S-Z

N-R

H-M

D-G

BC

A

Apotosis

6/3/2007/-19

Invest Ophthalmol Vis Sci. 2007 Jun;48(6):2634-43. Links

Synergism of TNF and IL-1 in the Induction of Matrix Metalloproteinase-3 in Trabecular Meshwork.

Kelley MJ, Rose AY, Song K, Chen Y, Bradley JM, Rookhuizen D, Acott TS.

Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.

PURPOSE: TNF and IL-1 increase matrix metalloproteinase-3 (MMP-3) expression in the trabecular meshwork (TM). TNF-alpha, in combination with IL-1alpha or IL-1beta, produces highly synergistic MMP-3 increases. Possible mechanisms for this synergism in TM cells were investigated. METHODS: Porcine and human TM cells were treated with TNF-alpha, IL-1alpha, IL-1beta and their combinations. Western immunoblots were used to evaluate MMP-3, MMP-9, MMP-12, TNF-alpha, IL-1alpha, IL-1beta, IL-6, TNF receptor I (RI), IL-1 RI, and IL-1 RII levels and the phosphorylation of Erk, JNK, and p38 MAP kinases. Dose-response effects for TNF-alpha, IL-1alpha and IL-1beta on MMP-3 were evaluated. Microarray and quantitative RT-PCR were used to determine mRNA levels. MMP-3 transcription rate was assessed by transfecting TM cells with an MMP-3 promoter/reporter construct. Combined cytokine effects on outflow facility were appraised in perfused anterior segment organ culture. RESULTS: TNF-alpha, IL-1alpha, and IL-1beta each individually increased MMP-3 levels, whereas TNF-alpha in combination with IL-1alpha or IL-1beta produced highly synergistic increases. MMP-9 and MMP-12 levels were also elevated, but only MMP-12 showed synergism. IL-1alpha, IL-1beta, and IL-6, but not TNF-alpha mRNA or protein level, were elevated by these cytokines. Maximum MMP-3 production for individual cytokines, even at high doses, was far less than with dual cytokine doses. Erk 1 and 2, JNK 1 and 2, and p38 alpha and beta phosphorylation increased, but not synergistically. However, phosphorylation of novel isoforms of JNK and p38 delta and gamma did show synergism. MMP-3 mRNA levels and transcription rates also demonstrated synergism. TNF-alpha significantly increased IL-1 RI levels. Synergism in outflow facility was observed with TNF-alpha and IL-1alpha. CONCLUSIONS: TNF-alpha, in combination with IL-1alpha or IL-1beta, produced intense synergistic increases in MMP-3 and MMP-12 but not in MMP-9. Induction of IL-1 RI by TNF-alpha partially explains the synergism. Responses of novel JNK and p38 MAP kinase delta and gamma isoforms also partially account for the synergism. Understanding this strong synergistic effect may provide useful insight into optimizing therapeutic regulation of intraocular pressure in glaucoma.

PMID: 17525194 [PubMed - in process]

Exp Cell Res. 2007 Apr 1;313(6):1069-79. Epub 2007 Jan 8. Links

Regulation of the human SOX9 promoter by Sp1 and CREB.

Piera-Velazquez S, Hawkins DF, Whitecavage MK, Colter DC, Stokes DG, Jimenez SA.

Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Jefferson Medical College, 233 S. 10th Street, Room 509 BLSB, Philadelphia, PA 19107-5541, USA.

The transcription factor SOX9 is essential for multiple steps during skeletal development, including mesenchymal cell chondrogenesis and endochondral bone formation. We recently reported that the human SOX9 proximal promoter region is regulated by the CCAAT-binding factor through two CCAAT boxes located within 100 bp of the transcriptional start site. Here we report that the human SOX9 proximal promoter is also regulated by the cyclic-AMP response element binding protein (CREB) and Sp1. We show by DNaseI protection and EMSA analysis that CREB and Sp1 interact with specific sites within the SOX9 proximal promoter region. By transient transfection analysis we also demonstrate that mutations of the CREB and Sp1 binding sites result in a profound reduction of SOX9 promoter activity. Chromatin immunoprecipitation (ChIP) assay demonstrated that both Sp1 and CREB interact with the SOX9 promoter in vivo. Finally, we demonstrate that IL-1beta treatment of chondrocytes isolated from human normal and osteoarthritic (OA) cartilage down-regulates SOX9 promoter activity, an effect accompanied by a reduction of Sp1 binding to the SOX9 proximal promoter.

PMID: 17289023 [PubMed - indexed for MEDLINE]

Biochemistry. 2006 Aug 8;45(31):9615-23. Links

Cyclic AMP response element-binding protein (CREB) and CAAT/enhancer-binding protein beta (C/EBPbeta) bind chimeric DNA sites with high affinity.

Flammer JR, Popova KN, Pflum MK.

Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.

