PIAS1 protects against myocardial ischemia-reperfusion injury by stimulating PPARγ SUMOylation.

BACKGROUND: Myocardial ischemia-reperfusion injury (IRI) has become one of the most serious complications after reperfusion therapy in patients with acute myocardial infarction. Small ubiquitin-like modification (SUMOylation) is a reversible process, including SUMO E1-, E2-, and E3-mediated SUMOylation and SUMO-specific protease-mediated deSUMOylation, with the latter having been shown to play a vital role in myocardial IRI previously. However, little is known about the function and regulation of SUMO E3 ligases in myocardial IRI. RESULTS: In this study, we found dramatically decreased expression of PIAS1 after ischemia/reperfusion (I/R) in mouse myocardium and H9C2 cells. PIAS1 deficiency aggravated apoptosis and inflammation of cardiomyocytes via activating the NF-κB pathway after I/R. Mechanistically, we identified PIAS1 as a specific E3 ligase for PPARγ SUMOylation. Moreover, H9C2 cells treated with hypoxia/reoxygenation (H/R) displayed reduced PPARγ SUMOylation as a result of down-regulated PIAS1, and act an anti-apoptotic and anti-inflammatory function through repressing NF-κB activity. Finally, overexpression of PIAS1 in H9C2 cells could remarkably ameliorate I/R injury. CONCLUSIONS: Collectively, our findings demonstrate the crucial role of PIAS1-mediated PPARγ SUMOylation in protecting against myocardial IRI.

Pias1 is essential for erythroid and vascular development in the mouse embryo.

The protein inhibitor of activated STAT-1 (PIAS1) is one of the few known SUMO E3 ligases. PIAS1 has been implicated in several biological processes including repression of innate immunity and DNA repair. However, PIAS1 function during development and tissue differentiation has not been studied. Here, we report that Pias1 is required for proper embryonic development. Approximately 90% of Pias1 null embryos die in utero between E10.5 and E12.5. We found significant apoptosis within the yolk sac (YS) blood vessels and concomitant loss of red blood cells (RBCs) resulting in profound anemia. In addition, Pias1 loss impairs YS angiogenesis and results in defective capillary plexus formation and blood vessel occlusions. Moreover, heart development is impaired as a result of loss of myocardium muscle mass. Accordingly, we found that Pias1 expression in primary myoblasts enhances the induction of cardiac muscle genes MyoD, Myogenin and Myomaker. PIAS1 protein regulation of cardiac gene transcription is dependent on transcription factors Myocardin and Gata-4. Finally, endothelial cell specific inactivation of Pias1 in vivo impairs YS erythrogenesis, angiogenesis and recapitulates loss of myocardium muscle mass. However, these defects are not sufficient to recapitulate the lethal phenotype of Pias1 null embryos. These findings highlight Pias1 as an essential gene for YS erythropoiesis and vasculogenesis in vivo.

PIAS1 is a determinant of poor survival and acts as a positive feedback regulator of AR signaling through enhanced AR stabilization in prostate cancer.

Novel drugs like Abiraterone or Enzalutamide, which target androgen receptor (AR) signaling to improve androgen deprivation therapy (ADT), have been developed during the past years. However, the application of these drugs is limited because of occurrence of inherent or acquired therapy resistances during the treatment. Thus, identification of new molecular targets is urgently required to improve current therapeutic prostate cancer (PCa) treatment strategies. PIAS1 (protein inhibitor of activated STAT1 (signal transducer and activator of transcription-1)) is known to be an important cell cycle regulator and PIAS1-mediated SUMOylation is essential for DNA repair. In this context, elevated PIAS1 expression has already been associated with cancer initiation. Thus, in the present study, we addressed the question of whether PIAS1 targeting can be used as a basis for an improved PCa therapy in combination with anti-androgens. We show that PIAS1 significantly correlates with AR expression in PCa tissue and in cell lines and demonstrate that high PIAS1 levels predict shorter relapse-free survival. Our patient data are complemented by mechanistic and functional in vitro experiments that identify PIAS1 as an androgen-responsive gene and a crucial factor for AR signaling via prevention of AR degradation. Furthermore, PIAS1 knockdown is sufficient to decrease cell proliferation as well as cell viability. Strikingly, Abiraterone or Enzalutamide treatment in combination with PIAS1 depletion is even more effective than single-drug treatment in multiple PCa cell models, rendering PIAS1 as a promising target protein for a combined treatment approach to improve future PCa therapies.

Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15.

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.

Site-specific ubiquitination is required for relieving the transcription factor Miz1-mediated suppression on TNF-α-induced JNK activation and inflammation.

The transcription factor zinc-finger protein Miz1 represses TNF-α-induced JNK activation and the repression is relieved upon TNF-α stimulation. However, the underlying mechanism is incompletely understood. Here we report that Miz1 interferes with the ubiquitin conjugating enzyme (E2) Ubc13 for binding to the RING domain of TNF-receptor associated factor 2 (TRAF2), thereby inhibiting the ubiquitin ligase (E3) activity of TRAF2 and suppressing TNF-α-induced JNK activation. Upon TNF-α stimulation, Miz1 rapidly undergoes K48-linked polyubiquitination at Lys388 and Lys472 residues and subsequent proteasomal degradation in a TRAF2-dependent manner. Replacement of Lysine 388 and Lysine 472 by arginines generates a nondegradable Miz1 mutant, which significantly suppresses TNF-α-induced JNK1 activation and inflammation. Thus, our results reveal a molecular mechanism by which the repression of TNF-α-induced JNK activation by Miz1 is de-repressed by its own site-specific ubiquitination and degradation, which may account for the temporal control of TNF-α-JNK signaling.

Miz1 is a signal- and pathway-specific modulator or regulator (SMOR) that suppresses TNF-alpha-induced JNK1 activation.

The proinflammatory cytokine TNF-alpha exerts its pleiotropic functions through activation of multiple downstream effectors, including JNK1. Yet, the underlying regulatory mechanism is incompletely understood. Here, we report that the transcription factor Myc-interacting zinc-finger protein 1 (Miz1) selectively suppresses TNF-alpha-induced JNK1 activation and cell death independently of its transcription activity. Proteomics analysis and yeast two-hybrid screening reveal that Miz1 is a JNK-associated protein. The TNF-alpha-induced activation of JNK1 is augmented in Miz1-deficient mouse embryonic fibroblasts (Miz1(-/-) MEFs), but the augmentation is abrogated by reintroduction of Miz1 or its transcription-deficient mutant. The regulation by Miz1 is highly specific, because it regulates TNF-alpha-induced TRAF2 K63-linked polyubiquitination. Neither JNK1 activation by IL-1beta or UV nor TNF-alpha-induced activation of p38, ERK, or IkappaB kinase complex is affected by the loss of Miz1. The TNF-alpha-induced cell death also is accelerated in Miz1(-/-) MEFs. Upon TNF-alpha stimulation, Miz1 is degraded rapidly by the proteasome, relieving its suppression on JNK1 activation. Thus, our results show that in addition to being a transcription factor Miz1 acts as a signal- and pathway-specific modulator or regulator that specifically regulates TNF-alpha-induced JNK1 activation and cell death.

Substantially reduced expression of PIAS1 is associated with colon cancer development.

PURPOSE: Protein inhibitors of activated STATs (PIAS) regulate the interferon-gamma (IFN-gamma) signaling pathway, which has numerous effects on tumor development and tumor cell biology. PIAS's also regulate STAT family members not directly involved in IFN-gamma signaling. This project was designed to assess PIAS1 expression in colon cancer. METHODS: To determine whether PIAS1, one of the PIAS family members, or IFN-gamma signaling pathway components could be used to stratify colon tumors, we stained tissue microarrays for PIAS1, interferon regulatory factor-1 (IRF-1) and STAT1alpha. RESULTS: PIAS1 staining of the colon cancer tissue microarrays indicated a strong correlation of normal colon cells, and adenomas, with high expression of both PIAS1 and IRF-1. CONCLUSION: The PIAS1 results in particular may represent a basis for new approaches for efficiently distinguishing adenomas from colon cancer.

Spatial interplay between PIASy and FIP200 in the regulation of signal transduction and transcriptional activity.

