Aryl hydrocarbon receptor-dependent induction of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells from mouse

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR-/-) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC(50) of 127±21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and α-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS.     

Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling

Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.     

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.     

A remarkable new target gene for the dioxin receptor

The dioxin receptor (AhR) is possibly the best characterized xenobiotic receptor because of its essential role in mediating the harmful effects of highly toxic environmental pollutants. Despite the fact that AhR-dependent toxicity is a major environmental concern, compelling evidence is been lately produced unveiling novel and remarkable endogenous functions of AhR in cell physiology and tissue homeostasis. Adding to its role in cell proliferation and differentiation, AhR is also involved in the control of cell adhesion and migration, both highly relevant tasks in development and in disease states such as cancer. Interestingly, the effect of AhR on cell migration is cell-type specific because it can sustain or slow down cell motility. Here, I will comment on our recent report showing that AhR is a positive regulator of fibroblast cells migration. Besides characterizing the phenotype of such mesenchymal cells, the most important single finding of our study is that AhR uses the cytoskeleton regulator and oncogen Vav3 to signal through small Rho GTPases, ultimately leading to the physiological control of cell adhesion and migration. These data reveal that AhR activity is required to maintain signaling pathways governing normal cell function and open the question of whether AhR plays a role in cell migration during development and in pathological conditions such as tumor metastasis.     

Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone- dependent steroidogenesis

We have recently succeeded in immortalizing rat granulosa cells by co- transfection with SV-40 DNA and the Ha-ras oncogene. These cells lost their response to gonadotropins, but expressed the cytochrome P450scc mitochondrial system enzymes and produced progesterone and 20 alpha- hydroxy-4-pregnan-3-one (20 alpha-OH-P) upon cAMP stimulation (Suh, B. S., and A. Amsterdam. 1990. Endocrinology. 127:2489-2500; Hanukoglu, I., B. S. Suh, S. Himmelhoch, and A. Amsterdam. 1990. J. Cell Biol. 111:1973-1981). In an attempt to restore the steroidogenic response to gonadotropins in immortalized cells, lutropin/choriogonadotropin (LH/CG- R) receptor expression plasmid was prepared by introducing the complete coding region of LH receptor cDNA (McFarland, K. C., R. Sprengel, H. S. Phillips, M. Kohler, N. Rosemblit, K. Nikolics, D. L. Segaloff, and P. H. Seeburg. 1989. Science (Wash. DC). 245:494-499) into a SV-40 early promoter based eucaryotic expression vector. Granulosa cells from preovulatory follicles were transfected with this LH receptor expression plasmid, together with SV-40 DNA and the Ha-ras oncogene. Cell lines obtained after this triple transfection accumulated cAMP in a dose-dependent manner in response to hCG. Moreover, they produced progesterone and 20 alpha-OH-P upon hCG stimulation with an ED50 of 125 pM and 75 pM, respectively, which is within the physiological range. Concomitantly with hCG induced differentiation, inhibition of cell proliferation was evident following stimulation with hormone concentrations as low as 40 pM. The number of hCG receptor sites per cell after numerous passages and several freezing and thawing cycles was 1.9 x 10(4), they showed a Kd of 180 pM. Stimulation with hCG induced pronounced morphological and biochemical changes in these cells including formation of mitochondrial located adrenodoxin, a marker enzyme for enhanced steroidogenesis. These findings make possible the expression in immortalized granulosa cells, of selectively mutated receptor molecules which preserve their steroidogenic potential, thereby opening the way to analysis of structure-function relationships of the receptor molecule.     

SV-40 transformation: effect on GM2 ganglioside in cultured cell lines.

In previous work, we observed the presence of substantially elevated levels of GM2 after Simian Virus 40 (SV-40) transformation of human fetal brain cells. This elevated level of GM2 contrasted with the reports of many other investigators who had often observed decreased levels of GM2 and a simplification of ganglioside pattern in various non-neural rodent cell lines. In order to determine if the increase in GM2 in the transformed human brain cells would also be found in transformed rodent brain cells, we analyzed ganglioside changes after transformation in mouse brain cell lines and observed the increase in GM3 and low levels or lack of GM2 usually noted in rodent SV-40 transformed cell lines. In addition, we analyzed changes after SV-40 transformation in three human fibroblast lines and found that all three lines contained substantially elevated levels of GM2 after SV-40 transformation. As a result of this study, our earlier work on SV-40 transformed human brain cells, and occasional other reports of high levels of GM2 in human SV-40 transformed cell lines, elevated levels of GM2 may be considered a marker for SV-40 transformed human cells of both fibroblastic and neural origin.     

