Cornelia Dietrich

The aryl hydrocarbon receptor (AhR) in the regulation of cell–cell contact and tumor growth

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor, which is activated by a large group of environmental pollutants including polycyclic aromatic hydrocarbons, dioxins and planar polychlorinated biphenyls. Ligand binding leads to dimerization of the AhR with aryl hydrocarbon receptor nuclear translocator and transcriptional activation of several xenobiotic phase I and phase II metabolizing enzymes, such as cytochrome P4501A1 and glutathione-S-transferase, respectively. Since phase I enzymes convert inert carcinogens to active genotoxins, the AhR plays a key role in tumor initiation. Besides this classical route, the AhR mediates tumor promotion and recent evidence suggests that the AhR also plays a role in tumor progression. To date, no mechanistic link could be established between the canonical pathway involving xenobiotic metabolism and AhR-dependent tumor promotion and progression. A hallmark of tumor promotion is unbalanced proliferation, whereas tumor progression is characterized by dedifferentiation, increased motility and metastasis of tumor cells. Tumor progression and presumably also tumor promotion are triggered by loss of cell-cell contact. Cell-cell contact is known to be a critical regulator of proliferation, differentiation and cell motility in vitro and in vivo. Increasing evidence suggests that activation of the AhR may lead to deregulation of cell-cell contact, thereby inducing unbalanced proliferation, dedifferentiation and enhanced motility. In line with this is the finding of increased AhR expression and malignancy in some animal and human cancers. Here, we summarize our current knowledge on non-canonical AhR-driven pathways being involved in deregulation of cell-cell contact and discuss the data with respect to tumor initiation, promotion and progression.

TCDD induces c-jun expression via a novel Ah (dioxin) receptor-mediated p38-MAPK-dependent pathway

The aryl hydrocarbon receptor (AhR) has a fundamental role during postnatal liver development and is essential for mediating dioxin toxicity. However, the genetic programs mediating, both, the toxic and physiological effects downstream of the transcription factor AhR are in major parts unknown. We have identified the proto-oncogene c-jun as a novel target gene of AhR. Induction of c-jun depends on activation of p38–mitogen-activated protein kinase (MAPK) by an AhR-dependent mechanism. None of the kinases that are known to phosphorylate p38-MAPK is activated by AhR. Neither the dephosphorylation rate of p38–MAPK is reduced. Furthermore, increased p38–MAPK phosphorylation in response to dioxins does not require ongoing transcription. These findings establish activating ‘cross-talk’ with MAPK signaling as a novel principle of AhR action, which is apparently independent of the AhRs function as a DNA-binding transcriptional activator.

Differences in the mechanisms of growth control in contact-inhibited and serum-deprived human fibroblasts

In the present work we studied mechanisms of growth control in contact-inhibited and serum-deprived human diploid fibroblasts. The observation that the effects on [3H]thymidine incorporation and reduction of retinoblastoma gene product-phosphorylation were additive when contact-inhibition and serum-deprivation were combined led us to the conclusion that the underlying mechanisms might be different. Both contact-inhibition and serum-deprivation led to a strong decrease of cdk4-kinase-activity and cdk2-phosphorylation at Thr 160, while the total amounts of cdk4 and cdk2 remained constant. In contact-inhibited cells, we revealed a strong protein accumulation of the cdk2-inhibitor p27 and a slight, but significant increase of the cdk4-inhibitor p16. In serum-deprived cells, the protein levels in p27 and p16 remained low. In contrast, we detected a rapid decrease of cyclin D1 and cyclin D3 which did not occur in contact-inhibited cells. These results indicate that serum-deprivation and contact-inhibition have different mechanisms although they affect the same pathway cyclin D – cdk4, pRB, cyclin E – cdk2.

p16INK4 mediates contact-inhibition of growth.

