Hypochlorous acid stimulation of the mitogen-activated protein kinase pathway enhances cell survival

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAP kinase), the extracellular regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), by the myeloperoxidase-derived oxidant HOCl, in human umbilical vein endothelial cells (HUVEC) and human skin fibroblasts. Treatment of fibroblasts with 10-30 microM HOCl induced a dose-dependent increase in the tyrosine phosphorylation of several proteins. ERK1/2 was activated by exposure to sublethal concentrations of reagent HOCl or by HOCl generated by myeloperoxidase as shown by immune complex kinase assays. Maximum activation was seen at 20 microM and peak activation occurred within 10 min. Western blot analysis demonstrated activation of p38 with 30 microM HOCl, occurring at 15-30 min. No activation of JNK was detected in the concentration range investigated. These results show that HOCl is able to activate MAP kinases. Effective doses were considerably lower than with H2O2 and the lack of JNK activation contrasts with the activation frequently seen with H2O2. Exposure to HOCl caused a loss of viability in HUVEC that was markedly enhanced when ERK1/2 activation was inhibited by U0126. This suggests that the activation of ERK promotes cell survival in response to the oxidative challenge.     

Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase

Human collagenase-3 (matrix metalloproteinase 13 (MMP-13)) is characterized by exceptionally wide substrate specificity and restricted tissue specific expression. Human skin fibroblasts in culture express MMP-13 only when they are in three-dimensional collagen (Ravanti, L., Heino, J., López-Otín, C., and K?h?ri. V.-M. (1999) J. Biol. Chem. 274, 2446-2455). Here we show that MMP-13 is expressed by fibroblasts during normal human gingival wound repair. Expression of MMP-13 by human gingival fibroblasts cultured in monolayer or in collagen gel was induced by transforming growth factor-beta1 (TGF-beta1). Treatment of gingival fibroblasts with TGF-beta1 activated two distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2) in 15 min and p38 MAPK in 1 and 2 h. Induction of MMP-13 expression by TGF-beta1 was blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a selective inhibitor of ERK1/2 activation. Adenovirus-mediated expression of dominant negative p38alpha and c-Jun potently inhibited induction of MMP-13 expression in gingival fibroblasts by TGF-beta1. Infection of gingival fibroblasts with adenovirus for constitutively active MEK1 resulted in activation of ERK1/2 and JNK1 and up-regulation of collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) production but did not induce MMP-13 expression. In addition, activation of p38 MAPK by constitutively active MKK6b or MKK3b was not sufficient to induce MMP-13 expression. These results show that TGF-beta-elicited induction of MMP-13 expression by gingival fibroblasts is dependent on the activity of p38 MAPK and the presence of functional AP-1 dimers. These observations demonstrate a fundamental difference in the regulation of collagenolytic capacity between gingival and dermal fibroblasts and suggest a role for MMP-13 in rapid turnover of collagenous matrix during repair of gingival wounds, which heal with minimal scarring.     

A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways.

A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant. This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs. In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro. Overexpression of a dominant-negative MTK1 mutant [MTK1(K/R)] strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha. The dominant-negative MTK1(K/R) had no effect on the activation of the JNK pathway or the ERK pathway. These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway.     

Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells

Heat shock proteins (HSPs) require no adjuvant to confer immunogenicity to bound peptides, as if they possessed an intrinsic "danger" signature. To understand the proinflammatory nature of HSP, we analyzed signaling induced by human and chlamydial HSP60. We show that both HSP60s activate the stress-activated protein kinases p38 and JNK1/2, the mitogen-activated protein kinases ERK1/2, and the I-kappaB kinase (IKK). Activation of JNK and IKK proceeds via the Toll/IL-1 receptor signaling pathway involving MyD88 and TRAF6. Human fibroblasts transfected with TLR2 or TLR4 plus MD-2 gain responsiveness to HSP60, while TLR2- or TLR4-defective cells display impaired responses. Initiation of signaling requires endocytosis of HSP60 that is effectively inhibited by serum component(s). The results revealed that adjuvanticity of HSP60 operates similar to that of classical pathogen-derived ligands.     

