Recruitment of CREB1 and Histone Deacetylase 2 (HDAC2) to the Mouse Ltbp-1 Promoter Regulates its Constitutive Expression in a Dioxin Receptor-dependent Manner

Abstract

Latent TGFbeta-binding protein 1 (LTBP-1) is a key regulator of TGFbeta targeting and activation in the extracellular matrix. LTBP-1 is recognized as a major docking molecule to localize, and possibly to activate, TGFbeta in the extracellular matrix. Despite this relevant function, the molecular mechanisms regulating Ltbp-1 transcription remain largely unknown. Previous results from our laboratory revealed that mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR) had increased Ltbp-1 mRNA expression and elevated TGFbeta activity, suggesting that AhR repressed Ltbp-1 transcription. Here, we have cloned the mouse Ltbp-1 gene promoter and analysed its mechanism of transcriptional repression by AhR. Reporter gene assays, AhR over-expression and site-directed mutagenesis showed that basal Ltbp-1 transcription is AhR-dependent. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi) revealed that AhR regulates Ltbp-1 transcription by a mechanism involving recruitment of co-activators such as CREB1 and co-repressors such as HDAC2 to the Ltbp-1 promoter. In AhR-expressing (AhR+/+) MEF cells, the recruitment of HDAC1, 2 and 4 correlated with decreased K8H4 acetylation and impaired binding of pCREB(Ser133) to the Ltbp-1 promoter, likely maintaining a constitutive repressed state. AhR-/- MEF cells had the opposite pattern of HDACs and pCREB1(Ser133) binding to Ltbp-1 promoter, and therefore, over-expressed Ltbp-1 mRNA. In agreement, siRNA for HDAC2 increased Ltbp-1 expression and K8H4 acetylation in AhR+/+ but not in AhR-/- MEF cells. We suggest that HDAC2 binding keeps Ltbp-1 promoter repressed in AhR+/+ MEF cells, whereas in AhR-null MEF cells the absence of HDAC2 and the binding of pCREB(Ser133) allow Ltbp-1 transcription. Thus, epigenetics can contribute to constitutive Ltbp-1 repression by a mechanism requiring AhR activity.

Chronic TCDD exposure results in the dysregulation of gene expression in splenic B-lymphocytes and in the impairments in T-cell and B-cell differentiation in mouse model

“…The promoter analysis done with Mapper database (Marinescu et al, 2005b) showed that all but one of the most highly dysregulated genes had CREM and/or CREB1 binding sites in their promoter regions. Dysregulated pattern of gene expression associated with chronic TCDD exposure is consistent with existing evidence of the AhR involvement in chromatin remodeling (Gomez-Duran et al, 2008;Oesch-Bartlomowicz and Oesch, 2009a;Tian, 2009), potentially in concert with CREB1 in the context of cAMP/PKA signaling (Oesch-Bartlomowicz and Oesch, 2009b;Saeed et al, 2014). Involvement of cAMP/PKA signaling in the AhR signaling pathway has been demonstrated in a number of contexts including central nervous system, reproductive toxicology and wasting syndrome (Landers and Bunce, 1991;Vogel et al, 1998).…”

Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1

“…12 Noteworthy, a nuclear-localized constitutively active AHR that blocks gene expression, probably by inducing local epigenetic modifications, has also been previously described. 43,44 Whether the AHR directly binds the p27 KIP1 promoter to repress transcription is not known up-to-now. Alternatively, the AHR may affect p27 KIP1 expression indirectly by modulating other transcription factors, such as HES-1 or c-myc, which are known to inhibit p27 KIP1 expression.…”

Loss of dioxin-receptor expression accelerates wound healing in vivo by a mechanism involving TGFβ

“…It is well known that many cytokines and growth factors regulate the efficiency of wound healing Scheid et al, 2000;Singer and Clark, 1999). We focused our attention on TGFβ for several reasons: (1) mouse models with targeted inactivation of the genes encoding β3 integrin (Reynolds et al, 2005) and Dpr2 (Meng et al, 2008) showed accelerated re-epithelialization and an enhanced response to TGFβ, whereas decreased TGFβ activity correlated with impaired wound healing in PKCε-null mice (Leask et al, 2008) and (2) we have extensively shown that absence of AhR expression results in increased TGFβ activity in certain cell types such as fibroblasts and hepatocytes (Corchero et al, 2004;Elizondo et al, 2000;Gomez-Duran et al, 2008a;Gomez-Duran et al, 2006;Santiago-Josefat et al, 2004). Fibroblasts produce many components of the ECM and are also a relevant source of TGFβ.…”

“…In addition, AhR has a role in TGFβ activation because AhR-null mice produce increased levels of active cytokine (Corchero et al, 2004;Elizondo et al, 2000;Gomez-Duran et al, 2008a;Gomez-Duran et al, 2008b;Gomez-Duran et al, 2006;Santiago-Josefat et al, 2004). Based on this information, we performed experiments aimed to analyze the contribution of TGFβ signaling in the AhR-dependent wound-healing phenotype.…”

“…Among the different cell functions requiring AhR, the control of TGFβ activation appears particularly relevant. In cell culture systems, AhR activity has been functionally related to increased secretion and activation of TGFβ in primary hepatocytes (Zaher et al, 1998) and mouse embryo fibroblasts (Elizondo et al, 2000;Gomez-Duran et al, 2008a;Gomez-Duran et al, 2006;Santiago-Josefat et al, 2004) and, consistently, with diminished cell proliferation and increased apoptosis. In mice, knockdown of AhR expression results in fibrotic lesions in the liver (Corchero et al, 2004;FernandezSalguero et al, 1995;Peterson et al, 2000), heart and skin (Fernandez-Salguero et al, 1997), which, at least for the hepatic tissue, colocalized to the portal areas with increased levels of TGFβ (Corchero et al, 2004).…”