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Dioxin-Induced Adseverin Expression in the Mouse Thymus Is Strictly Regulated and Dependent on the Aryl Hydrocarbon Receptor
Biochemical and Biophysical Research Communications 291, 1194 –1200 (2002) doi:10.1006/bbrc.2002.6582, available online at http://www.idealibrary.com on
Dioxin-Induced Adseverin Expression in the Mouse Thymus Is Strictly Regulated and Dependent on the Aryl Hydrocarbon Receptor 1 Camilla Svensson,* ,2 Allen E. Silverstone,† Zhi-Wei Lai,† and Katarina Lundberg* *Department of Pharmaceutical Biosciences, Division of Toxicology, Biomedical Centre, Uppsala University, Uppsala, Sweden; and †Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York 13210
Received January 21, 2002
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ligand for the ubiquitous, intracellular aryl hydrocarbon receptor (AhR), up-regulates the actin-modulating protein adseverin in mouse lymphoid tissues, a response that may be correlated to the immunotoxicity of TCDD. Here, by using chimeric mice with TCDDresponsive (AhR ⴙ/ⴙ) hematopoietic cells and TCDDunresponsive (AhR ⴚ/ⴚ) thymic stroma, or the reverse, we show that TCDD-induced expression of adseverin in thymus is dependent on AhR expression in hematopoietic cells but not in stroma. The use of fetal thymic organ cultures also indicates that TCDD-induced expression of adseverin is confined to the thymocytes. The thymic stroma showed no induction of adseverin expression after TCDD exposure, although TCDD clearly activated the AhR in these cells, as indicated by the induction of CYP1A1. Adseverin was not induced in the thymus of normal adult C57BL/6 mice exposed to -estradiol or dexamethasone, two other agents, which also cause thymic atrophy. This further supports that adseverin induction is a specific gene regulatory effect by TCDD on thymocytes. © 2002 Elsevier Science (USA)
Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; adseverin; AhR; TCDD; thymocyte; thymus; chimera.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other structurally related compounds are persistent environmental contaminants that induce a variety of biological and toxic effects in mammals, the immune system being particularly sensitive (1). Most of the 1 Supported by the Swedish Council for Work Life Research, Dnr 95-0437 and U.S. NIEHS ES07216 (AES). 2 To whom correspondence and reprint requests should be addressed at Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, P.O. Box 594, SE-751 24 Uppsala, Sweden. Fax: ⫹46 18 471 42 53. E-mail: [email protected].
0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
effects of TCDD and its related congeners are mediated by the aryl hydrocarbon receptor (AhR), a basic helix– loop– helix protein which upon TCDD-binding acts as a transcription factor and binds to dioxin responsive elements in target genes, thereby regulating their expression and a number of cellular processes (2). We have recently shown that one of the genes that are regulated by TCDD is the actin-modulating protein adseverin (3). The TCDD-induced expression of adseverin seems to be an effect restricted to the immune system, most pronounced in the thymus, and mediated via the AhR (3). Adseverin is a calcium-dependent, actin-binding protein which severs and caps filamentous actin (4, 5). Studies in different in vitro systems has shown that adseverin is important for exocytosis (6, 7). In addition, it was recently shown that expression of adseverin in megakaryoblastic leukemia cells activates signaling pathways and subsequently inhibits cell proliferation and induces differentiation of these cells to a mature phenotype with an apoptotic fate (8). In line with this study, we have previously hypothesized that TCDD-induced expression of adseverin leads to F-actin disassembly which may disturb normal transduction pathways which are important for normal cell development and thus for a normal sized thymus (3). The induction of adseverin gene expression can be observed in thymocytes at an early time-point after TCDD exposure (3). This suggests that thymocytes might be a primary target of this TCDD-mediated effect. A direct effect of TCDD on thymic hematopoietic cells has previously been shown by Staples and coworkers (9). However, earlier studies have suggested that the thymic stroma is the main target of TCDD and that the effect on thymocytes was indirect (10, 11). To determine if the TCDD-induced adseverin expression in thymocytes is a direct effect of TCDD on thymocytes or a secondary response to a targeted stroma, we have
