Abstract
Rho-associated coiled-coil kinase (ROCK)2 targeting down-regulates autoimmune responses in animal models and patients, however the underlying molecular mechanism is still an enigma. We report that ROCK2 binds phosphorylated-STAT3 and its kinase activity controls the formation of ROCK2/STAT3/JAK2 complex and optimal STAT3 phosphorylation in human CD4+ T cells during T helper 17 (TH17)-skewing. Moreover, chromatin-immunoprecipitation sequencing (ChIP-seq) analysis revealed that, genome-wide, about 70% of ROCK2 and STAT3 peaks overlapped and co-localized to several key genes controlling TH17 and T follicular helper (TFH) cell functions. Specifically, the co-occupancy of ROCK2 and STAT3 on the Irf4 and Bcl6 genes was validated by ChIP-qPCR analysis. Furthermore, the binding of ROCK2 to both the Irf4 and Bcl6 promoters was attenuated by STAT3 silencing as well as by selective ROCK2 inhibitor. Thus, the present study demonstrated previously unidentified evidence that ROCK2-mediated signaling in the cytosol provides a positive feed-forward signal for nuclear ROCK2 to be recruited to the chromatin by STAT3 and potentially regulates TH17/TFH gene transcription.
Highlights
Rho-associated coiled-coil kinases (ROCKs) play central role in the control of actin cytoskeleton assembly and cellular functions, such as proliferation, adhesion, migration and phagocytosis[1,2,3,4]
Using human peripheral blood CD4+ T cells stimulated under T helper 17 (TH17)-skewing conditions we found that a selective ROCK2 inhibitor (KD025) inhibits the phosphorylation of myosin light chain (MLC), one of the ROCK targets, in a concentration-dependent manner with an IC50 of 1.97 ± 1.2 μM
We report that ROCK2, but not ROCK1, interacts with STAT3 in primary human T cells activated by TH17-skewing
Summary
Rho-associated coiled-coil kinases (ROCKs) play central role in the control of actin cytoskeleton assembly and cellular functions, such as proliferation, adhesion, migration and phagocytosis[1,2,3,4]. ROCKs are expressed in both cytoplasmic and nuclear compartments and have been associated with JAK/STAT8–10 and p300 signaling pathways in cells[11]. The ROCK2-dependent regulation of TH17 pathway was mediated through STAT3 phosphorylation. The ROCK2 kinase activity is required for the formation of JAK-STAT complex leading to optimal STAT3 phosphorylation. The critical role of STAT3 in the recruitment of ROCK2 to chromatin was demonstrated by using siRNA-mediated inhibition of STAT3 as well as by treatment of human T cells with a potent JAK inhibitor
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