A low dose of daidzein acts as an ERbeta-selective agonist in trabecular osteoblasts of young female piglets

The role of estrogens and estrogen-like molecules, including isoflavones, in regulating bone cell activities is essential in understanding the etiology and treatment of post-menopausal osteoporosis. Although estrogen replacement (HRT) has been the main therapy used to prevent and treat osteoporosis, there are concerns about its safety. Isoflavones have attracted attention to their potential roles in osteoporosis prevention and treatment. We have compared the effects of the isoflavone daidzein (1 nM), which has no effect on tyrosine kinases, and 17beta-estradiol (1 nM) on the development and function of cultured osteoblasts isolated from long bones of young female piglets. Daidzein increased ALP activity, osteocalcin secretion, and mineralization, while E2 increased only ALP activity. The content of ERbeta and osteoprotegerin secretion by control cells gradually increased during osteoblast differentiation, whereas the ERalpha and RANK-L content decreased. Daidzein enhanced only the nuclear ERbeta whereas estradiol increased both ERalpha and ERbeta. Daidzein and estradiol increased osteoprotegerin and RANK-L secretion. Daidzein had a more pronounced effect than did estradiol. Daidzein and estradiol increased the membrane content of RANK-L and the nuclear content of runx2/Cbfa1. Daidzein enhanced the nuclear content of progesterone and vitamin D receptors but not as much as did estradiol. All the effects of daidzein were blocked by ICI 182,780. We conclude that a low concentration of daidzein may exert its anti-resorptive action by increasing the activity of porcine mature osteoblasts via ERbeta, by regulating runx2/Cbfa1 production, and by stimulating the secretion of key proteins involved in osteoclastogenesis, such as osteoprotegerin and RANK-ligand.     

Galpha(q/11) and gbetagamma proteins and membrane signaling of calcitriol and estradiol.

17beta-estradiol and 1,25-dihydroxyvitamin D(3)()(calcitriol) rapidly increase (< 5 sec) the concentration of intracellular calcium by mobilizing Ca(2+) from the endoplasmic reticulum and forming inositol 1,4,5-trisphosphate (InsP(3)) and diacylglycerol. Calcitriol increases InsP(3) formation via activation of phospholipase C (PLC)-beta1 linked to a pertussis toxin (PTX)-insensitive G-protein, and estradiol via activation of PLC-beta2 linked to a PTX-sensitive G-protein. Since PLC are effectors of different subunits of various G-proteins, we looked for and identified several G-subunits (Galpha(q/11), Galphas, Galphai, Gbeta and Ggamma) in female rat osteoblasts using Western immunoblotting. The action of calcitriol on InsP(3) formation and Ca(2+) mobilization in Fura-2-loaded confluent osteoblasts involved Galpha(q/11). The membrane effects of estradiol involved Gbetagamma; subunits, and principally Gbeta subunits, but not alpha-subunits. These results may provide additional evidence for membrane receptors of steroid hormones. Since PLC-beta1 is the target effector of Galpha(q/11), whereas PLC-beta2 is only activated by betagamma subunits, this specificity may help to generate membrane receptor-specific responses in vivo.     

Daidzein induces neuritogenesis in DRG neuronal cultures

ABSTRACT: BACKGROUND: Daidzein, a phytoestrogen found in isoflavone, is known to exert neurotrophic and neuroprotective effects on the nervous system. Using primary rat dorsal root ganglion (DRG) neuronal cultures, we have examined the potential neurite outgrowth effect of daidzein. METHODS: Dissociated dorsal root ganglia (DRG) cultures were used to study the signaling mechanism of daidzein-induced neuritogenesis by immunocytochemistry and Western blotting. RESULTS: In response to daidzein treatment, DRG neurons showed a significant increase in total neurite length and in tip number per neuron. The neuritogenic effect of daidzein was significantly hampered by specific blockers for Src, protein kinase C delta (PKCdelta) and mitogen-activated protein kinase/extracellular signal-regulated kinase kinases (MEK/ERK), but not by those for estrogen receptor (ER). Moreover, daidzein induced phosphorylation of Src, PKCdelta and ERK. The activation of PKCdelta by daidzein was attenuated in the presence of a Src kinase inhibitor, and that of ERK by daidzein was diminished in the presence of either a Src or PKCdelta inhibitor. CONCLUSION: Daidzein may stimulate neurite outgrowth of DRG neurons depending on Src kinase, PKCdelta and ERK signaling pathway.     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.     

Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells

Expression of the PRL gene is regulated by many factors, including cAMP, estradiol (E2), phorbol esters, epidermal growth factor (EGF), and TRH. The promoter region of the rat PRL gene has been shown to contain DNA sequences that are thought to support the direct interaction of estrogen receptors (ERs) with DNA. It is by this direct ER/DNA interaction that estrogen is thought to modulate expression of PRL. We report here that estrogeninduced PRL expression requires an intact mitogen-activated protein kinase (MAPK) signal transduction pathway in cultured rat pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines). Interfering with the MAPK signaling cascade by inhibiting the activity of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA and protein. In these cell lines, estrogen activates extracellular regulated protein kinases ERK-1 and ERK-2 enzyme activities maximally within 10 min of 1 nM E2 treatment. This activity is blocked by pretreatment of the cells with the MEK inhibitors PD98059 and UO126. The mechanism by which ERKs-1 and -2 are activated by estrogen appears to be independent of c-Src since the effects of estrogen on PRL gene expression are not affected by herbimycin A or PP1 administration. c-Raf-1 may be involved in the effects of E2 because estrogen causes the rapid and transient tyrosine phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both ERK-1 and ERK-2 activation in addition to PRL protein and mRNA, implying a central role for the classical ER in the activation of the MAPK pathway resulting in PRL gene expression.     

Negative regulation of RANKL-induced osteoclastic differentiation in RAW264.7 Cells by estrogen and phytoestrogens

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.     

Estrogen-mediated neuroprotection against beta-amyloid toxicity requires expression of estrogen receptor alpha or beta and activation of the MAPK pathway

It is well documented that estrogen can activate rapid signaling pathways in a variety of cell types. These non-classical effects of estrogen have been reported to be important for cell survival after exposure to a variety of neurotoxic insults. Since direct evidence of the ability of the estrogen receptors (ERs) alpha and/or beta to mediate such responses is lacking, the hippocampal-derived cell line HT22 was stably transfected with either ERalpha (HTERalpha) or ERbeta (HTERbeta). In HTERalpha and HTERbeta cells, but not untransfected cells, an increase in ERK2 phosphorylation was measured within 15 min of 17beta-estradiol treatment. The ER antagonist ICI 182, 780 (1 microm) and the MEK inhibitor, PD98059 (50 microm) blocked this increase in ERK2 phosphorylation. Treatment of HT22, HTERalpha and HTERbeta cells with the beta-amyloid peptide (25-35) (10 micro m) resulted in a significant decrease in cell viability. Pre-treatment for 15 min with 10 nm 17beta-estradiol resulted in a 50% increase in the number of living cells in HTERalpha and HTERbeta cells, but not in HT22 cells. Finally, ICI 182, 780 and PD98059 prevented 17beta-estradiol-mediated protection. This study demonstrates that both ERalpha and ERbeta can couple to rapid signaling events that mediate estrogen-elicited neuroprotection.     

Estradiol‐induced phosphorylation of ERK1/2 in explants of the mouse cerebral cortex: The roles of heat shock protein 90 (Hsp90) and MEK2

