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Steroid hormone receptors (SHRs) are ligand-activated transcription factors that regulate gene expression. SHRs also mediate rapid, nongenomic cellular activation by steroids. In vascular endothelial cells, the SHR for estrogen, estrogen receptor (ER) [alpha], is targeted by unknown mechanisms to a functional signaling module in membrane caveolae that enables estrogen to rapidly activate the mitogen-activated protein kinase and phosphatidylinositol 3-Akt kinase pathways, and endothelial NO synthase (eNOS). Here we identify the 110-kDa caveolin-binding protein striatin as the molecular anchor that localizes ER[alpha] to the membrane and organizes the ER[alpha]-eNOS membrane signaling complex. Striatin directly binds to amino acids 183-253 of ER[alpha], targets ER[alpha] to the cell membrane, and serves as a scaffold for the formation of an ER[alpha]-G[alpha]i complex. Disruption of complex formation between ER[alpha] and striatin blocks estrogen-induced rapid activation mitogen-activated protein kinase, Akt kinase, and eNOS, but has no effect on ER-dependent regulation of an estrogen response element-driven reporter plasmid. These findings identify striatin as a molecular scaffold required for rapid, nongenomic estrogen-mediated activation of downstream signaling pathways. Furthermore, by demonstrating independent regulation of nongenomic vs. genomic ER-dependent signaling, these findings provide conceptual support for the potential development of 'pathway-specific' selective ER modulators.

Steroid hormone receptors (SHRs) are ligand-activated transcription factors that regulate gene expression. SHRs also mediate rapid, nongenomic cellular activation by steroids. In vascular endothelial cells, the SHR for estrogen, estrogen receptor (ER) α, is targeted by unknown mechanisms to a functional signaling module in membrane caveolae that enables estrogen to rapidly activate the mitogen-activated protein kinase and phosphatidylinositol 3-Akt kinase pathways, and endothelial NO synthase (eNOS). Here we identify the 110-kDa caveolin-binding protein striatin as the molecular anchor that localizes ERα to the membrane and organizes the ERα-eNOS membrane signaling complex. Striatin directly binds to amino acids 183-253 of ERα, targets ERα to the cell membrane, and serves as a scaffold for the formation of an ERα-Gαi complex. Disruption of complex formation between ERα and striatin blocks estrogen-induced rapid activation mitogen-activated protein kinase, Akt kinase, and eNOS, but has no effect on ER-dependent regulation of an estrogen response element-driven reporter plasmid. These findings identify striatin as a molecular scaffold required for rapid, nongenomic estrogen-mediated activation of downstream signaling pathways. Furthermore, by demonstrating independent regulation of nongenomic vs. genomic FR-dependent signaling, these findings provide conceptual support for the potential development of "pathway-specific" selective ER modulators. [ABSTRACT FROM AUTHOR]

Studies the binding of the catalytic subunit of protein phosphatase 2A (PP2A) by estrogen receptor alpha. Role of PP2A in the transcriptional activation of the receptor; Model for the regulation of ligand-independent transcriptional activation.

