Protein Inhibitor Of Activated STAT Research Articles

T cell recognition of novel shared breast cancer antigens is frequently observed in peripheral blood of breast cancer patients

ABSTRACTAdvances within cancer immunotherapy have fueled a paradigm shift in cancer treatment, resulting in increasing numbers of cancer types benefitting from novel treatment options. Despite originally being considered an immunologically silent malignancy, recent studies encourage the research of breast cancer immunogenicity to evaluate immunotherapy as a treatment strategy. However, the epitope landscape in breast cancer is minimally described, limiting the options for antigen-specific, targeted strategies. Aromatase, never in mitosis A-related kinase 3 (NEK3), protein inhibitor of activated STAT3 (PIAS3), and prolactin are known as upregulated proteins in breast cancer. In the present study, these four proteins are identified as novel T cell targets in breast cancer.From the four proteins, 147 peptides were determined to bind HLA-A*0201 and -B*0702 using a combined in silico/in vitro affinity screening. T cell recognition of all 147 peptide-HLA-A*0201/-B*0702 combinations was assessed through the use of a novel high-throughput method utilizing DNA barcode labeled multimers.T cell recognition of sequences within all four proteins was demonstrated in peripheral blood of patients, and significantly more T cell responses were detected in patients compared to healthy donors for both HLA-A*0201 and -B*0702. Notably, several of the identified responses were directed toward peptides, with a predicted low or intermediate binding affinity. This demonstrates the importance of including low-affinity binders in the search for epitopes within shared tumor associated antigens (TAAs), as these might be less subject to immune tolerance mechanisms.The study presents four novel TAAs containing multiple possible targets for immunotherapy of breast cancer.

STAT3 and STAT5 Activation in Solid Cancers.

The Signal Transducer and Activator of Transcription (STAT)3 and 5 proteins are activated by many cytokine receptors to regulate specific gene expression and mitochondrial functions. Their role in cancer is largely context-dependent as they can both act as oncogenes and tumor suppressors. We review here the role of STAT3/5 activation in solid cancers and summarize their association with survival in cancer patients. The molecular mechanisms that underpin the oncogenic activity of STAT3/5 signaling include the regulation of genes that control cell cycle and cell death. However, recent advances also highlight the critical role of STAT3/5 target genes mediating inflammation and stemness. In addition, STAT3 mitochondrial functions are required for transformation. On the other hand, several tumor suppressor pathways act on or are activated by STAT3/5 signaling, including tyrosine phosphatases, the sumo ligase Protein Inhibitor of Activated STAT3 (PIAS3), the E3 ubiquitin ligase TATA Element Modulatory Factor/Androgen Receptor-Coactivator of 160 kDa (TMF/ARA160), the miRNAs miR-124 and miR-1181, the Protein of alternative reading frame 19 (p19ARF)/p53 pathway and the Suppressor of Cytokine Signaling 1 and 3 (SOCS1/3) proteins. Cancer mutations and epigenetic alterations may alter the balance between pro-oncogenic and tumor suppressor activities associated with STAT3/5 signaling, explaining their context-dependent association with tumor progression both in human cancers and animal models.

Immune‐Informed Mucin Hydrogels Evade Fibrotic Foreign Body Response In Vivo

AbstractThe immune‐mediated foreign body response to biomaterial implants can trigger the formation of insulating fibrotic capsules that can compromise implant function. To address this challenge, the intrinsic bioactivity of the mucin biopolymer, a heavily glycosylated protein that forms the protective mucus gel covering mucosal epithelia, is leveraged. By using a bioorthogonal inverse electron demand Diels–Alder reaction, mucins are crosslinked into implantable hydrogels. It is shown that mucin hydrogels (Muc‐gels) modulate the immune response driving biomaterial‐induced fibrosis. Muc‐gels do not elicit fibrosis 21 days after implantation in the peritoneal cavity of C57Bl/6 mice, whereas medical‐grade alginate hydrogels are covered by fibrous tissues. Further, Muc‐gels dampen the recruitment of innate and adaptive immune cells to the gel and trigger a pattern of very mild activation marked by a noticeably low expression of the fibrosis‐stimulating transforming growth factor beta 1 cytokine. Macrophages recruited to Muc‐gels upregulate the gene expression of the protein inhibitor of activated STAT 1 (PIAS1) and SH2‐containing phosphatase 1 (SHP‐1) cytokine regulatory proteins, which likely contributes to their low cytokine expression profiles. With this advance in mucin materials, an essential tool is provided to better understand mucin bioactivities and to initiate the development of new mucin‐based and mucin‐inspired “immune‐informed” materials for implantable devices subject to fibrotic encapsulation.

