A novel ligand-independent function of the estrogen receptor is essential for osteocyte and osteoblast mechanotransduction

Bone senses and adapts to meet mechanical needs by means of an extensive mechanotransduction network comprising osteocytes (former osteoblasts entrapped in mineral) and their cytoplasmic projections through which osteocytes communicate with osteoblasts and osteoclasts on the bone surface. Mechanical stimulation promotes osteocyte (and osteoblast) survival by activating the extracellular signal-regulated kinases, ERKs. Estrogens have similar effects and, intriguingly, the adaptive response of bone to mechanical forces is defective in mice lacking estrogen receptor (ER) alpha or ERbeta. We report that ERKs are not activated by stretching in osteocytic and osteoblastic cells in which both ERalpha and ERbeta have been knocked out or knocked down and this is reversed partially by transfection of either one of the two human ERs and fully by transfection of both receptors. ERK activation in response to stretching is also recovered by transfecting the ligand-binding domain (E) of either receptor or an ERalpha mutant that does not bind estrogens. Furthermore, mechano-responsiveness is restored by transfecting the Ealpha targeted to the plasma membrane, but not to the nucleus, whereas ERalpha mutants with impaired plasma membrane localization or binding to caveolin-1 fail to confer ERK activation in response to stretching. Lastly, the ER antagonist ICI 182,780 abrogates ERK activation and the anti-apoptotic effect of mechanical stimulation. We conclude that in addition to their role as ligand-dependent mediators of the effects of estrogens, the ERs participate in the transduction of mechanical forces into pro-survival signaling in bone cells, albeit in a ligand-independent manner.     

PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity.

Expression of oncogenic ras in PC12 cells causes neuronal differentiation and sustained protein tyrosine phosphorylation and activity of extracellular signal-regulated kinases (ERKs), p42erk2 and p44erk1. Oncogenic N-ras-induced neuronal differentiation is inhibited by compounds that block ERK protein tyrosine phosphorylation or ERK activity, indicating that ERKs are not only activated by p21ras but serve as the primary downstream effectors of p21ras. Treatment of PC12 cells with nerve growth factor or fibroblast growth factor results in neuronal differentiation and in a sustained elevation of p21ras activity, of ERK activity, and of ERK tyrosine phosphorylation. Epidermal growth factor, which does not cause neuronal differentiation, stimulates only transient (< 1 hr) activation of p21ras and ERKs. These data indicate that transient activation of p21ras and, consequently, ERKs is not sufficient for induction of neuronal differentiation. Prolonged ERK activity is required: a consequence of sustained activation of p21ras by the growth factor receptor protein tyrosine kinase.     

Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.     

Dynamics of proximal signaling events after TCR/CD8-mediated induction of proliferation or apoptosis in mature CD8+ T cells

Engagement of the TCR can induce different functional outcomes such as activation, proliferation, survival, or apoptosis. How the TCR-mediated signaling cascades generating these distinct cellular responses are organized on the molecular level is so far not completely understood. To obtain insight into this question, we analyzed TCR/CD8-mediated signaling events in mature OT-I TCR transgenic T cells under conditions of stimulation that lead to either proliferation or apoptosis. These experiments revealed major differences in the phosphorylation dynamics of LAT, ZAP70, protein kinase B, phospholipase C-gamma1, protein kinase D1, and ERK1/2. Moreover, input signals leading to apoptosis induced a strong, but transient activation of ERK1/2 mainly at sites of TCR-engagement. In contrast, stimuli promoting survival/proliferation generated a low and sustained activation of ERK1/2, which colocalizes with Ras in recycling endosomal vesicles. The transient activation of ERK1/2 under pro-apoptotic conditions of stimulation is at least partially due to the rapid polyubiquitination and subsequent degradation of ZAP70, whereas the sustained activation of ERK1/2 under survival promoting conditions is paralleled by the induction/phosphorylation of anti-apoptotic molecules such as protein kinase B and Bcl-x(L). Collectively, our data provide signaling signatures that are associated with proliferation or apoptosis of T cells.     

Neuronal Differentiation Dictates Estrogen-Dependent Survival and ERK1/2 Kinetic by Means of Caveolin-1

Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E₂ stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival.     

Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.     

