Phytoestrogens Enhance the Vascular Actions of the Endocannabinoid Anandamide in Mesenteric Beds of Female Rats

In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular adrenergic hyperactivity that usually precedes the onset of primary hypertension, the oral administration (3 daily doses) of either 10 mg/kg genistein or 20 mg/kg daidzein potentiated the anandamide-induced reduction of contractility to NA in female but not in male rats. Oral treatment with phytoestrogens also restored the vascular effects of anandamide as well as the mesenteric content of calcitonin gene-related peptide (CGRP) that were reduced after ovariectomy. The enhancement of anandamide effects caused by phytoestrogens was prevented by the concomitant administration of the estrogen receptor antagonist fulvestrant (2.5 mg/kg, s.c., 3 daily doses). It is concluded that, in the vasculature of female rats, phytoestrogens produced an estrogen-receptor-dependent enhancement of the anandamide-vascular actions that involves the modulation of CGRP levels and appears to be relevant whenever an adrenergic hyperactivity occurs

A Mitochondrial Kinase Complex Is Essential to Mediate an ERK1/2-Dependent Phosphorylation of a Key Regulatory Protein in Steroid Biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein –a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser232. Directed mutagenesis of Ser232 to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined

Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis

Mitochondrial fusion and ERK activity regulate steroidogenic acute regulatory protein localization in mitochondria.

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR

Phytoestrogens Enhance the Vascular Actions of the Endocannabinoid Anandamide in Mesenteric Beds of Female Rats

In rat isolated mesenteric beds that were contracted with NA as an in vitro model of the vascular adrenergic hyperactivity that usually precedes the onset of primary hypertension, the oral administration (3 daily doses) of either 10 mg/kg genistein or 20 mg/kg daidzein potentiated the anandamide-induced reduction of contractility to NA in female but not in male rats. Oral treatment with phytoestrogens also restored the vascular effects of anandamide as well as the mesenteric content of calcitonin gene-related peptide (CGRP) that were reduced after ovariectomy. The enhancement of anandamide effects caused by phytoestrogens was prevented by the concomitant administration of the estrogen receptor antagonist fulvestrant (2.5 mg/kg, s.c., 3 daily doses). It is concluded that, in the vasculature of female rats, phytoestrogens produced an estrogen-receptor-dependent enhancement of the anandamidevascular actions that involves the modulation of CGRP levels and appears to be relevant whenever an adrenergic hyperactivity occurs

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

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

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