Functional characterization of a protein inhibitor of activated STAT (PIAS) gene in Litopenaeus vannamei

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.

Negative regulation of eNOS-NO signaling by over-SUMOylation of PPARγ contributes to insulin resistance and dysfunction of vascular endothelium in rats

SUMOylation of peroxisome proliferator-activated receptor gamma (PPAR γ) plays important regulatory role in its transcriptional activity. Our recent studies in vitro found that over-SUMOylation of PPARγ, like high glucose and high fat (HG/HF), induced endothelial insulin resistance (IR). However, whether such an event occurs in rats remains unclear. Therefore, our study aimed at investigating whether PPARγ over-SUMOylation could mimic high sucrose/fat diet (HFD) to induce endothelial IR and dysfunction and explored its underlying mechanisms. Normal chow-fed rats were intravenously infected with adenoviruses carrying the wild type cDNAs encoding PPARγ, SUMO1 and PIAS1 (protein inhibitor of activated STAT1). HFD-fed rats were regarded as a positive control. Body physical and biochemical parameters, glucose tolerance and vessel function were detected. The expression and SUMOylation levels of PPARγ were measured by western blotting and co-immunoprecipitation. Our results showed that like HFD, PPARγ over-SUMOylation induced endothelial IR and dysfunction via a negative regulation of eNOS-NO pathway. More importantly, we found that PPARγ over-SUMOylation induced endogenous SUMOylation cascade and exacerbated endothelial IR and dysfunction.The findings will deepen the understanding on PPARγ SUMOylation-regulating insulin signaling network and offer a potential target for prevention and cure of diabetic vascular complications.

Protein inhibitor of activated STAT genes are differentially expressed in breast tumor tissues.

Aim: Protein inhibitor of activated STAT (PIAS) family includes transcriptional regulator proteins with SUMO E3 ligase activity. They regulate expression of several genes involved in cell proliferation, differentiation and survival. Method: We evaluated expression of PIAS1-4 genes in 54 breast cancer tissues and their paired adjacent noncancerous tissues. Results:PIAS2 and PIAS3 genes were significantly downregulated in tumoral tissues compared with adjacent noncancerous tissues. PIAS1-3 expressions were significantly lower in estrogen receptor (ER+) samples compared with ER- samples while PIAS4 had the opposite trend. PIAS3 expression was significantly higher in grade 1 samples compared with grade 2 samples. Conclusion: These findings highlight the role of PIAS genes in the pathogenesis of breast cancer and their association with determinants of response to antihormone therapies.

DPPA2/4 and SUMO E3 ligase PIAS4 opposingly regulate zygotic transcriptional program.

