Non-classical testosterone signaling is mediated by a G-protein-coupled receptor interacting with Gnα11

AbstractTestosterone is known to mediate its effects by two different mechanisms of action. In the so-called “classical” pathway testosterone binds to cytosolic androgen receptors (AR), which essentially function as ligand-activated transcription factors. Once activated, these receptors bind to DNA and activate the expression of target genes. In the “non-classical” pathway, the steroid hormone binds to receptors associated with the plasma membrane and induces signaling cascades mediated through activation of Erk1/2. The precise nature of the membrane-associated AR, however, remains controversial. Although some assume that the membrane and cytosolic AR are identical, others propose that the AR of the membrane is a G-protein-coupled receptor (GPCR). To evaluate these two possibilities we first searched for testosterone-induced signaling cascades in the spermatogenic cell line GC-2. Testosterone was found to cause phosphorylation (activation) of Erk1/2, CREB, and ATF-1, consistent with its non-classical mechanism of action. Silencing of AR expression by means of siRNA did not influence testosterone-induced activation of Erk1/2, CREB, or ATF-1, indicating that this pathway is not activated by the classical cytosolic/nuclear AR. In contrast, when the expression of the G-protein Gnα11 is suppressed, the activation of these signaling molecules is abolished, suggesting that these responses are elicited through a membrane-bound GPCR. The results presented here and the identification of the testosterone-specific GPCR in future investigations will help to reveal and characterize new testosterone-mediated mechanisms associated not only with fertility and reproduction but perhaps also with other physiological processes

Dehydroepiandrosterone sulfate stimulates expression of blood-testis-barrier proteins claudin-3 and -5 and tight junction formation via a Gna11-coupled receptor in Sertoli cells

Dehydroepiandrosterone sulfate (DHEAS) is a circulating sulfated steroid considered to be a pro-androgen in mammalian physiology. Here we show that at a physiological concentration (1 µM), DHEAS induces the phosphorylation of the kinase Erk1/2 and of the transcription factors CREB and ATF-1 in the murine Sertoli cell line TM4. This signaling cascade stimulates the expression of the tight junction (TJ) proteins claudin-3 and claudin-5. As a consequence of the increased expression, tight junction connections between neighboring Sertoli cells are augmented, as demonstrated by measurements of transepithelial resistance. Phosphorylation of Erk1/2, CREB, or ATF-1 is not affected by the presence of the steroid sulfatase inhibitor STX64. Erk1/2 phosphorylation was not observed when dehydroepiandrosterone (DHEA) was used instead of DHEAS. Abrogation of androgen receptor (AR) expression by siRNA did not affect DHEAS-stimulated Erk1/2 phosphorylation, nor did it change DHEAS-induced stimulation of claudin-3 and claudin-5 expression. All of the above indicate that desulfation and conversion of DHEAS into a different steroid hormone is not required to trigger the DHEAS-induced signaling cascade. All activating effects of DHEAS, however, are abolished when the expression of the G-protein Gna11 is suppressed by siRNA, including claudin-3 and -5 expression and TJ formation between neighboring Sertoli cells as indicated by reduced transepithelial resistance. Taken together, these results are consistent with the effects of DHEAS being mediated through a membrane-bound G-protein-coupled receptor interacting with Gna11 in a signaling pathway that resembles the non-classical signaling pathways of steroid hormones. Considering the fact that DHEAS is produced in reproductive organs, these findings also suggest that DHEAS, by acting as an autonomous steroid hormone and influencing the formation and dynamics of the TJ at the blood-testis barrier, might play a crucial role for the regulation and maintenance of male fertility

Author Correction: Deficiency of Axl aggravates pulmonary arterial hypertension via BMPR2.

Abstract: Pulmonary arterial hypertension (PAH), is a fatal disease characterized by a pseudo-malignant phenotype. We investigated the expression and the role of the receptor tyrosine kinase Axl in experimental (i.e., monocrotaline and Su5416/hypoxia treated rats) and clinical PAH. In vitro Axl inhibition by R428 and Axl knock-down inhibited growth factor-driven proliferation and migration of non-PAH and PAH PASMCs. Conversely, Axl overexpression conferred a growth advantage. Axl declined in PAECs of PAH patients. Axl blockage inhibited BMP9 signaling and increased PAEC apoptosis, while BMP9 induced Axl phosphorylation. Gas6 induced SMAD1/5/8 phosphorylation and ID1/ID2 increase were blunted by BMP signaling obstruction. Axl association with BMPR2 was facilitated by Gas6/BMP9 stimulation and diminished by R428. In vivo R428 aggravated right ventricular hypertrophy and dysfunction, abrogated BMPR2 signaling, elevated pulmonary endothelial cell apoptosis and loss. Together, Axl is a key regulator of endothelial BMPR2 signaling and potential determinant of PAH

