Mitochondrial StAR protein <i>per se</i> is not enough to sustain steroidogenesis stimulated by cAMP.

<p>Design of two-phase experiments. <b>A.</b> Cells were incubated in the absence or presence of 8Br-cAMP (cAMP) (1 mM) and CCCP (5 µM) for 1 h or 2 h (Phase I; Treatment). Here, the proteasome inhibitor MG132 (MG; 5 µM) was used to avoid cytoplasmatic StAR degradation. Then, these additives were removed (wash-out) with fresh media (Phase II; Recovery). Among cells incubated with cAMP plus CCCP, those treated for 2 h in Phase I recovered after 2 h in Phase II, while those treated for 1 h in Phase I recovered after 3 h in Phase II, reaching in both cases a 4 h final experimental time. <b>B.</b> Mitochondrial proteins from cells in Phase I were obtained and western blotting was performed. Membranes were sequentially blotted for StAR and III Complex. A representative western blot is shown. For each band, the OD of expression levels of StAR protein were quantified (arbitrary units) and normalized to the corresponding III Complex protein. The relative levels of StAR protein are shown: ¥¥¥ <i>p</i><0.001 vs. control; ***<i>p</i><0.001 vs. cAMP alone. <b>C.</b> In another set of experiments, cells were sequentially subjected to Phase I and II, thereafter mitochondrial proteins were isolated and western blotting was performed as indicated in panel B. The relative levels of StAR protein are shown: ### <i>p</i><0.001 vs. 1 h cAMP +CCCP in Phase I; ## <i>p</i><0.01 vs. 2 h cAMP +CCCP in Phase I. <b>D.</b> P4 production in the culture media from Phase I and II was measured by RIA and data are shown as P4 concentration (ng/ml): ¥¥¥ <i>p</i><0.001 vs. control; ***<i>p</i><0.001 vs. cAMP alone in Phase I, **<i>p</i><0.01 vs. cAMP alone in Phase I, ### <i>p</i><0.001 vs. recovery from cAMP alone. <b>E.</b> Mitochondrial proteins from cells in Phase I were obtained and western blotting was performed. Membranes were sequentially blotted for phospho-ERK1/2 (pERK) and total ERK1/2 (tERK). A representative western blot is shown. For each band, the OD of expression levels of pERK were quantified (arbitrary units) and normalized to the corresponding tERK protein. The relative levels of pERK are shown: ***<i>p</i><0.001 vs. control, **<i>p</i><0.01 vs. control, ### <i>p</i><0.001 vs. 1 h cAMP alone, ## <i>p</i><0.01 vs. 2 h cAMP alone. <b>F.</b> In another set of experiments, cells were sequentially subjected to Phase I and II, thereafter mitochondrial proteins were isolated and western blotting was performed as indicated in panel <b>E.</b> The relative levels of pERK are shown: ns <i>p</i>>0.05 vs. cAMP +CCCP in Phase I. Results are expressed as mean ± SEM of three independent experiments.</p

ERK phosphorylation of StAR is required for the correct association of StAR with the mitochondria.

<p>Cells were transfected with an empty pRc/CMVi vector (mock) or containing the StAR wt cDNA (StAR wt) or StAR mutant form (StAR S232A). After 48 h, cells were stimulated for 1 h with <b>A.</b> hCG (20 ng/ml) or <b>B.</b> 8Br-cAMP (cAMP) (0.5 mM). Membranes were sequentially blotted for StAR and III Complex. Representative western blots are shown. For each band, the OD of the expression levels of StAR protein were quantified (arbitrary units) and normalized to the corresponding III Complex protein. The relative levels of StAR protein are shown: <b>A.</b> ***<i>p</i><0.001 vs. control mock; **<i>p</i><0.01 vs. control StAR wt; ### <i>p</i><0.001 vs. hCG StAR wt. <b>B.</b> ***<i>p</i><0.001 vs. control mock; **<i>p</i><0.01 vs. control StAR wt; ## <i>p</i><0.01 vs. cAMP StAR wt. Results are expressed as mean ± SEM of three independent experiments. Progesterone production was determined by RIA in the incubation media. Results are indicated in a Table at the bottom of each panel. Data are expressed as ng/ml of P4.</p

Summary of the proposed mechanisms for the association of StAR to the OMM in MA-10 Leydig cells following hormone stimulation.

<p>After hormone stimulation, mitochondrial fusion induction through Mfn2 up-regulation in mitochondria is needed for increasing StAR mRNA levels. Also, mitochondrial fusion is required post-transcriptionally for StAR localization at the OMM and subsequent binding to a cholesterol molecule. Then, cholesterol-bound StAR is available for ERK and PKA mitochondrial phosphorylation, increasing its activity and cholesterol transport. After the 2 h temporal frame of ERK activity, StAR is dephosphorylated, de-activated and translocated to the mitochondrial matrix where it is degraded by mitochondrial proteases. Abbreviations: pERK1/2 (phospho-ERK1/2), pStAR: chol (phospho-StAR bound to a cholesterol molecule), PKA(c) (PKA catalytic subunit), StAR (m) (StAR in the mitochondrial matrix).</p

Mitochondrial fusion modulates StAR mRNA levels.