The molecular mechanism controlling the zygotic genome activation (ZGA) in mammals remains poorly understood. The 2-cell (2C)-like cells spontaneously emerging from cultures of mouse embryonic stem cells (ESCs) share some key transcriptional and epigenetic programs with 2C-stage embryos. By studying the transition of ESCs into 2C-like cells, we identified developmental pluripotency associated 2 and 4 (Dppa2/4) as important regulators controlling zygotic transcriptional program through directly up-regulating the expression of double homeobox (Dux). In addition, we found that DPPA2 protein is sumoylated and its activity is negatively regulated by small ubiquitin-like modifier (Sumo) E3 ligase protein inhibitor of activated STAT 4 (PIAS4). PIAS4 is down-regulated during ZGA process and during transitioning of ESCs into 2C-like cells. Depleting Pias4 or overexpressing Dppa2/4 is sufficient to activate 2C-like transcriptional program, whereas depleting Dppa2/4 or forced expression of Pias4 or Sumo2–Dppa2 inhibits 2C-like transcriptional program. Furthermore, ectopic expression of Pias4 or Sumo2–Dppa2 impairs early mouse embryo development. In summary, our study identifies key molecular rivals consisting of transcription factors and a Sumo2 E3 ligase that regulate zygotic transcriptional program upstream of Dux.

Protein inhibitor of activated STAT1 Ser503 phosphorylation‐mediated Elk‐1 SUMOylation promotes neuronal survival in APP/PS1 mice

Protein inhibitor of activated STAT1 (PIAS1) is phosphorylated by IKKα at Ser90 in a PIAS1 E3 ligase activity-dependent manner. Whether PIAS1 is also phosphorylated at other residues and the functional significance of these additional phosphorylation events are not known. The transcription factor Elk-1 remains SUMOylated under basal conditions, but the role of Elk-1 SUMOylation in brain is unknown. Here, we examined the functional significance of PIAS1-mediated Elk-1 SUMOylation in Alzheimer's disease (AD) using the APP/PS1 mouse model of AD and amyloid β (Aβ) microinjections in vivo. Novel phosphorylation site(s) on PIAS1 were identified by LC-MS/MS, and MAPK/ERK-mediated phosphorylation of Elk-1 demonstrated using in vitro kinase assays. Elk-1 SUMOylation by PIAS1 in brain was determined using in vitro SUMOylation assays. Apoptosis in hippocampus was assessed by measuring GADD45α expression by western blotting, and apoptosis of hippocampal neurons in APP/PS1 mice was assessed by TUNEL assay. Using LC-MS/MS, we identified a novel MAPK/ERK-mediated phosphorylation site on PIAS1 at Ser503 and showed this phosphorylation determines PIAS1 E3 ligase activity. In rat brain, Elk-1 was SUMOylated by PIAS1, which decreased Elk-1 phosphorylation and down-regulated GADD45α expression. Moreover, lentiviral-mediated transduction of Elk-1-SUMO1 reduced the number of hippocampal apoptotic neurons in APP/PS1 mice. MAPK/ERK-mediated phosphorylation of PIAS1 at Ser503 determines PIAS1 E3 ligase activity. Moreover, PIAS1 mediates SUMOylation of Elk-1, which functions as an endogenous defence mechanism against Aβ toxicity in vivo. Targeting Elk-1 SUMOylation could be considered a novel therapeutic strategy against AD.

YB1 protects cardiac myocytes against H2O2‑induced injury via suppression of PIAS3 mRNA and phosphorylation of STAT3.

Oxidative stress serves important roles in cardiac injury during the process of ischemia/reperfusion (I/R). Y-box protein 1 (YB1), a member of the highly conserved Y-box protein family, is closely associated with inflammation and stress responses by regulating gene transcription, RNA splicing and mRNA translation. However, the roles of YB1 in oxidative stress-induced myocardial-I/R (M-I/R) injury are unknown. The aim of the present study was to examine the effects of YB1 on H2O2-induced cardiomyocyte injury and its underlying mechanism. The results demonstrated that YB1 expression was upregulated during H2O2-induced myocardial injury. YB1 knockdown through transfection of small interfering RNA significantly aggravated cardiac cell apoptosis. Furthermore, YB1 knockdown significantly reversed the H2O2-mediated increase in phosphorylated signal transducer and activator of transcription (STAT)3, but did not affect the phosphorylation of P38, extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinases, P65, Janus kinase 1 and 2 or STAT1. Moreover, protein co-immunoprecipitation and RNA-binding protein immunoprecipitation assays revealed that YB1 interacted with protein inhibitor of activated STAT 3 (PIAS3) mRNA but not its translated protein. YB1 overexpression may have promoted PIAS3 mRNA decay, decreasing PIAS3 protein levels, and therefore increased the levels of phosphorylated STAT3. Finally, YB1 knockdown, mediated by a lentivirus carrying YB1 targeted short hairpin RNA, significantly decreased left ventricle percentage fractional shortening and ejection fraction values, while increasing the infarct sizes in a rat model of M-I/R injury. These results demonstrated for the first time (to the best of our knowledge) that YB1 may protect cardiac myocytes against H2O2 or M-I/R-induced injury by binding to PIAS3 mRNA and resulting in the phosphorylation of STAT3.