Role of the Purinergic P2Y2 Receptor in Pulmonary Hypertension

Pulmonary arterial hypertension (PAH), group 1 pulmonary hypertension (PH), is a fatal disease that is characterized by vasoconstriction, increased pressure in the pulmonary arteries, and right heart failure. PAH can be described by abnormal vascular remodeling, hyperproliferation in the vasculature, endothelial cell dysfunction, and vascular tone dysregulation. The disease pathomechanisms, however, are as yet not fully understood at the molecular level. Purinergic receptors P2Y within the G-protein-coupled receptor family play a major role in fluid shear stress transduction, proliferation, migration, and vascular tone regulation in systemic circulation, but less is known about their contribution in PAH. Hence, studies that focus on purinergic signaling are of great importance for the identification of new therapeutic targets in PAH. Interestingly, the role of P2Y2 receptors has not yet been sufficiently studied in PAH, whereas the relevance of other P2Ys as drug targets for PAH was shown using specific agonists or antagonists. In this review, we will shed light on P2Y receptors and focus more on the P2Y2 receptor as a potential novel player in PAH and as a new therapeutic target for disease management

Dehydroepiandrosterone sulfate mediates activation of transcription factors CREB and ATF-1 via a Gα11-coupled receptor in the spermatogenic cell line GC-2

AbstractDehydroepiandrosterone sulfate (DHEAS) is a circulating steroid produced in the adrenal cortex, brain, and gonads. Whereas a series of investigations attest to neuroprotective effects of the steroid in the brain, surprisingly little is known about the physiological effects of DHEAS on cells of the reproductive system. Here we demonstrate that DHEAS acting on the spermatogenic cell line GC-2 induces a time- and concentration-dependent phosphorylation of c-Src and Erk1/2 and activates the transcription factors activating transforming factor-1 (ATF-1) and cyclic AMP-responsive element binding protein (CREB). These actions are consistent with the non-classical signaling pathway of testosterone and suggest that DHEAS is a pro-androgen that is converted into testosterone in order to exert its biological activity. The fact, however, that steroid sulfatase mRNA was not detected in the GC-2 cells and the clear demonstration of DHEAS-induced activation of Erk1/2, ATF-1 and CREB after silencing the androgen receptor by small interfering RNA (siRNA) clearly contradict this assumption and make it appear unlikely that DHEAS has to be converted in the cytosol into a different steroid in order to activate the kinases and transcription factors mentioned. Instead, it is likely that the DHEAS-induced signaling is mediated through the interaction of the steroid with a membrane-bound G-protein-coupled receptor, since silencing of Guanine nucleotide-binding protein subunit alpha-11 (Gnα11) leads to the abolition of the DHEAS-induced stimulation of Erk1/2, ATF-1, and CREB. The investigation presented here shows a hormone-like activity of DHEAS on a spermatogenic cell line. Since DHEAS is produced in male and female reproductive organs, these findings could help to define new roles for DHEAS in the physiology of reproduction

DHEAS-induced stimulation of claudin-3 and claudin-5 expression after silencing AR expression.

<p>The conditions are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150143#pone.0150143.g006" target="_blank">Fig 6</a>, with the exception that the cells were incubated with either negative control siRNA (nc-siRNA) or with AR-specific siRNA (AR-siRNA) to prevent its expression before treatment with DHEAS. Nuclei of the cells were labeled with DAPI and appear blue. (A-F) Stimulation of claudin-3 expression by DHEAS after incubation of cells with either nc-siRNA (A, B, and C) or AR-siRNA (D, E and F). (G-L) Stimulation of claudin-5 expression by DHEAS after treatment of the cells with either nc-siRNA (G, H, and I) or with AR-siRNA (J, K, and L). For the statistics shown in C, F, I, and L: n = 45; means ±SEM; **p≤0.01.</p

DHEAS-induced phosphorylation of Erk1/2 after silencing AR expression by siRNA.