Caspase activation in etoposide-treated fibroblasts is correlated to ERK phosphorylation and both events are blocked by polyamine depletion

Activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 is correlated to cell survival, but in some cases ERKs can act in signal transduction pathways leading to apoptosis. Treatment of mouse fibroblasts with 20 microM etoposide elicited a sustained phosphorylation of ERK 1/2, that increased until 24 h from the treatment in parallel with caspase activity. The inhibitor of ERK activation PD98059 abolished caspase activation, but caspase inhibition did not reduce ERK 1/2 phosphorylation, suggesting that ERK activation is placed upstream of caspases. Both ERK and caspase activation were blocked in cells depleted of polyamines by the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO). In etoposide-treated cells, DFMO also abolished phosphorylation of c-Jun NH(2)-terminal kinases triggered by the drug. Polyamine replenishment with exogenous putrescine restored the ability of the cells to undergo caspase activation and ERK 1/2 phosphorylation in response to etoposide. Ornithine decarboxylase activity decreased after etoposide, indicating that DFMO exerts its effect by depleting cellular polyamines before induction of apoptosis. These results reveal a role for polyamines in the transduction of the death signal triggered by etoposide.     

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.     

Endosulfan decreases cell growth and apoptosis in human HaCaT keratinocytes: partial ROS-dependent ERK1/2 mechanism

Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen-activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 microM endosulfan led to persistent extracellular signal-regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan-treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N-acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS-dependent mechanism. Therefore, to assess the putative pro-apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP-ribose)-polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine-induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects.     

Estrogen induces phospholipase A2 activation through ERK1/2 to mobilize intracellular calcium in MCF-7 cells

The principal secreted estrogen, 17beta-estradiol rapidly activates signaling cascades that regulate important physiological processes including ion transport across membranes, cytosolic pH and cell proliferation. These effects have been extensively studied in the MCF-7 estrogen-responsive human breast carcinoma cell line. Here, we demonstrate that a physiological concentration of 17beta-estradiol caused a rapid, synchronous and transient increase in intracellular calcium concentration in a confluent monolayer of MCF-7 cells 2-3 min after treatment. This response was abolished when cells were pre-incubated with the phospholipase A(2) (PLA(2)) inhibitor quinacrine or with the cyclooxygenase inhibitor indomethacin. The translocation of GFP-cPLA(2)alpha to perinuclear membranes occurred 1-2 min after 17beta-estradiol treatment; this translocation was concurrent with the transient phosphorylation of cPLA(2)alpha at serine residue 505. The phosphorylation and translocation of cPLA(2) were sensitive to inhibition of the extracellular signal regulated kinase (ERK) signaling cascade and occurred simultaneously with a transient activation of ERK. The phosphorylation of cPLA(2) could be stimulated by membrane impermeable 17beta-estradiol conjugated to bovine serum albumen and was blocked by an antagonist of the classical estrogen receptor. Here we show, for the first time, that PLA(2) and the eicosanoid biosynthetic pathway are involved in the 17beta-estradiol induced rapid calcium responses of breast cancer cells.     

Epidermal growth factor receptor and protein kinase C signaling to ERK2: spatiotemporal regulation of ERK2 by dual specificity phosphatases

Spatiotemporal aspects of ERK activation are stimulus-specific and dictate cellular consequences. They are dependent upon dual specificity phosphatases (DUSPs) that bind ERK via docking domains and can both inactivate and anchor ERK in cellular compartments. Using high throughput fluorescence microscopy in combination with a system where endogenous ERKs are removed and replaced with wild-type or mutated ERK2-green fluorescent protein (GFP), we show that ERK2 activation responses to epidermal growth factor (EGF) and protein kinase C (PKC) are transient and sustained, respectively. PKC-mediated ERK2 activation is associated with prolonged nuclear localization in the dephosphorylated form, whereas EGF-stimulated ERK2 activation mediates only transient nuclear accumulation. By using short inhibitory RNAs to nuclear inducible DUSP1, -2, or -4 (alone or in combination), we demonstrate that all three of these enzymes contribute to the dephosphorylation of PKC (but not EGF)-activated ERK2 in the nucleus but that they have opposing effects on localization. DUSP2 and -4 inactivate and anchor ERK2, whereas DUSP1 dephosphorylates ERK in the nucleus but allows its traffic back to the cytoplasm. Overexpression of DUSP1, -2, or -4 prevented ERK2 activation, but only DUSP2 and -4 caused ERK2-GFP nuclear accumulation or could be immunoprecipitated with ERK2. Furthermore, protein synthesis inhibition or replacement of wild-type ERK2-GFP with docking domain mutants selectively increased PKC effects on ERK activity and altered ERK2-GFP localization. These mutations also impaired the ability of ERK2-GFP to bind DUSP2 and -4. Together, our data reveal a novel, stimulus-specific, and phosphatase-specific mechanism of ERK2 regulation in the nucleus by DUSP1, -2, and -4.     

IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation

IEX-1 is an early response and NF-kappaB target gene implicated in the regulation of cellular viability. We show here that IEX-1 is a substrate for ERKs and that IEX-1 and ERK regulate each other's activities. IEX-1 was isolated by phosphorylation screening with active ERK2 and found subsequently phosphorylated in vivo upon ERK activation. IEX-1 interacts with phosphorylated ERKs but not with c-jun N-terminal kinase (JNK) or p38. Upon phosphorylation by ERKs, IEX-1 acquires the ability to inhibit cell death induced by various stimuli. In turn, IEX-1 potentiates ERK activation in response to various growth factors. By using various IEX-1 mutants in which the ERK phosphoacceptor and/or ERK docking sites were mutated, we show that the IEX-1 pro-survival effect is dependent on its phosphorylation state but not on its ability to potentiate ERK activation. Conversely, IEX-1-induced modulation of ERK activation requires ERK-IEX-1 association but is independent of IEX-1 phosphorylation. Thus, IEX-1 is a new type of ERK substrate that has a dual role in ERK signaling by acting both as an ERK downstream effector mediating survival and as a regulator of ERK activation.     

Sustained activation of the extracellular signal-regulated kinase pathway is required for extracellular calcium stimulation of human osteoblast proliferation

Elevated levels of [Ca(2+)](o) in bone milieu as a result of the resorptive action of osteoclasts are implicated in promoting proliferation and migration of osteoblasts during bone remodeling. However, mitogenic effects of [Ca(2+)](o) have only been shown in some, but not all, clonal osteoblast-like cells, and the molecular mechanisms underlying [Ca(2+)](o)-induced mitogenic signaling are largely unknown. In this study we demonstrated for the first time that [Ca(2+)](o) stimulated proliferation of primary human osteoblasts and selectively activated extracellular signal-regulated kinases (ERKs). Neither p38 mitogen-activated protein (MAP) kinase nor stress-activated protein kinase was activated by [Ca(2+)](o). Treatment of human osteoblasts with a MAP kinase kinase inhibitor, PD98059, impaired both basal and [Ca(2+)](o)-stimulated phosphorylation of ERKs and also reduced both basal and [Ca(2+)](o)-stimulated proliferation. [Ca(2+)](o) treatment resulted in two distinctive phases of ERK activation: an acute phase and a sustained phase. An inhibition time course revealed that it was the sustained phase, not the acute phase, that was critical for [Ca(2+)](o)-stimulated osteoblast proliferation. Our results demonstrate that mitogenic responsiveness to [Ca(2+)](o) is present in primary human osteoblasts and is mediated via prolonged activation of the MAP kinase kinase/ERK signal pathway.     

Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.     

beta-Adrenergic-responsive activation of extracellular signal-regulated protein kinases in salivary cells: role of epidermal growth factor receptor and cAMP

The -adrenergic receptor agonist isoproterenol exerts growth-promoting effects on salivary glands. In this study, activation of ERKs, members of the mitogen-activated protein kinase family, by isoproterenol was examined in a human salivary gland cell line (HSY). Immunoblot analysis indicated that isoproterenol (10^–5 M) induced transient activation of ERK1/2 (4.4-fold relative to basal at 10 min) similar to that caused by EGF (6.7 fold). Isoproterenol, like EGF, also induced phosphorylation of the EGF receptor. However, inhibition of EGF receptor phosphorylation by the tyrphostin AG-1478 only partially attenuated isoproterenol-induced ERK phosphorylation, whereas EGF-responsive ERK activation was completely blocked. The G_i inhibitor pertussis toxin also caused partial inhibition of isoproterenol-stimulated ERK activation. The cAMP analog 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and the cAMP-elevating agents IBMX and cholera toxin produced transient ERK1/2 activation, similar to the effect of isoproterenol, in HSY cells. The stimulatory effects of isoproterenol and cAMP on ERK phosphorylation were not reduced by the PKA inhibitor H-89, whereas the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidase (PP2) and transfection of a dominant-negative Src construct diminished isoproterenol-induced ERK activation. Isoproterenol induced marked overexpression of the cell growth-related adhesion molecule CD44, and this effect of isoproterenol was abolished by the ERK pathway inhibitor PD-98059. In summary, we show a dual mechanism of isoproterenol-induced ERK phosphorylation in HSY cells—one pathway mediated by EGF receptor transactivation and the other by an EGF receptor-independent pathway possibly mediated by cAMP. Our results also suggest that isoproterenol-induced growth of salivary tissue may involve ERK-mediated CD44 expression. mitogen-activated protein kinase; CD44