Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.     

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha1B-adrenergic receptor/Galphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha1B-adrenergic receptor. Together, these results suggest that the alpha1B-adrenergic receptor/Galphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.     

Prolonged nuclear retention of activated extracellular signal-regulated protein kinase promotes cell death generated by oxidative toxicity or proteasome inhibition in a neuronal cell line.

In the HT22 mouse hippocampal cell line and primary immature embryonic rat cortical neurons, glutamate-induced oxidative toxicity is associated with a delayed but chronic activation of extracellular signal-regulated kinase-1/2 (ERK-1/2). ERK-1/2 is also activated in HT22 cells that undergo caspase-dependent cell death upon inhibition of proteasome-dependent protein degradation brought about by MG132 treatment. As in glutamate-treated HT22 cells and primary neurons, inhibition of MEK-1, an upstream activator of ERK-1/2 protects against MG132-induced toxicity. Furthermore, activated ERK-1/2 is retained within the nucleus in glutamate- and MG132-treated HT22 cells. Although previous studies suggested that ERK-1/2 activation was downstream of many cell death-inducing signals in HT22 cells, we show here that cycloheximide, the Z-vad caspase inhibitor, and a nonlethal heat shock protect against glutamate- and MG132-induced toxicity without diminishing ERK-1/2 activation. In these cases, ERK-1/2, although chronically activated, is not retained within the nucleus but accumulates within the cytoplasm. Thus, persistent nuclear retention of activated ERK-1/2 may be a critical factor in eliciting proapoptotic effects in neuronal cells subjected to oxidative stress or proteasome inhibition.     

Inhibition of protein kinase CK2 sensitizes non-small cell lung cancer cells to cisplatin via upregulation of PML

Dexamethasone is a potent and widely used anti-inflammatory and immunosuppressive drug. However, recent evidences suggest that dexamethasone cause pathologic cardiac remodeling, which later impairs cardiac function. The mechanism behind the cardiotoxic effect of dexamethasone is elusive. The present study aimed to verify if dexamethasone-induced cardiotoxicity would be associated with changes in the cardiac net balance of calcium handling protein and calcineurin signaling pathway activation. Wistar rats (~400 g) were treated with dexamethasone (35 µg/g) in drinking water for 15 days. After dexamethasone treatment, we analyzed cardiac function, cardiomyocyte diameter, cardiac fibrosis, and the expression of proteins involved in calcium handling and calcineurin signaling pathway. Dexamethasone-treated rats showed several cardiovascular abnormalities, including elevated blood pressure, diastolic dysfunction, cardiac fibrosis, and cardiomyocyte apoptosis. Regarding the expression of proteins involved in calcium handling, dexamethasone increased phosphorylation of phospholamban at threonine 17, reduced protein levels of Na⁺/Ca²⁺ exchanger, and had no effect on protein expression of Serca2a. Protein levels of NFAT and GATA-4 were increased in both cytoplasmic and nuclear faction. In addition, dexamethasone increased nuclear protein levels of calcineurin. Altogether our findings suggest that dexamethasone causes pathologic cardiac remodeling and diastolic dysfunction, which is associated with impaired calcium handling and calcineurin signaling pathway activation.     

Inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon cancer cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.     

HSP70 deficiency results in activation of c-Jun N-terminal Kinase, extracellular signal-regulated kinase, and caspase-3 in hyperosmolarity-induced apoptosis

In this study we examined the function of heat shock protein 70 (HSP70) in the hyperosmolarity-induced apoptotic pathway using hsp70.1-/-mouse embryonic fibroblasts (MEFs). When the cells were exposed to hyperosmotic stress, an absence of HSP70 negatively affected cell viability. Caspase-9 and caspase-3 were rapidly activated, and extensive cleavage occurred in focal adhesion and cytoskeletal molecules in the hsp70.1-/-MEFs. In contrast, hsp70.1+/+ MEFs exhibited no caspase-9 or caspase-3 activation and finally recovered intact cell morphology when cells were shifted back to an isosmotic state. Because HSP70 might be involved in the regulation of mitogen-activated protein kinase (MAPK) activities with regard to various cellular activities, we also monitored MAPK phosphorylation. The absence of HSP70 affected c-Jun N-terminal kinase phosphorylation. However, it had no effect on p38. Sustained phosphorylation of extracellular signal-regulated kinase (ERK) was observed during the hyperosmolarity-induced apoptosis of hsp70.1-/-MEFs. Inhibition of ERK activity by the treatment of PD98059 accelerated the apoptotic pathway. ERK phosphorylation was precisely correlated with shift of mitogen-activated protein kinase phosphatase-3 from the soluble to insoluble fraction. Our results demonstrate that the inhibitory effect of HSP70 on caspase-3 activation is sufficient to inhibit apoptosis and that HSP70 exhibits regulatory functions to c-Jun N-terminal kinase and ERK phosphorylation in hyperosmolarity-induced apoptosis.     