<p>(A) AR expression, indicated by the green fluorescence of the secondary antibody, in TM4 cells exposed to negative control siRNA (nc-siRNA). (B) Abrogation of AR expression after treatment of the cells with AR-specific siRNA (AR-siRNA). In both A and B, nuclei are stained blue. (C) Western blot showing that the expression of AR in the presence of AR-siRNA is reduced by 94 ± 5% (n = 3). (D) At the same time expression of actin is not affected by AR-siRNA, indicating that the reduction of AR in (C) is specific and not due to an overall suppression of protein expression. (E) Erk1/2 phosphorylation in response to DHEAS after abrogation of AR expression with AR-specific siRNA. (F) Total Erk1/2 levels after treatment of cells with either nc-siRNA or AR-siRNA. The western blot shown was generated from the western blot shown in (E) which was first stripped of the original antibodies and then reprobed with appropriate antibodies to detect total Erk1/2. (G) Statistical analysis of Erk1/2 activation in the presence of either nc-siRNA or AR-siRNA (n = 3; mean ±SEM; **p≤0.01).</p

DHEAS-induced activation of Erk1/2, CREB and ATF-1 detected in western blots.

<p>TM4 cells were treated for 120 min with the indicated concentrations of DHEAS. Proteins in cell lysates were then separated on SDS polyacrylamide gels and subsequently probed in a western blot using a monoclonal antibody against either total Erk1/2 (t-Erk1/2) (A), as a loading control, or phosphorylated (activated) Erk1/2 (p-Erk1/2) (B). The western blots in (A) and (B) show typical results for the Erk1/2 bands of 42/44 kDa. (C) Statistical analysis of Erk1/2 activation as a function of DHEAS concentration in several identical experiments in which the chemiluminescence was quantified by gel image analysis software (n = 4; mean ±SEM; *p≤0.05; **p≤0.01). (D) Detection of total actin served as loading control in further western blots for the detection of either phosphorylated CREB or ATF-1 (E). The western blots in (D) and (E) show representative results from several identical experiments using the indicated concentrations of DHEAS; the quantification and statistical analysis of these results are shown in (F) and (G) (n = 4; mean±SEM; *p≤0.05; **p≤0.01).</p

Silencing expression of Gnα11 by means of siRNA.

<p>Cells were treated for 3 days with OptiMem plus Lipofectamine RNAiMAX alone (1) or OptiMem plus Lipofectamine RNAiMAX plus the siRNA negative control (2) or OptiMem plus Lipofectamine RNAiMAX plus the Gnα11-specific siRNA (3). Cells were then used to either isolate mRNA for RT-PCR, for immunofluorescence or for western blot experiments. (A) RT-PCR for the detection of GAPDH-, Gnα11- or Gnαq-specific mRNA. Effects on GAPDH mRNA/cDNA (430 bp) are shown on the left, results for Gnα11-specific mRNA/cDNA (917 bp) are shown in the center, and effects on Gnαq (688 bp) are shown on the right. (B) Detection of Gnα11 by immunofluorescence. The green fluorescence indicates Gnα11, and the blue refers to DAPI-stained nuclei. In both western blotting (A) and immunofluorescence (B) experiments, treatment of cells with Gnα11-specific siRNA (panel labeled “3”) abrogates Gnα11 expression. (C) Results of western blotting using an antibody against Gnα11 on lysates from cells treated with either nc-siRNA or Gnα11-siRNA. Effects on expression of actin (left panel) and Gnα11 (right panel, upper band, 42 kDa). The lower band also recognized by the antibody is Gnαq (40 kDa). All results shown are representative of n = 3 similar experiments.</p

DHEAS-stimulated expression of claudin-3 and claudin-5 proteins as detected by immunofluorescence.

<p>TM4 Sertoli cells were cultured to 80% confluence and were further incubated for 2 days in the absence or presence of 1 μM DHEAS. Nuclei were labeled with DAPI and appear blue; the Alexa fluor 488-labeled secondary antibody shows the localization of claudin-3 or -5. (A and D) Fluorescence in the absence of DHEAS; (B and E) Fluorescence after exposure of cells to 1 μM DHEAS. (C) Quantification and statistical analysis of results shown in panels (A) and (B); (F) Quantification and statistical analysis of results shown in panels (D) and (E) (in each case n = 45; means ±SEM; **p≤0.01).</p