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used chimeric mice with TCDD-responsive (AhR ⫹/⫹) hematopoietic cells and TCDD-unresponsive (AhR ⫺/⫺) thymic stroma, or the reverse. We have also used fetal thymic organ cultures (FTOCs) to compare the induction of adseverin in thymocytes and the thymic stroma. In addition, we investigated the ability of -estradiol (E2) and dexamethasone (DEX), two other substances also causing thymic atrophy (12, 13), to induce adseverin gene expression in the thymus of normal adult C57BL/6 mice. Our results show that TCDD-induced expression of adseverin in the thymus is dependent on AhR expression in thymocytes but not in the stroma. We also show that in contrast to what we see in thymocytes, TCDD does not induce adseverin in the thymic stroma. Finally, E2 and DEX do not regulate the expression of adseverin in the thymus. MATERIALS AND METHODS Chemicals. TCDD (98.4% pure, Larodan Fine Chemicals, Malmo¨ , Sweden) was dissolved in 1,4-dioxane (Merck, Darmstadt, Germany), and subsequently diluted with either corn oil or culture medium. -Estradiol 17-valerate (E2) (ICN Biomedicals, Costa Mesa, CA) and dexamethasone 21-phosphate (DEX) (Sigma, St. Louis, MO) were dissolved in corn oil and PBS, respectively. Experimental animals. C57BL/6 mice used for generating FTOCs and for in-vivo treatment with TCDD, DEX, and E2 were bred at the animal facility at Uppsala Biomedical Centre with original breeding pairs obtained from B&K Universal, Solna, Sweden. The strains used for the construction of the chimeric mice were the 129/Sv ⫻ C57BL/6N AhR ⫺/⫺ and AhR ⫹/⫹ mice (B6Ly5.2 phenotype), originally obtained from P. Fernandez-Salguero and F. Gonzalez (National Cancer Institute, National Institutes of Health, Bethesda, MD), and C57BL/6J (B6Ly5.1) mice, originally obtained from Dr. E. A. Boyse (Memorial Sloan-Kettering Cancer Center, New York). All experimental animals were housed and cared for according to The Guide for the Care and Use of Laboratory animals (14). Production and TCDD treatment of chimeric mice. AhR ⫹/⫹ or AhR ⫺/⫺ bone marrow chimeras were constructed and maintained at the SUNY Upstate Medical University, Syracuse, NY, USA, as previously described (9). The different chimeric mice constructed were (1) AhR ⫹/⫹ bone marrow donor into AhR ⫹/⫹ recipient (AhR ⫹/⫹ 3 AhR ⫹/⫹), (2) AhR ⫹/⫹ bone marrow donor into AhR ⫺/⫺ recipient (AhR ⫹/⫹ 3 AhR ⫺/⫺), (3) AhR ⫺/⫺ bone marrow donor into AhR ⫹/⫹ recipient (AhR ⫺/⫺ 3 AhR ⫹/⫹), and (4) AhR ⫺/⫺ bone marrow donor into AhR ⫺/⫺ recipient (AhR ⫺/⫺ 3 AhR ⫺/⫺). Irradiation and bone marrow reconstitution did not alter the thymic response to TCDD as compared to nonmanipulated mice (9). At 8 weeks of age, chimeric male mice were age-matched, randomly divided into 3– 6 animals per treatment group and injected ip with either 30 g/kg of TCDD (in this case from Cambridge Isotopes, Cambridge, MA) in olive oil or olive oil alone (0.1 ml/20 g). All mice were killed 10 days after the injection by CO 2 asphyxiation and cell suspensions of thymocytes prepared as previously described (9). Thymocytes were then subjected to RNA isolation with TRIzol (Invitrogen, Groningen, The Netherlands) according to the manufacturer’s recommendations. Fetal thymus organ culture. FTOCs were prepared as described previously (15). Thymus lobes from C57BL/6 fetuses on day 15 of gestation were placed on cellulose acetate filters (Sartorius AG, Goettingen, Germany), resting on metallic grids in culture plates with 2.5 ml RPMI (SBL, Uppsala, Sweden) supplemented with 10%
