Rapid vitamin D-dependent PKC signaling shares features with estrogen-dependent PKC signaling in cartilage and bone

Our work is based on the hypothesis that steroid hormones regulate cells through traditional cytoplasmic and nuclear receptor-mediated mechanisms, as well as by rapid effects that are mediated by membrane-associated pathways. We have used the rat costochondral growth plate chondrocyte culture model to study the signaling mechanisms used by steroid hormones to elicit rapid responses and to modulate gene expression in target cells. Our studies show that the secosteroids 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and 24,25-dihydroxyvitamin D3 [24R,25(OH)2D3], and the steroid hormone 17beta-estradiol, cause rapid increases in protein kinase C alpha (PKCalpha) activity, and many of the physiological responses of the cells to these regulators are PKC-dependent. Target cell specificity and the mechanisms by which PKCalpha is activated vary with each hormone. PKC activation initiates a signaling cascade that results in activation of the ERK1/2 family of mitogen activated protein kinases (MAPK), providing an alternate method for the steroids to modulate gene expression other than by traditional steroid hormone receptor-mediated pathways. In addition to their effects on growth plate chondrocytes, steroid hormones secreted by the cells also control events in the extracellular matrix through direct non-genomic regulation of matrix vesicles.     

Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon

Studies from our laboratory have demonstrated rapid ( < 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC activity and a PKC-dependent Ca(2+) entry through L-type Ca(2+) channels specifically by mineralocorticoids in distal colon. Aldosterone directly stimulates the activity of the PKC alpha isoform (but not PKC delta, PKC epsilon and PKC zeta) in a cell-free assay system containing only purified commercially available enzyme, appropriate substrate peptide, co-factors and lipid vesicles. The primary ion transport target of the non-genomic signal transduction cascade elicited by aldosterone in epithelia is the Na(+)-H(+) exchanger. In isolated colonic crypts, aldosterone produced a PKC alpha sensitive intracellular alkalinisation within 1 min of hormone addition. Intracellular alkalinisation upregulates an ATP-dependent K(+) channel, which is involved in K(+) recycling to maintain the electrical driving force for Na(+) absorption, while inhibiting a Ca(2+) -dependent K(+) channel, which generates the charge balance for Cl(-) secretion. The non-genomic response to aldosterone in distal colon appears to enhance the capacity for absorption while down-regulating the potential for secretion. We have also demonstrated rapid (< 1 min) non-genomic activation of Na(+)-H(+) exchange, K(+) recycling, PKC alpha activity, and a PKC delta- and PKA-dependent Ca(2+) entry through di-hydropyridine-blockable Ca(2+) channels specifically by 17beta-estradiol in distal colon. These rapid effects are female gender specific and are insensitive to inhibitors of the classical estrogen receptor (ER). 17 beta-Estradiol directly stimulated the activity of both PKC delta and PKC alpha (but not PKC epsilon or PKC zeta) in a cell-free assay system. E2 rapidly inhibited basolateral K(Ca) channel activity which would be expected to result in an acute inhibition of Cl(-) secretion. Physiological concentrations of E2 (0.1-10 nM) reduced both basal and secretagogue-induced Cl(-) secretion. This anti-secretory effect of E2 is sensitive to PKC inhibition, intracellular Ca(2+) chelation, and is female gender specific and insensitive to inhibitors of the classical ER. These observations link rapid non-genomic activation of second messengers with a rapid gender-specific physiological effect in the whole tissue. Aldosterone and E2 differ in their protein kinase signal transduction and both hormones stimulate specific PKC isoforms indicating both common and divergent signalling systems for salt-retaining steroid hormones. The physiological function of non-genomic effects of aldosterone and estradiol is to shift the balance from net secretion to net absorption in a pluripotential epithelium.     

The direct action of estrone on vascular tissue involves genomic and non-genomic actions

A two step model mechanism of steroid action has been recently postulated. In this study, we test the hypothesis that, the biochemical action of estrone (E(1)) on vascular tissue could be performed via genomic and non-genomic actions. Rat aortic rings or vascular smooth muscle cell cultures (VSMC) were used to test the effect of the hormone on nitric oxide (NO) production, protein kinases activities and cell proliferation. Our data showed that estrone increased NO synthesis between 30 s and 20 min treatment, and this stimulatory effect was dependent on MAPK cascade activation, since it was prevented in the presence of a MAPK inhibitor (PD98059). Using a phosphorylation assay, we also showed that E(1) significantly increased MAPK activity. The effect of the hormone on PKC activity was measured in concentrations and time course studies. Direct treatment of rat aortic homogenates with E(1) significantly enhanced PKC activity (1-10 fold increase, p<0.01) at all concentrations (1; 10; 50 nM) and time tested (1-10 min). We demonstrated that 24 h of E(1) treatment markedly increased VSMC proliferation (53% above control), and this effect was suppressed by a PKC inhibitor. The rapid and the long term effects of the hormone were completely suppressed in the presence of an estradiol receptor antagonist (ICI 182780). In summary, we provided evidence that, the steroid exerts both non-genomic and genomic actions, the former associated with MAPK kinase dependent on NO production, and the latter related with induction of VSMC proliferation involving PKC pathway activation.     