Inhibition of arachidonate 5-lipoxygenase triggers prostate cancer cell death through rapid activation of c-Jun N-terminal kinase

Previously, we reported that inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in both androgen-sensitive (LNCaP) and androgen-refractory (PC3) human prostate cancer cells within hours of treatment [Proc. Natl. Acad. Sci. USA 95 (1998) 13182-13187]. Apoptosis was prevented by exogenous 5(S)-HETE, a product of 5-lipoxygenase, indicating a role of this eicosanoid as an essential survival/anti-apoptotic factor for prostate cancer cells. However, nothing was clearly known about details of the underlying molecular mechanisms or events mediating the induction of fulminating apoptosis in these cells. This report documents the fact that inhibition of arachidonate 5-lipoxygenase induces rapid activation of c-Jun N-terminal kinase (JNK) in human prostate cancer cells which is prevented by the 5-lipoxygenase metabolite, 5(S)-HETE. Activation of JNK is unaffected by the cell-permeable tetra-peptide inhibitors of caspase 8 or caspase 3 (IETD-FMK and DEVD-FMK), though these inhibitors effectively blocked apoptosis triggering, suggesting that activation of JNK is independent or upstream of caspase activation. Both 5-lipoxygenase inhibition-induced activation of JNK and induction of apoptosis are prevented by curcumin, an inhibitor of JNK-signaling pathway. Apoptosis is also blocked by SP600125, a specific inhibitor of JNK activity, indicating that JNK activity is required for the induction of apoptosis in these cells. These findings suggest that the metabolites of arachidonate 5-lipoxygenase promote survival of prostate cancer cells involving down-regulation of stress-activated protein kinase.     

The Drosophila cell shape regulator c-Jun N-terminal kinase also functions as a stress-activated protein kinase.

Mammalian c-Jun N-terminal kinases (JNKs) are members of a group of stress-activated intracellular signalling molecules within the MAP kinase family. Molecular genetic analysis of a highly evolutionarily conserved Drosophila JNK homologue, DJNK, has demonstrated that this molecule plays an essential developmental role in cell shape regulation. However, it remains to be determined whether DJNK also responds to the broad range of cellular stresses and other stimuli that affect its mammalian counterpart. Here we demonstrate that c-Jun, a substrate for mammalian JNKs, is a specific substrate for DJNK and that an antiserum that cross-reacts with activated mammalian JNK at the conserved threonyl-prolyl-tyrosyl (TPY) motif within the activation loop also specifically recognises the activated form of DJNK. Using these two assays, we show that DJNK activity is stimulated in cultured cells by several treatments that activate mammalian JNKs, including addition of arsenite, vanadate and ceramide derivatives. It is therefore concluded that in addition to its essential developmental functions, DJNK plays an important role in stress responses that mirrors its mammalian counterpart.     

Noncanonical function of MEKK2 and MEK5 PB1 domains for coordinated extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase signaling

MEKK2 and MEK5 encode Phox/Bem1p (PB1) domains that heterodimerize with one another. MEKK2, MEK5, and extracellular signal-related kinase 5 (ERK5) form a ternary complex through interactions involving the MEKK2 and MEK5 PB1 domains and a 34-amino-acid C-terminal extension of the MEK5 PB1 domain. This C-terminal extension encodes an ERK5 docking site required for MEK5 activation of ERK5. The PB1 domains bind in a front-to-back arrangement, with a cluster of basic amino acids in the front of the MEKK2 PB1 domain binding to the back-end acidic clusters of the MEK5 PB1 domain. The C-terminal moiety, including the acidic cluster of the MEKK2 PB1 domain, is not required for MEK5 binding and binds MKK7. Quiescent MEKK2 preferentially binds MEK5, and MEKK2 activation results in ERK5 activation. Activated MEKK2 binds and activates MKK7, leading to JNK activation. The findings define how the MEKK2 and MEK5 PB1 domains are uniquely used for differential binding of two mitogen-activated protein kinase kinases, MEK5 and MKK7, for the coordinated control of ERK5 and c-Jun N-terminal kinase activation.     

