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1.  Protein Inhibitor of Activated STAT 1 (PIAS1) Is Identified as the SUMO E3 Ligase of CCAAT/Enhancer-Binding Protein β (C/EBPβ) during Adipogenesis 
Molecular and Cellular Biology  2013;33(22):4606-4617.
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.
PMCID: PMC3838193  PMID: 24061474
2.  Interaction of Protein Inhibitor of Activated STAT (PIAS) Proteins with the TATA-binding Protein, TBP* 
The Journal of biological chemistry  2006;281(18):12260-12269.
Transcription activators often recruit promoter-targeted assembly of a pre-initiation complex; many repressors antagonize recruitment. These activities can involve direct interactions with proteins in the pre-initiation complex. We used an optimized yeast two-hybrid system to screen mouse pregnancy-associated libraries for proteins that interact with TATA-binding protein (TBP). Screens revealed an interaction between TBP and a single member of the zinc finger family of transcription factors, ZFP523. Two members of the protein inhibitor of activated STAT (PIAS) family, PIAS1 and PIAS3, also interacted with TBP in screens. Endogenous PIAS1 and TBP co-immunoprecipitated from nuclear extracts, suggesting the interaction occurred in vivo. In vitro-translated PIAS1 and TBP coimmunopreciptated, which indicated that other nuclear proteins were not required for the interaction. Deletion analysis mapped the PIAS-interacting domain of TBP to the conserved TBPCORE and the TBP-interacting domain on PIAS1 to a 39-amino acid C-terminal region. Mammals issue seven known PIAS proteins from four pias genes, pias1, pias3, piasx, and piasy, each with different cell type-specific expression patterns; the TBP-interacting domain reported here is the only part of the PIAS C-terminal region shared by all seven PIAS proteins. Direct analyses indicated that PIASx and PIASy also interacted with TBP. Our results suggest that all PIAS proteins might mediate situation-specific regulatory signaling at the TBP interface and that previously unknown levels of complexity could exist in the gene regulatory interplay between TBP, PIAS proteins, ZFP523, and other transcription factors.
PMCID: PMC2030495  PMID: 16522640
3.  PIAS1 Mediates TGFβ-Induced SM α-Actin Gene Expression Through Inhibition of KLF4 Function-Expression by Protein Sumoylation 
TGFβ and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) α-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFβ activates SM α-actin through PIAS1.
Methods and Results
An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFβ-induced expression of SM α-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM α-actin promoter activity in a TGFβ control element (TCE)-dependent manner. Because the TCE within the SM α-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmuno-precipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM α-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation.
These results provide evidence that PIAS1 promotes TGFβ-induced activation of SM α-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.
PMCID: PMC3322553  PMID: 18927467
transforming growth factor β; protein inhibitor of activated STAT1; vascular smooth muscle cells; Krüppel-like factor
4.  NF-κB Repression by PIAS3 Mediated RelA SUMOylation 
PLoS ONE  2012;7(5):e37636.
Negative regulation of the NF-κB transcription factor is essential for tissue homeostasis in response to stress and inflammation. NF-κB activity is regulated by a variety of biochemical mechanisms including phosphorylation, acetylation, and ubiquitination. In this study, we provide the first experimental evidence that NF-κB is regulated by SUMOylation, where the RelA subunit of NF-κB is SUMOylated by PIAS3, a member of the PIAS (protein inhibitor of activated STAT) protein family with E3 SUMO ligase activity. PIAS3-mediated NF-κB repression was compromised by either RelA mutant resistant to SUMOylation or PIAS3 mutant defective in SUMOylation. PIAS3-mediated SUMOylation of endogenous RelA was induced by NF-κB activation thus forming a negative regulatory loop. The SUMOylation of endogenous RelA was enhanced in IκBα null as compared with wild type fibroblasts. The RelA SUMOylation was induced by TNFα but not leptomycin B mediated RelA nuclear translocation. Furthermore, RelA mutants defective in DNA binding were not SUMOylated by PIAS3, suggesting that RelA DNA binding is a signal for PIAS3-mediated SUMOylation. These results support a novel negative feedback mechanism for NF-κB regulation by PIAS3-mediated RelA SUMOylation.