The members of the protein inhibitor of activated STAT (PIAS) family of proteins are implicated in fundamental cellular processes, including transcriptional regulation, either through action as E3 SUMO ligases or through SUMO-independent effects. We report here the identification of FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) as a new PIASy-interacting protein. We show that the interaction depends on the integrity of the RING finger of PIASy and the carboxy terminus of FIP200. Both in vitro and in vivo sumoylation assays failed to reveal any sumoylation of FIP200, suggesting that FIP200 is not a bona fide SUMO substrate. Immunofluorescence microscopy and subcellular fractionation, either upon forced PIASy expression or in the absence of PIASy, revealed that interaction with PIASy redistributes FIP200 from the cytoplasm to the nucleus, correlating with abrogation of FIP200 regulation of TSC/S6K signaling. Conversely, FIP200 enhances the transcriptional activation of the p21 promoter by PIASy whereas PIASy transcription activity is severely reduced upon FIP200 depletion by RNA interference. Chromatin immunoprecipitation analysis demonstrates that endogenous PIASy and FIP200 are corecruited to the p21 promoter. Altogether, these results provide the first evidence for the existence of a close-spatially controlled-mode of regulation of FIP200 and PIASy nucleocytoplasmic functions.

PIASx is a MEF2 SUMO E3 ligase that promotes postsynaptic dendritic morphogenesis.

Postsynaptic morphogenesis of dendrites is essential for the establishment of neural connectivity in the brain, but the mechanisms that govern postsynaptic dendritic differentiation remain poorly understood. Sumoylation of the transcription factor myocyte enhancer factor 2A (MEF2A) promotes the differentiation of postsynaptic granule neuron dendritic claws in the cerebellar cortex. Here, we identify the protein PIASx as a MEF2 SUMO E3 ligase that represses MEF2-dependent transcription in neurons. Gain-of-function and genetic knockdown experiments in rat cerebellar slices and in the postnatal cerebellum in vivo reveal that PIASx drives the differentiation of granule neuron dendritic claws in the cerebellar cortex. MEF2A knockdown suppresses PIASx-induced dendritic claw differentiation, and expression of sumoylated MEF2A reverses PIASx knockdown-induced loss of dendritic claws. These findings define the PIASx-MEF2 sumoylation signaling link as a key mechanism that orchestrates postsynaptic dendritic claw morphogenesis in the cerebellar cortex and suggest novel functions for SUMO E3 ligases in brain development and plasticity.

Control of specificity and magnitude of NF-kappa B and STAT1-mediated gene activation through PIASy and PIAS1 cooperation.

NF-kappaB and STATs regulate multiple cellular processes through the transcriptional activation of genes with diversified functions. Although the molecular mechanisms that can turn on/off the overall NF-kappaB/STAT signaling have been extensively studied, how NF-kappaB/STAT-target genes can be differentially regulated is poorly understood. Here we report that PIASy, a member of the PIAS (for protein inhibitor of activated STAT) protein family, is a physiologically important transcriptional repressor of NF-kappaB and STAT1. Piasy deletion in dendritic cells resulted in enhanced expression of a subset of NF-kappaB and STAT1-dependent genes in response to LPS or IFN-gamma treatment, respectively. Consistently, Piasy null mice are hypersensitive to the LPS-induced endotoxic shock. Furthermore, PIASy and PIAS1 display specific as well as redundant effects on the regulation of NF-kappaB/STAT1 signaling. Pias1-/-Piasy-/- embryos died before day 11.5. The disruption of one allele of Pias1 in the Piasy-/- background significantly enhanced the effect of Piasy deletion on the transcriptional induction of NF-kappaB/STAT1-dependent genes, and vice versa. Our results demonstrate that PIASy cooperates with PIAS1 to regulate the specificity and magnitude of NF-kappaB/STAT1-mediated gene activation.

The E3 SUMO ligase PIASy is a regulator of cellular senescence and apoptosis.

Cellular senescence and apoptosis have evolved to restrain unwarranted proliferation of potentially tumorigenic cells. Here we show that overexpression of the E3 SUMO ligase PIASy in normal human fibroblasts recruits the p53 and Rb tumor suppressor pathways to provoke a senescence arrest. By contrast, in Rb-deficient fibroblasts, expression of PIASy leads to p53-dependent apoptosis. Induction of senescence requires PIASy E3 activity and is specific for this member of the PIAS ligase family. PIASy stimulates sumoylation and transcriptional activity of p53 and increases Rb-dependent corepression through recruitment to E2F-responsive promoters. Viral oncoprotein E6 suppresses both PIASy-induced senescence and sumoylation of PIASy substrates. Finally, we show that fibroblasts lacking PIASy exhibit a highly reduced propensity to undergo senescence in response to a prosenescence stimulus. Altogether, these data provide the first evidence for a direct role of an E3 SUMO ligase, and by implication of the SUMO pathway, in cellular senescence and apoptosis.