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Rheumatoid arthritis (RA) is a chronic autoimmune disorder that leads to systemic inflammation of diarthrodial joint, synovial hyperplasia, cartilage damage, and ultimately joint destruction and deformity. As the dominant non-immune cells in the synovium, fibroblast-like synoviocytes (FLSs) significantly contribute to the deterioration of RA. Our study aimed to explore the regulatory role of long non-coding RNA FOXD2-AS1 in RA progression. Compared to patients with joint trauma, the expression of FOXD2-AS1 was elevated in serum and synovial tissue samples of RA patients. FOXD2-AS1 knockdown inhibited the proliferation and invasion of rheumatoid FLSs but restored their apoptotic ability. Furthermore, FOXD2-AS1 acted as a sponge for microRNA (miR)-331-3p. The expressions of FOXD2-AS1 and miR-331-3p in synovial tissues of RA patients were negatively correlated. Protein inhibitor of activated STAT 3 (PIAS3) was predicted as a downstream target of miR-331-3p. The expressions of FOXD2-AS1 and PIAS3 in synovial tissues of RA patients were positively correlated, whereas a negative correlation was observed between the levels of miR-331-3p and PIAS3. Moreover, increased proliferation and invasion of rheumatoid FLSs induced by FOXD2-AS1 overexpression was inhibited by the knockdown of PIAS3. In summary, this study demonstrated that FOXD2-AS1 promoted RA progression via regulating the miR-331-3p/PIAS3 pathway, suggesting that therapeutic strategies based on the FOXD2-AS1/miR-331-3p/PIAS3 axis may represent a promising treatment approach for RA patients.

Introduction: PIAS1 and PIAS2 (protein inhibitor of activated STAT 1,2) play key roles in the pathogenesis of autoimmune and inflammatory diseases. This study aims to evaluate the gene expr...

Objectives Protein 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 methods Considering 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. Results Although 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. Conclusion The 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.

It is well recognized that PIAS1, a SUMO (small ubiquitin-like modifier) E3 ligase, modulates such cellular processes as cell proliferation, DNA damage responses, and inflammation responses. Recent studies have shown that PIAS1 also plays a part in cell differentiation. However, the role of PIAS1 in adipocyte differentiation remains unknown. CCAAT/enhancer-binding protein β (C/EBPβ), a major regulator of adipogenesis, is a target of SUMOylation, but the E3 ligase responsible for the SUMOylation of C/EBPβ has not been identified. The present study showed that PIAS1 functions as a SUMO E3 ligase of C/EBPβ to regulate adipogenesis. PIAS1 expression was significantly and transiently induced on day 4 of 3T3-L1 adipocyte differentiation, when C/EBPβ began to decline. PIAS1 was found to interact with C/EBPβ through the SAP (scaffold attachment factor A/B/acinus/PIAS) domain and SUMOylate it, leading to increased ubiquitination and degradation of C/EBPβ. C/EBPβ became more stable when PIAS1 was silenced by RNA interference (RNAi). Moreover, adipogenesis was inhibited by overexpression of wild-type PIAS1 and promoted by knockdown of PIAS1. The mutational study indicated that the catalytic activity of SUMO E3 ligase was required for PIAS1 to restrain adipogenesis. Importantly, the inhibitory effect of PIAS1 overexpression on adipogenesis was rescued by overexpressed C/EBPβ. Thus, PIAS1 could play a dynamic role in adipogenesis by promoting the SUMOylation of C/EBPβ.

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is a molecule that regulates STAT3 and has antiproliferative properties. Glioblastoma and squamous cell lung cancer lack PIAS3 expression. To test the hypothesis that PIAS3 transcriptional effects are STAT3-independent, we developed models for STAT3 knockdown and PIAS3 over-expression. PIAS3 expression results in a distinct transcriptional profile that does not occur with STAT3 knockdown. We identify novel transcription factor binding partners for PIAS3 including ETS, EGR1, NR1I2, and GATA1. PIAS3 binds to these factors and regulates their transcriptional effects resulting in alterations in canonical pathways including Wnt/β-catenin signaling and functions such as cell death and proliferation. A model is proposed by which PIAS3 effects EGR1 regulated pathways.

