Interferon-α (IFN-α) stimulates macrophages to produce an inflammatory response. Wang et al. show that the activation of signal transducer and activator of transcription 1 (STAT1), but not STAT2, downstream of the IFN-α receptor (IFNAR) is regulated by crosstalk from signals generated by immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as integrins (mediating cell adhesion) and CCR5 (a G protein-coupled receptor for the chemokines CCL3, CCL4, and CCL5). STAT activation requires tyrosine phosphorylation, which then allows STAT proteins to dimerize and translocate to the nucleus to regulate gene expression. Wang et al. found basal phosphorylation of STAT1 on Ser727 in unstimulated primary human macrophages. Ser727 phosphorylation, as well as tyrosine phosphorylation (Tyr701) stimulated by IFN-α, was inhibited by the calcium/calmodulin kinase (CaMK) inhibitor KN-93, the calmodulin inhibitor W-7, or the calcium chelator BAPTA. Thus, a basal calcium signal appears to be involved in this process of sensitizing cells to respond to IFN-α stimulation with STAT1 tyrosine phosphorylation. Although CaMK activity was likely to regulate STAT1 Ser727 phosphorylation, it was not directly responsible for the tyrosine phosphorylation. Instead the calcium-dependent tyrosine kinase Pyk2 was found to interact with the tyrosine kinases associated with the IFNAR, Jak1, and Tyk2, and a role for Pyk2 in enhancing IFN-α-stimulated STAT1 tyrosine phosphorylation was confirmed by experiments with pharmacological inhibitors, cells expressing dominant-negative mutants, cells in which Pyk2 was depleted, and Pyk2-knockout cells. STAT2 activation was not affected by the treatments that altered STAT1 activation, suggesting that these two STATs may have different sensitivities to IFN-α. Dose-response experiments with a Jak1 inhibitor indicated that phosphorylation of STAT2 required low Jak1 activity, whereas STAT1 tyrosine phosphorylation required higher Jak1 activity. Thus, the basal calcium signal may help boost the STAT1 activation in response to low IFN-α signaling. The IFNAR does not produce a calcium signal; however, receptors that couple to the ITAM adaptor DAP12, which is basally active in macrophages, do stimulate calcium signals. At least two receptors that couple to DAP12 appear to contribute to the basal calcium signal and priming of the STAT1 response: integrins and CCR5. In experiments with mouse bone marrow macrophages, which are more adherent than the primary human macrophages from blood, detachment of the cells from the substrate decreased Pyk2 phosphorylation and decreased STAT1 activation in response to IFN-α. Primary human macrophages were found to produce ligand for the CCR5 receptor, and blocking CCR5 activity with a function-blocking antibody decreased CaMK activation and decreased STAT1 activation in response to IFN-α. Finally, in a mouse model of systemic lupus erythematosus, STAT1 was basally phosphorylated at Ser727 in cells from the kidney (an affected organ in the mouse model). When the mice were treated with IFN-α, the kidney cells exhibited increased STAT1 activation compared with that in cells from control mice. The enhanced STAT1 activation and the basal Ser727 phosphorylation were reduced in mice treated with the CaMK inhibitor KN-93. Thus, crosstalk through receptors coupled to DAP12 contributes a signal that tunes the IFN-α response and may be amenable to therapeutic intervention for treatment of autoimmune disease.
L. Wang, I. Tassiulas, K.-H. Park-Min, A. C. Reid, H. Gil-Henn, J. Schlessinger, R. Baron, J. J. Zhang, L. B. Ivashkiv, 'Tuning' of type I interferon-induced Jak-STAT1 signaling by calcium-dependent kinases in macrophages. Nat. Immunol. 9, 186-193 (2008). [PubMed]