Ligand-Independent Regulation of Transforming Growth Factor β1 Expression and Cell Cycle Progression by the Aryl Hydrocarbon Receptor

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of its xenobiotic ligands and acts as an environmental checkpoint during the cell cycle. We expressed stably integrated, Tet-Off-regulated AHR variants in fibroblasts from AHR-null mice to further investigate the AHR role in cell cycle regulation. Ahr ؉/؉ fibroblasts proliferated significantly faster than Ahr ؊/؊ fibroblasts did, and exposure to a prototypical AHR ligand or deletion of the ligand-binding domain did not change their proliferation rates, indicating that the AHR function in cell cycle was ligand independent. Growth-promoting genes, such as cyclin and cyclin-dependent kinase genes, were significantly down-regulated in Ahr ؊/؊ cells, whereas growth-arresting genes, such as the transforming growth factor ␤1 (TGF-␤1) gene, extracellular matrix (ECM)-related genes, and cyclin-dependent kinase inhibitor genes, were up-regulated. Ahr ؊/؊ fibroblasts secreted significantly more TGF-␤1 into the culture medium than Ahr ؉/؉ fibroblasts did, and Ahr The Ah receptor (AHR) is a ligand-activated transcription factor that belongs to the basic region helix-loop-helix/PerArnt-Sim (PAS) family of proteins. Prototypical AHR ligands include many polycyclic and halogenated aromatic compounds, such as benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The unliganded AHR is considered to be a cytosolic protein that translocates to the nucleus upon ligand binding and dimerizes with a second basic region helixloop-helix/PAS protein, the AHR nuclear translocator (ARNT), to form a heterodimeric transcription factor (27). The AHR/ ARNT complex binds to canonical DNA recognition sequences (termed AhREs, XREs, or DREs) found in the genes coding for many phase I and phase II detoxification enzymes, such as the cytochromes P450 CYP1A1, CYP1A2, and CYP1B1, and initiates gene transcription (68).Besides its role in the regulation of drug metabolism, the AHR also has an important physiologic role in liver and vascular development, immune system function, and cell growth and differentiation (18,56). Evidence dating back more than 20 years has shown that TCDD, the prototypical AHR ligand, can inhibit cell proliferation and that this effect is AHR dependent. Confluent mouse epithelial cell cultures exhibited a diminished capacity for DNA replication in the presence of as little as 10 pM TCDD (22). TCDD also inhibited DNA synthesis in rat primary hepatocytes (30) and in rat and mouse liver following partial hepatectomy (3, 46). Inhibition of G 1 phase entry by TCDD was first reported for rat 5L hepatoma cells and was found to involve increases in the levels of the CDK2 inhibitor p27KIP1 (37). The activated Ah receptor has also been shown to form protein-protein complexes with the retinoblastoma protein RB (16,21,51) to slow down entry into S phase (51) and to silence the expression of S-phase-specific E2F target genes (42).These observations indicate that the activated AHR slows down cell proliferation, suppor...

Aryl hydrocarbon receptor inhibition downregulates the TGF-β/Smad pathway in human glioblastoma cells

“…In non-malignant cells of AhR null mice, the LTBP-1/TGF-b pathway by AhR is regulated by diverse mechanisms. These include post-translational regulatory mechanisms, as described for primary hepatocytes (Zaher et al, 1998), regulation of mRNA levels (Chang et al, 2007) and cytokine activation in the extracellular matrix of embryonic fibroblasts (Gomez-Duran et al, 2006). The fact that LTBP-1 may activate TGF-b in the absence of altering total TGF-b levels in LN-308 cells (Tritschler et al, 2009) adds complexity to this issue.…”

“…The wide range of toxic effects induced by these compounds, including birth defects, impaired reproductive capacity and altered immune responses, have encouraged research on the interaction of AhR with sex steroid receptors and the immune system (Ohtake et al, 2008;Quintana et al, 2008;Veldhoen et al, 2008). AhR regulates cellular differentiation during development (Huang et al, 2004;Lahvis et al, 2000) and may promote cell cycle progression in the absence of exogenous ligand (Chang et al, 2007;Marlowe and Puga, 2005). There is also an emerging evidence for a prominent role of AhR in tumorigenesis.…”

“…Depleting TGF-b by RNA interference or antagonizing the TGF-b receptor I kinase, which mediates Smad2 signaling, both inhibit growth and invasiveness and enhance the immunogenicity of murine and human glioma cells in vitro and in vivo (Friese et al, 2004;Uhl et al, 2004). Importantly, AhR and TGF-b signaling pathways are known to cross-regulate each other in a cell type-specific manner (Chang et al, 2007;Wolff et al, 2001). Moreover, latent TGF-b-binding protein-1 (LTBP-1), a protein that has been characterized as crucial in the process of TGF-b activation in gliomas (Tritschler et al, 2009), is a transcriptional target of AhR (Corchero et al, 2004;Santiago-Josefat et al, 2004).…”

The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

“…In addition to its role in the regulation of drug metabolism, evidence dating back more than 20 years shows that the AHR plays a central role in the regulation of cell proliferation. AHR activation by ligand or by deletion of the ligandbinding domain alters several cell cycle and signaling pathways, including those required for normal cell cycle regulation (Puga et al, 2002;Chang et al, 2007). Most evidence shows that AHR activation delays cell cycle progression and G 1 to S phase transition, although this effect seems to be cellular context specific, because in rat oval progenitor cells the AHR promotes rather than delays cell cycle progression (Weiss et al, 2008).…”

“…Most evidence shows that AHR activation delays cell cycle progression and G 1 to S phase transition, although this effect seems to be cellular context specific, because in rat oval progenitor cells the AHR promotes rather than delays cell cycle progression (Weiss et al, 2008). A canonical RB-binding cyclin D-like motif in the AHR protein sequence mediates a direct interaction between AHR and RB, and experimental work focused on the characterization of this interaction as the mediator of AHR-dependent cell cycle delay (Ge and Elferink, 1998;Puga et al, 2000;Marlowe et al, 2004;Huang and Elferink, 2005) has shown that the activated AHR cooperates with RB in its ability to repress E2F-dependent transcription and delay cell cycle progression Strobeck et al, 2000).Paradoxically, embryo fibroblasts from Ahr gene-knockout mice also show a relative delay in cell cycle progression, which has been primarily associated with posttranscriptional stabilization of Tgfb1 mRNA (Chang et al, 2007). In these cells, several E2F transcriptional targets with AHR and E2F binding motifs in their promoters, conserved in both the human and mouse sequences (Supplemental Tables S1 and S2), were also found to be AHR targets, being repressed by both E2f1 and Ahr ablation (Supplemental Figure S1).…”