UBC Theses and Dissertations
The roles of SH2-containing inositol 5'-phosphatase 1 (SHIP1) and signal transducer and activator of transcription 3 (STAT3) in interleukin-10 regulation of activated macrophages Tsz Ying Sylvia, Cheung
Inflammation is a protective mechanism against infection, but it must be appropriately regulated to prevent pathological consequences including inflammatory diseases. Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that inhibits the activation of many immune cell types, including the macrophage, to prevent exaggerated immune responses. Understanding the signalling pathway downstream of IL-10 and IL-10 receptor (IL-10R) is essential in developing therapeutics to treat inflammatory diseases. Canonically, IL-10R signalling is described as solely depending on the activation of the transcription factor STAT3 and the expression of STAT3-response genes. However, our laboratory has previously shown that IL-10 also activates the inositol 5'-phosphatase SHIP1 to mediate its anti-inflammatory responses, such as inhibiting the expression of pro-inflammatory cytokines. Moreover, we have now found that IL-10 is able to inhibit expression of miRNA-155 (miR-155) in macrophages activated by the bacterial product lipopolysaccharide (LPS). This inhibition by IL-10 requires both STAT3 and SHIP1, and occurs at the maturation step, but not at the transcription step, of miR-155. Consistent with SHIP1’s involvement in IL-10 function, a previously described SHIP1 activator, AQX-MN100, mimics IL-10 and inhibits LPS-induced miR-155. Next, we investigated the roles of STAT3 and SHIP1 in IL-10 regulation of global gene expression in activated macrophages. Among the genes identified as IL-10 regulated, we have found different subsets of genes potentially to be SHIP1-regulated, STAT3-regulated, and SHIP1-STAT3 regulated. Considering the importance of SHIP1 activation in IL-10 function, SHIP1 activators provide an alternate way to control inflammation. We have previously shown that SHIP1 activators bind to SHIP1’s C2 domain and regulate SHIP1 enzymatic activity allosterically. Using x-ray crystallography, we obtained the crystal structure of SHIP1’s phosphatase and C2 domains, the minimal region necessary for allosteric activation by SHIP1 activators. Analysis of the crystal structure revealed differences between SHIP1 and related phosphatases. Biochemical and biophysical methods have been employed to identify amino acid residues potentially interacting with SHIP1 activators. Together, this work strengthens the model that STAT3 and SHIP1 work together to mediate the anti-inflammatory function of IL-10. We also described the structure of SHIP1, providing the first step in rational drug design to generate better small molecule SHIP1 activators.
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