In cardiac myocytes the stimulation of p38 mitogenactivated 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 constitutively active MEKK-1 (MEKK-1 COOH ), 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, protected cells from either anisomycin-or MEKK-1 COOH -induced apoptosis. This protection was blocked by SB 203580, a selective p38 inhibitor. MKK6 (Glu) also activated transcription mediated by NF-B, a factor which protects other cell types from apoptosis. The activation of NF-B and the protection from apoptosis mediated by MKK6 (Glu) were both blocked by SB 203580. Interestingly, overexpression of a mutant form of I-B␣, which inhibits nuclear translocation of NF-B, completely blocked MKK6 (Glu)-activated NF-B but had 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-B 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.

In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-B, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IB kinase (IKK)-␤ and stimulated its abilities to phosphorylate IB and to activate NF-B. MKK6(Glu) induced NF-B-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-␣, which activates both NF-B and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-␣ was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-␣-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-␤ activated protein kinase (Tak1), which is known to activate p38 and NF-B in other cell types. Thus, by stimulating both p38 and NF-B, Tak1-activating cytokines, like TNF-␣, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38.

Many cell types mount elaborate, compensatory responses to stress that enhance survival; however, the intracellular signals that govern these responses are poorly understood. Cardiotrophin-1 (CT-1), a stress-induced cytokine, belongs to the interleukin-6/glycoprotein 130 receptor-coupled cytokine family. CT-1 is released from the heart in response to hypoxic stress, and it protects cardiac myocytes from hypoxia-induced apoptosis, thus establishing a central role for this cytokine in the cardiac stress response. In the present study, CT-1 activated p38 and ERK MAPKs as well as Akt in cultured cardiac myocytes; these three pathways were activated in a parallel manner. CT-1 also induced the degradation of the NF-B cytosolic anchor, IB, as well as the translocation of the p65 subunit of NF-B to the nucleus and increased expression of an NF-B-dependent reporter gene. Inhibitors of the p38, ERK, or Akt pathways each partially reduced CT-1-mediated NF-B activation, as well as the cytoprotective effects of CT-1 against hypoxic stress. Together, the inhibitors completely blocked CT-1-dependent NF-B activation and cytoprotection. A cellpermeable peptide that selectively disrupted NF-B activation also completely inhibited the cytoprotective effects of CT-1. These results indicate that CT-1 signals through p38, ERK, and Akt in a parallel manner to activate NF-B and that NF-B is required for CT-1 to mediate its full cytoprotective effects in cardiac myocytes.