The role of sphingosine 1‐phosphate type 2 receptor on IL‐4/IL‐13‐stimulated STAT6 phosphorylation in phorbol12‐myristate13‐acetate‐treated THP‐1 cells

Abstract

Background/Purpose We previously observed reduced activation of STAT6 in broncho-alveolar lavage fluids (BALF) cells from bleomycin-administered sphingosine 1-phosphate receptor type 2 (S1P2) knockout mice compared to BALF cells from bleomycin-administered wild-type mice (PLoS One, 2018). In this study, we examined the role of S1P2 on STAT6 activation using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) as a model for macrophages (MΦs). Methods THP-1 cells were cultured in RPMI-1640 containing 10% fetal bovine serum. THP-1 cells were treated with PMA to differentiate into adherent MΦs. S1P2knockout THP-1 cells were established using CRISPR-Cas9 gene editing system. Quantification of mRNA expression was performed by quantitative RT-PCR using specific primers. Jak/STAT6 activation was performed by Western blot analysis using antibodies against tyrosine-phosphorylated Jak1, Jak2 and STAT6. Results Quantitative PCR analysis showed that THP-1 cells expresses S1P2. We examined the responses of PMA-treated THP-1 MΦs to stimulation with IL-4/IL-13 in the presence of S1P. IL-4/IL-13 stimulation induced a strong increase in STAT6 phosphorylation in PMA-treated THP-1 MΦs. The major signaling pathway of S1P2 is a Rho–Rho kinase pathway. Pretreatment of PMA-treated THP-1 MΦs with the S1P2 inhibitor JTE-013 and the Rho kinase inhibitor Y27632 inhibited IL-4/IL-13–induced increase in STAT6 phosphorylation. PMA-treated S1P2knockout THP-1 MΦs also showed a lower extent of STAT6 phosphorylation in response to IL-4/IL-13 stimulation than PMA-treated wild-type THP-1 MΦs. mRNA expression of IL-4/IL-13 receptors was not different between wild-type and S1P2knockout THP-1 MΦs. No difference in mRNA and protein expression of STAT6 was observed between wild-type and S1P2knockout THP-1 MΦs. The phosphorylation of Jak1 and Jak2 in S1P2knockout THP-1 MΦs was diminished compared with wild-type THP-1 MΦs. Finally, S1P2 knockout in THP-1 MΦs attenuated IL-4/IL-13-induced increase in the mRNA expression of the M2 marker arginase 1. Conclusion These results suggested that S1P2/Rho kinase pathway is necessary for full activation of STAT6 by IL-4 or IL-13 in MΦs and could be a drug target for Th2-associated diseases.