Growth of non-transformed cells in vitro is regulated by density-dependent mechanisms via cell-cell contacts, leading to arrest in late G1-phase at confluency (contact-inhibition of growth). In the present study it is shown that this results from p16INK4-mediated dissociation of the complex cdk4-cyclin D1, which is responsible for the inactivation of the gate keeper of G1-S transition, the retinoblastoma protein pRb. As a consequence of the inactivation of cdk4, downstream the activation of cdk2 and hyperphosphorylation and thus inactivation of pRb was impaired. Direct evidence for the central role of p16INK4 in growth control comes from the observation that a competitive inhibitor of p16INK4 repressed contact inhibition of growth. These findings provide an explanation for the high incidence of mutation or loss of INK4 in human tumours.

p38α MAPK is required for contact inhibition

Proliferation of nontransformed cells is regulated by cell–cell contacts, which are referred to as contact-inhibition. Despite its generally accepted importance for cell cycle control, knowledge about the intracellular signalling pathways involved in contact inhibition is scarce. In the present work we show that p38α mitogen-activated protein kinase (MAPK) is involved in the growth-inhibitory signalling cascade of contact inhibition in fibroblasts. p38α activity is increased in confluent cultures of human fibroblasts compared to proliferating cultures. Time course studies show a sustained activation of p38α in response to cell–cell contacts in contrast to a transient activation after serum stimulation. The induction of contact inhibition by addition of glutaraldehyde-fixed cells is impaired by pharmacological inhibition of p38 as well as in p38α−/− fibroblasts. Further evidence for a central role of p38α in contact inhibition comes from the observation that p38α−/− fibroblasts show a higher saturation density compared to wild-type (wt) fibroblasts, which is reversed by reconstituted expression of p38α. In agreement with a defect in contact inhibition, p27Kip1 accumulation is impaired in p38α−/− fibroblasts compared to wt fibroblasts. Hence, our work shows a new role for p38α in contact inhibition and provides a mechanistic basis for the recently proposed tumour suppressive function of this MAPK pathway.

Involvement of protein kinase Cδ in contact-dependent inhibition of growth in human and murine fibroblasts

There is evidence that protein kinase C δ (PKCδ) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell–cell contacts we studied whether PKCδ is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell–cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms α, δ, ε, and μ after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 μM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCδ-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCδ in contact-inhibition. In accordance, the PKCδ specific inhibitor Rottlerin (1 μM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCδ to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCδ in response to cell–cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCδ-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCδ induced a transformed phenotype. We conclude that PKCδ is involved in contact-dependent inhibition of growth.

Differential p38-dependent signalling in response to cellular stress and mitogenic stimulation in fibroblasts

Abstractp38 MAP kinase is known to be activated by cellular stress finally leading to cell cycle arrest or apoptosis. Furthermore, a tumour suppressor role of p38 MAPK has been proposed. In contrast, a requirement of p38 for proliferation has also been described. To clarify this paradox, we investigated stress- and mitogen-induced p38 signalling in the same cell type using fibroblasts. We demonstrate that - in the same cell line - p38 is activated by mitogens or cellular stress, but p38-dependent signalling is different. Exposure to cellular stress, such as anisomycin, leads to a strong and persistent p38 activation independent of GTPases. As a result, MK2 and downstream the transcription factor CREB are phosphorylated. In contrast, mitogenic stimulation results in a weaker and transient p38 activation, which upstream involves small GTPases and is required for cyclin D1 induction. Consequently, the retinoblastoma protein is phosphorylated and allows G1/S transition. Our data suggest a dual role of p38 and indicate that the level and/or duration of p38 activation determines the cellular response, i.e either proliferation or cell cycle arrest.

TCDD‐dependent downregulation of γ‐catenin in rat liver epithelial cells (WB‐F344)