PIAS genes as disease markers in bipolar disorder.

The protein inhibitors of activated STAT (PIAS) are involved in regulation of many transcription factors and signaling pathways that contribute to the pathogenesis of bipolar disease (BD). In the current study, we evaluated the expression of four PIAS genes (PIAS1-4) in peripheral blood of BD patients and healthy subjects to explore their contribution in the pathogenesis of BD and their suitability as peripheral biomarkers for this disorder. All PIAS genes were significantly upregulated in total BD patients compared with total controls. The sex-based analysis confirmed upregulation of PIAS1-4 genes in male BD patients compared with male controls (P < 0.001). However, PIAS1 was significantly downregulated in female patients compared with female controls (P = 0.02). Expression levels of other PIAS genes were not significantly different between female patients and female controls. There were no significant correlations between expression levels of PIAS genes and any of the clinical data of study participants after adjustment of the effects of the sex. On the basis of the area under the curve (AUC) values in receiver operating characteristic curves, PIAS4 had the best performance in the differentiation of disease status between study participants (AUC = 0.81). PIAS3 and PIAS4 genes had the best sensitivity and specificity values, respectively. Combination of expression levels of four genes resulted in the improvement of diagnostic power (AUC = 0.82). The current data implies the role of PIAS genes in the pathogenesis of BD and denotes their suitability as peripheral markers for this disorder.

Identification of the targets of hematoporphyrin derivative in lung adenocarcinoma using integrated network analysis

BackgroundHematoporphyrin derivative (HPD) has a sensibilization effect in lung adenocarcinoma. This study was conducted to identify the target genes of HPD in lung adenocarcinoma.MethodsRNA sequencing was performed using the lung adenocarcinoma cell line A549 after no treatment or treatment with X-ray or X-ray + HPD. The differentially expressed genes (DEGs) were screened using Mfuzz package by noise-robust soft clustering analysis. Enrichment analysis was carried out using “BioCloud” online tool. Protein–protein interaction (PPI) network and module analyses were performed using Cytoscape software. Using WebGestalt tool and integrated transcription factor platform (ITFP), microRNA target and transcription factor (TF) target pairs were separately predicted. An integrated regulatory network was visualized with Cytoscape software.ResultsA total of 815 DEGs in the gene set G1 (continuously dysregulated genes along with changes in processing conditions [untreated—treated with X-ray—X-ray + treated with HPD]) and 464 DEGs in the gene set G2 (significantly dysregulated between X-ray + HPD-treated group and untreated/X-ray-treated group) were screened. The significant module identified from the PPI network for gene set G1 showed that ribosomal protein L3 (RPL3) gene could interact with heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1). TFs AAA domain containing 2 (ATAD2) and protein inhibitor of activated STAT 1 (PIAS1) were separately predicted for the genes in gene set G1 and G2, respectively. In the integrated network for gene set G2, ubiquitin-specific peptidase 25 (USP25) was targeted by miR-200b, miR-200c, and miR-429.ConclusionRPL3, HSP90AA1, ATAD2, and PIAS1 as well as USP25, which is targeted by miR-200b, miR-200c, and miR-429, may be the potential targets of HPD in lung adenocarcinoma.

