Synergistic Transactivation of the Sis-Inducible Element by STAT4/STAT3 Heterodimer in Cytokine Signal Cascade.
Authors: Higashi Takehiro , Tsukada Junichi , Mizobe Takamitsu , Mouri Fumihiko , Matsuura Ai , Minami Yasuhiro , Yoshida Yasuhiro , Tanaka Yoshiya 
Published: April 2020

Abstract
Signal transducer and activator of transcription (STAT) proteins are activated in response to cytokine stimulation. STAT3 is activated in response to interleukin (IL)-6 family of cytokines, following activation of Janus family of tyrosine kinases JAK1 and JAK2, which in turn phosphorylate STAT3 on tyrosine 705 (tyr-705). On the other hand, STAT4 is activated in response to IL-12 following activation of Janus family of tyrosine kinases JAK2 and Tyk2, which in turn phosphorylate STAT4 on tyrosine 693 (tyr-693). p38/MKK6 signaling pathway activated by IL-12 further phosphorylates STAT4 on serine 721 (ser-721). STAT4 plays a crucial role in the development of type-1 helper T (Th1) cells and production of interferon (IFN)-γ in response to IL-12. STAT4 is activated by IFN-α as well as IL-12. Several STAT heterodimer complexes have been demonstrated. Selective interactions of individual STATs upon complex formation might contribute to the transcriptional target specificity of cytokine and growth factor signaling.
In the present study, we demonstrated that STAT4 forms a heterodimer with STAT3 to synergistically transactivate the high-affinity sis-inducible element (hSIE). A wild-type STAT3 expression vector Rc/CMV-STAT3WT and/or a wild-type STAT4 expression vector Rc/CMV-STAT4WT were cotransfected along with a hSIE reporter pGLmfoshSIE into Hep3B cells, and cells were treated with IL-6 and/or IFN-α. In the absence of overexpression of STAT3 and STAT4, little increase in hSIE activities was observed following treatment with the cytokines, However, when Rc/CMV-STAT3WT and -STAT4WT were cotransfected, transcriptional activities were synergistically enhanced following simultaneous stimulation with IL-6 and IFN-α. γ-interferon activated site (GAS) activity was not enhanced by the two STATs. No synergistic activation of hSIE was detected between STAT1 and STAT4, when a wild-type STAT1 expression vector was used instead of Rc/CMV-STAT3WT. Our mutational analyses further revealed that the synergy between STAT3 and STAT4 was almost completely abrogated by either mutation of STAT4 at tyr-693 or STAT3 at tyr-705, suggesting direct interaction of STAT3 with STAT4 as well as the importance of STAT tyrosine phosphorylation. However, mutation of STAT4 at ser-721 caused only a little suppression of the synergy. Similar results were obtained from mutation of STAT3 at ser-727. Our immunoprecipitation (IP)/western blot (WB) analyses exhibited that STAT4 forms a heterodimer with STAT3 in response to stimulation with IL-6 and IFN-α. Moreover, in electrophoretic mobility shift assay (EMSA), hSIE preferentially bound STAT3/STAT4 heterodimer.
Based upon our results, STAT3 and STAT4 can form a heterodimer, which exhibits significantly different transcripitional activities and sequence preferences to bind its target DNA. Thus, the specificity in STAT signaling is at least partly dependent on selective interactions between individual STATs upon complex formation. STAT3/STAT4 heterodimer may play an important role in immune and inflammatory reactions in NK and T-cells in response to IL-12 and IFN-α.