PMCID: PMC3359287  PMID: 22649547
5.  Association and nuclear translocation of PIAS3-STAT3 complex is ligand and time dependent 
Molecular cancer research : MCR  2009;7(11):1854-1860.
The Epidermal Growth Factor Receptor (EGFR) activation of downstream Signal Transducers and Activators of Transcription 3 (STAT3) plays a crucial role in the pathogenesis of lung cancer. STAT3 transcriptional activity can be negatively regulated by Protein Inhibitor of Activated STAT3 (PIAS3). We investigated PIAS3 time-dependent shuffling and binding to STAT3 in an EGF-dependent model in lung cancer by using confocal microscopy, immunoprecipitation, luciferase reporter assay and protein analysis of segregated cellular components. We also explored the role of phosphorylation at Tyr705 of STAT3 in the formation of PIAS3/STAT3 complex and intracellular shuffling. In a growth factor-free state, PIAS3 was localized to the cytoplasm and unbound to STAT3 in both H520 and A549 cells. Upon exposure to EGF, we observed STAT3 phosphorylation and rapid formation of the PIAS3/STAT3 complex. Within 5 minutes there was a progressive translocation of the complex to the nucleus and by 10 minutes PIAS3 was uniquely localized to the nuclear compartment. Thirty minutes after, PIAS3 returned to the cytoplasm. Using site directed mutagenesis, we substituted Tyr705 of STAT3 with a phenylalanine. Despite EGF stimulation, we observed a significant decrease in PIAS3 and STAT3 binding and a significant reduction in nuclear translocation of PIAS3. Furthermore, there was a significant reduction in PIAS3 capacity to reduce STAT3 mediated gene transcription. In wild type STAT3 cells, increasing concentrations of PIAS3 resulted in a proportional decrease in STAT3 phosphorylation. These data suggest an important role for the negative regulatory effect of PIAS3 on STAT3 in epidermal growth factor driven tumors.
PMCID: PMC2783234  PMID: 19903771
PIAS3; STAT3; non small cell lung cancer
6.  Combinatorial Recruitment of CREB, C/EBPβ and c-Jun Determines Activation of Promoters upon Keratinocyte Differentiation 
PLoS ONE  2013;8(11):e78179.
Transcription factors CREB, C/EBPβ and Jun regulate genes involved in keratinocyte proliferation and differentiation. We questioned if specific combinations of CREB, C/EBPβ and c-Jun bound to promoters correlate with RNA polymerase II binding, mRNA transcript levels and methylation of promoters in proliferating and differentiating keratinocytes.
Induction of mRNA and RNA polymerase II by differentiation is highest when promoters are bound by C/EBP β alone, C/EBPβ together with c-Jun, or by CREB, C/EBPβ and c-Jun, although in this case CREB binds with low affinity. In contrast, RNA polymerase II binding and mRNA levels change the least upon differentiation when promoters are bound by CREB either alone or in combination with C/EBPβ or c-Jun. Notably, promoters bound by CREB have relatively high levels of RNA polymerase II binding irrespective of differentiation. Inhibition of C/EBPβ or c-Jun preferentially represses mRNA when gene promoters are bound by corresponding transcription factors and not CREB. Methylated promoters have relatively low CREB binding and, accordingly, those which are bound by C/EBPβ are induced by differentiation irrespective of CREB. Composite “Half and Half” consensus motifs and co localizing consensus DNA binding motifs are overrepresented in promoters bound by the combination of corresponding transcription factors.
Correlational and functional data describes combinatorial mechanisms regulating the activation of promoters. Colocalization of C/EBPβ and c-Jun on promoters without strong CREB binding determines high probability of activation upon keratinocyte differentiation.
PMCID: PMC3820678  PMID: 24244291
7.  SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin 
Nucleic Acids Research  2014;43(2):848-861.
Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth.