FBS, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The lobes were cultured with or without the addition of 135 mM 2-deoxyguanosine (Sigma), a base analogue that eliminates proliferating cells, here mainly thymocytes, while leaving the nonproliferating epithelial cells intact (16), at 37°C and 5% CO 2 in a water-saturated atmosphere. At day 4 a fraction of deoxyguanosinetreated lobes were homogenized and examined in a hemocytometer to check for the absence of thymocytes. No thymocytes could be detected at this time point. Deoxyguanosine-treated and non-treated thymic lobes were then transferred into fresh media, again with or without deoxyguanosine. TCDD was added to half of the cultures to a final concentration of 10 nM TCDD/0.1% 1,4-dioxane and control groups received an equal volume of 1.4-dioxane. Twenty-four hours after addition of TCDD the lobes were harvested and whole thymuses, thymocytes and thymic stroma were subjected to RNA isolation with TRIzol (Invitrogen). Thymocytes were prepared from lobes that had not been treated with deoxyguanosine by gently pressing the lobes through a steel net in PBS with 3% FCS and passing the cell suspension through a sterile cotton mesh. The viability and purity of the cells was found to be ⬎95% by eosin exclusion. We used ten to twenty thymus lobes, depending on treatment, to generate one RNA sample and three RNA samples were collected from each treatment group. In vivo treatment of C57BL/6 with TCDD, E2 or DEX. At 5 to 6 weeks of age, female C57BL/6J mice were weight-matched and randomly allocated into treatments groups of 3– 4 mice per group. The mice were injected with either TCDD (10 g/kg, ip), E2 (5 mg/kg, sc) or DEX (5 mg/kg, ip). Control mice were injected with an equal volume of the pertinent vehicle. At the indicated times of exposure (Figs. 3 and 4) the mice were killed by CO 2 asphyxiation and their thymuses were removed. Thymocytes were isolated as described in the FTOC section, counted and subjected to RNA isolation with TRIzol (Invitrogen). Reverse transcription. One microgram of total RNA isolated from thymocytes of AhR chimeras, from thymocytes of in vivo treated C57BL/6 mice and from whole thymic lobes, isolated thymocytes and stroma from FTOCs, were treated with 1 U DNase I Amp grade (Life Technologies) according to the manufacturer’s instruction and subjected to reverse transcription (RT) to produce cDNA. The RT was performed in a total volume of 40 l containing 1 g oligo(dT) primer (Amersham Pharmacia Biotech, Uppsala, Sweden), 8 l 5 ⫻ RTbuffer (Promega, Madison, WI), 1 mM dNTP, 20 U RNase inhibitor (Amersham Pharmacia Biotech) and 400 U of Moloney murine leukemia virus reverse transcriptase (Promega). The samples were incubated at 37°C for 60 min and then heated to 70°C for 10 min to stop the reactions. Polymerase chain reaction (PCR). Five microliters of the cDNA samples were amplified in a total volume of 50 l containing 0.5 M of 5⬘ and 3⬘ primers, 0.2 mM dNTPs, 1⫻ PCR Buffer (Clontech, Palo Alto, CA) and 1 l Advantage polymerase mix (Clontech). The samples were incubated at 94°C for 4 min and then amplified with 25 and 30 cycles for adseverin and CYP1A1 respectively: 94°C for 15 s, 62°C for 30 s and 68°C for 30 s with a final extension at 68°C for 7 min. As an internal control of the amount of cDNA used, the housekeeping gene hypoxanthine phosphoribosyl nucleotide phosphate (HPRT) was amplified for 25 cycles along with the target gene. The following primers were used: 5⬘-TGGCTTTGGGAAGGTGTACATCA-3⬘ and 5⬘-GCCATTGTTTCGTGGCAGTTTTA-3⬘ for adseverin; 5⬘-GGCATTCATCCTTCGTCCCCTT-3⬘ and 5⬘-TCACAGCGGGCGTGTTTTAAAGT-3⬘ for CYP1A1; and 5⬘-CACAGGACTAGAACACCTGC-3⬘ and 5⬘-GCTGGTGAAAAGGACTCT-3⬘ for HPRT. After amplification, 15 l of each PCR sample was run on a 1.5% agarose gel containing 0.4 g ethidium bromide (EtBr)/ml. The gel was photographed in a gel documentation system (GDS 5000 from UVP Ltd, Cambridge, England) and the bands were analyzed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov).
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FIG. 1. Effects of TCDD on the mRNA levels of adseverin (Ads) (A and B) and CYP1A1 (CYP) (C and D) in thymocytes from AhR chimeric mice. The EtBr-stained gel images (A and C) where each lane represents one out of three to five individual samples are shown. The data shown (B and D) are the mean of three to five independent experiments expressed as target gene/HPRT ratios in vehicle (䊐) or TCDD (■)-exposed thymocytes. Error bars indicate SE. *P ⬍ 0.05; **P ⬍ 0.01, with respect to vehicle-treated, matched control.
