Regulation of STAT4 by ETS1 in Sjögren syndrome pathogenesis

Abstract

<h3>Objectives</h3> Primary Sjögren syndrome (pSS) is a chronic autoimmune disease mainly affecting women over the age of 40, causing dry mouth and eyes. The etiology of pSS is complex and includes the overexpression of inflammatory cytokines from affected salivary gland epithelial cells (SGECs). Our group has previously identified candidate biomarkers overexpressed in salivary gland tissues of patients with pSS. We showed that ETS proto-oncogene 1 (ETS1) had higher mRNA and protein expression in pSS salivary glands that correlated with higher levels of lymphocytic infiltration. It has also been shown that the signal transducer and activator of transcription 4 (STAT4) gene contains pSS susceptibility polymorphisms and is a significant mediator of cytokine (ie, interferon, tumor necrosis factor-α) production. Additionally, in previous studies unrelated to pSS, ETS1 was shown to regulate STAT4 expression at the transcription level. Thus, we hypothesized that ETS1 plays a significant role in regulating STAT4 transcription within SGECs of patients with pSS. Our objectives were as follows: (1) to determine whether downregulation of ETS1 mRNA by siRNA knockdown results in decreased mRNA and protein expression of STAT4 within the salivary gland cell line A253 and (2) to demonstrate the regulation of STAT4 mRNA expression by ETS1 siRNA knockdown in cultured SGECs from control patients with pSS and non-pSS sicca. <h3>Methods</h3> The salivary gland cell line, A253, was transfected with siRNA targeting ETS1 or a scrambled siRNA control. After 48 hours, mRNA and protein were isolated. cDNA synthesis was performed using 1 µg of mRNA. Expression of ETS1 and STAT4 mRNA was quantified by quantitative reverse transcription–polymerase chain reaction using the delta cycle threshold method to determine the relative fold changes normalized to glyceraldehyde 3-phosphate dehydrogenase in triplicate experiments. In addition, downregulation of STAT4 protein by ETS1 siRNA was determined by Western blot analysis and semiquantified by densitometry using cofilin as the loading control. SGECs were cultured from labial salivary gland biopsies of 2 patients with pSS (focus score [FS] ≥1) and 4 patients with non-pSS sicca (FS = 0, n = 2; 0 < FS < 1; n = 2) and transfected with siRNA targeting ETS1 or a scrambled siRNA control. After 48 hours, mRNA was isolated for cDNA synthesis and used to determine the mRNA expression of ETS1 and STAT4 as described above. <h3>Results</h3> In A253 cells, STAT4 mRNA expression was changed by −2.15-fold, on average, when treated with siRNA targeting ETS1. STAT4 protein expression was measured by Western blot analysis 48 hours post-ETS1 siRNA knockdown showing an average −1.25-fold change in A253 total cell lysate. SGECs cultured from patients with lymphocytic infiltration (ie, FS >0) showed a significantly higher expression of STAT4 (<i>P</i> = .0485) than patients with non-pSS sicca with FS = 0. However, we did not find a significant change in STAT4 mRNA expression (average, +1.14-fold) in cultured SGECs treated with siRNA targeting ETS1. <h3>Conclusions</h3> STAT4 is overexpressed by cultured SGECs in patients with pSS and those without pSS with focal lymphocytic infiltrates. STAT4 overexpression in SGECs of patients with pSS and those without pSS could be a result of inflammatory cytokine exposure by neighboring cells or effects of infiltrating lymphocyte exposure. Thus, our results suggest a separate regulatory mechanism of STAT4 in pSS bypassing ETS1 regulation, which might be due to pathogenic pathways involving other transcriptional regulation.