Regulation of STAT-1 Phosphorylation by Histamine Through H1 and H2 Receptors: Inverse Agonism and PKA-PKC Interaction in STAT-1 Phosphorylation

Abstract

Signal transducer and activator of transcription-1 (STAT1) is a latent signal transducer protein which, on phosphorylation, is translocated from the cytoplasm to the nucleus and is subsequently activated. This work was designed to determine the role of histamine in the regulation of ST ATI phosphorylation. Histamine mediates the regulation of cytokines such as IL-5, IL-6, IL-10 and IL-13. However, it is not clear whether histamine regulates the cytokine-induced transcription factors such as STATs. Histamine is a key mediator of the airway inflammatory events including goblet cell hypersecretion, airway hyperresponsiveness, eosinophilia and smooth muscle hyperplasia. Histamine has been shown to induce the expression of adhesion molecule intracellular adhesion molecule- 1(ICAM-1) and chemokine RANTES, the genes expressed by phosphorylated STAT1. STAT1 has been shown to be spontaneously phosphorylated in asthmatic airways and has been shown to be dormant in non-asthmatics. Studies have demonstrated that ST ATI phosphorylation mediates IL-13-induced subepithelial fibrosis, eosinophil infiltration and transcriptional regulation of nitric oxide thus signifying its inflammatory role in asthma. It was therefore interesting to determine if histamine could affect the activation of STAT1 at some juncture. The actions of histamine are mediated through HI and H2 receptors which are dependent on their respective downstream pathways, Ca2+-PKC and cAMP-PKA respectively. The histamine- induced elevation of ICAM-1 was mediated through HI receptors and PKC. Role of PKC in ST ATI phosphorylation has been established however role of PKA has not been studied yet. In this study we delineated the role of HI and H2 receptors and investigated the significance of cAMP-PKA pathway in STAT1 phosphorylation. It is know that cytokines relay their signals through tyrosine kinase and we have previously demonstrated that histamine regulated the levels of various cytokines which phosphorylate STAT1. We were therefore interested in finding out if the effect of histamine on STAT1 phosphorylation is mediated through cytokines. We therefore determined the role of tyrosine kinase in the effect of histamine on ST ATI phosphorylation. C57BL/6 mouse splenocytes were isolated and treated with histamine (10'7 - 10"4 M) and then activated with PMA (phorbol 12 myristate 13- acetate) plus ionomycin. The phosphorylated ST ATI levels were analyzed by immunoblotting. Agents used to identify receptors and upstream signaling kinases included histamine receptor agonists amthamine and betahistine; histamine receptor antagonists pyrilamine maleate, tripelennamine, ranitidine, cimetidine and thioperamide; cAMP agonists N6, 2 -0-dibutyryladenosine-3,5 -cyclic monophosphate sodium salt (db-cAMP) and forskolin; protein kinase A inhibitors N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide (H-89) and Rp diastereomer of adenosine cyclic 3,5 - phosphorothioate (RpcAMPs); and tyrosine kinase inhibitor tyrphostin. We observed that histamine augmented the phosphorylation of STAT1 through both HI and H2 receptors. Furthermore, HI and H2 receptor antagonists displayed inverse agonism whereby HI inverse agonists inhibited the phosphorylation of STAT1 in the absence of histamine. However H2 inverse agonists exhibited effects similar to the H2 agonists and augmented the phosphorylation of STAT1. Similar effects were reported by other investigators where they observed that H2 inverse agonists caused upregulation of H2 receptors. Tyrosine kinase inhibitor, tyrphostin, did not affect the phosphorylation of STAT1 thus suggesting that cytokines may not be mediating the effect of histamine on STAT1 phosphorylation. Ca2+-PKC-induced phosphorylation of STAT1 was completely inhibited by H89 and significantly inhibited by RpcAMPs in the presence and absence of histamine. Further, dbcAMP and forskolin augmented the Ca2+-PKC-induced STAT1 phosphorylation. These observations suggested a convergent interaction between the two histamine receptor signaling pathways, PKA and PKC, where PKC required PKA to phosphorylate STAT1. Studies have suggested that ST ATI is a crucial inflammatory mediator. Our study supported these findings by demonstrating that the phosphorylation of STAT1 was augmented by a key inflammatory mediator, histamine. Our study further suggested factors involved in the phosphorylation of STAT1 thus suggesting possible ways to inhibit this inflammatory signaling pathway. STAT1 phosphorylation and pathways regulating STAT1 phosphorylation could thus be important targets for asthma.