17ß-Estradiol Regulates mTORC2 Sensitivity to Rapamycin in Adaptive Cardiac Remodeling

Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17β-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)α- and ERβ-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured female cardiomyocytes. These data highlight a novel modulatory function of E2 on rapamycin effect on mTORC2 in female cardiomyocytes and regulation of SERCA2A expression by mTORC2. Conceivably, rapamycin abrogates the premenopausal “female advantage”

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1RAbs- induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis

mTOR complex protein expressions and rictor phosphorylation at Ser1235.

<p>Cardiomyocytes were pretreated for 30 min with 20 nM rapamycin and then stimulated with 10 nM IGF-1 in presence or absence of 10 nM E2 for 24 h. A, shown are representative western blots for mTOR, rictor and raptor and B,C,D, quantitative analysis with mean ± SEM of fold stimulation by IGF-1 of at least 3 independently performed experiments (B,C,D). * p < 0.05, **p < 0.0095. Exposure to rapamycin lead to downregulation of mTOR and rictor, which were more pronounced under culture conditions without E2. E, Western blots for GSK3β-pS9, GSK3β, rictor pS1235 and rictor and F,G,H,I quantitative analysis of at least 3 independently performed experiments indicate that rictor phosphorylation at S1235 by GSK-3β which has been reported to interfere with Akt-substrate binding to mTORC2, thereby downregulating mTORC2 activity. IGF-1 induced strong phosphorylation of GSK-3β at Ser9 in the absence and presence of E2, however, rapamycin pretreatment only reduced this increased phosphorylation in E2 co-treated cardiomyocytes, indicating increased activity of GSK-3β. This higher activity was not associated with increased phosphorylation of rictor at S1235.</p

Rapamycin does not impair E2 induced ERK activation.

<p>A, ERK phosphorylation was assessed by immunoblots from lysates of HL-1 cells cultured in the presence of 10 nM E2 and treated with 20 nM rapamycin and IGF-1 for 24 h. B, Results from densitometric analysis of blots and determination of IGF-1 induced increase in protein phosphorylation levels from at least 3 independently performed experiments. C, Immunoblots and D, E, densitometric analyses of cardiomyocytes with MEK1/2 inhibition by 1μM PD 184352 1 h prior to IGF-1 stimulation resulted in increased mTORC2 activity as indicated by increased Akt-pS473 in E2 cotreated cells. * p < 0.04,** p < 0.007, *** p < 0.0007. F, Inhibition of Erk phosphorylation by MEK1/2 inhibitor PD 184352 did not inverse rapamycin effect on Akt-pS473 in E2 cultured cardiomyocytes as investigated by western blotting and G-I, Densitometric analyses; mean ± SEM of fold stimulation by IGF-1 is shown of at least 3 independently performed experiments. * p < 0.05.</p

E2 regulates rapamycin effects on mTORC2 activity.

<p>A-H, Rapamycin lowers mTORC1 activity independent of presence of E2, ERα- or ERβ-agonist, however lowers mTORC2 activity dependent on presence of E2. A-D, HL-1 cells were grown to near confluence in medium containing 10 nM E2, and E-H, 10 nM ERα-agonist PPT or 1 nM ERβ-agonist DPN and serum starved for 24 hours prior to incubation with 20 nM rapamycin and IGF-1 for 24 h. A and E show representative westernblots. Equal loading was verified by blotting with antibodies against β-actin or α-tubulin. For B,C,D and F,G,H, labeled bands were quantified with ImageJ software, normalized to loading and IGF-1 induced phosphorylation of indicated proteins was determined by ratio to the value of non-IGF-1 stimulated control cells. Mean ± SEM of fold stimulation by IGF-1 is shown of at least 3 independently performed experiments. * p < 0.05,** p < 0.009, *** p < 0.0001. If not indicated differently, significances are related to respective non-IGF-1 stimulated cells.</p

Sexual dimorphism of mTORC2 activation in response to rapamycin <i>in vivo</i>.

<p>Male and female C57Bl/6J mice were treated with rapamycin (“low concentration” 1.5 mg/kg, administered i.p. every third day) or vehicle (control) for 42 days (n = 6-8/group). mTORC2 activity was assessed by assessment of Akt phosphorylation at S473 and its nuclear localization. A, representative western blots are shown for Akt-pS473, Akt and GAPDH of mice treated as described above. B shows results from quantitative analysis of western blots for Akt-pS473 normalized to Akt (mean ± SEM; * p < 0.035) from female and male mouse hearts. C, Immunostaining for Akt-pS473 of male and female cardiac tissue sections treated with either vehicle (control) or rapamycin for 42 days and D, quantification of % cardiomyocyte nuclei stained for Akt-pS743 (mean ± SEM; *** p < 0.0001). Male mice had lower basal mTORC2 activity, yet responded to rapamycin with an increase in phosphorylation of Akt at S473, associated with increased nuclear localization important for induction of cardioprotective mechanisms. In contrast, female mice responded to rapamycin with reduced phosphorylation of Akt at S473 and loss of cardioprotective nuclear Akt.</p

Rapamycin disturbs adaptive cardiomyocyte responses in E2 and disrupts E2-induced increase in SERCA2A expression.

<p>A, Cells underwent cell volume measurements by FACS analysis. Bars indicate mean GeoMean of flow cytometry forward scatter (FSC-H) from vital cells stimulated with 20 nM IGF-1 for 48 h as indicated from at least 3 independently performed experiments. * p < 0,00, ** p < 0,00. Rapamycin had no significant influence on cardiomyocyte cell size when E2 was absent. However, in presence of E2, rapamycin significantly decreased cardiomyocyte cell size at basal conditions and in response to IGF-1. B, representative histograms for FSC-H from cardiomyocytes cultured with 10 nM E2 and stimulated for 24 h with IGF-1 with or without prior 30 min incubation with 20 nM rapamycin showing left shift of cell volume distribution in rapamycin pretreated cells indicating decrease in cell size of all cardiomyocytes under this treatment. C, D and E, HL-1- cells were cultured with or without E2 and stimulated for 24 h with 10 nM IGF-1 with or without preincubation with 20 nM rapamycin. C-E, SERCA2A expression was assessed on C, mRNA level by qRT-PCR (values were normalized to GAPDH) and D, protein expression of cell lysates stimulated as described above. E, Densitometric analysis of immunoblots from 3 independently performed experiments shown as mean ± SEM. * p < 0.02, ** p < 0.006 for analyses as indicated; # p < 0.04, ## p < 0.006 for comparison of IGF-1 stimulated cells with E2 compared to IGF-1 stimulated cells without E2. IGF-1 alone did not significantly affect SERCA2A expression in female cardiomyocytes. However, rapamycin significantly increased SERCA2A gene expression followed by minor increases in SERCA2A protein predominantly in the IGF-1 treated cells without E2. E2 itself significantly induced SERCA2A gene expression irrespective of additional IGF-1 treatment compared to control cells without E2 (# p< 0.05). However, co-treatment with rapamycin abrogated these increases.</p

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1RAbs- induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis