The p38 mitogen-activated protein kinase is activated by treatment of cells with cytokines and by exposure to environmental stress. The effects of these stimuli on p38 MAP kinase are mediated by the MAP kinase kinases (MKKs) MKK3, MKK4, and MKK6. The function of the p38 MAP kinase signaling pathway was examined by investigating the effect of targeted disruption of the Mkk3 gene. Mkk3 gene disruption causes a selective defect in the response of fibroblasts to the proinflammatory cytokine tumor necrosis factor, including reduced p38 MAP kinase activation and cytokine expression. These data demonstrate that the MKK3 protein kinase is a critical component of a tumor necrosis factor-stimulated signaling pathway that causes increased expression of inflammatory cytokines (Wysk, 1999).

The p38 mitogen-activated protein kinase (MAPK) pathway, like the c-Jun N-terminal kinase (JNK) MAPK pathway, is activated in response to cellular stress and inflammation and is involved in many fundamental biological processes. To study the role of the p38 MAPK pathway in vivo, homologous recombination in mice was used to inactivate the Mkk3 gene, one of the two specific MAPK kinases (MAPKKs) that activate p38 MAPK. Mkk3(-/-) mice are viable and fertile; however, they are defective in interleukin-12 (IL-12) production by macrophages and dendritic cells. Interferon-gamma production following immunization with protein antigens and in vitro differentiation of naive T cells is greatly reduced, suggesting an impaired type I cytokine immune response. The effect of the p38 MAPK pathway on IL-12 expression is at least partly transcriptional, since inhibition of this pathway blocks IL-12 p40 promoter activity in macrophage cell lines and IL-12 p40 mRNA is reduced in MKK3-deficient mice. It is concluded that the p38 MAP kinase, activated through MKK3, is required for the production of inflammatory cytokines by both antigen-presenting cells and CD4(+) T cells (Lu, 1999).

The inflammatory cytokine interleukin-1beta (IL-1beta) induces cyclooxygenase-2 (Cox-2) expression with a concomitant release of prostaglandins from glomerular mesangial cells. IL-1beta rapidly activates the c-Jun NH2-terminal/stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein kinase (MAPK) and also induces Cox-2 expression and prostaglandin E2 (PGE2) production. Overexpression of the dominant negative form of JNK1 or p54 JNK2/SAPKbeta reduces Cox-2 expression and PGE2 production stimulated by IL-1beta. Similarly, overexpression of the kinase-dead form of p38 MAPK also inhibits IL-1beta-induced Cox-2 expression and PGE2 production. These results suggest that activation of both JNK/SAPK and p38 MAPK is required for Cox-2 expression after IL-1beta activation. Furthermore, these experiments confirm that IL-1beta activates MAP kinase kinase-4 (MKK4)/SEK1, MKK3, and MKK6 in renal mesangial cells. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta- induced Cox-2 expression with inhibition of both JNK/SAPK and p38 MAPK phosphorylation. Overexpression of the kinase-dead form of MKK3 or MKK6 demonstrates that either of these two mutant kinases inhibit IL-1beta-induced p38 MAPK phosphorylation and Cox-2 expression but not JNK/SAPK phosphorylation and activation. This study suggests that the activation of both JNK/SAPK and p38 MAPK signaling cascades is required for IL-1beta-induced Cox-2 expression and PGE2 synthesis (Guan, 1998).

Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C subfamily of chemokines, is important for the local recruitment of leukocytes to sites of inflammatory challenge. Endothelial signaling pathways involving members of the mitogen-activated protein (MAP) kinase superfamily have been investigated and their role for MCP-1 expression in endothelium has been studied. Tumor necrosis factor-alpha (TNF-alpha), a potent inflammatory activator of endothelium, leads to activation of MAP kinases ERK, p38, and JNK in human umbilical vein endothelial cells (HUVEC). Contribution of MAP kinase pathways to TNF-alpha-induced synthesis of endothelial MCP-1 was studied by pharmacologic inhibition and transient expression of dominant negative or constitutively active kinase mutants. Inhibition of Raf/MEK/ERK or SEK/JNK pathways has no significant effect on MCP-1 levels, whereas blocking the MKK6/p38 pathway by p38 inhibitors SB203580 or SB202190 or by a dominant negative mutant of MKK6, the upstream activator of p38, strongly inhibits TNF-alpha-induced expression of MCP-1. Consistent with that finding, expression of wild-type or constitutively active MKK6 significantly enhances the effect of limiting TNF-alpha concentrations on MCP-1 synthesis. These data suggest a crucial role for the MKK6/p38 stress kinase cascade in TNF-alpha-mediated endothelial MCP-1 expression (Goebeler, 1999).

TNF-alpha regulates the expression of many proinflammatory and profibrogenic gene products in macrophages, and hence plays a vital role in controlling the inflammatory response. Exposure of macrophages to TNF-alpha stimulates the activation of members of the mitogen-activated protein kinase (MAPK) family. The mechanism of activation of the p38mapk by TNF-alpha has been investigated in mouse bone marrow-derived macrophages. Exposure to TNF-alpha results in the activation of p38mapk, as measured by (1) the trans-phosphorylation of recombinant activating transcription factor-2 substrate by immunoprecipitated p38mapk and (2) specific tyrosine phosphorylation of immunoprecipitated p38mapk. In addition, selective ligation of the TNF-alpha receptor CD120a (p55) with human TNF-alpha is sufficient to induce p38mapk activation. Using an in vitro kinase assay with recombinant kinase-inactive p38mapk as substrate in the presence of [gamma-32P]ATP, the upstream kinases MKK3 (mitogen-activated protein kinase kinase 3) and MKK4 have been found to be activated in response to TNF-alpha. These findings suggest that TNF-alpha transiently phosphorylates and activates the three members of the MAPK family [namely p42(mapk/erk2), p46 c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38mapk] following cross-linking of CD120a (p55), and that MKK3 and MKK4 are capable of phosphorylating p38mapk (Winston, 1997).

IL-1beta converting enzyme (ICE) family cysteine proteases are subdivided into three groups: ICE-, CPP32-, and Ich-1-like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases, which is thought to be essential for execution of the cell death. Two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and MKK6 is a major activator for p38 in Fas signaling. To dissect various cellular responses induced by Fas, several peptide inhibitors for ICE family proteases were used in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK (which inhibits almost all the Fas-induced cellular responses) blocks the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases (which inhibit the Fas-induced chromatin condensation and DNA fragmentation) do not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors do not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by their dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses (Toyoshima, 1987).

The Fas receptor mediates a signaling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. Fas activates the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlates temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiates Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK have shown that Fas requires ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis. CrmA antagonizes, and YVAD-CMK and Z-VAD-FMK completely inhibits, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide is resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in this study indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signaling cascades (Juo, 1997).

The persistent activation of p42/p44(MAPK) is required to pass the G1 restriction point in fibroblasts and it has been postulated that MAPKs control the activation of G1 cyclin-dependent complexes. The mitogen-dependent induction of cyclin D1 expression, one of the earliest cell cycle-related events to occur during the G0/G1 to S-phase transition, was examined as a potential target of MAPK regulation. Effects exerted either by the p42/p44(MAPK) or the p38/HOGMAPK cascade on the regulation of cyclin D1 promoter activity or cyclin D1 expression were compared in CCL39 cells, using a co-transfection procedure. Inhibition of the p42/p44(MAPK) signaling by expression of dominant-negative forms of either mitogen-activated protein kinase kinase 1 (MKK1) or p44(MAPK), or by expression of the MAP kinase phosphatase, MKP-1, strongly inhibits expression of a reporter gene driven by the human cyclin D1 promoter as well as the endogenous cyclin D1 protein. Conversely, activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically increases cyclin D1 promoter activity and cyclin D1 protein expression, in a growth factor-independent manner. Moreover, the use of a CCL39-derived cell line that stably expresses an inducible chimera of the estrogen receptor fused to a constitutively active Raf-1 mutant (DeltaRaf-1:ER) reveals that in absence of growth factors, activation of the Raf > MKK1 > p42/p44MAPK cascade is sufficient to fully induce cyclin D1. In marked contrast, the p38(MAPK) cascade shows an opposite effect on the regulation of cyclin D1 expression. In cells co-expressing high levels of the p38(MAPK) kinase (MKK3) together with the p38(MAPK), a significant inhibition of mitogen-induced cyclin D1 expression is observed. Furthermore, inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, enhances cyclin D1 transcription and protein level. Altogether, these results support the notion that MAPK cascades drive specific cell cycle responses to extracellular stimuli, at least in part, through the modulation of cyclin D1 expression and associated cdk activities (Lavoie, 1996).

Differentiation of muscle cells is regulated by extracellular growth factors that transmit largely unknown signals into the cells. Some of these growth factors induce mitogen-activated protein kinase (MAPK) cascades within muscle cells. The kinase activity of p38 MAPK is induced early during terminal differentiation of L8 cells. Addition of a specific p38 inhibitor (SB 203580) to myoblasts blocks their fusion to multinucleated myotubes and prevents the expression of MyoD and MEF2 family members and myosin light chain 2. The expression of MKK6, a direct activator of p38, or of p38 itself enhances the activity of MyoD in converting 10T1/2 fibroblasts to muscle, whereas treatment with SB 203580 inhibits MyoD. Several lines of evidence are presented suggesting that the involvement of p38 in MyoD activity is mediated via its co-activator MEF2C, a known substrate of p38. MEF2C protein and MEF2-binding sites are necessary for the p38 MAPK pathway to regulate the transcription of muscle creatine kinase reporter gene. These results indicate that the p38 MAPK pathway promotes skeletal muscle differentiation at least in part via activation of MEF2C (Zetser, 1999).

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1-adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen-activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augments cell size and effects relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase confers sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activates p38, augments cell size, induces NP and alpha-Ska promoter activities by up to 130-fold, and elicits sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE are blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy (Zechner, 1997).

In cardiac myocytes the stimulation of p38 mitogen-activated protein kinase activates a hypertrophic growth program and the induction of the cardiac-specific genes associated with this program. This study focused on determining whether these novel growth-promoting effects are accompanied by the p38-mediated inhibition of apoptosis, and if so, what signaling pathways might be responsible. Primary neonatal rat ventricular myocytes were driven into apoptosis by treatments known to induce apoptosis in other cell types, e.g. incubation with anisomycin or overexpression of constitutively active MEKK-1 (MEKK-1COOH), a protein that strongly activates extracellular signal-regulated kinase and N-terminal c-Jun kinase, but not p38. Overexpression of constitutively active MKK6, MKK6 (Glu), which selectively activates p38 in cardiac myocytes, protects cells from either anisomycin- or MEKK-1COOH-induced apoptosis. This protection is blocked by SB 203580, a selective p38 inhibitor. MKK6 (Glu) also activates transcription mediated by NF-kappaB, a factor that protects other cell types from apoptosis. The activation of NF-kappaB and the protection from apoptosis mediated by MKK6 (Glu) are both blocked by SB 203580. Interestingly, overexpression of a mutant form of I-kappaBalpha, which inhibits nuclear translocation of NF-kappaB, completely blocks MKK6 (Glu)-activated NF-kappaB but has little effect on MKK6s anti-apoptotic effects. These findings suggest that, in part, the overexpression of MKK6 (Glu) may foster growth and survival of cardiac myocytes by protecting them from apoptosis in a p38-dependent manner. Additionally, while NF-kappaB is activated in myocardial cells by p38, this does not appear to be the major mechanism by which MKK6 (Glu) exerts its anti-apoptotic effects in this cell type, suggesting a novel pathway for p38-mediated protection from apoptosis (Zechner, 1998).