibitory interactions via a two-step mechanism that is accelerated by the presence of Rho-GTP (45). In this study, we investigated the mechanism by which ACTH signaling controls mitochondrial trafficking during steroid hormone production in human H295R adrenocortical cells.

Materials and Methods

Reagents

Dibutyryl cAMP (Bt2cAMP) was obtained from Sigma (St. Louis, MO) and MitoTracker Red from Invitrogen (Carlsbad, CA). ACTH was purchased from American Peptide, Inc. (Sunnyvale, CA) and solubilized in 5% acetic acid. Colchicine, nocodazole, latrunculin B, and paclitaxel were obtained from EMD Biosciences (La Jolla, CA).

Cell culture

H295R adrenocortical cells (46,47) were generously donated by Dr. William E. Rainey (Medical College of Georgia, Augusta, GA) and cultured in DMEM/F12 medium (Mediatech, Manassas, VA) supplemented with 10% Nu-Serum I (BD Biosciences, Palo Alto, CA), 1% ITS Plus (BD Biosciences), antibiotics, and antimycotics.

Quantification of intracellular cAMP

Cells were treated for 5–30 min with 5 nm ACTH. The concentration of intracellular cAMP formed in response to ACTH stimulation was determined by ELISA (Assay Designs, Ann Arbor, MI), according to the manufacturer’s protocol. Data obtained were normalized to the total cellular protein concentration of each sample.

Confocal microscopy and time-lapsed video imaging

Cells were plated onto coverslips and confocal images were collected in control and treated cells using an LSM510 confocal microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a helium-neon coherent laser. Emissions were collected with a C-apochromat 40 1.3 NA oil immersion objective (Zeiss) using a 560-nm long-pass filter. Some cells were transfected with 50 ng pcDNA3-EGFP-RhoA, pcDNA3-EGFP-RhoA(T19N), pEGFP-DIAPH1, or pEGFP-DIAPH1(ΔN3) using Gene Juice (EMD Biosciences) and Optimem (Invitrogen). Transfection efficiency for green fluorescent protein (GFP)-tagged plasmids ranged from 64% for RhoA plasmids to 85% for DIAPH1 constructs. RhoA expression plasmids were obtained from Addgene (Cambridge, MA), and DIAPH1 expression plasmids were generously donated by Dr. Shuh Narumiya (Kyoto University Faculty of Medicine, Kyoto, Japan). The Zeiss imaging physiology platform was used to determine the velocity of movement of individual mitochondria from video-taped recordings. To quantify the rate of movement, the time of exposure of each frame was set to 1 sec, with a 3-sec interval between successive frames. The track of mitochondria was obtained by subtracting the change in position after each frame interval. Only mitochondria that changed position during a given time interval were calculated. For these mobile mitochondria, the translocation between neighbor frames was measured and the mean rate of movement was calculated. Each experiment was performed at least three times and the movement of at least 20 mitochondria was analyzed in each experimental condition.

Cortisol and dehydroepiandrosterone (DHEA) assays

Cells were cultured in 12-well plates and transfected and/or treated with 0.4 mm Bt2cAMP, 5 nm ACTH, 1 μm nocodazole, 1 μm colchicine, 1 μm latrunculin B, and 2 μm paxitaxel for 24 h. Cortisol or DHEA released into the media was determined in triplicate against standards made up in DME/F12 medium by ELISA (Diagnostic Systems Corp., Houston, TX). Results are expressed as picograms per milligram cellular protein.

Rho activation assays

Cells were treated for time periods ranging from 5 to 30 min with 50 nm ACTH or 0.4 mm Bt2cAMP. The amount of activated Rho in cell extracts was determined using a Rho activation kit (Assay Designs) according to the manufacturer’s instructions.

