Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and upregulate c-Jun protein in neonatal rat ventricular myocytes.

Pro-inflammatory cytokines may be important in the pathophysiological responses of the heart. We investigated the activation of the three mitogen-activated protein kinase (MAPK) subfamilies ¿c-Jun N-terminal kinases (JNKs), p38-MAPKs and extracellularly-responsive kinases (ERKs) by interleukin-1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) in primary cultures of myocytes isolated from neonatal rat ventricles. Both cytokines stimulated a rapid (maximal within 10 min) increase in JNK activity. Although activation of JNKs by IL-1 beta was transient returning to control values within 1 h, the response to TNF alpha was sustained. IL-1 beta and TNF alpha also stimulated p38-MAPK phosphorylation, but the response to IL-1 beta was consistently greater than TNF alpha. Both cytokines activated ERKs, but to a lesser degree than that induced by phorbol esters. The transcription factors, c-Jun and ATF2, are phosphorylated by the MAPKs and are implicated in the upregulation of c-Jun. IL-1 beta and TNF alpha stimulated the phosphorylation of c-Jun and ATF2. However, IL-1 beta induced a greater increase in c-Jun protein. Inhibitors of protein kinase C (PKC) (Ro318220, GF109203X) and the ERK cascade (PD98059) attenuated the increase in c-Jun induced by IL-1 beta, but LY294002 (an inhibitor of phosphatidylinositol 3' kinase) and SB203580 (an inhibitor of p38-MAPK, which also inhibits certain JNK isoforms) had no effect. These data illustrate that some of the pathological effects of IL-1 beta and TNF alpha may be mediated through the MAPK cascades, and that the ERK cascade, rather than JNKs or p38-MAPKs, are implicated in the upregulation of c-Jun by IL-1 beta.

Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c-jun expression.

Cardiac myocyte hypertrophy is associated with an increase in expression of immediate early genes (e.g. c-jun) via activation of pre-existing transcription factors. The activity of CREB transcription factor is regulated through phosphorylation of Ser-133 by one of several protein kinases (e.g. protein kinase A (PKA), p90 ribosomal S6 kinases (RSKs) and the related kinase, MSK1). A cell-permeable form of cAMP, hypertrophic agonists (endothelin-1 (ET-1), phenylephrine (PE)) and hyperosmotic shock all promoted phosphorylation of CREB(Ser-133) in rat neonatal cardiac myocytes. The response to endothelin-1 required the extracellular signal-regulated kinase cascade which stimulates both RSKs and MSK1. Phosphorylation of CREB(Ser-133) in response to ET-1 was not associated with any increase in DNA binding to a consensus cAMP-response element (CRE). The rat c-jun promoter contains elements which may bind either c-Jun/ATF2 or CREB/ATF1 dimers. Using extracts from rat cardiac myocytes, we identified at least two complexes which bind to the most proximal of these elements, one of which contained CREB and the other c-Jun. Thus, phosphorylation and activation of CREB in cardiac myocytes may be effected by a range of different stimuli to influence the expression of immediate early genes such as c-jun.

Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes.

Cardiac hypertrophy is associated with hypertrophic growth of cardiac myocytes and increased fibrosis. Much is known of the stimuli which promote myocyte hypertrophy and the changes associated with the response, but the links between the two are largely unknown. Using subtractive hybridization, we identified three genes which are acutely (<1 h) upregulated in neonatal rat ventricular myocytes exposed to the alpha-adrenergic agonist, phenylephrine. One represented connective tissue growth factor (CTGF) which is implicated in fibrosis and promotes hypertrophy in other cells. We further examined the expression of CTGF mRNA and protein in cardiac myocytes using quantitative PCR and immunoblotting, confirming that phenylephrine increased CTGF mRNA (maximal within 1 h) and protein (increased over 4 - 24 h). Endothelin-1 promoted a greater, though transient, increase in CTGF mRNA, but the increase in CTGF protein was sustained over 8 h. Neither agonist increased CTGF mRNA in cardiac non-myocytes. By increasing the expression of CTGF in cardiac myocytes, hypertrophic agonists such as phenylephrine and endothelin-1 may promote fibrosis. CTGF may also propagate the hypertrophic response initiated by these agonists.