p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38alpha is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38alpha, we utilized newly established mouse fibroblast cell lines originated from a p38alpha null mouse, namely, a parental cell line without p38alpha gene locus, knockout of p38alpha (KOP), Zeosin-resistant (ZKOP), revertant of p38alpha (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38alpha. The loss of MAPKAPK2 expression accompanied by the defect of p38alpha is confirmed in an embryonic extract prepared from p38alpha null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in analyzing the functions of MAPKs, especially p38alpha, and show that p38 is indispensable for MAPKAPK2 expression.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Role of mitogen-activated protein kinases in S-nitrosoglutathione-induced macrophage apoptosis

The inflammatory mediator nitric oxide (NO*) promotes apoptotic cell death based on morphological evidence, accumulation of the tumor suppressor p53, caspase-3 activation, and DNA fragmentation in RAW 264.7 macrophages. Since nitrosothiols may actually be the predominant form of biologically active NO* in vivo, we used S-nitrosoglutathione (GSNO) to study activation of extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and p38 kinases. Moreover, we determined the role of mitogen-activated protein kinase signaling in the apoptotic transducing ability of GSNO. ERK1/2 became activated in response to GSNO after 4 h and remained active for the next 20 h. Blocking the ERK1/2 pathway by the mitogen-activated protein kinase kinase inhibitor PD 98059 enhanced GSNO-elicited apoptosis. p38 was activated as well, but inhibition of p38 with SB 203580 left apoptosis unaltered. Activation of JNK1/2 by GSNO showed maximal kinase activities between 2 and 8 h. Attenuating JNK1/2 by antisense-depletion eliminated the pro-apoptotic action of low GSNO concentrations (250 microM), whereas apoptosis proceeded independently of JNK1/2 at higher doses of the NO donor (500 microM). Decreased apoptosis by JNK1/2 depletion prevented p53 accumulation after the addition of GSNO, which positions JNK1/2 upstream of the p53 response at low agonist concentrations. In line, JNK1/2 activation proceeded unaltered in p53-antisense transfected macrophages. However, with higher GSNO concentrations apoptotic transducing pathways, including p53 accumulation, were JNK1/2 unrelated. The regulation of mitogen-activated protein kinases by GSNO may help to define cell protective and destructive actions of reactive nitrogen species.     

Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Polymeric Immunoglobulin Receptor-mediated Invasion of Streptococcus pneumoniae into Host Cells Requires a Coordinate Signaling of SRC Family of Protein-tyrosine Kinases,ERK, and c-Jun N-terminal Kinase

Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.     

Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on g protein-mediated activation of mitogen-activated protein kinases

Hypoxia has been shown to act as a proliferative stimulus for adventitial fibroblasts of the pulmonary artery. The signaling pathways involved in this growth response, however, remain unclear. We tested the hypothesis that hypoxia-induced proliferation of fibroblasts would be dependent on distinct (compared with serum) activation and utilization patterns of mitogen-activated protein (MAP) kinases initiated by Galpha(i/o) proteins. We found that hypoxia stimulated increases in DNA synthesis and growth of quiescent fibroblasts in the absence of exogenous mitogens and also markedly augmented serum-stimulated growth responses. Hypoxia caused a transient activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the time course and pattern of which was somewhat similar to that induced by serum but which was of lesser magnitude. On the other hand, hypoxia-induced activation of p38 MAP kinase was biphasic, whereas serum-stimulated activation of p38 MAP kinase was transient, and the magnitude of activation was greater for hypoxia compared with that of serum stimulation. ERK1/2, JNK1, and p38 MAP kinase but not JNK2 were necessary for hypoxia-induced proliferation because PD98059, SB202190, and JNK1 antisense oligonucleotides nearly ablated the growth response. JNK2 appeared to act as a negative modulator of hypoxia-induced growth because JNK2 antisense oligonucleotides led to an increase in DNA synthesis. In serum-stimulated cells, antisense JNK1 oligonucleotides and PD98059 had inhibitory effects on proliferation, whereas SB202190 led to an increase in DNA synthesis. Pertussis toxin, which blocks Galpha(i/o)-mediated signaling, markedly attenuated hypoxia-induced DNA synthesis and activation of ERK and JNK but not p38 MAP kinase. We conclude that hypoxia itself can act as a growth promoting stimulus for subsets of bovine neonatal adventitial fibroblasts largely through Galpha(i/o)-mediated activation of a complex network of MAP kinases whose specific contributions to hypoxia-induced proliferation differ from traditional serum-induced growth signals.     