Coimmunoprecipitation and Western blotting

H295R cells were cultured in 100-mm dishes, treated with ACTH (50 nm), Bt2cAMP (0.4 mm), and/or H89 (10 μm) for 15, 30, or 60 min, and whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (containing 1× protease inhibitors; EMD Biosciences). Precleared lysates were incubated overnight with 30 μl protein A/G Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and 5 μg anti-DIAPH1 (Millipore, Bedford, MA) or anti-GFP (Sigma). Beads were washed twice in radioimmunoprecipitation assay and twice in PBS, separated by SDS-PAGE, and transferred to polyvinyl difluoride membranes (Millipore). Western blotting was carried out using antiphospho Ser-188 RhoA antibody (Santa Cruz). Blots were developed with enhanced chemifluorescence (Amersham Biosciences, Piscataway, NJ).

RNA interference

Cells plated onto coverslips or 12-well dishes and transfected with 75 nm nonspecific small interfering RNA (siRNA) oligonucleotides or siRNA oligonucleotides directed against DIAPH1 (Dharmacon, Lafayette, CO) for 72 h using HiPerfect (QIAGEN, Valencia, CA) in Optimem (Invitrogen). The amount of steroid hormones produced was determined as described above. Protein expression of DIAPH1 was assessed in lysates isolated from transfected cells by SDS-PAGE and Western blotting.

Statistical analysis

One-way ANOVA, Tukey-Kramer multiple comparison, and unpaired Student t tests were performed using GraphPad InStat software (GraphPad Software Inc., San Diego, CA). Significant differences from a compared value were defined as P < 0.05, P < 0.01, or P < 0.001 and denoted by asterisks (*) or carats (^).

Results

ACTH rapidly increases mitochondrial movement in H295R cells

To examine the mechanism by which ACTH regulates substrate trafficking between mitochondria and ER, we carried out real-time video imaging on MitoTracker Red-labeled (Invitrogen) H295R human adrenocortical cells. The rate of mitochondrial movement was quantified in untreated, ACTH-, or Bt2cAMP-treated cells over 5-min time periods. We calculated the rate of movement of mitochondria exhibiting saltatory, unidirectional movement (48,49,50). Both ACTH and Bt2cAMP increased mitochondrial movement by approximately 2.7-fold (Fig. 1A and Supplemental Videos 1 and 2, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Experiments using live cell imaging to track the rate of mitochondrial movement indicated that neither ACTH nor Bt2cAMP increased the rate of movement of all mitochondria. We postulate that this may be due to local concentrations of stimulus when treating the cells; however, the precise reason for the lack of increased mitochondrial movement in all cells examined is unknown.

Figure 1.

Figure 1

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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.

Although recent work by Janes et al. (51) demonstrated that H295R cells express the melanocortin 2 receptor and respond to ACTH by increasing intracellular cAMP and activating ERK (51), the production of cAMP in the H295R cell line has been shown to be modest in comparison with the Y1 murine adrenal cell line (52,53). To determine whether the stimulatory effects of ACTH are mediated by cAMP, we quantified the concentrations of intracellular cAMP released from H295R cells treated for time periods ranging from 5 to 30 min with 5 nm ACTH. As shown in Fig. 1B, within 5 min, ACTH maximally increases intracellular cAMP. Although the concentrations of the second-messenger decline at the 15- and 30-min time points, intracellular cAMP levels remain significantly higher than the untreated control group. The ability of ACTH to increase the rate of mitochondrial movement requires protein kinase A (PKA) activity, as evidenced by the ability of H89 to attenuate ACTH-stimulated mitochondrial movement (Fig. 1C).

cAMP-stimulated mitochondrial trafficking requires microtubule polymerization

We next sought to define the role of the cytoskeleton in mediating increased mitochondrial movement in response to cAMP. Time-lapsed video imaging was performed on MitoTracker-labeled cells (Invitrogen) treated with Bt2cAMP and chemicals that alter the polymerization of tubulin and actin. Colchicine and nocodazole promote microtubule depolymerization, latrunculin B inhibits actin polymerization, and paclitaxel promotes microtubule stabilization. Figure 2A shows that both colchicine and nocodazole retard mitochondrial movement, in the absence and presence of Bt2cAMP (Supplemental Video 3). This effect of tubulin polymerization inhibitors is in direct contrast to the stimulatory effect of the microtubule stabilizer paclitaxel, which increased the rate of mitochondrial movement by 3.5-fold. Latrunculin B also increased the rate of mitochondrial movement, and this stimulatory effect was further increased in the presence of Bt2cAMP (Fig. 1B and Supplemental Video 4).