Confocal laser scanning microscopy was used to identify the cells within organotypic slice cultures of the developing mouse cerebral cortex that respond to estradiol treatment by phosphorylation of ERK1 and ERK2. Estrogen‐responsive cells resembled neurons morphologically and expressed the neuronal marker microtubule‐associated protein 2B. The intracellular distribution of the phospho‐ERK signal was both cytoplasmic and nuclear, but inhibition of protein synthesis abolished the appearance of the nuclear signal. ERK1and ERK2 also coimmunoprecipitated with heat shock protein 90 (Hsp90) in the cerebral cortical explants. Geldanamycin effectively disrupted this association and prevented ERK phosphorylation. Surprisingly, MEK2 but not MEK1 was the principal mediator of estradiol‐induced activation of ERK. Our data demonstrate the requirement for Hsp90 in estrogen‐induced activation of ERK1 and ERK2 by MEK2 in the developing mouse cerebral cortex and also provide insight into alternative mechanisms by which estradiol may influence cytoplasmic and nuclear events in responsive neurons via the MAP kinase cascade. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 1–12, 2002     

Invasion inhibition by a MEK inhibitor correlates with the actin-based cytoskeleton in lung cancer A549 cells

Metastasis remains the primary cause of lung cancer. The molecules involved in metastasis may be candidates for new targets in the therapy of lung cancer. The MEK/ERK signaling pathway has been highlighted in a number of studies on invasiveness and metastasis. In this paper, we show that the MEK inhibitor U0126 induces flattened morphology, remodels the actin-based cytoskeleton, and potently inhibits chemotaxis and Matrigel invasion in the human lung cancer A549 cell line. Furthermore, downregulation of ERK by small interfering RNA significantly inhibits the invasion of A549 cells and induces stress fiber formation. Taken together, our findings provide the first evidence that the inhibition of invasion of lung cancer A549 cells by inhibiting MEK/ERK signaling activity is associated with remodeling of the actin cytoskeleton, suggesting a novel link between MEK/ERK signaling-mediated cell invasion and the actin-based cytoskeleton.     

Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1

Integrin-mediated adhesion to the extracellular matrix permits efficient growth factor-mediated activation of extracellular signal-regulated kinases (ERKs). Points of regulation have been localized to the level of receptor phosphorylation or to activation of the downstream components, Raf and MEK (mitogen-activated protein kinase/ERK kinase). However, it is also well established that ERK translocation from the cytoplasm to the nucleus is required for G1 phase cell cycle progression. Here we show that phosphorylation of the nuclear ERK substrate, Elk-1 at serine 383, is anchorage dependent in response to growth factor treatment of NIH 3T3 fibroblasts. Furthermore, when we activated ERK in nonadherent cells by expression of active components of the ERK cascade, subsequent phosphorylation of Elk-1 at serine 383 and Elk-1-mediated transactivation were still impaired compared with adherent cells. Elk-1 phosphorylation was dependent on an intact actin cytoskeleton, as discerned by treatment with cytochalasin D (CCD). Finally, expression of active MEK failed to predominantly localize ERK to the nucleus in suspended cells or adherent cells treated with CCD. These data show that integrin-mediated organization of the actin cytoskeleton regulates localization of activated ERK, and in turn the ability of ERK to efficiently phosphorylate nuclear substrates.     

Peroxisome proliferator-activated receptor gamma promotes epithelial to mesenchymal transformation by Rho GTPase-dependent activation of ERK1/2

Peroxisome proliferator-activated receptor gamma (PPARgamma) causes epithelial to mesenchymal transformation (EMT) in intestinal epithelial cells, as evidenced by reorganization of the actin cytoskeleton, acquisition of a polarized, mesenchymal cellular morphology, increased cellular motility, and colony scattering. This response is due to activation of Cdc42, resulting in p21-activated kinase-dependent phosphorylation and activation of MEK1 Ser(298) and activation of ERK1/2. Dominant negative MEK1, MEK2, and ERK2 block PPARgamma-induced EMT, whereas constitutively active MEK1 and MEK2 induce a mesenchymal phenotype similar to that evoked by PPARgamma. PPARgamma also stimulates ERK1/2 phosphorylation in the intestinal epithelium in vivo. PPARgamma induces the p110alpha subunit of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K blocks PPARgamma-dependent phosphorylation of MEK1 Ser(298), activation of ERK1/2, and EMT. We conclude that PPARgamma regulates the motility of intestinal epithelial cells through a mitogen-activated protein kinase cascade that involves PI3K, Cdc42, p21-activated kinase, MEK1, and ERK1/2. Regulation of cellular motility through Rho family GTPases has not been previously reported for nuclear receptors, and elucidation of the mechanism that accounts for the role of PPARgamma in regulating motility of intestinal epithelial cells provides fundamental new insight into the function of this receptor during renewal of the intestinal epithelium.     