<p>Cells were transfected with an empty pSUPER.Retro vector (mock) or containing a Mfn2-shRNA. After 48 h, cells were incubated with or without 8Br-cAMP (cAMP) (0.5 mM) for 1 h. Total RNA was isolated; reverse transcribed and subjected to Real-Time PCR.using specific primers. StAR mRNA expression levels were normalized to mouse 18S RNA expression, performed in parallel as endogenous control. Real-time PCR data were analyzed by calculating the 2^−ΔΔCt value (comparative Ct method) for each experimental sample. The relative expression levels of StAR are shown: ### <i>p</i><0.001 vs. control mock; ***<i>p</i><0.001 vs. cAMP mock. Results are expressed as mean ± SEM of three independent experiments.</p

Mfn2 protein is necessary for StAR and pERK mitochondrial localization.

<p>Cells were transfected with an empty pSUPER.Retro vector (mock) or containing a Mfn2-shRNA. After 48 h, cells were stimulated with hCG (20 ng/ml) or 8Br-cAMP (cAMP) (0.5 mM) for 1 h. Mitochondrial proteins were obtained and western blotting was performed. Membranes were sequentially blotted with anti-StAR, phospho-ERK (pERK) or anti-Mfn2 antibodies, and III Complex antibody as loading control. An image of a representative western blot is shown. For each band, the OD of expression levels of StAR and pERK proteins were quantified (arbitrary units) and normalized to the corresponding III Complex protein. <b>A.</b> The relative levels of StAR protein are shown: ¥¥¥ <i>p</i><0.001 and ¥¥ <i>p</i><0.01 vs. control mock; **<i>p</i><0.01 vs. cAMP mock; # <i>p</i><0.05 vs. hCG mock. <b>B.</b> The relative levels of pERK protein are shown: ¥¥¥ <i>p</i><0.001 vs. control mock; **<i>p</i><0.01 vs. cAMP mock; ### <i>p</i><0.001 vs. hCG mock. Results are expressed as mean ± SEM of three independent experiments.<b>C.</b> An image of a representative western blot is shown to assess shRNA knockdown efficiency. <b>D.</b> Progesterone production was determined by RIA in the incubation media. Data are expressed as ng/ml of P4.</p

ERK phosphorylation of StAR in Ser232 drives StAR retention on the OMM.

<p>pRc/CMVi vector containing the StAR wt cDNA (StAR wt) or StAR mutant form (StAR S232A) were subjected to a transcription/translation import assay and <i>in vitro</i> phosphorylation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100387#s2" target="_blank">Materials and Methods</a>). Mitochondrial proteins were separated by SDS-PAGE, transfered to a PVDF membrane and then autoradiography and immunoblot were performed. <b>A.</b> Levels of phosphorylated StAR (pStAR) are shown in the presence or absence of active ERK1 (upper panel). The autoradiography displays a representative result of three independent experiments. The immunoblots (lower panels) show total mitochondrial StAR and phospho-ERK (pERK) levels. Representative western blots are shown. <b>B.</b> For each band, the OD of pStAR levels were quantified (arbitrary units) and normalized to the corresponding total StAR protein levels. The relative levels of pStAR are shown: ***<i>p</i><0.001 vs. StAR wt without ERK1; ### <i>p</i><0.001 vs. StAR wt with ERK1. Results are expressed as mean ± SEM of three independent experiments.</p

ERK and PKA are strictly required as mediators to achieve maximal steroidogenesis in the presence of mitochondrial StAR.

<p><b>A.</b> Design of two-phase experiments. Cells were incubated in the absence or presence of 8Br-cAMP (cAMP) (1 mM) and CCCP (5 µM) for 2 h (Phase I; Treatment). Here, the proteasome inhibitor MG132 (MG; 5 µM) was used to avoid cytoplasmatic StAR degradation. Then, these additives were removed (wash-out) with fresh media (Phase II; Recovery) for 3 h. For panels B and C, mitochondria from cells subjected to the two-phase assay were isolated and incubated, with cholesterol (50 µM) as substrate, in the presence (grey bars) or absence (black bars) of constitutively active His-tagged ERK1 together with PKA catalytic subunit. After the above incubations, mitochondria were pelleted, media was collected and mitochondrial P4 production was measured by RIA. Data are shown as P4 concentration (ng/ml): <b>B.</b> Phase I: ns <i>p</i>>0.05 b vs. a; ***<i>p</i><0.001 c vs. a; ## <i>p</i><0.01 d vs. c; € <i>p</i><0.05 e vs. c; ns <i>p</i>>0.05 f vs. e. <b>C.</b> Phase II: ns <i>p</i>>0.05 h vs. g; §§§ <i>p</i><0.001 i vs. g; ¥¥ <i>p</i><0.01 j vs. i. Results are expressed as mean ± SEM of three independent experiments. <b>D.</b> In another set of experiments, cells were sequentially subjected to Phase I and II. During Phase II, incubation was conducted in the presence or absence of H89 (20 µM) or PD98059 (PD; 50 µM). Then, media was collected and P4 production was measured by RIA. Data are shown as P4 concentration (ng/ml): ns <i>p</i>>0.05 with inhibitors alone or both vs. without inhibitors.</p