Figure 2.

Figure 2

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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).

cAMP-stimulated steroid hormone biosynthesis requires microtubule polymerization

To define the physiological significance of perturbing the rate of mitochondrial movement, we collected media from cells treated with agents that alter microtubule and actin polymerization and determined the effect on cortisol and the adrenal androgen DHEA secretion. Both colchicine and nocodazole attenuated basal and Bt2cAMP-stimulated cortisol secretion and promoted the production of DHEA (Fig. 2B). In contrast, latrunculin B and paclitaxel increased basal secretion of cortisol but decreased basal and Bt2cAMP-stimulated DHEA biosynthesis.

cAMP-stimulated mitochondrial trafficking requires RhoA activation

Based on the data shown above demonstrating that ACTH promotes microtubule-dependent mitochondrial trafficking, we next sought to identify proteins involved in this process. Mitochondrial transport, particularly in neurons, has been linked to several cytoplasmic factors, including kinesins, dynamine, and the Rho family of GTPases (23,31,54,55,56,57). To identify components of the signaling pathway that couple ACTH to microtubule-dependent mitochondrial trafficking, we determined the effect of ACTH signaling on the activation of RhoA. As shown in Fig. 3A, the amount of active Rho (GTP bound) increased in cells treated with ACTH or Bt2cAMP within 5 min, with maximal activation for both stimuli at 15 min. Normalization of GTP-bound Rho to total RhoA revealed that Bt2cAMP elicited a 2- and 2.9-fold increase in active Rho at the 5- and 15-min time points, respectively (Fig. 3B). ACTH significantly increased active Rho after 15 min; however, the magnitude of the effects of ACTH were less than observed in response to Bt2cAMP. Neither ACTH nor Bt2cAMP increased cell division cycle 42, GTP-binding protein /Rac activity (data not shown).

Figure 3.

Figure 3

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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.

To further explore the role of Rho in mediating mitochondrial trafficking and steroidogenesis, we assessed the effect of a dominant-negative RhoA T19N mutant (58) on Bt2cAMP-stimulated mitochondrial movement. Transfection of the dominant-negative mutant attenuated both basal and Bt2cAMP-dependent mitochondrial trafficking (Fig. 3C). Notably, although cells expressing the RhoA T19N mutant secreted lower amounts of cortisol, unstimulated cells produced 2.9-fold more DHEA (Fig. 3D).

cAMP signaling induces the interaction between RhoA and DIAPH1

Because cAMP-dependent PKA phosphorylates RhoA at Ser-188 (59), we assessed the effect of ACTH/Bt2cAMP on the cellular levels of Ser-188 phosphorylated RhoA. As shown in Fig. 4, A and B, both ACTH and Bt2cAMP also increased the amount of phosphorylated RhoA in H295R lysates, with maximal effects occurring at the 30-min time point. Next we examined the contribution of RhoA effector proteins in this process. Activated Rho promotes changes in varied cellular functions, including cell migration, organelle trafficking, and cytoplasmic streaming, by interacting with effector proteins. Coimmunoprecipitation studies revealed that cAMP stimulates the interaction between RhoA and DIAPH1 in a time-dependent manner (Fig. 4C) and that the PKA inhibitor H89 prevents this interaction (Fig. 4D).

Figure 4.

Figure 4

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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).

