Stress kinase MKK7: savior of cell cycle arrest and cellular senescence

The c-Jun N-terminal kinase (JNK/SAPK) signaling cascade controls a spectrum of cellular processes, including cell growth, differentiation, transformation, and apoptosis. We recently demonstrated that stress kinase MKK7, a direct activator of JNKs, couples stress signaling to G2/M cell cycle progression, CDC2 expression, and cellular senescence. We further explored other molecules involved in JNK pathway and found that both MKK4, another direct activator of JNK, and c-Jun, a direct substrate of JNK, have similar roles to MKK7. Here we discuss the importance of the MKK4/MKK7-JNK-c-Jun pathway linking stress and developmental signals to cell proliferation, cell cycle progression, cellular senescence, and apoptosis including recent unpublished data from our lab.     

Stress Kinase MKK7: Saviour of Cell Cycle Arrest and Cellular Senescence

The c-Jun N-terminal kinase (JNK/SAPK) signaling cascade controls a spectrum ofcellular processes, including cell growth, differentiation, transformation, and apoptosis.We recently demonstrated that stress kinase MKK7, a direct activator of JNKs, couplesstress signaling to G2/M cell cycle progression, CDC2 expression, and cellularsenescence. We further explored other molecules involved in JNK pathway and foundthat both MKK4, another direct activator of JNK, and c-Jun, a direct substrate of JNK,have similar roles to MKK7. Here we discuss the importance of the MKK4/MKK7-JNKc-Jun pathway linking stress and developmental signals to cell proliferation, cell cycleprogression, cellular senescence, and apoptosis including recent unpublished data fromour lab.     

MKK7 couples stress signalling to G2/M cell-cycle progression and cellular senescence

During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4-5, mkk7(-/-) mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7-JNK-c-Jun pathway. These data show that the MKK7-JNK-c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.     

Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.     

Differentiation-associated genes regulated by TPA-induced c-Jun expression via a PKC/JNK pathway in KYSE450 cells

A group of potential differentiation-associated genes had been identified by microarray analysis as c-Jun/AP-1 target genes essential for epithelial differentiation program. Our previous study showed that c-Jun/AP-1 could bind and activate these gene promoters in vivo using chromatin immunoprecipitation. To further understand how the mitogen-activated protein kinase signaling pathways regulate AP-1 activity and expression of c-Jun target genes, our strategy was based on the use of 12-o-tetradecanoylophorbol-13-acetate (TPA) and pharmacological reagents to induce or block c-Jun expression. The mRNA and protein expression of these genes increased in response to TPA-induced c-Jun/AP-1 expression. Inhibitors of JNK (SP600125) and PKC (GF109203X) mainly blocked expression and phosphorylation of c-Jun, while inhibition of MEK-ERK activity with PD98059 (an inhibitor of MEK) had little effect. Expression of involucrin and keratin 4 in response to TPA was attenuated by pretreatments with GF109203X and SP600125, but not PD98059, suggesting involvement of PKC and JNK in this response. Taken together, these results suggested that differentiation-associated genes were regulated by TPA-induced c-Jun/AP-1 mainly via a PKC/JNK pathway in esophageal cancer cell line KYSE450.     

Differential gene regulation by specific gain-of-function JNK1 proteins expressed in Swiss 3T3 fibroblasts

The c-Jun N-terminal kinases (JNKs) are encoded by three genes that yield 10 isoforms through alternative mRNA splicing. The roles of each JNK isoform in the many putative biological responses where the JNK pathway is activated are still unclear. To examine the cellular responses mediated by different JNK isoforms, gain-of-function JNK1 polypeptides were generated by fusing the upstream mitogen-activated protein kinase kinase, MKK7, with p46JNK1alpha or p46JNK1beta. The MKK7-JNK fusion proteins, which exhibited constitutive activity in 293T cells, were stably expressed in Swiss 3T3 fibroblasts using retrovirus-mediated gene transfer. Swiss 3T3 cells expressing either of the MKK7-JNK polypeptides were equally sensitized to induction of cell death following serum withdrawal. To search for other cellular responses that may be selectively regulated by the JNK1 isoforms, the gene expression profiles of Swiss 3T3 cells expressing MKK7-JNK1alpha or MKK7-JNK1beta were compared with empty vector-transfected control cells. Affymetrix Genechips identified 46 genes for which expression was increased in MKK7-JNK-expressing cells relative to vector control cells. Twenty genes including those for c-Jun, MKP-7, interluekin-1 receptor family member ST2L/ST2, and c-Jun-binding protein were induced similarly by MKK7-JNK1alpha and MKK7-JNK1beta proteins, whereas 13 genes were selectively increased by MKK7-JNK1alpha and 13 genes were selectively increased by MKK7-JNK1beta. The set of genes selectively induced by MKK7-JNK1beta included a number of known interferon-stimulated genes (ISG12, ISG15, IGTP, and GTPI). Consistent with these gene expression changes, Swiss 3T3 cells expressing MKK7-JNK1beta exhibited increased resistance to vesicular stomatitis virus-induced cell death. These findings reveal evidence for JNK isoform-selective gene regulation and support a role for distinct JNK isoforms in specific cellular responses.     

Phorbol ester-induced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059.