Special AT-rich sequence-binding protein 1 (SATB1) is a tissue-restricted genome organizer that provides a key link between DNA loop organization, chromatin modification/remodeling, and transcription factor association at matrix attachment regions (MARs). The SUMO E3 ligase PIAS1 enhances SUMO conjugation to SATB1 lysine-744, and this modification regulates caspase-6 mediated cleavage of SATB1 at promyelocytic leukemia nuclear bodies (PML NBs). Since this regulated caspase cleavage occurs on only a subset of SATB1, and the products are relatively stable, proteolysis likely mediates cellular processes other than programmed cell death. However, the mechanism for the spatial and temporal regulation of SATB1 sumoylation and caspase cleavage is not known. Here we report that these processes are controlled by SATB1 phosphorylation; specifically, PIAS1 interaction with SATB1 is inhibited by phosphorylation. Mutagenesis studies identified interaction of the PIAS SAP (scaffold attachment factor-A/B/acinus/PIAS) motif with SATB1 N-terminal sequences. Notably, phosphorylation of SATB1 at threonine-188 regulates its interaction with PIAS1. Sequences near this phosphorylation site, LXXLL (residues 193 to 197), appear to be conserved among a subset of SUMO substrate proteins. Thus, this motif may be commonly involved in interaction with the PIAS SAP, and phosphorylation may similarly inhibit some of these substrates by preventing their interaction with the ligase.

Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.

PIAS (protein inhibitor of activated STAT) proteins interact with and modulate the activities of various transcription factors. In this work, we demonstrate that PIAS proteins xalpha, xbeta, 1, and 3 interact with the small ubiquitin-related modifier SUMO-1 and its E2 conjugase, Ubc9, and that PIAS proteins themselves are covalently modified by SUMO-1 (sumoylated). PIAS proteins also tether other sumoylated proteins in a noncovalent fashion. Furthermore, recombinant PIASxalpha enhances Ubc9-mediated sumoylation of the androgen receptor and c-Jun in vitro. Importantly, PIAS proteins differ in their abilities to promote sumoylation in intact cells. The ability to stimulate protein sumoylation and the interaction with sumoylated proteins are dependent on the conserved PIAS RING finger-like domain. These functions are linked to the activity of PIASxalpha on androgen receptor-dependent transcription. Collectively, our results imply that PIAS proteins function as SUMO-1-tethering proteins and zinc finger-dependent E3 SUMO protein ligases, and these properties are likely to explain their ability to modulate the activities of various transcription factors.

Small ubiquitin-like modifier (SUMO) is a post-translational modifier peptide that is involved in several biological processes in eukaryotes. Arabidopsis SIZ1, a SUMO E3 ligase, is an ortholog of the mammalian PIAS (Protein Inhibitor of Activated STAT) and yeast SIZ (SAP/Miz) proteins. SIZ1 contains all of the typical domains of PIAS/SIZ-type proteins, such as the PINIT, SAP, SP-RING, and plant-specific PHD domains. SIZ1 plays a pivotal role in controlling SUMOylation, and disruption of its function has been reported to affect stress responses, growth, and development. We performed a structural and functional analysis of SIZ1 by determining the phenotypes of siz1 knockout mutants transformed with SIZ1 alleles carrying point mutations in predicted SIZ1 domains. This study establishes that the diverse properties characteristic of SIZ1 are associated with specific domains and that they can be separated.

The first STAT-containing transcription factor to be studied, the alpha-interferon-induced ISGF3, is composed of a Stat1:2 heterodimer and a weak DNA-binding protein, p48, that is a member of a growing family of proteins similar to the so-called interferon regulatory factor (IRF-1). The p48 and Stat1:2 heterodimer do not associate stably in the absence of DNA, but we show that amino acids approximately 150 to 250 of Stat1 and a COOH-terminal portion of p48 exhibit physical interaction, implying contact that stabilizes ISGF3. Moreover, amino acid exchanges within the Stat1 contact region diminish or abolish the functional activity of Stat1. This protein interaction domain may be important in other STAT proteins to recruit partners to multiprotein transcription factors.