The integrated band intensities were normalized to the mRNA expression of the HPRT gene (target gene/HPRT) and the fold of induction calculated:((target gene treated ⫻ HPRT control)/(target gene control ⫻ HPRT treated)) (17). Statistics. A two-tailed Student’s t test for paired variables, in the MicrosoftExcel ’98 program, was used to evaluate differences between treatment and control groups. Results were considered significant at P ⬍ 0.05.
RESULTS Thymocytes are direct targets of TCDD. Using RTPCR we determined the mRNA level of adseverin in thymocytes isolated from different AhR chimeras treated with a single injection of either 30 g TCDD/kg or vehicle 10 days earlier. There was a low constitutive level of adseverin mRNA in thymocytes from all vehicle treated mice. Upon TCDD treatment the level of adseverin mRNA was increased, but only in thymocytes with the AhR ⫹/⫹ genotype and irrespective of the genotype of the thymic stroma (Figs. 1A and 1B). Thymocytes from AhR ⫹/⫹ 3 AhR ⫹/⫹ mice showed a 22-fold increase of adseverin mRNA after TCDD treatment and in thymocytes from AhR ⫹/⫹ 3 AhR ⫺/⫺ mice the increase of adseverin mRNA was 9-fold. The lower relative increase in thymocytes from AhR ⫹/⫹ 3 AhR ⫺/⫺ mice was due to a higher constitutive level of adseverin in the controls of these mice. This difference between the controls was however not significant (P ⫽ 0.11). In contrast, no induction of adseverin was observed in
AhR ⫺/⫺ thymocytes isolated from AhR ⫺/⫺ 3 AhR ⫹/⫹ or AhR ⫺/⫺ 3 AhR ⫺/⫺ chimeras treated with TCDD (Figs. 1A and 1B). In parallel, we also measured the induction of CYP1A1, another AhR regulated gene, and it followed the pattern of adseverin (Figs. 1C and 1D). Adseverin gene expression can be regulated by TCDD in thymocytes but not in the thymic stroma. To investigate if adseverin is also induced in the thymic stroma upon TCDD treatment we used the FTOC technique to determine the levels of adseverin and CYP1A1 mRNA in whole thymus lobes, thymocytes and thymic stroma after TCDD-exposure. There was a constitutive level of adseverin mRNA in both thymocytes and stroma (Figs. 2A and 2B). TCDD treatment increased the levels of adseverin mRNA in whole thymus 2.6-fold and in isolated thymocytes 2.9-fold (Figs. 2A and 2B). However, TCDD failed to induce adseverin in thymic stroma (Figs. 2A and 2B). This is in contrast to CYP1A1 gene expression, which was induced in both thymocytes and stroma (Figs. 2C and 2D). Induction of adseverin is not a common effect of agents causing thymus atrophy. Although the TCDDregulated induction of adseverin seems to be mediated via the AhR, we considered the possibility that increased levels of adseverin in thymocytes could be a common early effect of different agents that cause thymus atrophy. This was investigated by using RT-PCR and comparing the effects of TCDD, E2, and DEX on
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FIG. 2. Effects of TCDD on the mRNA levels of adseverin (Ads) (A and B) and CYP1A1 (CYP) (C and D) in isolated thymocytes, whole thymus and thymic stroma from FTOCs. The EtBr-stained gel images (A and C) where each lane represents one out of three individual samples are shown. The data shown (B and D) are the mean of three independent experiments expressed as target gene/HPRT ratios in vehicle (䊐) or TCDD-exposed (■) thymocytes, whole thymus, and stroma. Error bars indicate SE. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001, with respect to vehicle-treated, matched control.
the adseverin mRNA levels in thymocytes. There was a marked, 8.3-fold, increase in adseverin mRNA levels in thymocytes 24 h after exposure to 10 g TCDD/kg (Fig. 3). At this time-point there was no effect on cellularity in the thymus by TCDD (Fig. 4). However, at 1 week of exposure the cell number was reduced to 40% of control (Fig. 4). E2 is known to induce thymus atrophy with
FIG. 3. Effects of TCDD, E2, and DEX on the level of adseverin mRNA in thymocytes revealed by RT-PCR. Gel images of the separated PCR products were quantified by densitometric analysis and the data shown are the mean of three independent experiments expressed as the fold of induction of adseverin above control. The ratio of adseverin/HPRT in the controls was defined as 1 (broken line). Error bars indicate SE *P ⬍ 0.05, with respect to vehicletreated, matched control.