The purpose of this study was to evaluate whether the mitogen-activated protein kinase (MAPK) signaling pathway contributes to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mononuclear differentiation in the human myeloblastic leukemia ML-1 cells. Upon TPA treatment, the activity of ERK1 and ERK2 rapidly increased, with maximal induction between 1 and 3 h, while ERK2 protein levels remained constant. The activity of JNK1 was also significantly induced, with JNK1 protein levels increasing moderately during exposure to TPA. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited TPA-induced ERK2 activity. Furthermore, PD98059 completely blocked the TPA-induced differentiation of ML-1 cells, as assessed by a number of features associated with mononuclear differentiation including changes in morphology, nonspecific esterase activity, phagocytic ability, NADPH oxidase activity, mitochondrial respiration, and c-jun mRNA inducibility. We conclude that activation of the MEK/ERK signaling pathway is necessary for TPA-induced mononuclear cell differentiation.     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

Antagonistic control of cell fates by JNK and p38-MAPK signaling

During the development and organogenesis of all multicellular organisms, cell fate decisions determine whether cells undergo proliferation, differentiation, or aging. Two independent stress kinase signaling pathways, p38-MAPK, and JNKs, have evolved that relay developmental and environmental cues to determine cell responses. Although multiple stimuli can activate these two stress kinase pathways, the functional interactions and molecular cross-talks between these common second signaling cascades are poorly elucidated. Here we report that JNK and p38-MAPK pathways antagonistically control cellular senescence, oncogenic transformation, and proliferation in primary mouse embryonic fibroblasts (MEFs). Similarly, genetic inactivation of the JNK pathway results in impaired proliferation of fetal hepatoblasts in vitro and defective adult liver regeneration in vivo, which is rescued by inhibition of the p38-MAPK pathway. Thus, the balance between the two stress-signaling pathways, MKK7-JNK and MKK3/6-p38-MAPK, determines cell fate and links environmental and developmental stress to cell cycle arrest, senescence, oncogenic transformation, and adult tissue regeneration.     

Bovine type I collagen inhibits Raw264.7 cell proliferation through phosphoinositide 3-kinase- and mitogen-activated protein kinase-dependent down-regulation of cyclins D1, A and B1

Bovine type I collagen (BIC), which is widely used as a fibrous extracellular matrix component in cell culture models, inhibits the progression of melanoma cell cycle via p27 up-regulation. BIC also induces nitric oxide synthase in macrophages through JunB/AP-1 and NF-kappaB activation. Given the previous observations, this study investigates the effect of BIC on the cell cycle progression and regulatory function of Raw264.7 macrophage cells and the responsible signaling pathways. Cell cycle analysis revealed that BIC completely suppressed proliferation of Raw264.7 cells with inhibition of the percentage of cells in the S phase and the reciprocal decrease in the G0/G1 phase. DNA synthesis was also inhibited by BIC, as evidenced by a decrease in the cellular incorporation of [3H]thymidine. The G1/S arrest induced by BIC was reversed by chemical inhibition of phosphatidylinositol 3-kinase (PI3-kinase) or overexpression of the p85 subunit of PI3-kinase. Either PD98059 or stable transfection with mitogen-activated protein kinase kinase-1 [MKK1(-)] or c-Jun N-terminal kinase 1 [JNK1(-)] also released the cell cycle arrest. Immunoblot analyses revealed that the levels of cyclins D1, A and B1 were partly or completely down-regulated by BIC, but cyclin E, p21 and p27 were minimally changed. Chemical inhibition and dominant negative mutant overexpression experiments revealed that either PI3-kinase inhibition or JNK1(-) transfection prevented the decreases in cyclin D1, A and B1 by BIC, indicating that the PI3-kinase and JNK1 pathways were associated with disruption of the cyclins. The pathway involving MKK1-extracellular signal-regulated kinase-1/2 (ERK1/2) was responsible for the suppression of cyclin A and B1, but not that of cyclin D1. The present study showed that BIC inhibited proliferation of Raw264.7 cells and that the pathways involving PI3-kinase and mitogen-activated protein kinases regulate the cell cycle arrest.     

ASK1 regulates influenza virus infection-induced apoptotic cell death

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.     

12-O-tetradecanoylphorbol-13-acetate (TPA)-induced c-Jun N-terminal kinase (JNK) phosphatase renders immortalized or transformed epithelial cells refractory to TPA-inducible JNK activity

c-Jun N-terminal kinase (JNK) regulates gene expression in response to various extracellular stimuli. JNK can be activated by the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) in normal human oral keratinocytes but not in human keratinocytes that have been immortalized (HOK-16B and HaCaT) or transformed (HOK-16B-Bap-T) nor in a cervical carcinoma cell line (HeLa). The refractory JNK activation response to TPA is not due a defect in the JNK pathway, because JNK can be activated by other stimuli, e.g. UV irradiation and an alkylating agent N-methyl-N'-nitrosoguanidine in these immortalized or transformed cells. More importantly, the refractory JNK and JNKK activation response to TPA can be restored by treatment of the cells with a combination of TPA and a protein-tyrosine phosphatase inhibitor, sodium orthovanadate. Furthermore, pretreatment of cells with TPA partially inhibited UV- or N-methyl-N'-nitrosoguanidine-induced JNK activity. These results suggest that a TPA-inducible, orthovanadate-sensitive protein-tyrosine phosphatase may specifically down-regulate JNK signaling pathway in these immortalized/transformed epithelial cells. In contrast, ERK and p38/Mpk2 are not regulated by this TPA-induced phosphatase. This putative protein-tyrosine phosphatase appears to be JNK pathway-specific.     

Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with -754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38alpha, beta, and delta decreased and p38gamma increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (-668/-654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (-47/-42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediates HO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.