Regulation of STAT1 and STAT4 Expression by Growth Factor and Interferon Supplementation in Sjögren's Syndrome Cell Culture Models.

Our goal was to investigate the effects of epidermal growth factor (EGF) and interferons (IFNs) on signal transducer and activator of transcription STAT1 and STAT4 mRNA and active phosphorylated protein expression in Sjögren's syndrome cell culture models. iSGECs (immortalized salivary gland epithelial cells) and A253 cells were treated with EGF, IFN-alpha, -beta, -gamma, or mitogen-activated protein kinase p38 alpha (p38-MAPK) inhibitor for 0-24-48-72 h. STAT1 and STAT4 mRNA expression was quantified by qRT-PCR. Untreated and treated cells were compared using the delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized relative fold changes. phospho-tyrosine-701-STAT1 and phospho-serine-721-STAT4 were detected by Western blot analysis. STAT4 mRNA expression decreased 48 h after EGF treatment in A253 cells, immortalized salivary gland epithelial cells iSGECs nSS2 (sicca patient origin), and iSGECs pSS1 (anti-SSA negative Sjögren's Syndrome patient origin). EGF and p38-MAPK inhibitor decreased A253 STAT4 mRNA levels. EGF combined with IFN-gamma increased phospho-STAT4 and phospho-STAT1 after 72 h in all cell lines, suggesting additive effects for phospho-STAT4 and a major effect from IFN-gamma for phospho-STAT1. pSS1 and nSS2 cells responded differently to type I and type II interferons, confirming unique functional characteristics between iSGEC cell lines. EGF/Interferon related pathways might be targeted to regulate STAT1 and STAT4 expression in salivary gland epithelial cells. Further investigation is required learn how to better target the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) pathway-mediated inflammatory response in Sjögren's syndrome.

Regional heritability mapping identifies several novel loci (STAT4, ULK4, and KCNH5) for primary biliary cholangitis in the Japanese population.

While the advent of GWAS more than a decade ago has ushered in remarkable advances in our understanding of complex traits, the limitations of single-SNP analysis have also led to the development of several other approaches. Simulation studies have shown that the regional heritability mapping (RHM) method, which makes use of multiple adjacent SNPs jointly to estimate the genetic effect of a given region of the genome, generally has higher detection power than single-SNP GWAS. However, thus far its use has been mostly limited to agricultural settings, and its potential for the discovery of new genes in human diseases is yet to be fully exploited. In this study, by applying the RHM method to primary biliary cholangitis (PBC) in the Japanese population, we identified three novel loci (STAT4, ULK4, and KCNH5) at the genome-wide significance level, two of which (ULK4 and KCNH5) have not been found associated with PBC in any population previously. Notably, these genes could not be detected by using conventional single-SNP GWAS, highlighting the potential of the RHM method for the detection of new susceptibility loci in human diseases. These findings thereby provide strong empirical evidence that RHM is an effective and practical complementary approach to GWAS in this context. Also, liver tissue mRNA microarray analysis revealed higher gene expression levels in ULK4 in PBC patients (P < 0.01). Lastly, we estimated the common SNP heritability of PBC in the Japanese population (0.210 ± 0.026).

Aqueous extract of Fritillariae cirrhosae induces cellular apoptosis through activation of STATs-mediated immunomodulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae cirrhosae (FC), referred to'Chuan beimu'in China. As an important edible and medicinal plant, the bulbs of F.cirrhosae is used traditionally in the treatment of pulmonary diseases associated with lung heat, inflammation and tumors. In the study, we investigated the effect of aqueous extract of FC (FC-AE) and elucidated its mechanism in non-small cell lung cancer A549 cells and a xenograft model of nude mice. MATERIALS AND METHODS: CCK-8 and plate colony formation assay were used to evaluate the effect of FC-AE in A549 cells in vitro, and the gene expression profile of FC-AE on A549 cells was assessed by RNA sequencing system. Then, the effects of FC-AE on cell cycle and apoptosis of A549 cells were analyzed by flow cytometry. In combination with RNA-seq data, RT-PCR and western blot were used to evaluate the expression of proteins related to apoptosis and immune regulation. A xenograft model of nude mice was used to assess the effect of FC-AE in vivo. RESULTS: CCK-8 and plate cloning assays showed that FC-AE inhibited the proliferation and colony formation of A549 cells. A549 cells treated with FC-AE can triggered apoptosis. GO and KEGG pathway enrichment analysis of RNA-seq data showed that most of the differentially expressed genes (DEGs) were related to immune response, apoptosis and cell cycle process. Several immune and apoptotic DEGs were identified by qRT-PCR which were consistented with RNA-seq data. In nude mice, FC-AE reduced the tumor size and promoted the secretion of cytokines IL12 and IFNγ. FC-AE up-regulated the two members (STAT1 and STAT4) of STATs and their target genes (IFNγ and IL-12, respectively) protein expressions, and actively regulates Bcl-2/Bax family proteins which resulted in cellular apoptosis in A549 cells. CONCLUSION: Our finding suggests that FC-AE mediates apoptosis through a STAT1 and STAT4-mediated co-regulatory network, which may be the key novel mechanism for its antitumor activity. The F. cirrhosa may be a promising antitumor drug for modulating immune responses to improve cancer therapy.