StAR mitochondrial levels depend on post-transcriptional events.

<p>Cells were transfected with a pRc/CMVi vector containing the cDNA of wild type StAR full length (StAR wt). <b>A.</b> Cells were incubated in the absence or presence of 8Br-cAMP (cAMP) (1 mM) and CCCP (5 µM) for 1 h or 2 h (Phase I; Treatment). Here, the proteasome inhibitor MG132 (MG; 5 µM) was used to avoid cytoplasmatic StAR degradation. Then, these additives were removed (wash-out) with fresh media (Phase II; Recovery). Among cells incubated with cAMP plus CCCP, those treated for 2 h in Phase I recovered after 2 h in Phase II, while those treated for 1 h in Phase I recovered after 3 h in Phase II, reaching in both cases a 4 h final experimental time. <b>B.</b> Mitochondrial proteins from cells in Phase I were obtained and western blotting was performed. Membranes were sequentially blotted for StAR and III Complex. A representative western blot is shown. For each band, the OD of expression levels of StAR protein were quantified (arbitrary units) and normalized to the corresponding III Complex protein. The relative levels of StAR protein are shown: ¥¥¥ <i>p</i><0.001 and ¥¥ <i>p</i><0.01 vs. control; ### <i>p</i><0.001 vs. cAMP alone. <b>C.</b> In another set of experiments, cells were sequentially subjected to Phase I and II, thereafter mitochondrial proteins were isolated and western blotting was performed as indicated in panel B. The relative levels of StAR protein are shown. <b>D.</b> cAMP stimulated- and CCCP treated-cells were incubated with or without MG132 (5 µM) during Phase II. Mitochondrial proteins were obtained and western blotting was performed as indicated in panel B: ### <i>p</i><0.001 vs. 3 h Recovery Time (Phase II) without MG. Results are expressed as mean ± SEM of three independent experiments.</p

Double phosphorylation of ERK by MEK relies almost completely on mitochondrial phospho-Thr183.

<p>LP07 cells were transiently transfected with wild type ERK2-V5 (WT), Y183A or T183A ERK mutants, using Lipofectamine 2000 reagent. Forty-eight hours post-transfection LP07 were stimulated with 1 μM H₂O₂ during 0–30 min and then incubated in the presence or absence of 1 μM H₂O₂ for the indicated times. Then, the cytosolic, mitochondrial and nuclear fractions were obtained and the detection of ERK2 was analyzed by western blot using anti-V5 antibody. Representative images of cytosolic and mitochondrial fractions ERK2-WT (Panel A) and nuclear fraction ERK2-WT (panel D) distribution and representative images of Y185A and T185A distribution in the cytosolic (Panel B), mitochondrial (Panel C) and nuclear (Panel D) fractions, from three independent experiments. Relative levels of ERK mutants in cytosol and mitochondria relative to basal conditions, arbitrarily defined as 1 (Panels B and C). Data are expressed as the mean ± SD of three independent experiments * p<0.05, **p<0.01 vs. 0 min with H₂O₂.</p

Mitochondrial ERK localization depends on differential phosphorylation.

<p>Representative confocal images of LP07 tumor lung transfected cells with wild type ERK/V5 WT (left panel), Y183A/V5 (middle panel) and T183A/V5 (right panel). Forty-eight hours post transfection, cells were incubated with 1 μM H₂O₂ during 0–30 min. From top to bottom of the panels images show increasing H₂O₂ incubating times. Mitochondria were visualized in red by staining with Mitotracker Deep Red. Cells were fixed and incubated with anti-V5 antibody and a secondary antibody conjugated with Cy2, and analyzed in an Olympus FV1000 confocal microscope. Co-localization was highlighted in green on the grayscale images (last column of each panel). Images directly exported from Olympus Fluoview acquisition program. Fold changes relative to control images of combined MitoTracker and Cy2 intensity for ERK2 WT (Panel A), Y185A (Panel B) and T183A (Panel C). Basal conditions without H₂O₂ arbitrarily defined as 1. Bar = 10 μm.</p