LTBP-1 blockade in dioxin receptor-null mouse embryo fibroblasts decreases TGF-beta activity: Role of extracellular proteases plasmin and elastase

In mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR), high levels of latent transforming growth factor-beta (TGF-beta)-binding protein-1 (LTBP-1) correlated with increased TGF-beta1 activity, an observation suggesting that LTBP-1 could contribute to maintain TGF-beta1 levels. Here, using small interfering RNAs (siRNA), we have first analyzed if LTBP-1 expression affected TGF-beta1 activity in MEF cells. We have then determined how LTBP-1 levels could alter the activity of extracellular proteases known to activate TGF-beta1, and finally, whether protease inhibition could reduce TGF-beta1 activation. LTBP-1 inhibition by siRNA in AhR-/- MEF decreased the amount of active TGF-beta1 and reduced plasminogen activators (PA)/plasmin and elastase activities and thrombospondin-1 (TSP-1) expression, without significantly affecting their mRNA levels. On the contrary, LTBP-1 siRNA restored matrix metalloproteinase-2 (MMP-2) activity in AhR-/- MEF. Interestingly, whereas a TGF-beta1 neutralizing antibody mimicked many of the LTBP-1 siRNA effects on extracellular proteases, addition of recombinant TGF-beta1 protein increased proteases activity over basal levels in AhR-/- MEF. These proteases contributed to TGF-beta activation since their specific inhibitors reduced active TGF-beta levels in these cells. These results suggest that LTBP-1 contributes to TGF-beta1 activation in MEF, possibly by influencing the activities of PA/plasmin, elastase, TSP-1, and MMP-2. TGF-beta1, on the other hand, could be also involved in maintaining the activity of these extracellular proteases. Thus, LTBP-1 appears to play a role in TGF-beta1 activation through a process involving extracellular protease activities, which, in turn, could be affected by TGF-beta1 levels.     

Transgenic mouse models that explore the multistep hypothesis of intestinal neoplasia

SV-40 T antigen (TAg), human K-rasVal12, and a dominant negative mutant of human p53 (p53Ala143) have been expressed singly and in all possible combinations in postmitotic enterocytes distributed throughout the duodenal-colonic axis of 1-12-mo-old FVB/N transgenic mice to assess the susceptibility of this lineage to gene products implicated in the pathogenesis of human gut neoplasia. SV-40 TAg produces re-entry into the cell cycle. Transgenic pedigrees that produce K-rasVal12 alone, p53Ala143 alone, or K-rasVal12 and p53Ala143 have no detectable phenotypic abnormalities. However, K-rasVal12 cooperates with SV-40 TAg to generate marked proliferative and dysplastic changes in the intestinal epithelium. These abnormalities do not progress to form adenomas or adenocarcinomas over a 9-12-mo period despite sustained expression of the transgenes. Addition of p53Ala143 to enterocytes that synthesize SV-40 TAg and K-rasVal12 does not produce any further changes in proliferation or differentiation. Mice that carry one, two, or three of these transgenes were crossed to animals that carry Min, a fully penetrant, dominant mutation of the Apc gene associated with the development of multiple small intestinal and colonic adenomas. A modest (2-5-fold) increase in tumor number was noted in animals which express SV-40 TAg alone, SV-40 TAg and K-rasVal12, or SV-40 TAg, K-rasVal12 and p53Ala143. However, the histopathologic features of the adenomas were not altered and the gut epithelium located between tumors appeared similar to the epithelium of their single transgenic, bi-transgenic, or tri-transgenic parents without Min. These results suggest that (a) the failure of the dysplastic gut epithelium of SV-40 TAg X K-rasVal12 mice to undergo further progression to adenomas or adenocarcinomas is due to the remarkable protective effect of a continuously and rapidly renewing epithelium, (b) initiation of tumorigenesis in Min mice typically occurs in crypts rather than in villus-associated epithelial cell populations, and (c) transgenic mouse models of neoplasia involving members of the enterocytic lineage may require that gene products implicated in tumorigenesis be directed to crypt stem cells or their immediate descendants. Nonetheless, directing K-rasVal12 production to proliferating and nonproliferating cells in the lower and upper half of small intestinal and colonic crypts does not result in any detectable abnormalities.     