PMCID: PMC4333416  PMID: 25552417
8.  Modulation of inducible nitric oxide synthase expression by sumoylation 
In astrocytes, the inflammatory induction of Nitric Oxide Synthase type 2 (NOS2) is inhibited by noradrenaline (NA) at the transcriptional level however its effects on specific transcription factors are not fully known. Recent studies show that the activity of several transcription factors including C/EBPβ, which is needed for maximal NOS2 expression, is modulated by conjugation of the small molecular weight protein SUMO. We examined whether the expression of SUMO Related Genes (SRGs: SUMO-1, the conjugating enzyme Ubc9, and the protease SENP1) are affected by inflammatory conditions or NA and whether SUMO-1 regulates NOS2 through interaction with C/EBPβ.
Bacterial endotoxin lipopolysaccharide (LPS) was used to induce inflammatory responses including NOS2 expression in primary astrocytes. The mRNA levels of SRGs were determined by QPCR. A functional role for SUMOylation was evaluated by determining effects of over-expressing SRGs on NOS2 promoter and NFκB binding-element reporter constructs. Interactions of SUMO-1 and C/EBPβ with the NOS2 promoter were examined by chromatin immunoprecipitation assays. Interactions of SUMO-1 with C/EBPβ were examined by immunoprecipitation and Western blot analysis and by fluorescence resonance energy transfer (FRET) assays.
LPS decreased mRNA levels of SUMO-1, Ubc9 and SENP1 in primary astrocytes and a similar decrease occurred during normal aging in brain. NA attenuated the LPS-induced reductions and increased SUMO-1 above basal levels. Over-expression of SUMO-1, Ubc9, or SENP1 reduced the activation of a NOS2 promoter, whereas activation of a 4 × NFκB binding-element reporter was only reduced by SUMO-1. ChIP studies revealed interactions of SUMO-1 and C/EBPβ with C/EBP binding sites on the NOS2 promoter that were modulated by LPS and NA. SUMO-1 co-precipitated with C/EBPβ and a close proximity was confirmed by FRET analysis.
Our results demonstrate that SUMOylation regulates NOS2 expression in astrocytes, and point to modification of C/EBPβ as a possible mechanism of action. Targeting the SUMOylation pathway may therefore offer a novel means to regulate inflammatory NOS2 expression in neurological conditions and diseases.
PMCID: PMC2667488  PMID: 19323834
9.  CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation 
PLoS ONE  2014;9(8):e104547.
CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh) also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2) was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation.
Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between −214 and −210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression.
C/EBPβ and RUNX2 cooperatively stimulate expression of Ihh through direct interactions with a C/EBPβ binding element, which further promotes hypertrophic differentiation of chondrocytes during the chondrocyte differentiation process.
PMCID: PMC4126692  PMID: 25105964
10.  Regulation of Basal and Induced Expression of C-Reactive Protein through an Overlapping Element for OCT-1 and NF-κB on the Proximal Promoter 
C-reactive protein (CRP) is an acute phase protein produced by hepatocytes. A minor elevation in the baseline levels of serum CRP is considered an indicator of chronic inflammation. In hepatoma Hep3B cells, IL-6 induces CRP expression by activating transcription factors STAT3 and C/EBPβ. IL-1 synergistically enhances the effects of IL-6. The first 157 bp of the CRP promoter are sufficient for IL-1 synergy. Previously, NF-κB, a transcription factor activated by IL-1β in Hep3B cells, has been shown to increase endogenous CRP expression. The purpose of this study was to investigate the possible action of NF-κB on the 157 bp of the proximal promoter. In this study we show that NF-κB requires and acts synergistically with C/EBPβ on the CRP-proximal promoter to regulate CRP expression. We located the regulatory element that consisted of overlapping binding sites for NF-κB (p50-p50 and p50-p65) and OCT-1. The κB site was responsible for the synergy between NF-κB and C/EBPβ and was also necessary for the CRP transactivation by C/EBPβ through the C/EBP site. Mutation of the κB site decreased the synergistic effect of IL-1β on IL-6-induced CRP expression. Basal CRP expression increased dramatically when binding of both OCT-1 and NF-κB was abolished. Combined data from luciferase transactivation assays and EMSA lead us to conclude that the binding of OCT-1 to the promoter, facilitated by p50-p50 in a novel way, represses, whereas replacement of OCT-1 by p50-p65 induces CRP transcription in cooperation with C/EBPβ. This model for CRP expression favors the variation seen in baseline serum CRP levels in a normal healthy population.