Inhibition of the CSF‐1 receptor sensitizes ovarian cancer cells to cisplatin

Ovarian cancer is one of the most common female malignancies, and cisplatin‐based chemotherapy is routinely used in locally advanced ovarian cancer patients. Acquired or de novo cisplatin resistance remains the barrier to patient survival, and the mechanisms of cisplatin resistance are still not well understood. In the current study, we found that colony‐stimulating‐factor‐1 receptor (CSF‐1R) was upregulated in cisplatin‐resistant SK‐OV‐3 and CaoV‐3 cells. Colony‐stimulating‐factor‐1 receptor knockdown suppressed proliferation and enhanced apoptosis in cisplatin‐resistant SK‐OV‐3 and CaoV‐3 cells. However, CSF‐1R overexpression had inverse effects. While parental SK‐OV‐3 and CaoV‐3 cells were more resistant to cisplatin after CSF‐1R overexpression, CSF‐1R knockdown in SK‐OV‐3 and CaoV‐3 cells promoted cisplatin sensitivity. Overexpression and knockdown studies also showed that CSF‐1R significantly promoted active AKT and ERK1/2 signalling pathways in cisplatin‐resistant cells. Furthermore, a combination of cisplatin and CSF‐1R inhibitor effectively inhibited tumour growth in xenografts. Taken together, our results provide the first evidence that CSF‐1R inhibition can sensitize cisplatin‐refractory ovarian cancer cells. This study may help to increase understanding of the molecular mechanisms underlying cisplatin resistance in tumours.     

Cutting edge: CTLA-4 (CD152) differentially regulates mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) in CD4+ T cells from receptor/ligand-deficient mice

Although CTLA-4 (CD152) has potent inhibitory effects on T cell function, the signaling events affected by this coreceptor remain to be fully defined. Mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) act as crucial regulators of multiple aspects of cell function. Ab ligation studies have reported an inhibitory effect of CTLA-4 on TCR-induced ERK and JNK activation. In this study, we have re-examined the specificity of CTLA-4 inhibition of MAPKs by using natural ligand with ex vivo-purified CD4(+) T cells deficient in CD80 and CD86 (double knockout), or CTLA-4, CD80, and CD86 (triple knockout). Under these conditions, CTLA-4 ligation was found to up-regulate and sustain JNK activation, while inhibiting ERK activity. At the same time, JNK activation could not account for CTLA-4 induction of TGF-beta production. Our findings demonstrate that CTLA-4 cosignaling is more complex than previously appreciated, with an ability to differentially regulate members of the MAPK family in T cells.     

Cytokinetic effects of carboplatin and cisplatin on a human ovarian cancer cell line

The cytokinetic effects of carboplatin(CBDCA) on a human ovarian cancer cell line(KF-1) were examined by means of cell survival rate and flow cytometry in comparison with cisplatin(CDDP). CBDCA and CDDP exhibited dose dependent cytotoxicity on KF-1, and CBDCA showed compatible cell growth inhibition to that of 15 times concentration of CDDP in comparison with IC50 of 72 hrs after drug addition. From the analysis of cell cycle, CBDCA and CDDP inhibited cell cycle progression at G2 + M phase. CBDCA exhibited G2 + M phase block to that of 15 to 20 times the concentration of CDDP. We suggested that CBDCA had potential therapeutic activity against ovarian cancer, but should be evaluated carefully in the clinical use.     

Maintenance of differentiated phenotype of articular chondrocytes by protein kinase C and extracellular signal-regulated protein kinase.

The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signal-regulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC alpha was essentially the same as that of type II collagen during de- and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC alpha, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC alpha in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC alpha did not affect type II collagen levels, suggesting that PKC alpha expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by down-regulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of N-cadherin, alpha-catenin, and beta-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP.

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10(-8) M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.