Tetrachlorodibenzo-p-dioxin exposure alters radial arm maze performance and hippocampal morphology in female AhR mice

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been reported to alter spatial learning in rats tested on a radial arm maze (RAM). TCDD is believed to exert most of its effects through binding to the aryl hydrocarbon receptor (AhR). To determine whether the AhR mediates TCDD-induced alterations in spatial learning, we tested male and female AhR-knockout (AhR-/-), heterozygous (AhR+/-) and wild-type (AhR+/+) mice on the RAM. AhR+/- male and female mice were time mated, and treated dams were dosed with 5 microg TCDD/kg body weight on day 13 of gestation. When offspring reached adulthood, male and female AhR+/+, AhR+/- and AhR-/- mice from TCDD-exposed and unexposed litters were tested on the eight-arm RAM. After testing, we examined hippocampal morphology as visualized by the Timm's silver sulfide stain. TCDD-exposed female AhR+/- mice made more errors than their respective controls on the RAM and exhibited a decrease in the size of the intra- and infrapyramidal mossy fiber (IIP-MF) field of the hippocampus. None of the other TCDD-exposed groups differed from their respective control groups with regard to maze performance or hippocampal morphology. The reduction of IIP-MF field indicates a possible morphological basis for the learning deficit that was observed in the female AhR+/- mice. It is hypothesized that the effect of TCDD exposure is AhR dependent and that TCDD may alter GABAergic activity in the hippocampus of female mice during development.     

Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector

We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, greater than 90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein. The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.     

microRNA-501-5p promotes cell proliferation and migration in gastric cancer by downregulating LPAR1

In spite of the achievement in treatment, the gastric cancer (GC) mortality still remains high. MicroRNAs (miRNAs) are a group of small noncoding RNAs that play a crucial part in tumor progression. In this study, we explored the expression and function of microRNA-501-5p (miR-501-5p) in GC cell lines. Quantitative real-time polymerase chain reaction assay results suggested that miR-501-5p was significantly upregulated in GC tissues and cell lines. And, the Cell Counting Kit-8 colony formation and cell migration assay results showed that the downregulation of miR-501-5p decreased GC cell proliferation and migration. Besides that, we found that GC cell cycle was arrested in G2 phase and cell apoptosis rate was increased by silencing the expression of miR-501-5p in GC cell lines using the flow cytometry. We also found that miR-501-5p could directly target lysophosphatidic acid receptor 1 (LPAR1) and negatively regulate LPAR1 expression in GC cell lines by performing dual-luciferase reporter gene assay and Western blot analysis. And, LPAR1 was significantly downregulated in GC tissues and inversely correlated with miR-501-5p expression. Furthermore, LPAR1 downregulation promoted cell proliferation and migration, which were attenuated by cotransfection of miR-501-5p inhibitor in GC cells. In conclusion, miR-501-5p can promote GC cell proliferation and migration by targeting and downregulating LPAR1. miR-501-5p/LPAR1 may become a potential therapeutic target for GC treatment.     

Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation

The contrasting control of lysosomal proteinases, protein turnover and proliferation was studied in 3T3 and SV-3T3 (SV-40-virus-transformed 3T3) cells. 1. In 3T3 cells, net protein accumulation proceeded from 5%/h (doubling time, T(d)=14h) in growing cells to 0%/h as cells became quiescent. SV-3T3 cells never ceased to gain protein, but rather decreased their protein accumulation rate from 6-7%/h (T(d)=10-12h) to 2%/h (T(d)=35-40h) just before culture death in unchanged medium. 2. In both cell types the rates of protein synthesis per unit of protein (a) were proportional to the initial serum concentration from 0 to 6%, and (b) declined under progressive depletion of undefined serum growth factors. In depleted growth medium, leucine incorporation per unit of protein in 3T3 and SV-3T3 cells declined to almost equal synthetic rates while the 3T3 cell existed in a steady state of zero net gain, and the SV-3T3 cell continued to gain protein at a rate of 2%/h. 3. Whereas a large fraction of the control of 3T3-cell net protein accumulation can be accounted for by an increase in degradation from 1%/h to 3%/h, the SV-3T3 cell did not exhibit a growth-related increase in degradation appreciably above 1%/h. 4. Thus, by using first-order kinetics, the continued net protein accumulation of the transformed cell can be accounted for by a failure to increase protein degradation, whereas fractional synthesis can be made to decline to a rate similar to that in the quiescent non-transformed cell. 5. Upon acute serum deprivation, both cell types similarly exhibited small rapid increases in proteolysis independent of cell growth state or lysosomal enzyme status. 6. The 3T3 cell increased its lysosomal proteinase activity in conjunction with increase in the growth-state-dependent proteolytic mechanism; however, the SV-3T3 cell failed to increase lysosomal proteinases or the growth-state-dependent proteolytic mechanism.