Direct binding and activation of protein kinase C isoforms by aldosterone and 17beta-estradiol

Protein kinase C (PKC) is a signal transduction protein that has been proposed to mediate rapid responses to steroid hormones. Previously, we have shown aldosterone directly activates PKCalpha whereas 17beta-estradiol activates PKCalpha and PKCdelta; however, neither the binding to PKCs nor the mechanism of action has been established. To determine the domains of PKCalpha and PKCdelta involved in binding of aldosterone and 17beta-estradiol, glutathione S-transferase fusion recombinant PKCalpha and PKCdelta mutants were used to perform in vitro binding assays with [(3)H]aldosterone and [(3)H]17beta-estradiol. 17beta-Estradiol bound both PKCalpha and PKCdelta but failed to bind PKC mutants lacking a C2 domain. Similarly, aldosterone bound only PKCalpha and mutants containing C2 domains. Thus, the C2 domain is critical for binding of these hormones. Binding affinities for aldosterone and 17beta-estradiol were between 0.5-1.0 nM. Aldosterone and 17beta-estradiol competed for binding to PKCalpha, suggesting they share the same binding site. Phorbol 12,13-dybutyrate did not compete with hormone binding; furthermore, they have an additive effect on PKC activity. EC(50) for activation of PKCalpha and PKCdelta by aldosterone and 17beta-estradiol was approximately 0.5 nM. Immunoblot analysis using a phospho-PKC antibody revealed that upon binding, PKCalpha and PKCdelta undergo autophosphorylation with an EC(50) in the 0.5-1.0 nm range. 17beta-Estradiol activated PKCalpha and PKCdelta in estrogen receptor-positive and -negative breast cancer cells (MCF-7 and HCC-38, respectively), suggesting estrogen receptor expression is not required for 17beta-estradiol-induced PKC activation. The present results provide first evidence for direct binding and activation of PKCalpha and PKCdelta by steroid hormones and the molecular mechanisms involved.     

MNAR plays an important role in ERa activation of Src/MAPK and PI3K/Akt signaling pathways

Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.     

Estradiol modulates the synapsins phosphorylation by various protein kinases in the rat brain under in vitro and in vivo conditions.

Synapsins are the neuronal phosphoproteins which play very important role in processes of synaptic neurotransmission. They are physiological substrates for Ser/Thr protein kinases. The reversible phosphorylation of synapsins may be modified by several compounds including steroid hormones. The aim of our study was to investigate, if the one of neuroactive steroid--17beta-estradiol--could modulate the phosphorylation of synapsins by PKA, CaM-PK and PKC in rat brain and what type of mechanism of their action is possible. The activity of kinases was evaluated as phosphorylation of synapsin in cerebral cortex and hippocampus in vivo and in vitro conditions. We conclude that 17E2 has inhibitory effect on synapsins phosphorylation by all tested kinases in vitro and in vivo conditions. The lack of nuclei in synaptosomal membrane fraction and short time of hormone exposure can be evidence of direct, non-genomic mechanism of estradiol action.     

Non-genomic effects of thyroid hormone in adult cardiac myocytes: relevance to gene expression and cell growth

Besides the well-characterized genomic action of thyroid hormone (TH), mediated by thyroid hormone receptors (TRs), accumulating data support the so-called non-genomic action of TH, which is often related to activation of signalling pathways. In this study, we sought to determine whether TH activates intracellular signalling pathways in the adult cardiac myocytes and whether such activation modulates cell growth and the expression of target proteins important in cardiac function. We demonstrate that TH promotes a rapid increase in the phosphorylation of several kinases, ERK1/2, PKCδ, p38-MAPK and Akt. This activation is inhibited by triiodothyroacetic acid (triac), which is a TH analogue known to displace the hormone from membrane bound receptors, indicating that this TH effect is mediated through a cell membrane-initiated mechanism. Furthermore, using specific inhibitors of the TH-activated kinases, we show that the long-term effects of TH on the expression of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), α- and β-myosin heavy chain (MHC) and cell growth are reverted, implying that what is initiated as a non-genomic action of the hormone interfaces with genomic effects. These data provide further insights into the underlying mechanisms of TH action in the heart with potentially important implications in the management of cardiac pathology.     

Modulation of LH release by estradiol. Involvement of protein kinase C in the action mechanism of GnRH

The involvement of PKC in GnRH action is still controversial. Discrepancies between different results could be due to the endocrine status of cells used for the studies. In order to determine a putative role for PKC in GnRH action and if gonadal steroids could be implicated in the PKC contribution to GnRH action, we have conducted a study of LH release in response to GnRH and to PMA, an activator of PKC, using an anterior pituitary cell culture system. The direct effects of E2 were considered coupled or not with the effect of PKC depletion. GnRH and PMA induced LH releases in a dose-dependent manner. Both are increased by E2. The PKC depletion had no effect on GnRH stimulated LH release in cells deprived of gonadal steroid influence but induced a significant decrease in cells which had been treated by E2. These results indicate that E2 alters cell sensitivity to GnRH by affecting post-receptor intracellular pathways such as PKC activation.