PMCID: PMC3818712  PMID: 16116232
11.  Protein Inhibitor of Activated STAT-3 Expression in Lung Cancer 
Molecular oncology  2011;5(3):256-264.
Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is an endogenous inhibitor of STAT3 transcriptional activity. We have previously demonstrated the concentration-dependent negative regulatory effect of PIAS3 on STAT3 signaling and its capacity to decrease lung cancer proliferation and synergize with epidermal growth factor inhibition. We now investigate PIAS3 expression in both non-small cell lung cancer (NSCLC) cell lines and human resected NSCLC specimens. We also investigated the mechanism by which some lung cancers have significantly decreased PIAS3 expression. Expression of PIAS3 is variable in lung cancer cells lines with 2 of 3 squamous cell carcinoma (SCC) cell lines having no or little PIAS3 protein expression. Similarly, the majority of human SCCs of the lung lack PIAS3 expression by immunohistochemistry; this despite the finding that SCCs have significantly higher levels of PIAS3 mRNA compared to adenocarcinomas. High PIAS3 expression generally correlates with decreased phosphorylated STAT3 in both SCC cell lines and human specimens compatible with the negative regulatory effect of this protein on STAT3 signaling. To investigate this variable expression of PIAS3 we first performed sequencing of the PIAS3 gene that demonstrated single nucleotide polymorphisms but no mutations. Exposure of lung cancer cells to 5-azacytidine and trichostatin A results in a significant increase in PIAS3 mRNA and protein expression. However, methylation-specific PCR demonstrates a lack of CpG island methylation in the promoter region of PIAS3. Exposure of cells to an agent blocking proteosomal degradation results in a significant increase in PIAS3. Our data thus shows that SCC of the lung commonly lacks PIAS3 protein expression and that post-translational modifications may explain this finding in some cases. PIAS3 is a potential therapeutic molecule to target STAT3 pathway in lung cancer.
PMCID: PMC3104085  PMID: 21497567
12.  ATF4 licenses C/EBPβ activity in human mesenchymal stem cells primed for adipogenesis 
eLife  null;4:e06821.
A well-established cascade of transcription factor (TF) activity orchestrates adipogenesis in response to chemical cues, yet how cell-intrinsic determinants of differentiation such as cell shape and/or seeding density inform this transcriptional program remain enigmatic. Here, we uncover a novel mechanism licensing transcription in human mesenchymal stem cells (hMSCs) adipogenically primed by confluence. Prior to adipogenesis, confluency promotes heterodimer recruitment of the bZip TFs C/EBPβ and ATF4 to a non-canonical C/EBP DNA sequence. ATF4 depletion decreases both cell-density-dependent transcription and adipocyte differentiation. Global profiling in hMSCs and a novel cell-free assay reveals that ATF4 requires C/EBPβ for genomic binding at a motif distinct from that bound by the C/EBPβ homodimer. Our observations demonstrate that C/EBPβ bridges the transcriptional programs in naïve, confluent cells and early differentiating pre-adipocytes. Moreover, they suggest that homo- and heterodimer formation poise C/EBPβ to execute diverse and stage-specific transcriptional programs by exploiting an expanded motif repertoire.
eLife digest
Human body fat consists mostly of fat-storing cells called adipocytes. These cells develop in two main steps. First, mesenchymal stem cells—which can potentially become one of many different types of cell—commit to becoming pre-adipocyte cells. These pre-adipocytes then develop into mature adipocytes. Proteins called transcription factors control both steps of this process by binding to particular sites in the DNA of the cell and activating certain genes that control the cell's identity and activity.
Various different transcription factors are known to stimulate the development of mesenchymal stem cells into adipocytes. Experiments performed on cells that have been grown in the laboratory suggest that the cells must also be tightly packed together to become adipocytes.
Cohen et al. have now investigated the role a protein called C/EBPβ plays in the development of adipocytes, and have found that it plays different roles at different stages of development. When mesenchymal stem cells become tightly packed, more of another protein called ATF4 is produced. This protein binds to C/EBPβ, and the resulting two-protein complex then binds to sites on the DNA of the mesenchymal stem cell to activate genes that turn the stem cell into a pre-adipocyte. Reducing the amount of ATF4 in mesenchymal stem cells reduces the number of pre-adipocytes that develop.