Effect of daidzein, a soy isoflavone, on bone metabolism in Cd-treated ovariectomized rats

This study compared the ability of daidzein, a soy isoflavone, with that of 17beta-estradiol to prevent bone loss in cadmium (Cd)-exposed ovariectomized (OVX) rats during growth. Four week-old female Wistar rats were randomly assigned to five treatment groups of 9 rats each, either (1) sham-operated (SH); (2) OVX and placed on experimental diets (OVX); (3) OVX fed 50 ppm of CdCl2 (OVX-Cd); (4) OVX fed 50 ppm of CdCl2 and 10 microg of daidzein per kg of body mass (OVX-CD-D); or (5) OVX fed 50 ppm of CdCl2 and 10 microg of estrogen per kg of body mass (OVX-CD-E). All rats were given free access to AIN-76 modified diet and drinking water, with or without Cd, for 8 weeks. The OVX groups gained more (P < 0.05) body mass than the SH group. Femoral mass was increased by feeding daidzein and estradiol, whereas femoral length was not (P > 0.05) significantly different among groups. Femoral breaking force was not significantly different among groups, however, femoral BMD was significantly lower in OVX-Cd than in the SH and OVX groups. Morphologically proliferative cartilage and hypertrophic cells in femur showed normal distribution in OVX-Cd-D and OVX-Cd-E groups unlike those in OVX-Cd group. These findings suggest that Cd-OVX-induced osteopenia or osteoporosis probably results from an increase in bone turnover.     

Phospholipase C-beta and ovarian sex steroids in pig granulosa cells.

We compared the membrane effects of estradiol, progesterone, and androstenedione in a single experimental model, the ovarian granulosa cells collected from immature Large White sows. We measured changes in cytosolic free calcium concentration ([Ca2+]i) in confluent Fura-2 loaded cells. We used pharmacological tools and polyclonal phospholipase C-beta (PLC-beta) antibodies. Each steroid (0.1 pM to 1 nM) transiently increased intracellular calcium concentration ([Ca2+]i) within 5 sec. They mobilized Ca2+ from the endoplasmic reticulum as shown by using two phospholipase C inhibitors, neomycin and U-73122. Ca2+ mobilization involved PLC-beta1 for progesterone, PLC-beta2 for estradiol and PLC-beta4 for androstenedione. A pertussis toxin-insensitive G protein was involved in the effects of progesterone on Ca2+ mobilization whereas estradiol and androstenedione effects were mediated via a pertussis toxin-sensitive G-protein. Ca2+ influx from the extracellular milieu was involved in the increase in [Ca2+]i induced by progesterone and estradiol, but not by androstenedione. Influx of Ca2+ was independent of Ca2+ mobilization from calcium stores, and it was suggested that L-type Ca2+ channels for estradiol and T-type Ca2+ channels for progesterone were involved. The three steroids had no effect on cAMP. Rapid effects of progesterone, estradiol, and androstenedione involved a direct action on cell membrane elements such as PLC-beta, G-proteins, and calcium channels, and these mechanisms were hormone-specific.     

Phospholipase C-beta1 directly accelerates GTP hydrolysis by Galphaq and acceleration is inhibited by Gbeta gamma subunits.