Abstract B036: T-cell recognition of breast cancer antigens

Abstract Breast cancer remains the leading cause of cancer death among women worldwide, rendering conventional therapies insufficient despite decades of research. Despite originally considered an immunologically silent malignancy, recent studies have shown encouraging results, from treatment of breast cancer with checkpoint inhibition to enhance the immunogenicity and tumor cell killing mediated by immune cells. The epitope landscape in breast cancer is minimally described; thus it is necessary to identify T-cell targets to develop cancer-specific immune-mediated therapies. This project investigates four proteins commonly upregulated in breast cancer and thus probable tumor-associated antigens (TAAs), namely, aromatase, prolactin, never in mitosis a related kinase 3 (NEK3), and protein inhibitor of activated STAT3 (PIAS3). Each contributes to increase growth, survival, and motility of malignant cells. To uncover novel epitopes for cytotoxic T-cells, a reverse immunology approach is applied. In silico screening via NetMHC was used to predict peptides within the full length of each of the four proteins that bind to HLA-A*0201 and HLA-B*0702. Via in silico screening 415 HLA-A*0201 or HLA-B*0702 binding peptides were predicted An MHC ELISA was applied to experimentally confirm which of the peptides are true HLA-A*0201 and HLA-B*0702 binders, reducing the library from 415 to 147. The 147 peptides were evaluated for T-cell recognition utilizing DNA barcode labeled MHC multimers to screen peripheral blood lymphocytes from 24 breast cancer patients and 18 healthy donor samples. Significantly more TAA specific T-cell responses were detected in breast cancer patients than healthy donors for both HLA-A*0201 (p=0.0039) and HLA-B*0702 (p&amp;lt;0.001) restricted peptides. Thus, the inspected proteins aromatase, prolactin, NEK3 and PIAS3 indeed contain targets for T-cell reactivity. Importantly, several of the responses were identified towards peptides with a predicted intermediate HLA-binding affinity. This emphasizes the importance of including such weaker affinity ligands in the search for epitopes within shared TAAs. Citation Format: Nadia Viborg Petersen, Sofie Ramskov, Rikke Sick Andersen, Özcan Met, Per thor Straten, Amalie Kai Kai Bentzen, Sine Reker Hadrup. T-cell recognition of breast cancer antigens [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B036.

Protein inhibitor of activated STAT3 reduces peripheral arthritis and gut inflammation and regulates the Th17/Treg cell imbalance via STAT3 signaling in a mouse model of spondyloarthritis

BackgroundSpondyloarthritis (SpA) is chronic inflammatory arthritis, and interleukin (IL)-17 is crucial in SpA pathogenesis. Type 17 helper T (Th17) cells are one of major IL-17-secreting cells. Signal transducer and activator of transcription (STAT)-3 signaling induces Th17 differentiation. This study investigated the effects of protein inhibitor of activated STAT3 (PIAS3) on SpA pathogenesis. Curdlan was injected into SKG ZAP-70W163C mice for SpA induction.MethodsThe PIAS3 or Mock vector was inserted into mice for 10 weeks. Clinical and histologic scores of the paw, spine, and gut were evaluated. The expression of IL-17, tumor necrosis factor-α (TNF-α), STAT3, and bone morphogenic protein (BMP) was measured. Confocal microscopy and flow cytometry were used to assess Th cell differentiation.ResultsPIAS3 significantly diminished the histologic scores of the paw and gut. PIAS3-treated mice displayed decreased expression of IL-17, TNF-α, and STAT3 in the paw, spine, and gut. BMP-2/4 expression was lower in the spines of PIAS3-treated mice. Th cell differentiation was polarized toward the upregulation of regulatory T cells (Tregs) and the downregulation of Th17 in PIAS3-treated mice.ConclusionPIAS3 had beneficial effects in mice with SpA by reducing peripheral arthritis and gut inflammation. Pro-inflammatory cytokines and Th17/Treg differentiation were controlled by PIAS3. In addition, BMPs were decreased in the spines of PIAS3-treated mice. These findings suggest that PIAS3 could have therapeutic benefits in patients with SpA.