The DIAPH1 effector mediates cAMP-stimulated mitochondrial movement and cortisol production

Next, we determined whether DIAPH1 affected cAMP-stimulated mitochondrial movement by transfecting cells with WT or mutant effector. The constitutively active DIAPH1 mutant (ΔN3) lacks the RhoA interaction domain and has been shown to align microtubules in parallel to F-actin bundles (44). As shown in Fig. 5A, overexpression of a GFP-tagged mutant DIAPH1 significantly altered the morphology of mitochondria whereby mitochondria appeared rounded and aggregated (compare mitochondria in transfected cells to mitochondria in adjacent untransfected cells). Consistent with previous findings (44), the DIAPH1 mutant also promoted longitudinal elongation of the H295R cells (Fig. 5A). Significantly, transfection with the constitutively active mutant DIAPH1 rendered mitochondria virtually immobile in both control and Bt2cAMP-stimulated cells (Fig. 5B and Supplemental Video 5). The loss of mitochondrial trafficking in cells transfected with the DIAPH1 mutant was restored by treatment with the actin polymerization inhibitor latrunculin B, but not with inhibitors of microtubule polymerization (Fig. 5B). Untreated cells overexpressing the constitutively active DIAPH1 mutant secreted approximately 50% less cortisol, compared with cells overexpressing wild-type DIAPH1 (Fig. 5C). Treatment of cells expressing mutant DIAPH1 with Bt2cAMP exhibited increased DHEA secretion but did not secrete cortisol. Finally, as shown in Fig. 5B, although latrunculin B increased cortisol production in cells expressing wild-type DIAPH1, the microfilament disruptor was unable to promote cortisol secretion in cells transfected with the constitutively active DIAPH1 mutant.

Figure 5.

Figure 5

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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.

DIAPH1 is required for mitochondrial movement and cortisol biosynthesis

To further explore the role of DIAPH1 in cortisol production, we transfected cells with siRNA oligonucleotides targeted against DIAPH1. Suppressing DIAPH1 translation reduced expression of the protein by 88% (Fig. 5D) and decreased both basal and Bt2cAMP-stimulated cortisol production, with a concomitant increase in DHEA secretion (Fig. 5E).

Discussion

In this study, we identified key roles for RhoA and DIAPH1 in regulating optimal production of cortisol in human adrenocortical cells. We also uncovered a novel mechanism through which ACTH promotes hormone production, by controlling the rate of mitochondrial movement in steroidogenic cells. We found that stabilizing microtubules or inhibiting actin polymerization promoted both mitochondrial movement and cortisol production, whereas preventing the assembly of microtubules resulted in impaired mitochondrial trafficking and suppressed cortisol secretion (Fig. 2). Our initial hypothesis was that impairing mitochondrial movement would decrease both DHEA and cortisol secretion. However, our data indicate that the delivery of pregnenolone is not dependent on cytoskeletal proteins or DIAPH1. It would be of interest to comprehensively quantify the effect of chemicals that perturb the assembly of microtubules and microfilaments on organellar concentrations of all steroid metabolites. Studies are underway to optimize mass spectrometric conditions to simultaneously quantify both cellular (in specific organelles) and secreted steroid metabolites. Interestingly, the loss of mitochondrial movement increased the amount of DHEA produced in the cells. As mentioned earlier, roles for cytoskeletal protein steroidogenesis have been previously demonstrated, whereby chemical agents that perturb microtubule and actin polymerization alter steroid hormone production in gonadal and adrenal cells (10,11,17,18,19,20,21). However, these studies have yielded conflicting data with regard to the importance of microtubules and microfilaments. For example, colchicine has been shown to impair progesterone production in ovine follicular cells (60) and stimulate progesterone secretion from porcine luteal cells (61). Although species differences may explain some of the discrepancies between these studies, similar conflicting results have been found in adrenocortical cells (15,16,20,21,62).

