Both the suppressor of cytokine signaling 1 (SOCS-1) kinase inhibitory region and SOCS-1 mimetic bind to JAK2 autophosphorylation site: implications for the development of a SOCS-1 antagonist

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by IFN-gamma by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS-1 mimetic. Tkip is compared in this study with the kinase inhibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN-gamma-induced biological activity. Tkip and a peptide corresponding to the KIR of SOCS-1, ((53))DTHFRTFRSHSDYRRI((68)) (SOCS1-KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001-1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr(1007), pJAK2(1001-1013). Dose-response competitions suggest that Tkip and SOCS1-KIR similarly recognize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN-gamma-induced STAT1-alpha phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1-alpha activation. Both Tkip and SOCS1-KIR inhibited IFN-gamma activation of Raw 264.7 murine macrophages and inhibited Ag-specific splenocyte proliferation. The fact that SOCS1-KIR binds to pJAK2(1001-1013) suggests that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFN-gamma activity, blocked SOCS-1-induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN-gamma activation site promoter activity, and enhanced Ag-specific proliferation. Furthermore, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS-1.     

Early target genes of IL-12 and STAT4 signaling in th cells

IL-12 signaling through STAT4 is essential for induction of optimal levels of IFN-gamma production and commitment of Th1 cells. The molecular mechanism that controls how IL-12 and STAT4 signaling induces Th1 differentiation is poorly described. To identify the early target genes of IL-12 and STAT4 signaling, oligonucleotide arrays were used to compare the gene expression profiles of wild-type and STAT4-knockout murine Th cells during the early Th1 differentiation. According to the results, 20 genes were regulated in an IL-12- and STAT4-dependent manner. Importantly, Ifngamma was clearly the first gene induced by IL-12 in a STAT4-dependent manner. Most of the other defects in gene expression in STAT4-knockout cells were seen after 48 h of Th1 polarization. In addition to IL-12 signaling mediated by STAT4, STAT4-independent induction of a number of genes was observed immediately in response to Th1 induction. This induction was at least in part driven by IFN-gamma independently of STAT4. Importantly, addition of exogenous IFN-gamma into Th1 cell cultures of STAT4-knockout cells restored the defect in IFN-gamma production further demonstrating the critical role of IFN-gamma in early Th1 differentiation.     

Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells

Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.     

Differential regulation of epithelial-derived C-C chemokine expression by IL-4 and the glucocorticoid budesonide.

Airway epithelial cells are a rich source of eosinophil-selective C-C chemokines. We investigated whether cytokines and the topical glucocorticoid budesonide differentially regulate RANTES, monocyte chemoattractant protein-4 (MCP-4), and eotaxin mRNA and protein expression in the human bronchial epithelial cell line BEAS-2B and in primary human bronchial epithelial cells by Northern blot analysis and ELISAs. Eotaxin and MCP-4 mRNA expression induced by TNF-alpha alone or in combination with IFN-gamma was near-maximal after 1 h, peaked at 4 and 8 h, respectively, remained unchanged up to 24 h, and was protein synthesis independent. In contrast, RANTES mRNA was detectable only after 2 h and slowly increased to a peak at 24 h, and was protein synthesis dependent. Induction of eotaxin and MCP-4 mRNA showed a 10- to 100-fold greater sensitivity to TNF-alpha compared with RANTES mRNA. IL-4 and IFN-gamma had selective effects on chemokine expression; IL-4 selectively up-regulated the expression of eotaxin and MCP-4 and potentiated TNF-alpha-induced eotaxin, while IFN-gamma markedly potentiated only the TNF-alpha-induced expression of RANTES. Although budesonide inhibited the expression of chemokine mRNA to a variable extent, it effectively inhibited production of eotaxin and RANTES protein. Budesonide inhibited both RANTES- and eotaxin promoter-driven reporter gene activity. Budesonide also selectively accelerated the decay of eotaxin and MCP-4 mRNA. These results point to IL-4 as a possible mediator by which Th2 cells may induce selective production of C-C chemokines from epithelium and indicate that glucocorticoid inhibit chemokine expression through multiple mechanisms of action.     

SOCS-3 is tyrosine phosphorylated in response to interleukin-2 and suppresses STAT5 phosphorylation and lymphocyte proliferation.

Members of the recently discovered SOCS/CIS/SSI family have been proposed as regulators of cytokine signaling, and while targets and mechanisms have been suggested for some family members, the precise role of these proteins remains to be defined. To date no SOCS proteins have been specifically implicated in interleukin-2 (IL-2) signaling in T cells. Here we report SOCS-3 expression in response to IL-2 in both T-cell lines and human peripheral blood lymphocytes. SOCS-3 protein was detectable as early as 30 min following IL-2 stimulation, while CIS was seen only at low levels after 2 h. Unlike CIS, SOCS-3 was rapidly tyrosine phosphorylated in response to IL-2. Tyrosine phosphorylation of SOCS-3 was observed upon coexpression with Jak1 and Jak2 but only weakly with Jak3. In these experiments, SOCS-3 associated with Jak1 and inhibited Jak1 phosphorylation, and this inhibition was markedly enhanced by the presence of IL-2 receptor beta chain (IL-2Rbeta). Moreover, following IL-2 stimulation of T cells, SOCS-3 was able to interact with the IL-2 receptor complex, and in particular tyrosine phosphorylated Jak1 and IL-2Rbeta. Additionally, in lymphocytes expressing SOCS-3 but not CIS, IL-2-induced tyrosine phosphorylation of STAT5b was markedly reduced, while there was only a weak effect on IL-3-mediated STAT5b tyrosine phosphorylation. Finally, proliferation induced by both IL-2- and IL-3 was significantly inhibited in the presence of SOCS-3. The findings suggest that when SOCS-3 is rapidly induced by IL-2 in T cells, it acts to inhibit IL-2 responses in a classical negative feedback loop.