Hypoxia-induced Slug SUMOylation enhances lung cancer metastasis

BackgroundThe Slug-E-cadherin axis plays a critical role in non-small-cell lung cancers (NSCLCs) where aberrant upregulation of Slug promotes cancer metastasis. Now, the post-translational modifications of Slug and their regulation mechanisms still remain unclear in lung cancer. Hence, exploring the protein linkage map of Slug is of great interest for investigating the scenario of how Slug protein is regulated in lung cancer metastasis.MethodsThe Slug associated proteins, Ubc9 and SUMO-1, were identified using yeast two-hybrid screening; and in vitro SUMOylation assays combined with immunoprecipitation and immunoblotting were performed to explore the detail events and regulations of Slug SUMOylation. The functional effects of SUMOylation on Slug proteins were examined by EMSA, reporter assay, ChIP assay, RT-PCR, migration and invasion assays in vitro, tail vein metastatic analysis in vivo, and also evaluated the association with clinical outcome of NSCLC patients.ResultsSlug protein could interact with Ubc9 and SUMO-1 and be SUMOylated in cells. Amino acids 130–212 and 33–129 of Slug are responsible for its binding to Ubc9 and protein inhibitor of activated STAT (PIAS)y, respectively. SUMOylation could enhance the transcriptional repression activity of Slug via recruiting more HDAC1, resulting in reduced expression of downstream Slug target genes and enhanced lung cancer metastasis. In addition, hypoxia could increase Slug SUMOylation through attenuating the interactions of Slug with SENP1 and SENP2. Finally, high expression Slug and Ubc9 levels were associated with poor overall survival among NSCLC patients.ConclusionsUbc9/PIASy-mediated Slug SUMOylation and subsequent HDAC1 recruitment may play a crucial role in hypoxia-induced lung cancer progression, and these processes may serve as therapeutic targets for NSCLC.

A nanogel based oral gene delivery system targeting SUMOylation machinery to combat gut inflammation.

Poor success rates and challenges associated with the current therapeutic strategies of inflammatory bowel disease (IBD) have accelerated the emergence of gene therapy as an alternative treatment option with great promise. However, oral delivery of nucleic acids (NAs) to an inflamed colon is challenged by multiple barriers presented by the gastrointestinal, extracellular and intracellular compartments. Therefore, we screened a series of polyaspartic acid-derived amphiphilic cationic polymers with varied hydrophobicity for their ability to deliver NAs into mammalian cells. Using the most effective TAC6 polymer, we then engineered biocompatible and stable nanogels composed of polyplexes (TAC6, NA) and an anionic polymer, sodium polyaspartate, that were able to deliver the NAs across mammalian cells using caveolae-mediated cellular uptake. We then utilized these nanogels for oral delivery of PIAS1 (protein inhibitor of activated STAT1), a SUMO 3 ligase, encoding plasmid DNA since PIAS1 is a key nodal therapeutic target for IBD due to its ability to control NF-κB-mediated inflammatory signaling. We show that plasmid delivery using TAC6-derived nanogels diminished gut inflammation in a murine colitis model. Therefore, our study presents engineering of orally deliverable nanogels that can target SUMOylation machinery to combat gut inflammation with very high efficacy.

Atractylenolide II Induces Apoptosis of Prostate Cancer Cells through Regulation of AR and JAK2/STAT3 Signaling Pathways.

Prostate cancer is the most common illness affecting men worldwide. Although much progress has been made in the study of prostate cancer prevention and treatment, less attention has been paid to the molecular mechanism of the disease. The molecular arrangement by which atractylenolide II (ATR II) induces human prostate cancer cytotoxicity was comprehensively examined in the present study. As indicated by the results, ATR II could inhibit prostate cancer cell proliferation and promote DU145 and LNCaP cell apoptosis through induced G2/M cell cycle arrest. The cell apoptosis process induced by ATR II in both DU145 and LNCaP cells was associated with its ability to inhibit androgen receptor (AR) with overexpression of protein inhibitor of activated STAT-1 (PIAS1) and the repression of Janus kinase (Jak2) signaling pathways. The data from the present study demonstrated the antitumor effects and the potential pharmacological application of ATR II as an efficient drug for prostate cancer treatment.

Expression analysis of protein inhibitor of activated STAT (PIAS) genes in IFNβ-treated multiple sclerosis patients.

ObjectivesProtein inhibitors of activated STAT (PIAS) are transcription co-regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway as well as nuclear factor-κB family of transcription factors. Both of them are involved in cytokine release during inflammatory response.Patients and methodsConsidering the role of cytokine imbalance in the pathogenesis of multiple sclerosis (MS), we compared blood expressions of PIAS1-4 genes in 48 interferon β (IFNβ)-treated MS patients with those of healthy subjects by means of real time PCR.ResultsAlthough the expression levels of these genes were not significantly different between MS patients and healthy subjects, significant inverse correlations have been found between PIAS1 expression and age at disease onset. PIAS2 and PIAS3 expressions were inversely correlated with Expanded Disability Status Scale (EDSS) in patients. Moreover, PIAS3 expression was correlated with disease duration in patients and with age in controls. In addition, PIAS4 expression was inversely correlated with EDSS and age at disease onset while it was positively correlated with disease duration.ConclusionThe present study provides evidences for altered expression of PIAS genes in IFNβ-treated MS patients compared with healthy subjects. However, future studies are needed for elaboration of their exact function in this disorder.

Different SUMO paralogues determine the fate of wild-type and mutant CFTRs: biogenesis versus degradation.

A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40–50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.

Protein Inhibitor of Activated STAT (PIAS) Negatively Regulates the JAK/STAT Pathway by Inhibiting STAT Phosphorylation and Translocation

Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.

JAK-1 Inhibition Suppresses Interferon-Induced BAFF Production in Human Salivary Gland: Potential Therapeutic Strategy for Primary Sjögren's Syndrome.

To examine whether a JAK inhibitor regulates functional responses of human salivary gland epithelial cells (SGECs) and disease parameters in an animal model of Sjögren's syndrome (SS). Common differentially expressed genes (DEGs) were analyzed among peripheral blood mononuclear cells from patients with primary SS and other data sets, using blood and SG tissue. Validation of expression in SGs was analyzed by focus score. Inhibition of messenger RNA expression of DEGs and BAFF by filgotinib was analyzed using reverse transcription-polymerase chain reaction in primary SGECs. SG organoid cultures were used to determine the association between DEGs and BAFF via knockdown using small interfering RNAs or to determine regulation of BAFF by JAK inhibitor. Filgotinib (1.5 mg/kg) was intraperitoneally injected into 8-week-old NOD/ShiLtJ mice 3 times per week to analyze manifestations of disease. Finally, STAT signaling was assessed in human and mouse SGECs. Expression of the DEGs IFNG and BAFF increased in SGs from patients with primary SS, as assessed by focus score. There was a significant correlation between IFIT2 and BAFF expression. JAK inhibitor suppressed interferon (IFN)-induced transcription of DEGs and BAFF in human primary SGECs. Knockdown of DEGs or inhibition of JAK caused reduced secretion of BAFF inhuman SG organoid cultures. In addition, filgotinib-treated mice exhibited increased salivary flow rates and marked reductions in lymphocytic infiltration of SGs. JAK inhibitor regulated IFNα- and IFNγ-induced pSTAT-1Y701 , pSTAT-3Y705 , and protein inhibitor of activated STAT-3 (PIAS-3) in human SGECs as well as IFNγ-induced pSTAT-1Y701 , pSTAT-3S727 , and PIAS-1 in mouse SGECs. JAK inhibition controls aberrant activation of SGECs and may be a novel therapeutic approach for primary SS.