similar kinetics to TCDD (18) and was accordingly also examined at 24 h of exposure. A dose of 5 mg E2/kg had no effect on the level of adseverin mRNA or the thymocyte number at 24 h, but still reduced the cell number to 26% of control, at 1 w of exposure (Figs. 3 and 4). In contrast to TCDD and E2, DEX induces a very rapid but transient thymic atrophy (19) and its effect was therefore analyzed at 3, 6, and 24 h of exposure. At the
FIG. 4. Effects of TCDD, E2 and DEX on thymic cell number. Data shown are the mean thymic cell number based on three individual mice, expressed as % of control (broken line) with the cell number in matched, vehicle-treated controls defined as 100%. Error bars indicate SE. *P ⬍ 0.05, with respect to vehicle-treated, matched control.
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dose of 5 mg DEX/kg no effect on the adseverin mRNA level was observed at 3 or 6 h of exposure (Fig. 3) while a reduction in the cell number was observed after 6 h (Fig. 4). At 24 h of exposure to DEX, there was a slight but not significant increase, 1.7-fold, in adseverin levels (Fig. 3). At this time-point there was marked thymus atrophy with the cell number being 40% of that in control thymi (Fig. 4). DISCUSSION Reduced proliferation and increased differentiation and apoptosis are effects that all have been associated with TCDD-induced thymic atrophy (15, 20, 21). Similar effects have also been demonstrated in megakaryoblastic leukemia cells by reintroduction of the adseverin expression (8). Considering that all stages in lymphocyte life—lymphocyte development, migration and activation—are associated with profound changes in cell morphology and cell signaling both of which require actin modulation (22–24), we believe that adseverin might be a mediator of some of the immunotoxic effects of TCDD. Here, we have further characterized the TCDD-induced adseverin expression in the thymus, reported previously by us (3), to strengthen the evidence for the role of adseverin in TCDD-induced thymic atrophy and precisely define the cellular target cells. By analyzing thymocytes from different AhRchimeric mice, we have shown that adseverin is induced in AhR-expressing thymocytes upon TCDD exposure, regardless of the AhR genotype of the thymic stroma. A similar observation was made for CYP1A1, the most frequently used marker for TCDD activation. The gene expression of adseverin was increased by TCDD to roughly the same level in thymocytes from AhR ⫹/⫹ 3 AhR ⫹/⫹ and AhR ⫹/⫹ 3 AhR ⫺/⫺ chimeras. However, for unknown reasons there was a higher variability in the levels of adseverin mRNA in thymocytes from both vehicle and TCDD-treated AhR ⫹/⫹ 3 AhR ⫺/⫺ chimeras, compared to the AhR ⫹/⫹ 3 AhR ⫹/⫹ thymocytes and therefore the fold of induction was lower in these mice. The constitutive level of adseverin in AhR ⫺/⫺ thymocytes was not significantly different from the levels in AhR ⫹/⫹ thymocytes (Fig. 1B). This could indicate that the AhR is not involved in the basal transcriptional regulation of adseverin in thymocytes under normal conditions. However, another interpretation is that the expression of adseverin is critical for normal cell development, as indicated in the study by Zunino and co-workers (8), and therefore alternative pathways to regulate adseverin gene expression have evolved. It is clear however, that presence of a functional AhR in thymocytes, is pivotal for the regulation of adseverin by TCDD, since no adseverin induction was observed in AhR ⫺/⫺ thymocytes after TCDD exposure. The adseverin induction observed here also coin-
cides with the TCDD-induced thymic effects previously reported by Staples and co-workers (9). In these chimeras they observed that TCDD induces thymus atrophy in mice with AhR ⫹/⫹ hematopoietic cells but not in mice with AhR ⫺/⫺ hematopoietic cells, irrespective of the AhR genotype of the stroma. Although the induction of adseverin in thymocytes and the reduction in thymic weight and cellularity is due to a direct effect on thymocytes it is also possible that TCDD can induce adseverin in the thymic stroma. To investigate this effect we used FTOCs in combination with deoxyguanosine. In controls the constitutive level of adseverin was similar for whole thymus, stroma and thymocytes. However, no