Regulation of Cytokine-inducible Nitric Oxide Synthase in Cardiac Myocytes and Microvascular Endothelial Cells

Adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC) both express an inducible nitric oxide synthase (iNOS or NOS2) following exposure to soluble inflammatory mediators. However, NOS2 gene expression is regulated differently in response to specific cytokines in each cell type. Interleukin-1β (IL-1β) induces NOS2 in both, whereas interferon γ (IFNγ) induces NOS2 expression in myocytes but not in CMEC. Therefore, we examined the specific signal transduction pathways that could regulate NOS2 mRNA levels, including activation of 44- and 42-kDa mitogen-activated protein kinases (MAPKs; ERK1/ERK2) and STAT1α, a transcriptional regulatory protein linked to cell membrane receptors. Although IL-1β treatment increased ERK1/ERK2 activities in both cell types, IFNγ activated these MAPKs only in myocytes. STAT1α phosphorylation, consistent with IFNγ-induced signaling, was readily apparent in both cell types, and binding of activated STAT1α from cytoplasmic or nuclear fractions from IFNγ-treated adult myocytes to a sis-inducible element could be demonstrated by gel-shift assay. The farnesyl transferase inhibitor BZA-5B blocked activation of ERK1/ERK2 and induction of NOS2 by IFNγ and IL-1β in myocytes. IL-1β and IFNγ-induced NOS2 gene expression in myocytes was also down-regulated by both protein kinase C (PKC) desensitization and by the PKC inhibitor bisindolylmaleimide, implicating PKC-linked activation of Ras or Raf in the induction of NOS2 by IL-1β and IFNγ in cardiac muscle cells. In CMEC, the MAPK kinase inhibitor PD 98059 blocked activation of ERK1/ERK2 and down-regulated IL-1β-mediated NOS2 induction, whereas activation of ERK2 in the absence of cytokines by okadaic acid, an inhibitor of phosphoserine protein phosphatases, also induced NOS2 mRNA. These data demonstrate that ERK1/ERK2 activation appears to be necessary for the induction of NOS2 by IL-1β and IFNγ in cardiac myocytes and CMEC. In the absence of ERK1/ERK2 activation by IFNγ in CMEC, phosphorylation of STAT1α is not sufficient for NOS2 gene expression. These overlapping yet distinct cellular responses to specific cytokines may serve to target NOS2 gene expression to specific cells or regions within the heart and also provide for rapid escalation of NO production if required for host defense. Adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC) both express an inducible nitric oxide synthase (iNOS or NOS2) following exposure to soluble inflammatory mediators. However, NOS2 gene expression is regulated differently in response to specific cytokines in each cell type. Interleukin-1β (IL-1β) induces NOS2 in both, whereas interferon γ (IFNγ) induces NOS2 expression in myocytes but not in CMEC. Therefore, we examined the specific signal transduction pathways that could regulate NOS2 mRNA levels, including activation of 44- and 42-kDa mitogen-activated protein kinases (MAPKs; ERK1/ERK2) and STAT1α, a transcriptional regulatory protein linked to cell membrane receptors. Although IL-1β treatment increased ERK1/ERK2 activities in both cell types, IFNγ activated these MAPKs only in myocytes. STAT1α phosphorylation, consistent with IFNγ-induced signaling, was readily apparent in both cell types, and binding of activated STAT1α from cytoplasmic or nuclear fractions from IFNγ-treated adult myocytes to a sis-inducible element could be demonstrated by gel-shift assay. The farnesyl transferase inhibitor BZA-5B blocked activation of ERK1/ERK2 and induction of NOS2 by IFNγ and IL-1β in myocytes. IL-1β and IFNγ-induced NOS2 gene expression in myocytes was also down-regulated by both protein kinase C (PKC) desensitization and by the PKC inhibitor bisindolylmaleimide, implicating PKC-linked activation of Ras or Raf in the induction of NOS2 by IL-1β and IFNγ in cardiac muscle cells. In CMEC, the MAPK kinase inhibitor PD 98059 blocked activation of ERK1/ERK2 and down-regulated IL-1β-mediated NOS2 induction, whereas activation of ERK2 in the absence of cytokines by okadaic acid, an inhibitor of phosphoserine protein phosphatases, also induced NOS2 mRNA. These data demonstrate that ERK1/ERK2 activation appears to be necessary for the induction of NOS2 by IL-1β and IFNγ in cardiac myocytes and CMEC. In the absence of ERK1/ERK2 activation by IFNγ in CMEC, phosphorylation of STAT1α is not sufficient for NOS2 gene expression. These overlapping yet distinct cellular responses to specific cytokines may serve to target NOS2 gene expression to specific cells or regions within the heart and also provide for rapid escalation of NO production if required for host defense.