Growth hormone-induced production of cytokines in murine peritoneal macrophages in vitro: Role of JAK/STAT, PI3K, PKC and MAP kinases

Abstract

The immunomodulatory properties of growth hormone (GH) are well recognized. Enhanced production of NO and cytokines by macrophages on treatment with GH was reported by us recently. The present investigation elucidates the signaling mechanism(s) by which GH activates macrophages in vitro. It is observed that GH induces the phosphorylation (activation) of JAK2, PI3K, PKC and MAP kinases. Studies with pharmacological inhibitors of various signaling molecules also indicated that GH-induced proinflammatory responses in macrophages are mediated by JAK2/PI3K/PKC/ERK1/2, JAK2/JNK and JAK/STAT signaling cascades. It was further observed that GH induced the enhanced expression/phosphorylation of transcription factors c-fos, c-jun, Elk-1 and Stat1.

It is also demonstrated that GH-induced ERK1/2 cascade regulates the production of TNF-α and IL-1β in macrophages, whereas JNK cascade mediated the production of TNF-α, IFN-γ and IL-12. These results suggest that JAK2 plays a central role in mediating proinflammatory responses of macrophages on GH treatment.

Introduction

Macrophages are phagocytic cells that recognize and kill microbial and tumor targets by cell-to-cell contact or through secretion of a wide array of products including reactive oxygen species, reactive nitrogen intermediates, cytokines, chemokines, etc. Immune modulation through the production of cytokines such as IL-1β, TNF-α, IFN-γ, IL-12 is central to their regulatory role. They are also important antigen-presenting cells and play a role in the regulation of T cell differentiation and activation (Nathan, 1987; Germain and Margulies, 1993; Adams and Hamilton, 1984; Sodhi and Chauhan, 2007; Stuehr and Nathan, 1989; Klostergaard et al., 1990). Exposure to macrophage-activating factors, like IFN-γ, primes or activates the cells to mount a vigorous response against the tumor cells or microbial challenge (Nathan et al., 1984; Murray, 1988). Growth hormone (GH) has long been known to possess macrophage-activating properties (Warwick-Davies et al., 1995). There are numerous reports that suggest a role for GH in the regulation of the immune system, its role as a cytokine and reconstitution of the immune system (Gala, 1991; Hooghe-Peters and Hooghe, 1995; Chappel, 1999). GH has been postulated to be an important mediator in thymic development and increases the thymic cellularity (Kelley et al., 1986; Murphy et al., 1993). Apart from the pituitary, production of immunoreactive GH from the cells of the immune system has been documented, indicating a paracrine/autocrine role of GH (Weigent et al., 1988; Kao et al., 1992; Lytras et al., 1993). The treatment of cells of the immune system with GH modulates antibody production by B cells, NK cell activity, phagocytosis and the killing capacity of neutrophils and macrophages (Kooijman et al., 1996; Weigent, 1996).

An understanding of the intracellular signal transduction pathways initiated by GH is necessary to know how GH may act as a growth factor or immunostimulant. The JAK/STAT pathway is the most studied cascade in GH signaling (Kopchick and Andry, 2000). JAK2, a non-receptor tyrosine kinase, was identified as growth hormone receptor (GHR)-associated tyrosine kinase. When GH binds to its receptor, JAK2 rapidly associates with the GHR, which leads to its activation and phosphorylation of tyrosines on JAK2 (Argetsinger et al., 1993). Apart from JAK/STAT, the MAP kinase pathway is also considered important for the regulation of GH-induced gene transcription (Smit et al., 1999). Recently, we have demonstrated the involvement of p-38 MAP kinase in the GH-induced production of cytokines by murine macrophages (Sodhi and Tripathi, 2008).

The signaling pathways involved in GH signaling in macrophages remain largely unknown. We have previously reported that GH induces production of NO and cytokines in macrophages; it was observed that tyrosine kinases, Ca²⁺ signaling and MAP kinases were involved in GH-induced NO production (Sodhi and Tripathi, 2008; Tripathi and Sodhi, 2007). We have also shown the involvement of tyrosine kinases and MAP kinases in PRL-induced macrophage activation (Tripathi and Sodhi, 2008). The present manuscript investigates the involvement of the JAK/STAT pathway, ERK1/2 and JNK MAP kinase signaling cascades in the GH-induced production of cytokines.