adseverin induction could be detected in the stroma while induction was observed in both whole thymus and thymocytes. The fold of adseverin induction in TCDD-treated FTOCs is lower than the fold of induction we have observed in TCDD-exposed adult thymocytes. It should also be noted that the ratio of adseverin/HPRT is higher in control thymocytes from FTOC as compared to adult thymocytes. This may be due to in vitro culture conditions or may reflect a real difference in adseverin expression between adult and fetal thymus. Preliminary data indicates that immature thymocytes express higher levels of adseverin than more mature subsets (our own unpublished data). In contrast to adseverin, CYP1A1 was induced in both stroma and thymocytes, and the level of induction was approximately the same in the two compartments of FTOCs. This result shows that with respect to CYP1A1 induction TCDD has an effect on the stroma. Thus, while CYP1A1 seems to be induced in most cell types upon TCDD exposure, the induction of adseverin is not only confined to lymphoid tissues, but more specifically to the hematopoietic compartment. This agrees with previous results from our group (3) and others showing that certain TCDDmediated effects on gene expression are tissue- or cellspecific (25). Although we have shown here that TCDD has an AhR-dependent effect on adseverin gene regulation in thymocytes, we also wanted to know if other agents causing thymic atrophy could induce adseverin gene expression in an AhR–independent way. We used DEX and E2, agents with well documented immunomodulatory properties, which also induce thymic atrophy, although by different mechanisms (19, 26). We have previously shown that the induction of adseverin by TCDD appears before any sign of thymus atrophy is evident (3). Therefore, we found it relevant to examine the effects of the different compounds early in the time course of atrophy. TCDD was used as a positive control and in agreement with our previous study (3), adseverin induction was observed in thymocytes before any signs of atrophy was evident, 24 h after exposure to TCDD. In contrast, E2— known to induce thymus atrophy with similar kinetics to TCDD (18, 26)—and
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DEX— known to induce atrophy faster than TCDD— did not induce adseverin expression significantly within 24 h. A small but not statistically significant adseverin induction was observed after 24 h of DEX exposure, a time when the thymocyte number was already reduced by approximately 60%. It is possible that this slight increase in adseverin level is due to an indirect effect of DEX, which selectively reduces the number of double positive thymocytes (18). Our preliminary data show that the DN subset has a higher constitutive expression of adseverin than DP cells (unpublished data). Thus, if the remaining cells have a higher constitutive level of adseverin, a relative increase would be detected. To summarize our findings, the TCDD-induced adseverin expression in the thymus is restricted to thymocytes and dependent on AhR activation in these cells. In addition, the induction of adseverin is not a common toxic effect of agents causing thymic atrophy but rather seems to be strictly regulated. The cellspecific induction of adseverin by TCDD indicates that although the presence of an AhR is obligatory for TCDD-induced adseverin expression, other factors are also required to mediate this response. Such factors remains to be identified but could be transcription factors specifically expressed in the developing thymocytes. Analysis of the promoter region of adseverin as well as functional studies of adseverin may be helpful in revealing such regulatory factors and hence new mechanistic aspects on TCDD-induced immunotoxicity.
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ACKNOWLEDGMENTS The authors thank Nancy Fiore, J. Erin Staples, and Mike Laiosa (all of the Department of Microbiology and Immunology, SUNY Upstate Medical University) for generating the original samples from chimeras, for organizing samples for shipment to Uppsala and for discussion of the results. We also thank Raili Engdahl and Lena Norgren (Department of Pharmaceutical Biosciences, Div. of toxicology, Uppsala University) for technical assistance.