Differential role of p38 and c-Jun N-terminal kinase 1 mitogen-activated protein kinases in NK cell cytotoxicity

The serine-threonine mitogen-activated protein kinase (MAPK) family includes extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinases. In NK cells, spontaneous or Ab-mediated recognition of target cells leads to activation of an ERK-2 MAPK-dependent biochemical pathway(s) involved in the regulation of NK cell effector functions. Here we assessed the roles of p38 and JNK MAPK in NK cell-mediated cytotoxicity. Our data indicate that p38 is activated in primary human NK cells upon stimulation with immune complexes and interaction with NK-sensitive target cells. FcgammaRIIIA-induced granule exocytosis and both spontaneous and Ab-dependent cytotoxicity were reduced in a dose-dependent manner in cells pretreated with either of two specific inhibitors of this kinase. Target cell-induced IFN-gamma and FcgammaRIIIA-induced TNF-alpha mRNA accumulation was similarly affected under the same conditions. Lack of inhibition of NK cell cytotoxicity in cells overexpressing an inactive form of JNK1 indicates that this kinase, activated only upon FcgammaRIIIA ligation, does not play a significant role in cytotoxicity. These data underscore the involvement of p38, but not JNK1, in the molecular mechanisms regulating NK cell cytotoxicity.     

Differential regulation of HSP70 expression by the JNK kinases SEK1 and MKK7 in mouse embryonic stem cells treated with cadmium

JNK, a member of the mitogen-activated protein kinases (MAPKs), is activated by the MAPK kinases SEK1 and MKK7 in response to environmental stresses. In the present study, the effects of CdCl2 treatment on MAPK phosphorylation and HSP70 expression were examined in mouse embryonic stem (ES) cells lacking the sek1 gene, the mkk7 gene, or both. Following CdCl2 exposure, the phosphorylation of JNK, p38, and ERK was suppressed in sek1-/- mkk7-/- cells. When sek1-/- or mkk7-/- cells were treated with CdCl2, JNK phosphorylation, but not the phosphorylation of either p38 or ERK, was markedly reduced, while a weak reduction in p38 phosphorylation was observed in sek1-/- cells. Thus, both SEK1 and MKK7 are required for JNK phosphorylation, whereas their role in p38 and ERK phosphorylation could overlap with that of another kinase. We also observed that CdCl2-induced HSP70 expression was abolished in sek1-/- mkk7-/- cells, was reduced in sek1-/- cells, and was enhanced in mkk7-/- cells. Similarly, the phosphorylation of heat shock factor 1 (HSF1) was decreased in sek1-/- mkk7-/- and sek1-/- cells, but was increased in mkk7-/- cells. Transfection with siRNA specific for JNK1, JNK2, p38, ERK1, or ERK2 suppressed CdCl2-induced HSP70 expression. In contrast, silencing of p38 or p38 resulted in further accumulation of HSP70 protein. These results suggest that HSP70 expression is up-regulated by SEK1 and down-regulated by MKK7 through distinct MAPK isoforms in mouse ES cells treated with CdCl2.     

Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases jun N-terminal kinase and p38

Collagenase-1 (matrix metalloproteinase-1, MMP-1) is expressed by several types of cells, including fibroblasts, and apparently plays an important role in the remodeling of collagenous extracellular matrix in various physiologic and pathologic situations. Here, we have examined the molecular mechanisms of the activation of fibroblast MMP-1 gene expression by a naturally occurring non-phorbol ester type tumor promoter okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatase 2A. We show that in fibroblasts OA activates three distinct subgroups of mitogen activated protein kinases (MAPKs): extracellular signal-regulated kinase 1,2 (ERK 1,2), c-Jun N-terminal-kinase/stress-activated protein kinase (JNK/SAPK) and p38. Activation of MMP-1 promoter by OA is entirely blocked by overexpression of dual-specificity MAPK phosphatase CL100. In addition, expression of kinase-deficient forms of ERK 1,2, SAPKβ, p38, or JNK/SAPK kinase SEK1 strongly inhibited OA-elicited activation of MMP-1 promoter. OA-elicited enhancement of MMP-1 mRNA abundance was also strongly prevented by two chemical MAPK inhibitors: PD 98059, a specific inhibitor of the activation of ERK1,2 kinases MEK1,2; and SB 203580, a selective inhibitor of p38 activity. Results of this study show that MMP-1 gene expression in fibroblasts is coordinately regulated by ERK1,2, JNK/SAPK, and p38 MAPKs and suggest an important role for the stress-activated MAPKs JNK/SAPK and p38 in the activation of MMP-1 gene expression. Based on these observations, it is conceivable that specific inhibition of stress-activated MAPK pathways may serve as a novel therapeutic target for inhibiting degradation of collagenous extracellular matrix.     

MAP kinases p38 and JNK are activated by the steroid hormone 1alpha,25(OH)2-vitamin D3 in the C2C12 muscle cell line

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1alpha,25(OH)2-vitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1alpha,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen-activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1Alpha,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c-Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced-p38 activation. Of relevance, 1alpha,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen-activated protein kinase activating protein kinase 2 (MAPKAP-kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation-dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1Alpha,25(OH)2D3 also promotes the phosphorylation of c-jun N-terminal protein kinases (JNK 1/2), the response is fast (0.5-1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1alpha,25(OH)2D3 (1 nM). The relative contribution of ERK-1/2, p38, and JNK-1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established.     

The conditional kinase DeltaMEKK1:ER* selectively activates the JNK pathway and protects against serum withdrawal-induced cell death

The conditional protein kinase DeltaMEKK3:ER* allows activation of the mitogen-activated and stress-activated protein kinases (MAPKs and SAPKs) without imposing a primary cellular stress or damage. Such separation of stress from stress-induced signalling is particularly important in the analysis of apoptosis. Activation of DeltaMEKK3:ER* in cycling CCl39 cells caused a rapid stimulation of the ERK1/2, JNK and p38 pathways but resulted in a slow, delayed apoptotic response. Paradoxically, activation of the same pathways inhibited the rapid expression of Bim(EL) and apoptosis following withdrawal of serum. Inhibition of the ERK1/2 pathway prevented the down-regulation of Bim(EL) but caused only a partial reversion of the cyto-protective effect of DeltaMEKK3:ER*. In contrast, inhibition of p38 had no effect, raising the possibility that activation of JNK might also exert a protective effect. To test this we used CCl39 cells expressing DeltaMEKK1:ER* which activates JNK but not ERK1/2, p38, PKB or IkappaB kinase. Activation of DeltaMEKK1:ER* inhibited serum withdrawal-induced conformational changes in Bax and apoptosis. These results suggest that in the absence of any overt cellular damage or chemical stress activation of JNK can act independently of the ERK1/2 or PKB pathways to inhibit serum withdrawal-induced cell death.