When not bound to ATF4, and in response to certain cell signals, C/EBPβ binds to different DNA sites and helps pre-adipocytes develop into mature adipocytes. Whether transcription factors other than ATF4 partner with C/EBPβ to change DNA-binding-site preferences remains unknown. Future studies will search for these, with the aim of providing a clearer molecular understanding for how C/EBPβ acts as a ‘bridge’ that links together the two stages of adipocyte development.
PMCID: PMC4501333  PMID: 26111340
adipogenesis; transcription factors; gene expression; human; mouse
13.  Regulation of Id2 expression by CCAAT/enhancer binding protein β 
Nucleic Acids Research  2005;33(6):1924-1934.
Mice deficient for Id2, a negative regulator of basic helix–loop–helix (bHLH) transcription factors, exhibit a defect in lactation due to impaired lobuloalveolar development during pregnancy, similar to the mice lacking the CCAAT enhancer binding protein (C/EBP) β. Here, we show that Id2 is a direct target of C/EBPβ. Translocation of C/EBPβ into the nucleus, which was achieved by using a system utilizing the fusion protein between C/EBPβ and the ligand-binding domain of the human estrogen receptor (C/EBPβ-ERT), demonstrated the rapid induction of endogenous Id2 expression. In reporter assays, transactivation of the Id2 promoter by C/EBPβ was observed and, among three potential C/EBPβ binding sites found in the 2.3 kb Id2 promoter region, the most proximal element was responsible for the transactivation. Electrophoretic mobility shift assay (EMSA) identified this element as a core sequence to which C/EBPβ binds. Chromatin immunoprecipitation (ChIP) furthermore confirmed the presence of C/EBPβ in the Id2 promoter region. Northern blotting showed that Id2 expression in C/EBPβ-deficient mammary glands was reduced at 10 days post coitus (d.p.c.), compared with that in wild-type mammary glands. Thus, our data demonstrate that Id2 is a direct target of C/EBPβ and provide insight into molecular mechanisms underlying mammary gland development during pregnancy.
PMCID: PMC1074397  PMID: 15809228
14.  Activation of multiple sites by adenosine analogues in the rat isolated aorta. 
British Journal of Pharmacology  1996;118(6):1509-1517.
1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin
PMCID: PMC1909666  PMID: 8832079
15.  PIAS3 Interacts with ATF1 and Regulates the Human Ferritin H Gene through an Antioxidant-responsive Element*s 
The Journal of biological chemistry  2007;282(31):22335-22343.
Gene transcription is coordinately regulated by the balance between activation and repression mechanisms in response to various external stimuli. Ferritin, composed of H and L subunits, is the major intracellular iron storage protein involved in iron homeostasis. We previously identified an enhancer, termed antioxidant-responsive element (ARE), in the human ferritin H gene and its respective transcriptional activators including Nrf2 and JunD. Here we found that ATF1 (activating transcription factor 1) is a transcriptional repressor of the ferritin H ARE. Subsequent yeast two-hybrid screening identified PIAS3 (protein inhibitor of activated STAT3) as an ATF1-binding protein. Further investigation of the human ferritin H ARE regulation showed that 1) PIAS3 reversed ATF1-mediated repression of the ferritin H ARE; 2) ATF1 was sumoylated, but PIAS3, a SUMO E3 ligase, did not appear to play a major role in SUMO1-mediated ATF1 sumoylation or ATF1 transcription activating function; 3) PIAS3 decreased ATF1 binding to the ARE; and 4) ATF1 knockdown with siRNA increased ferritin H expression, whereas PIAS3 knockdown decreased basal expression and oxidative stress-mediated induction of ferritin H. These results suggest that PIAS3 antagonizes the repressor function of ATF1, at least in part by blocking its DNA binding, and ultimately activates the ARE. Collectively our results suggest that PIAS3 is a new regulator of ATF1 that regulates the ARE-mediated transcription of the ferritin H gene.
