Dagmar Faust

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.

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.

Involvement of the transcription factor FoxM1 in contact inhibition.

Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Although it is generally accepted that contact inhibition plays a pivotal role in maintaining tissue homeostasis, the molecular mechanisms of contact inhibition are still not fully understood. FoxM1 is known as a proliferation-associated transcription factor and is upregulated in many cancer types. Vice versa, anti-proliferative signals, such as TGF-β and differentiation signals decrease FoxM1 expression. Here we investigated the role of FoxM1 in contact inhibition in fibroblasts. We show that protein expression of FoxM1 is severely and rapidly downregulated upon contact inhibition, probably by inhibition of ERK activity, which then leads to decreased expression of cyclin A and polo-like kinase 1. Vice versa, ectopic expression of FoxM1 prevents the decrease in cyclin A and polo-like kinase 1 and causes a two-fold increase in saturation density indicating loss of contact inhibition. Hence, we show that downregulation of FoxM1 is required for contact inhibition by regulating expression of cyclin A and polo-like kinase 1.

Regulation of ERK1/2 activity upon contact inhibition in fibroblasts

Contact inhibition is a crucial mechanism regulating proliferation in vitro and in vivo. Despite its generally accepted importance for maintaining tissue homeostasis knowledge about the underlying molecular mechanisms of contact inhibition is still scarce. Since the MAPK ERK1/2 plays a pivotal role in the control of proliferation, we investigated regulation of ERK1/2 phosphorylation which is downregulated in confluent NIH3T3 cultures. We found a decrease in upstream signaling including phosphorylation of the growth factor receptor adaptor protein ShcA and the MAPK kinase MEK1/2 in confluent compared to exponentially growing cultures whereas involvement of ERK1/2 phosphatases in ERK1/2 inactivation is unlikely. Treatment of confluent, serum-deprived cultures with PDGF-B resulted in similar phosphorylation of ERK1/2 and induction of DNA-synthesis as detected in sparse, serum-deprived cultures. In contrast, ERK1/2 phosphorylation and DNA-synthesis could not be stimulated in confluent, serum-deprived cultures exposed to EGF. Our data indicate that PDGFR- and EGFR signaling are differentially inhibited in confluent cultures of NIH3T3 cells.