Phospholipase C-beta, the principal effector protein regulated by Galphaq, has been shown to increase the agonist-stimulated, steady-state GTPase activity of Gq in proteoliposomes that contain both heterotrimeric Gq and m1 muscarinic receptor. We now use a moderately stable complex of R183C Galphaq bound to GTP to show that PLC-beta1 acts directly as a GTPase-activating protein (GAP) for isolated Galphaq in a membrane-free system. PLC-beta1 accelerated the hydrolysis of GalphaqR183C.GTP up to 20-fold. The Km was 1.5 nM, which is similar both to the EC50 with which R183C and wild type Galphaq activate PLC-beta1 and to the EC50 with which PLC-beta1 acts as a Gq GAP in the vesicle-based assay. The Galphaq GAP activity of RGS4 can also be quantitated by this assay; it accelerated hydrolysis of bound GTP about 100-fold. The Gq GAP activities of both PLC-beta1 and RGS4 are blocked by Gbeta gamma subunits, probably by a competitive mechanism. These data suggest either that the Gbeta gamma subunits are not continuously required for receptor-catalyzed GDP/GTP exchange during steady-state GTP hydrolysis or that GAPs, either PLC-beta or RGS proteins, can substitute for Gbeta gamma in this set of reactions.     

Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells.

To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that express a temperature sensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39 degrees C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (deltaRaf-1:ER). deltaRaf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the deltaRaf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. deltaRaf-1:ER, MEK, and MAP kinase activities in the deltaRaf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.     

FGFR-ERK signaling is an essential component of tissue separation

Formation of a constriction and tissue separation between parent and young polyp is a hallmark of the Hydra budding process and controlled by fibroblast growth factor receptor (FGFR) signaling. Appearance of a cluster of cells positive for double phosphorylated ERK (dpERK) at the late separation site indicated that the RAS/MEK/ERK pathway might be a downstream target of the Hydra Kringelchen FGFR. In fact, inhibition of ERK phosphorylation by the MEK inhibitor U0126 reversibly delayed bud detachment and prevented formation of the dpERK-positive cell cluster indicating de novo-phosphorylation of ERK at the late bud base. In functional studies, a dominant-negative Kringelchen FGFR prevented bud detachment as well as appearance of the dpERK-positive cell cluster. Ectopic expression of full length Kringelchen, on the other hand, induced a localized rearrangement of the actin cytoskeleton at sites of constriction, localized ERK-phosphorylation and autotomy of the body column. Our data suggest a model in which (i) the Hydra FGFR targets, via an unknown pathway, the actin cytoskeleton to induce a constriction and (ii) FGFR activates MEK/ERK signaling at the late separation site to allow tissue separation.     

Equol, a metabolite of the soybean isoflavone daidzein, inhibits neoplastic cell transformation by targeting the MEK/ERK/p90RSK/activator protein-1 pathway

Daidzein and genistein are isoflavones found in soybean. Genistein is known to exhibit anticarcinogenic activities and inhibit tyrosine kinase activity. However, the underlying molecular mechanisms of the chemopreventive activities of daidzein and its metabolite, equol, are not understood. Here we report that equol inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal cells by targeting the MEK/ERK/p90RSK/activator protein-1 signaling pathway. TPA-induced neoplastic cell transformation was inhibited by equol, but not daidzein, at noncytotoxic concentrations in a dose-dependent manner. Equol dose-dependently attenuated TPA-induced activation of activator protein-1 and c-fos, whereas daidzein did not exert any effect when tested at the same concentrations. The TPA-induced phosphorylation of ERK1/2, p90RSK, and Elk, but not MEK or c-Jun N-terminal kinase, was inhibited by equol but not by daidzein. In vitro kinase assays revealed that equol greatly inhibited MEK1, but not Raf1, kinase activity, and an ex vivo kinase assay also demonstrated that equol suppressed TPA-induced MEK1 kinase activity in JB6 P+ cell lysates. Equol dose-dependently inhibited neoplastic transformation of JB6 P+ cells induced by epidermal growth factor or H-Ras. Both in vitro and ex vivo pull-down assays revealed that equol directly bound with glutathione S-transferase-MEK1 to inhibit MEK1 activity without competing with ATP. These results suggested that the antitumor-promoting effect of equol is due to the inhibition of cell transformation mainly by targeting a MEK signaling pathway. These findings are the first to reveal a molecular basis for the anticancer action of equol and may partially account for the reported chemopreventive effects of soybean.