Our data showing that the dominant-negative mutant of a Rho family protein prevents the movement of mitochondria (Fig. 3C) are consistent with the findings of Gasman et al. (63), who found that GTPase-deficient RhoD impairs the movement of endosomes in HeLa cells. Interestingly, these researchers found that a constitutively active DIAPH2 mutant that lacked the Rho-binding domain mimicked the loss of endosome trafficking evoked by the GTPase-dead RhoD mutant. Similar results were obtained in the current study in which transfecting H295R adrenocortical cells with a dominant-negative RhoA mutant rendered mitochondrial largely immobile (Fig. 3C). Given that DIAPH proteins play pivotal roles in organizing the alignment of both actin filaments and microtubules (44), it is possible that expression of the constitutively active mutant alters the ratio and/or organization of microtubules and F-actin. Indeed, the movement of mitochondria is regulated by the dynamic interplay between actin filaments and microtubules (23,31,54,55,56,57). In contrast to the inhibitory effect of constitutively active DIAPH1 in endosome (63) and mitochondrial movement (this study), a stimulatory role for constitutively active DIAPH1 has been found in the induction transcripts targeted by the serum response factor-dependent pathway (64,65,66). Collectively these studies implicate different functional domains of DIAPH proteins in mediating distinct cellular processes, probably by interacting with unique effector proteins in a stimulus-dependent manner.

We found that ACTH and Bt2cAMP simultaneously increase the cellular levels of active GTP-bound RhoA (Fig. 3, A and B) and phosphorylated RhoA (Fig. 4, A and B). These findings are in contrast to the reported inhibitory effects of PKA on several Rho-mediated cellular processes. Phosphorylation of RhoA by PKA at Ser-188 decreases the binding of RhoA to Rho kinase Rho-associated serine/threonine kinase (67) and inhibits thrombin-induced RhoA activation and translocation (68). cAMP has also been shown to increase Rho phosphorylation at Ser-188 and inhibit lysophosphatidic acid-stimulated RhoA activation and F-actin formation in SGC-7901 human gastric cancer cells (69). In human natural killer cells, PKA-catalyzed phosphorylation of RhoA does not affect the ability of the protein to bind GTP or alter GTPAse activity but promotes RhoA translocation from the plasma membrane to the cytosol (59). Because nerve growth factor has been found to stimulate PKA-mediated phosphorylation of RhoA on Ser-188 and inhibit its binding to ROK but not to DIAPH1 or rhotekin in PC12 adrenal medullary cells (70), it is possible that ACTH signaling and PKA-catalyzed phosphorylation of RhoA may differentially affect partnering to effector proteins in H295R adrenocortical cells. Notably, phosphorylation of RhoA by PKA protects the GTPase, particularly GTP-bound RhoA, from ubiquitin-mediated proteasomal degradation (71). The simultaneous increase in active RhoA (Fig. 3A) and Ser-188 phosphorylated RhoA (Fig. 4A) in response to Bt2cAMP suggests that ACTH signaling may regulate cortisol biosynthesis by controlling the cellular levels of endogenous RhoA in H295R cells. However, further study is required to determine whether ACTH alters the stability of active RhoA.

Our data provide evidence for a role for DIAPH1 in conferring mitochondrial movement and cortisol secretion in response to ACTH/cAMP stimulation by coordinating microtubule organization. Interestingly, breakpoint mutations in DIAPH1 have been identified in a family of patients with infertility due to premature ovarian failure (72). This breakpoint was mapped to the last intron of the gene and has been postulated to lead to an unstable truncated transcript that lacks the last coding exon and 3′ untranslated region. Lartey et al. (73) found that elevated myometrial DIAPH1 results in spontaneous preterm labor. Elevated levels of adrenal androgens have been implicated in infertility and polycystic ovarian syndrome (74,75). Moreover, prenatal exposure to androgens is a critical factor in fetal programming (76,77,78). Given that suppressing DIAPH1 increases adrenal androgen secretion (Fig. 5E), future studies are warranted in examining the effects of mutations in DIAPH1 on circulating levels of adrenal androgens, particularly during ovulation and pregnancy. A role for Rho-associated, coiled-coil-containing protein kinase in regulating adrenal androgen production has recently been suggested in studies examining the effects of phosphorylating P450c17, in which inhibition of Rho-associated, coiled-coil-containing protein kinase activity decreased the lyase activity of P450c17 (79). Because P450c17 plays a critical role in determining the ratio of glucocorticoids to androgens produced, these findings along with our data presented herein identify novel roles for the RhoA pathway in regulating multiple steps in adrenocortical steroid hormone biosynthesis.

