Background/Purpose:  The transcription factor signal transducer and activator of transcription 3 (STAT3) mediates cytokine-induced changes in gene expression. STAT3 is classically activated by phosphorylation followed by homodimerization, nuclear translocation and DNA binding. STAT3 is downstream of numerous cytokines, including interleukin-6 (IL-6). IL-6 is a pro-inflammatory cytokine and therapeutic blockade of the IL-6 receptor using tocilizumab is approved for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Germline, gain-of-function (GOF) mutations in STAT3 have been shown to cause multi-organ autoimmunity, including juvenile-onset polyarthritis. Patients with STAT3 GOF also have short stature and decreased T regulatory cells. Specific mechanisms by which STAT3 GOF mutations impact the roles of STAT3 have not been determined.

Methods:  We investigated STAT3 activation using flow cytometry and Western blot in STAT3-deficient cells and cells engineered with heterozygous (het) and homozygous (homo) mutations in the STAT3 DNA binding domain (G421R), modeling a variant discovered in a GOF patient with polyarthritis, autoimmune hemolytic anemia, autoimmune hepatitis and scleroderma-like skin. Constructs containing STAT3 cDNA were modified and used in transfection experiments with a luciferase reporter. DNA binding was determined using an ELISA based assay. G421R STAT3 GOF mice were created using genetic engineering. Phenotypic characteristics were noted and the T cell compartments were analyzed by flow cytometry ex vivoand after polarization.

Results:  De-phosphorylation of G421R is delayed in a dose-dependent manner (homo>het>WT) in response to multiple cytokines, similar to primary patient cells. However, co-transfection of WT with G421R results in reduced transcriptional activity compared to G421R alone, suggesting G421R is not dominant. Cellular fractionation experiments reveal the prolonged phospho-G421R is retained in the nucleus. This was consistent with the finding that G421R displayed stronger DNA binding. After IL-6 stimulation, un-phosphorylated G421R increases compared to WT. Unlike WT, un-phosphorylated G421R is also found in the nucleus in the resting state. Further, un-phosphorylated G421R retains transcriptional activity, unlike un-phosphorylated WT. Interestingly, STAT3 GOF mice recapitulate the small size noted in STAT3 GOF patients, and in a dose-dependent fashion. After polarizing conditions, CD4 T cells from STAT3 GOF were less likely to develop into CD25+Foxp3+ regulatory cells, but produced increased IL-17 expressing cells.

Conclusion:  Our data suggests that G421R enhances STAT3 activity through increased DNA binding and enhanced transcriptional capacity of un-phosphorylated molecules. Initial characterization of STAT3 GOF mice suggests this model recapitulates the human disease. Future work will further characterize these mice, as well as investigate the mechanisms of other STAT3 GOF mutations. These studies will provide valuable information on the role of STAT3 in autoimmunity and arthritis, particularly given the potential impact of therapeutic inhibition of STAT3 in human disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mechanism-of-stat3-gain-of-function-in-a-patient-with-jia/