PMCID: PMC2409283  PMID: 17565989
16.  Inhibition of Corticosteroid-Binding Globulin Gene Expression by Glucocorticoids Involves C/EBPβ 
PLoS ONE  2014;9(10):e110702.
Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPβ, able to tether to the GR, as well as HNF3α involved in GR signaling, are present. C/EBPβ, but not HNF3α, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg. Furthermore, knockdown of C/EBPβ protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPβ’s involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPβ and GR to the Cbg promoter, while C/EBPβ knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPβ.
PMCID: PMC4205011  PMID: 25335188
17.  CCAAT/Enhancer-binding Protein β Inhibits Proliferation in Monocytic Cells by Affecting the Retinoblastoma Protein/E2F/Cyclin E Pathway but Is Not Directly Required for Macrophage Morphology* 
The Journal of Biological Chemistry  2011;286(26):22716-22729.
Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein β (C/EBPβ). Initially, we demonstrated a marked increase in nuclear C/EBPβ-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPβ effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPβ) cell lines stably overexpressing C/EBPβ isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPβ-long), but not those overexpressing LIP (C/EBPβ-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPβ-short cells. In C/EBPβWT macrophage-like cells (high endogenous C/EBPβ), we measured a reduced proliferation/cycling index compared with C/EBPβKO. The typical macrophage morphology was only observed in C/EBPβWT, whereas C/EBPβKO stayed round. C/EBPα did not compensate for C/EBPβ effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPβKO macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPβ-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPβ-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPβ-long and C/EBPβWT cells exhibited low E2F1 and cyclin E levels, and C/EBPβ overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPβ reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPβ may be important for coordinated monocytic differentiation.
PMCID: PMC3123039  PMID: 21558273
C/EBP Transcription Factor; Cell Differentiation; E2F Transcription Factor; Macrophage; Retinoblastoma (Rb); C/EBP beta; Cyclin E; Monocyte; Morphology; Proliferation
18.  Negative Regulation of NF-κB Signaling by PIAS1†  
Molecular and Cellular Biology  2005;25(3):1113-1123.
The NF-κB family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-κB activity is critical, since abnormal NF-κB signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-κB. Upon cytokine stimulation, the p65 subunit of NF-κB translocates into the nucleus, where it interacts with PIAS1. The binding of PIAS1 to p65 inhibits cytokine-induced NF-κB-dependent gene activation. PIAS1 blocks the DNA binding activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the binding of p65 to the promoters of NF-κB-regulated genes is significantly enhanced in Pias1−/− cells. Microarray analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-κB-mediated genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-κB.
PMCID: PMC544018  PMID: 15657437
19.  Type I Interferon Induces Inhibitory 16-kD CCAAT/ Enhancer Binding Protein (C/EBP)β, Repressing the HIV-1 Long Terminal Repeat in Macrophages: Pulmonary Tuberculosis Alters C/EBP Expression, Enhancing HIV-1 Replication  
The Journal of Experimental Medicine  1998;188(7):1255-1265.
We have previously observed that HIV-1 replication is suppressed in uninflamed lung and increased during tuberculosis. In vitro THP-1 cell–derived macrophages inhibited HIV-1 replication after infection with Mycobacterium tuberculosis. Suppression of HIV-1 replication was associated with inhibition of the HIV-1 long terminal repeat (LTR) and induction of ISGF-3, a type I interferon (IFN)–specific transcription factor. Repression of the HIV-1 LTR required intact CCAAT/enhancer binding protein (C/EBP) sites. THP-1 cell–derived macrophages infected with M. tuberculosis, lipopolysaccharide, or IFN-β induced the 16-kD inhibitory C/EBPβ isoform and coincidentally repressed HIV-1 LTR transcription. C/EBPβ was the predominant C/EBP family member produced in THP-1 macrophages during HIV-1 LTR repression. In vivo, alveolar macrophages from uninflamed lung strongly expressed inhibitory 16-kD C/EBPβ, but pulmonary tuberculosis abolished inhibitory C/EBPβ expression and induced a novel C/EBP DNA binding protein. Therefore, in vitro, proinflammatory stimulation produces an IFN response inhibiting viral replication by induction of a C/EBPβ transcriptional repressor. THP-1 cell–derived macrophages stimulated with type I IFN are similar to alveolar macrophages in the uninflamed lung in vivo. In contrast, the cellular immune response in active pulmonary tuberculosis disrupts this innate immunity, switching C/EBP expression and allowing high level viral replication.