Basic region leucine zipper (bZIP) proteins are transcription factors that interact selectively with duplex DNA to regulate gene expression. Specifically, the cAMP response element-binding protein (CREB) interacts with the cAMP response element (CRE) DNA site with high affinity, while it binds the CAAT/enhancer-binding protein (CEBP) DNA site with low affinity. Despite the selectivity of CREB for the CRE site, CREB-dependent transcription is observed via chimeric DNA sites with similarities to both CRE and CEBP sites. Because CRE/CEBP and CEBP/CRE chimeric DNA are relevant for transcription regulation but have not been rigorously characterized, quantitative electrophoretic mobility shift assays were used to characterize the binding affinity and specificity of CREB to the sites. In addition to CREB, C/EBPbeta was tested because chimeric DNA was shown to stabilize CREB-C/EBPbeta heterodimerization. Despite previous work, no CREB-C/EBPbeta heterodimer was observed in the presence of chimeric DNA; only CREB and C/EBPbeta homodimers were seen. The CREB homodimer bound to the chimeric sites with high affinity, demonstrating that the presence of one CRE half-site is sufficient for high-affinity interaction. A comparison of CREB and C/EBPbeta homodimers indicated that they bind the chimeric sites with similar, high affinity. Whereas the CRE and CEBP sites preferentially interact with CREB and C/EBPbeta, respectively, the chimeric sites bind CREB and C/EBPbeta competitively. Because DNA binding correlates with transcription regulation, the results suggest that gene expression from chimeric sites can be altered by small changes in relative bZIP concentrations or bZIP accessory factors.

PMID: 16878996 [PubMed - indexed for MEDLINE

J Virol. 2007 Feb;81(4):1543-53. Epub 2006 Dec 6. Links

Human T-cell leukemia virus type 1 (HTLV-1) bZIP protein interacts with the cellular transcription factor CREB to inhibit HTLV-1 transcription.

Lemasson I, Lewis MR, Polakowski N, Hivin P, Cavanagh MH, Thébault S, Barbeau B, Nyborg JK, Mesnard JM.

East Carolina University, Department of Microbiology and Immunology, Brody School of Medicine, 600 Moye Blvd., Greenville, NC 27834, USA. lemassoni@ecu.edu

The complex human T-cell leukemia virus type 1 (HTLV-1) retrovirus encodes several proteins that are unique to the virus within its 3'-end region. Among them, the viral transactivator Tax and posttranscriptional regulator Rex are well characterized, and both positively regulate HTLV-1 viral expression. Less is known about the other regulatory proteins encoded in this region of the provirus, including the recently discovered HBZ protein. HBZ has been shown to negatively regulate basal and Tax-dependent HTLV-1 transcription through its ability to interact with specific basic-leucine zipper (bZIP) proteins. In the present study, we found that HBZ reduces HTLV-1 transcription and virion production. We then characterized the interaction between HBZ and the cellular transcription factor CREB. CREB plays a critical role in Tax-mediated HTLV-1 transcription by forming a complex with Tax that binds to viral cyclic AMP-response elements (CREs) located within the viral promoter. We found that HBZ and CREB interact in vivo and directly in vitro, and this interaction occurs through the bZIP domain of each protein. We also found that CREM-Ia and ATF-1, which share significant homology in their bZIP domains with the bZIP domain of CREB, interact with HBZ-bZIP. The interaction between CREB and HBZ prevents CREB binding to the viral CRE elements in vitro and in vivo, suggesting that the reduction in HTLV-1 transcription by HBZ is partly due to the loss of CREB at the promoter. We also found that HBZ displaces CREB from a cellular CRE, suggesting that HBZ may deregulate CREB-dependent cellular gene expression.

PMID: 17151132 [PubMed - indexed for MEDLINE]

: J Neurochem. 2006 Aug;98(3):773-81. Links

GATA-3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with CREB.

Hong SJ, Huh Y, Chae H, Hong S, Lardaro T, Kim KS.

Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02478, USA.

The zinc finger transcription factor GATA-3 is a master regulator of type 2 T-helper cell development. Interestingly, in GATA-3-/- mice, noradrenaline (NA) deficiency is a proximal cause of embryonic lethality. However, neither the role of GATA-3 nor its target gene(s) in the nervous system were known. Here, we report that forced expression of GATA-3 resulted in an increased number of tyrosine hydroxylase (TH) expressing neurons in primary neural crest stem cell (NCSC) culture. We also found that GATA-3 transactivates the promoter function of TH via specific upstream sequences, a domain of the TH promoter residing at -61 to -39 bp. Surprisingly, this domain does not contain GATA-3 binding sites but possesses a binding motif, a cAMP response element (CRE), for the transcription factor, CREB. In addition, we found that site-directed mutation of this CRE almost completely abolished transactivation of the TH promoter by GATA-3. Furthermore, protein-protein interaction assays showed that GATA-3 is able to physically interact with CREB in vitro as well as in vivo. Based on these results, we propose that GATA-3 may regulate TH gene transcription via a novel and distinct protein-protein interaction, and directly contributes to NA phenotype specification.

PMID: 16893419 [PubMed - indexed for MEDLINE]

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