One important caveat to take into account with our findings is the cell culture system that was used to carry out the studies. Although these conditions (Nu-Serum I and ITS+ premix; BD Biosciences) have been established as optimal for investigating steroidogenesis that occurs in the zona fasciculata and zona reticularis (47,53), it is likely that the presence of cortisol and other steroid hormones in Nu-Serum I affects the rate of mitochondrial trafficking in response to ACTH. Indeed, we found that the rate of mitochondrial movement in serum-starved cells (48 h serum withdrawal) is approximately 1.3-fold higher than in cells grown in the presence of Nu-Serum I (Li, D., and M. B. Sewer, unpublished observations). However, because we also found that 48 h of serum starvation also markedly induces the expression of multiple steroidogenic genes [CYP17, CYP11A1, and CYP11B (Li, D., and M. B. Sewer, unpublished observations)], it is possible that increased gene expression may also contribute to an increase in hormone production that is independent of interorganelle substrate delivery. Perhaps more significant is the potential role for steroid hormones (particularly cortisol) present in Nu-Serum I in differentially regulating the efflux of cortisol and DHEA from adrenocortical cells. Organic anionic transporters have been shown to regulate cortisol secretion from H295R cells (80). Moreover, the expression of these transporters is regulated by forskolin and DHEA sulfate (81). Finally, we have preliminary mass spectrometric data that identify oxysterol-related-binding proteins as DIAPH1 binding partners (Li, D., and M. B. Sewer, unpublished observation). Given that oxysterol-related-binding proteins are implicated in the interorganelle transport of multiple classes of lipids (82), it is possible that the steroid hormones in Nu-Serum I may contribute to the rate of mitochondrial movement observed in the current study by controlling the expression and/or activity of lipid transporters and binding proteins.

In summary, we have identified RhoA and DIAPH1 as key regulators in mediating ACTH/cAMP-stimulated mitochondrial trafficking in H295R adrenocortical cells. We postulate that this movement is integral for assuring optimal cortisol secretion and maintaining appropriate levels of adrenal androgen production. Given that aldosterone biosynthesis requires interorganelle substrate delivery, it is possible that angiotensin II may regulate mitochondrial trafficking. However, future studies are required to determine the role of mitochondrial movement in angiotensin II-stimulated aldosterone secretion. Based on the data presented herein, we propose a model whereby ACTH signaling promotes RhoA activation and subsequent interaction with DIAPH1 (Fig. 6). The assembly of the RhoA/DIAPH1 complex may facilitate interorganelle substrate delivery by stimulating the microtubule-dependent movement of mitochondria. Finally, given the stimulatory effect of impairing mitochondrial trafficking on the secretion of adrenal androgens, our data herein coupled with previous studies (72) provide evidence for altered DIAPH1 function in pathologies such as nonclassical congenital adrenal hyperplasia and polycystic ovary syndrome.

Figure 6.

Figure 6

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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.

Supplementary Material

[Supplemental Data]
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Footnotes

This work was supported by National Institutes of Health/National Institute of General Medical Sciences Grant GM073241 and National Science Foundation Grant MCB1005815.

Disclosure Summary: D.L. and M.B.S. have nothing to declare.

First Published Online June 30, 2010

Abbreviations: Bt2cAMP, Dibutyryl cAMP; DAD, diaphanous-autoregulatory domain; DHEA, dehydroepiandrosterone; DIAPH, diaphanous-related homolog; GFP, green fluorescent protein; PKA, protein kinase A; siRNA, small interfering RNA.

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