PMCID: PMC2212491  PMID: 9763605
interferon β; CCAAT/enhancer binding protein β; HIV-1 long terminal repeat; tuberculosis; repression
20.  A Novel RBP-Jκ-Dependent Switch from C/EBPβ to C/EBPζ at the C/EBP Binding Site on the C-Reactive Protein Promoter1 
Regulation of basal and cytokine (IL-6 and IL-1β)-induced expression of C-reactive protein (CRP) in human hepatoma Hep3B cells occurs during transcription. A critical transcriptional regulatory element on the CRP promoter is a C/EBP binding site overlapping a NF-κB p50 binding site. In response to IL-6, C/EBPβ and p50 occupy the C/EBP-p50 site on the CRP promoter. The aim of this study was to identify the transcription factors occupying the C/EBP-p50 site in the absence of C/EBPβ. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPβ. Such treated nuclei contain only C/EBPζ (also known as CHOP10 and GADD153) because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBPζ. EMSA using this extract revealed formation of a C/EBPζ-containing complex at the C/EBP-p50 site on the CRP promoter. This complex also contained RBP-Jκ, a transcription factor known to interact with κB sites. RBP-Jκ was required for the formation of C/EBPζ-containing complex. The RBP-Jκ-dependent C/EBPζ-containing complexes were formed at the C/EBP-p50 site on the CRP promoter in the nuclei of primary human hepatocytes also. In luciferase transactivation assays, overexpressed C/EBPζ abolished both C/EBPβ-induced and (IL-6 + IL-1β)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBPζ occupies the C/EBP site, an action that requires RBP-Jκ. Under induced conditions, C/EBPζ is replaced by C/EBPβ and p50. We conclude that the switch between C/EBPβ and C/EBPζ participates in regulating CRP transcription. This process uses a novel phenomenon, that is, the incorporation of RBP-Jκ into C/EBPζ complexes solely to support the binding of C/EBPζ to the C/EBP site.
PMCID: PMC3831876  PMID: 17513780
21.  Contribution of C/EBP Proteins to Epstein-Barr Virus Lytic Gene Expression and Replication in Epithelial Cells 
Journal of Virology  2006;80(3):1098-1109.
The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPβ and had limited C/EBPα expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPα and C/EBPβ. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPβ expression and a prolonged increase in C/EBPα expression. In AGS/BX1 cells, endogenous C/EBPα and C/EBPβ proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPα and C/EBPβ proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPβ and C/EBPα expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.
PMCID: PMC1346937  PMID: 16414987
22.  Pias3-dependent SUMOylation directs rod photoreceptor development 
Neuron  2009;61(2):234-246.
The vertebrate retina is a tractable system in which to address the question of neuronal cell fate specification. Specification of retinal rod photoreceptors is determined by several different transcription factors that activate expression of rod-specific genes and repress expression of cone photoreceptor-specific genes. The mechanism by which this dual regulation occurs is unclear. We have found that Pias3, a transcriptional coregulator and E3 SUMO ligase that is selectively expressed in developing photoreceptors, promotes the differentiation of rod photoreceptors while preventing rods from adopting cone photoreceptor-like characteristics. Pias3 directly interacts with the photoreceptor-specific transcription factors Crx and Nr2e3 and is specifically targeted to the promoters of photoreceptor-specific genes. Pias3 SUMOylates Nr2e3, converting it into a potent repressor of cone-specific gene expression. Rod and cone-specific promoters are bound by hyperSUMOylated proteins in rod photoreceptors, and blocking SUMOylation in photoreceptors results in cells with morphological and molecular features of cones and an absence of rod-specific markers. Our data thus identifies Pias3-mediated SUMOylation of photoreceptor-specific transcription factors as a key mechanism of rod specification.