'Psoriasis 1' reduces psoriasis­like skin inflammation by inhibiting the VDR­mediated nuclear NF­κB and STAT signaling pathways.

'Psoriasis 1', a Chinese herbal medicine (CHM) formulation, is extensively used to treat psoriasis in China. Although this CHM formulation yields good therapeutic effect, the underlying mechanism of how this works remains unknown. The present study aimed to test the hypothesis that the CHM formulation 'psoriasis 1' inhibits vitamin D receptor (VDR)­mediated inflammation in psoriasis. To test this, a model of psoriasis was established by stimulating keratinocytes (HaCaT cells) with tumor necrosis factor (TNF)­α; these cells were subsequently transfected with a lentiviral VDR RNA interference expression vector. The expression levels of 25­hydroxyvitamin D3 (25HVD3), TNF­α, interleukin (IL)­4, IL­1, IL­17C, IL­23 and IL­6 were measured using ELISA, and the expression levels of VDR, inhibitor of nuclear factor (NF)­κB (IKK), NF­κB, signal transducer and activator of transcription (STAT) 3 and STAT4 were measured using reverse transcription­quantitative polymerase chain reaction analysis and western blotting. It was observed that 'psoriasis 1' downregulated the concentrations of TNF­α, IFN­Î³, IL­22, IL­17C, IL­1ß and IL­4, and upregulated the concentration of 25HVD3; furthermore, 'psoriasis 1' downregulated the expression levels of NF­κB, phosphorylated (p)­NF­κB, IKK, p­IKK, STAT3, p­STAT3, STAT4 and p­STAT4, and upregulated the expression level of VDR in TNF­α­induced HaCaT cells. These results suggested that 'psoriasis 1' suppressed the inflammatory response and the activation of the NF­κB and STAT signaling pathways. In addition, it was identified that silencing VDR expression decreased the levels of TNF­α, IFN­Î³, IL­22, IL­17C, IL­1ß and IL­4, and increased the level of 25HVD3; silencing VDR expression additionally downregulated the expression levels of NF­ÐºB, p­NF­ÐºB, IKK, p­IKK, STAT3, p­STAT3, STAT4 and p­STAT4, and upregulated the level of VDR in TNF­α­induced HaCaT cells. It was concluded that 'psoriasis 1' exerts inflammation­suppressive effects in psoriasis by suppressing the NF­ÐºB and STAT signaling pathways.

Gαq Regulates the Development of Rheumatoid Arthritis by Modulating Th1 Differentiation.

The Gαq-containing G protein, an important member of Gq/11 class, is ubiquitously expressed in mammalian cells. Gαq has been found to play an important role in immune regulation and development of autoimmune disease such as rheumatoid arthritis (RA). However, how Gαq participates in the pathogenesis of RA is still not fully understood. In the present study, we aimed to find out whether Gαq controls RA via regulation of Th1 differentiation. We observed that the expression of Gαq was negatively correlated with the expression of signature Th1 cytokine (IFN-γ) in RA patients, which suggests a negative role of Gαq in differentiation of Th1 cells. By using Gαq knockout (Gnaq-/-) mice, we demonstrated that loss of Gαq led to enhanced Th1 cell differentiation. Gαq negative regulated the differentiation of Th1 cell by modulating the expression of T-bet and the activity of STAT4. Furthermore, we detected the increased ratio of Th1 cells in Gnaq-/- bone marrow (BM) chimeras spontaneously developing inflammatory arthritis. In conclusion, results presented in the study demonstrate that loss of Gαq promotes the differentiation of Th1 cells and contributes to the pathogenesis of RA.