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and Trifaro, J. M. (2001) An antisense oligodeoxynucleotide targeted to chromaffin cell scinderin gene decreased scinderin levels and inhibited depolarization-induced cortical F-actin disassembly and exocytosis. J. Neurochem. 76, 768 –777. Vitale, M. L., Rodriguez Del Castillo, A., Tchakarov, L., and Trifaro, J. M. (1991) Cortical filamentous actin disassembly and scinderin redistribution during chromaffin cell stimulation precede exocytosis, a phenomenon not exhibited by gelsolin. J. Cell Biol. 113, 1057–1067. Zunino, R., Li, Q., Rose, S. D., Romero-Benitez, M. M., Lejen, T., Brandan, N. C., and Trifaro, J. M. (2001) Expression of scinderin in megakaryoblastic leukemia cells induces differentiation, maturation, and apoptosis with release of platelet-like particles and inhibits proliferation and tumorigenesis. Blood 98, 2210 –2219. Staples, J. E., Murante, F. G., Fiore, N. C., Gasiewicz, T. A., and Silverstone, A. E. (1998) Thymic alterations induced by 2,3,7,8tetrachlorodibenzo-p-dioxin are strictly dependent on aryl hydrocarbon receptor activation in hemopoietic cells [published erratum appears in J. Immunol. (1999) 163, 1092]. J. Immunol. 160, 3844 –3854. Kremer, J., Gleichmann, E., and Esser, C. (1994) Thymic stroma exposed to arylhydrocarbon receptor-binding xenobiotics fails to support proliferation of early thymocytes but induces differentiation. J. Immunol. 153, 2778 –2786. Greenlee, W. F., Dold, K. M., Irons, R. D., and Osborne, R. (1985) Evidence for direct action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic epithelium. Toxicol. Appl. Pharmacol. 79, 112–120. Erbach, G. T., and Bahr, J. M. (1988) Effect of chronic or cyclic exposure to estradiol on the humoral immune response and the thymus. Immunopharmacology 16, 45–51. Compton, M. M., Caron, L. A., and Cidlowski, J. A. (1987) Glucocorticoid action on the immune system. J. Steroid Biochem. 27, 201–208. (1996) The Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC. Lundberg, K., Gronvik, K. O., Goldschmidt, T. J., Klareskog, L., and Dencker, L. (1990) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters intrathymic T-cell development in mice. Chem.Biol. Interact. 74, 179 –193. Jenkinson, E. J., Franchi, L. L., Kingston, R., and Owen, J. J. (1982) Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments. Eur. J. Immunol. 12, 583–587. Lai, Z. W., Hundeiker, C., Gleichmann, E., and Esser, C. (1997) Cytokine gene expression during ontogeny in murine thymus on activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol. Pharmacol. 52, 30 –37. Staples, J. E., Fiore, N. C., Frazier, D. E., Jr., Gasiewicz, T. A., and Silverstone, A. E. (1998) Overexpression of the antiapoptotic oncogene, bcl-2, in the thymus does not prevent thymic atrophy induced by estradiol or 2,3,7, 8-tetrachlorodibenzo-pdioxin. Toxicol. Appl. Pharmacol. 151, 200 –210. Lundberg, K. (1991) Dexamethasone and 2,3,7,8-tetrachlorodibenzo-p-dioxin can induce thymic atrophy by different mechanisms in mice. Biochem. Biophys. Res. Commun. 178, 16 –23. Esser, C., and Welzel, M. (1993) Ontogenic development of murine fetal thymocytes is accelerated by 3,3⬘,4,4⬘-tetrachlorobiphenyl. Int. J. Immunopharmacol. 15, 841– 852. McConkey, D. J., Hartzell, P., Duddy, S. K., Hakansson, H., and Orrenius, S. (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca 2⫹-mediated endonuclease activation. Science 242, 256 –259. Dustin, M. L., and Cooper, J. A. (2000) The immunological syn-
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apse and the actin cytoskeleton: Molecular hardware for T cell signaling. Nat. Immunol. 1, 23. 23. Penninger, J. M., and Crabtree, G. R. (1999) The actin cytoskeleton and lymphocyte activation. Cell 96, 9 –12. 24. Serrador, J. M., Nieto, M., and Sanchez-Madrid, F. (1999) Cytoskeletal rearrangement during migration and activation of T lymphocytes. Trends Cell Biol. 9, 228 –233. 25. Jeon, M. S., and Esser, C. (2000) The murine IL-2 promoter
contains distal regulatory elements responsive to the Ah receptor, a member of the evolutionarily conserved bHLH-PAS transcription factor family. J. Immunol. 165, 6975– 6983. 26. Silverstone, A. E., Frazier, D. E., Jr., Fiore, N. C., Soults, J. A., and Gasiewicz, T. A. (1994) Dexamethasone, beta-estradiol, and 2,3,7,8-tetrachlorodibenzo-p-dioxin elicit thymic atrophy through different cellular targets. Toxicol. Appl. Pharmacol. 126, 248 –259.