TCDD (2,3,7,8‐tetrachlorodibenzo‐p‐dioxin) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AHR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact‐inhibition is a characteristic hallmark in tumorigenesis. In WB‐F344 cells, TCDD induces a release from contact‐inhibition manifested by a 2‐ to 3‐fold increase in DNA‐synthesis and the emergence of foci when TCDD (1 nM) is given to confluent cells. We focussed our interest on potential cell membrane proteins mediating contact‐inhibition in WB‐F344 cells, namely E‐cadherin, α,‐ β,‐ and γ‐catenin (plakoglobin). Using indirect immunofluorescence, E‐cadherin, α‐, β‐ and γ‐catenin were detected at cell adhesion sites in untreated, confluent cells. After TCDD‐exposure, γ‐catenin was exclusively localized in the cytoplasm whereas localization of E‐cadherin, α‐ and β‐catenin remained unaffected. Cytoplasmic γ‐catenin could be extracted by Triton X‐100 treatment, demonstrating that γ‐catenin was no longer bound to the actin cytoskeleton. Western blot analysis showed downregulation of γ‐catenin protein levels. This effect was not blocked by pre‐incubation with the selective proteasome inhibitor MG‐132, indicating that proteolytical degradation of γ‐catenin by the proteasome system was not increased by TCDD. Because mRNA‐levels of γ‐catenin were markedly diminished after TCDD‐exposure, we conclude that transcriptional downregulation or destabilization of the mRNA contributes to the decrease in γ‐catenin protein levels in response to TCDD. Because γ‐catenin is considered to be a tumor suppressor, our findings might give more insight into the tumor promoting actions of TCDD.

The Transcriptional Programme of Contact-Inhibition

Proliferation of non‐transformed cells is regulated by cell–cell contacts, which are referred to as contact‐inhibition. Vice versa, transformed cells are characterised by a loss of contact‐inhibition. Despite its generally accepted importance for cell‐cycle control, little is known about the intracellular signalling pathways involved in contact‐inhibition. Unravelling the molecular mechanisms of contact‐inhibition and its loss during tumourigenesis will be an important step towards the identification of novel target genes in tumour diagnosis and treatment. To better understand the underlying molecular mechanisms we identified the transcriptional programme of contact‐inhibition in NIH3T3 fibroblast using high‐density microarrays. Setting the cut off: ≥1.5‐fold, P ≤ 0.05, 853 genes and 73 cDNA sequences were differentially expressed in confluent compared to exponentially growing cultures. Importing these data into GenMAPP software revealed a comprehensive list of cell‐cycle regulatory genes mediating G0/G1 arrest, which was confirmed by RT‐PCR and Western blot. In a narrow analysis (cut off: ≥2‐fold, P ≤ 0.002), we found 110 transcripts to be differentially expressed representing 107 genes and 3 cDNA sequences involved, for example, in proliferation, signal transduction, transcriptional regulation, cell adhesion and communication. Interestingly, the majority of genes was upregulated indicating that contact‐inhibition is not a passive state, but actively induced. Furthermore, we confirmed differential expression of eight genes by semi‐quantitative RT‐PCR and identified the potential tumour suppressor transforming growth factor‐β (TGF‐β)‐1‐induced clone 22 (TSC‐22; tgfb1i4) as a novel protein to be induced in contact‐inhibited cells. J. Cell. Biochem. 110: 1234–1243, 2010. Published 2010 Wiley‐Liss, Inc.

Aryl hydrocarbon receptor-dependent cell cycle arrest in isolated mouse oval cells

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which mediates toxic responses to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Besides its well known role in induction of xenobiotic metabolizing enzymes, for instance CYP1A1, the AhR is also involved in tumor promotion in rodents although the underlying mechanisms are still poorly understood. Additionally, the AhR is known to regulate cellular proliferation, which might result in either inhibition or stimulation of proliferation depending on the cell-type studied. Potential targets in hepatocarcinogenesis are liver oval (stem/progenitor) cells. In the present work we analyzed the effect of TCDD on proliferation in oval cells derived from mouse liver. We show that TCDD inhibits proliferation in these cells. In line, the amount of G0/G1 cells increases in response to TCDD. We further show that the expression of cyclin D1 and cyclin A is decreased, while p27 is increased. As a result, the retinoblastoma protein is not phosphorylated thereby inducing G0/G1 arrest. Pharmacological inhibition of the AhR and knock-down of AhR expression by RNA interference decreased the inhibitory effect on cell cycle and protein expression, indicating that the AhR at least partially mediates cell cycle arrest.