Myocardial hypertrophy is an independent risk factor for heart failure (HF), yet the mechanisms underlying pathological cardiomyocyte growth are incompletely understood. The c-Jun NH 2 -terminal kinase (JNK) signaling cascade modulates cardiac hypertrophic remodeling, but the upstream factors regulating myocardial JNK activity remain unclear. In this study, we sought to identify JNK-activating molecules as novel regulators of cardiac remodeling in HF. We investigated mixed lineage kinase-3 (MLK3), a master regulator of upstream JNK-activating kinases, whose role in the remodeling process had not previously been studied. We observed increased MLK3 protein expression in myocardium from patients with nonischemic and hypertrophic cardiomyopathy and in hearts of mice subjected to transverse aortic constriction (TAC). Mice with genetic deletion of MLK3 (MLK3 -/- ) exhibited baseline cardiac hypertrophy with preserved cardiac function. MLK3 -/- mice subjected to chronic left ventricular (LV) pressure overload (TAC, 4 wk) developed worsened cardiac dysfunction and increased LV chamber size compared with MLK3 +/+ littermates ( n = 8). LV mass, pathological markers of hypertrophy ( Nppa, Nppb), and cardiomyocyte size were elevated in MLK3 -/- TAC hearts. Phosphorylation of JNK, but not other MAPK pathways, was selectively impaired in MLK3 -/- TAC hearts. In adult rat cardiomyocytes, pharmacological MLK3 kinase inhibition using URMC-099 blocked JNK phosphorylation induced by neurohormonal agents and oxidants. Sustained URMC-099 exposure induced cardiomyocyte hypertrophy. These data demonstrate that MLK3 prevents adverse cardiac remodeling in the setting of pressure overload. Mechanistically, MLK3 activates JNK, which in turn opposes cardiomyocyte hypertrophy. These results support modulation of MLK3 as a potential therapeutic approach in HF. NEW & NOTEWORTHY Here, we identified a role for mixed lineage kinase-3 (MLK3) as a novel antihypertrophic and antiremodeling molecule in response to cardiac pressure overload. MLK3 regulates phosphorylation of the stress-responsive JNK kinase in response to pressure overload and in cultured cardiomyocytes stimulated with hypertrophic agonists and oxidants. This study reveals MLK3-JNK signaling as a novel cardioprotective signaling axis in the setting of pressure overload.

Estrogen receptor alpha (ERalpha) mediates the effects of estrogen by altering gene expression following hormone binding. It has recently been shown that kinase-mediated phosphorylation of ERalpha also transcriptionally activates the receptor in the absence of estrogen. We now report that ERalpha-dependent gene expression also is regulated by protein phosphatase 2A (PP2A). ERalpha co-immunoprecipitates with enzymatically active PP2A. ERalpha binds directly to the catalytic subunit of PP2A, which dephosphorylates serine 118 of the receptor. Amino acids 176-182 in the A/B domain of ERalpha are required for the interaction between PP2A and the receptor. Phosphatase inhibition disrupts the ERalpha-PP2A complex and induces formation of an ERalpha-activated mitogen-activated protein kinase complex, phosphorylation of ERalpha on serine 118, and transcriptional activation. These findings demonstrate that estrogen receptors exist in complexes with phosphatases as well as kinases. We propose a new model of ligand-independent activation of estrogen receptors in which the level of phosphorylation of ERalpha, and hence its transcriptional activation, is determined by the net effect of these counterregulatory pathways.

In addition to mediating the classical transcriptional effects of estrogen, estrogen receptors (ERs) are now known to regulate gene expression in the absence of estrogen by ligand-independent activation pathways, and to mediate the rapid, non-genomic effects of estrogen as well. ERs have been shown to associate with the cell membrane, and recent studies demonstrate that this subpopulation of membrane-associated ER mediates the rapid effects of estrogen. To date, however, little is known regarding the pathways that regulate the distribution of the ER between the nuclear and membrane fractions. In the current study, we demonstrate membrane localization of transiently transfected ERalpha in human vascular smooth muscle cells, and translocation of ERalpha from the membrane to the nucleus in response to both estrogen-dependent and estrogen-independent stimulation. Mutational analyses identified serine 118 as the critical residue regulating nuclear localization following estrogen-independent stimulation, but not following estrogen stimulation. Induction of nuclear localization of ERalpha by estrogen-independent, but not estrogen-dependent stimulation was blocked by both pharmacologic and genetic inhibition of mitogen-activated protein (MAP) kinase activation. Furthermore, constitutive activation of MAP kinase resulted in nuclear translocation of ERalpha. These overexpression studies support that MAP kinase-mediated phosphorylation of ERalpha induces nuclear localization of the ER in response to estrogen-independent, but not estrogen-dependent stimulation, demonstrating stimulus-specific molecular pathways regulate the nuclear localization of the ER. These findings identify a previously unrecognized pathway that regulates the intracellular localization of the ER, and represent the first demonstration that the distribution of the ER between membrane and nuclear compartments is regulated by physiologic stimuli.