PMCID: PMC2701228  PMID: 19186166
23.  Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2. 
Journal of Virology  1997;71(9):6956-6966.
Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.
PMCID: PMC191980  PMID: 9261424
24.  Protein Inhibitor of Activated STAT1 Interacts with and Up-regulates Activities of the Pro-proliferative Transcription Factor Krüppel-like Factor 5* 
The Journal of biological chemistry  2006;282(7):4782-4793.
Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that regulates proliferation of various cell types, including fibroblasts, smooth muscle cells, and intestinal epithelial cells. To identify proteins that interact with KLF5, we performed a yeast two-hybrid screen of a 17-day mouse embryo cDNA library with KLF5 as bait. The screen revealed 21 preys clustered in four groups as follows: proteins mediating gene expression, metabolism, trafficking, and signaling. Among them was protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) ligase that regulates transcription factors through SUMOylation or physical interaction. Association between PIAS1 and KLF5 was verified by co-immunoprecipitation. Structural determination showed that the acidic domain of PIAS1 bound to both the amino- and carboxyl-terminal regions of KLF5 and that this interaction was inhibited by the amino terminus of PIAS1. Indirect immunofluorescence demonstrated that PIAS1 and KLF5 co-localized to the nucleus. Furthermore, the PIAS1-KLF5 complex was co-localized with the TATA-binding protein and was enriched in RNA polymerase II foci. Transient transfection of COS-7 cells by PIAS1 and KLF5 significantly increased the steady-state protein levels of each other. Luciferase reporter and chromatin immunoprecipitation assays showed that PIAS1 significantly activated the promoters of KLF5 and PIAS1 and synergistically increased the transcriptional activity of KLF5 in activating the cyclin D1 and Cdc2 promoters. Importantly, PIAS1 increased the ability of KLF5 to enhance cell proliferation in transfected cells. These results indicate that PIAS1 is a functional partner of KLF5 and enhances the ability of KLF5 to promote proliferation.
PMCID: PMC2212600  PMID: 17178721
25.  Transcriptional activation of histone H4 by C/EBPβ during the mitotic clonal expansion of 3T3-L1 adipocyte differentiation 
Molecular Biology of the Cell  2011;22(13):2165-2174.
Histone H4 is activated by C/EBPβ in mitotic clonal expansion during adipogenesis. C/EBP-binding sites are identified in histone H4 promoters, and H4 expression is suppressed when C/EBPβ is knocked down or its DNA-binding activity is inhibited by A-C/EBP. These results help in our understanding of how C/EBPβ plays important roles in the proliferation of other cells.
CCAAT enhancer binding protein β (C/EBPβ) is required for both mitotic clonal expansion (MCE) and terminal differentiation during the 3T3-L1 adipocyte differentiation program. Whereas the mechanism of C/EBPβ during terminal differentiation is well understood, the mechanism of C/EBPβ in MCE is not. We provide evidence that histone H4, the most conserved cell cycle–related histone, the change of which is strictly correlated with DNA content change during the cell cycle, is transcriptionally activated by C/EBPβ during MCE. Expression of histone H4 is increased at 16 h after induction when 3T3-L1 preadipocytes synchronously reenter S phase, which is correlated with the sequential phosphorylation and activation of C/EBPβ, and expression was partially suppressed when A-C/EBP (dominant negative for C/EBP protein) was overexpressed. One C/EBP-binding site was identified in one of the histone H4 gene promoters (hist4h4), confirmed by both electrophoretic mobility shift assay and chromatin immunoprecipitation assay. C/EBP-binding sites were also found in 9 of 11 other histone H4 promoters, which can also be transactivated by C/EBPβ. Knockdown of C/EBPβ by stealth small interfering RNA partially decreased H4 gene expression and arrested cells in G1 phase as indicated by bromodeoxyuridine incorporation and fluorescence-activated cell sorting analysis of DNA content. This study provides new insights into why C/EBPβ is required for MCE during 3T3-L1 adipocyte differentiation and why C/EBPβ plays important roles in the proliferation of other cell types.
PMCID: PMC3128520  PMID: 21562223

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