SUMOylation of protein substrates and the transcription factor NFκB family are both linked to a myriad of physiological and pathological processes.1–3 However, the known connections between SUMO and NFκB signaling are currently limited. Examples include SUMO-1 modification of IκBα, the main inhibitor of canonical NFκB dimers, which prevents signal-induced ubiquitination and degradation of IκBα and thus limits NFκB activation.4 SUMO-2/3 modification of the same lysine residue of IκBα, in contrast, seems to impart dissociation of NFκB from IκBα to induce NFκB activation in response to hypoxia.5 Similarly, basal SUMO-1 modification of p100 is thought to serve as an essential permissive mechanism to allow for non-canonical NFκB activation.6 In these cases, specific SUMO ligase or SUMO-specific protease (SENP) that mediates direct SUMOyation or deSUMOylation of respective substrate remains unclear. Another example is SUMO-1 modification of NEMO (NFκB Essential MOdulator) that mediates NFκB activation in response to genotoxic stress.7 In this case, the critical ligase is defined as PIASy (Protein Inhibitor of Activated Stat y, also known as PIASγ/PIAS4). Different nuclear proteins may also participate in this modification process, including PIDD (p53-induced protein with death domain), RIP1 (receptor-interacting protein 1) and PARP1 [poly-(ADP ribose) polymerase 1].7 In a recent study,8 we reported the identification of SENP2 (Sentin/SUMO-specific protease 2), among the six known SUMO pro-teases, as the major SUMO protease for NEMO. This study also provided a new concept that SUMO and NFκB ties are not simply one way but can be bidirectional, SUMO regulating NFκB signaling on one hand, and NFκB modulating SUMOylation via induction of SUMO proteases on the other, thereby implicating new roles for the NFκB system. One fundamental feature of the NFκB signaling system is the presence of negative feedback regulation.9 It appears that this control mechanism has evolved to tightly regulate its own activity, because uncontrolled NFκB activity (both excessive and deficient) contributes to pathological disease states.1 We revealed that the gene encoding SENP2 (and SENP1 but not PIASy) is a novel, direct transcriptional target of NFκB, which is induced after DNA damage signals to attenuate upstream IKK activation via deSU-MOylation of NEMO (Fig. 1A). This response is signal-specific and is coupled to DNA damage activation of the ATM (Ataxia telangiectasia mutated) protein kinase, ATM-dependent histone methylation of the SENP2 locus, and NFκB recruitment to regulatory κB binding sites located there. SENP2-deficient cells display both heightened and prolonged (biphasic) NFκB response accompanied by correspondingly increased NEMO SUMOylation and IKK activation. These cells also display increased cell survival in response to genotoxic stress in a manner dependent on NFκB activation. Thus, SENP2 induction is engaged to feedback attenuate NFκB-mediated cell survival response in the face of DNA damage stress. Figure 1 Feedback regulation of NFκB signaling induced by genotoxic agents. (A) NFκB activation by genotoxic stress involves SUMoylation of NEMO via PIASy (E3) and Ubc9 (E2), which leads to IKK activation. NFκB then activates the synthesis ... Given that post-translational modification of many protein substrates by SUMOylation impacts an array of processes and deSUMOylation needs to be mediated by a very small number of SUMO proteases, we speculate that NFκB induction of SENP2 (and SENP1) modulates other processes in addition to NEMO deSUMOylation (Fig. 1B). For example, activities of many transcriptional regulators are regulated by SUMOylation, including p53, Fos, Jun and HDAC1, among others.2,3 SENP2 is implicated in cell cycle regulation mediated by the p53-Mdm2 pathway, while SENP1 appears to regulate development of prostate cancer involving modulation of androgen receptor activity.2 Finally, the DNA damage responsive factors, BRCA1 and 53BP1, are SUMO modified to regulate DNA repair.10,11 While different proteins can be modified by SUMO-1 or −2/3 (or both), SENP2 and SENP1 can deconjugate SUMO-1 or −2/3, depending on the substrates.2,3 Thus, definition of SENP2/1 as direct NFκB transcriptional targets provides an initial mechanism for NFκB regulation of SUMO pathways. Elucidation of additional mechanisms for bidirectional regulation between SUMO and NFκB pathways will increase our understanding of their roles in normal and disease processes.