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Dioxin-Induced Adseverin Expression in the Mouse Thymus Is Strictly Regulated and Dependent on the Aryl Hydrocarbon Receptor 1 Camilla Svensson,* ,2 Allen E. Silverstone,† Zhi-Wei Lai,† and Katarina Lundberg* *Department of Pharmaceutical Biosciences, Division of Toxicology, Biomedical Centre, Uppsala University, Uppsala, Sweden; and †Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York 13210
Received January 21, 2002
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ligand for the ubiquitous, intracellular aryl hydrocarbon receptor (AhR), up-regulates the actin-modulating protein adseverin in mouse lymphoid tissues, a response that may be correlated to the immunotoxicity of TCDD. Here, by using chimeric mice with TCDDresponsive (AhR ⴙ/ⴙ) hematopoietic cells and TCDDunresponsive (AhR ⴚ/ⴚ) thymic stroma, or the reverse, we show that TCDD-induced expression of adseverin in thymus is dependent on AhR expression in hematopoietic cells but not in stroma. The use of fetal thymic organ cultures also indicates that TCDD-induced expression of adseverin is confined to the thymocytes. The thymic stroma showed no induction of adseverin expression after TCDD exposure, although TCDD clearly activated the AhR in these cells, as indicated by the induction of CYP1A1. Adseverin was not induced in the thymus of normal adult C57BL/6 mice exposed to -estradiol or dexamethasone, two other agents, which also cause thymic atrophy. This further supports that adseverin induction is a specific gene regulatory effect by TCDD on thymocytes. © 2002 Elsevier Science (USA)
Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; adseverin; AhR; TCDD; thymocyte; thymus; chimera.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other structurally related compounds are persistent environmental contaminants that induce a variety of biological and toxic effects in mammals, the immune system being particularly sensitive (1). Most of the 1 Supported by the Swedish Council for Work Life Research, Dnr 95-0437 and U.S. NIEHS ES07216 (AES). 2 To whom correspondence and reprint requests should be addressed at Department of Pharmaceutical Biosciences, Division of Toxicology, Uppsala University, P.O. Box 594, SE-751 24 Uppsala, Sweden. Fax: ⫹46 18 471 42 53. E-mail: [email protected].
0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
effects of TCDD and its related congeners are mediated by the aryl hydrocarbon receptor (AhR), a basic helix– loop– helix protein which upon TCDD-binding acts as a transcription factor and binds to dioxin responsive elements in target genes, thereby regulating their expression and a number of cellular processes (2). We have recently shown that one of the genes that are regulated by TCDD is the actin-modulating protein adseverin (3). The TCDD-induced expression of adseverin seems to be an effect restricted to the immune system, most pronounced in the thymus, and mediated via the AhR (3). Adseverin is a calcium-dependent, actin-binding protein which severs and caps filamentous actin (4, 5). Studies in different in vitro systems has shown that adseverin is important for exocytosis (6, 7). In addition, it was recently shown that expression of adseverin in megakaryoblastic leukemia cells activates signaling pathways and subsequently inhibits cell proliferation and induces differentiation of these cells to a mature phenotype with an apoptotic fate (8). In line with this study, we have previously hypothesized that TCDD-induced expression of adseverin leads to F-actin disassembly which may disturb normal transduction pathways which are important for normal cell development and thus for a normal sized thymus (3). The induction of adseverin gene expression can be observed in thymocytes at an early time-point after TCDD exposure (3). This suggests that thymocytes might be a primary target of this TCDD-mediated effect. A direct effect of TCDD on thymic hematopoietic cells has previously been shown by Staples and coworkers (9). However, earlier studies have suggested that the thymic stroma is the main target of TCDD and that the effect on thymocytes was indirect (10, 11). To determine if the TCDD-induced adseverin expression in thymocytes is a direct effect of TCDD on thymocytes or a secondary response to a targeted stroma, we have
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used chimeric mice with TCDD-responsive (AhR ⫹/⫹) hematopoietic cells and TCDD-unresponsive (AhR ⫺/⫺) thymic stroma, or the reverse. We have also used fetal thymic organ cultures (FTOCs) to compare the induction of adseverin in thymocytes and the thymic stroma. In addition, we investigated the ability of -estradiol (E2) and dexamethasone (DEX), two other substances also causing thymic atrophy (12, 13), to induce adseverin gene expression in the thymus of normal adult C57BL/6 mice. Our results show that TCDD-induced expression of adseverin in the thymus is dependent on AhR expression in thymocytes but not in the stroma. We also show that in contrast to what we see in thymocytes, TCDD does not induce adseverin in the thymic stroma. Finally, E2 and DEX do not regulate the expression of adseverin in the thymus. MATERIALS AND METHODS Chemicals. TCDD (98.4% pure, Larodan Fine Chemicals, Malmo¨ , Sweden) was dissolved in 1,4-dioxane (Merck, Darmstadt, Germany), and subsequently diluted with either corn oil or culture medium. -Estradiol 17-valerate (E2) (ICN Biomedicals, Costa Mesa, CA) and dexamethasone 21-phosphate (DEX) (Sigma, St. Louis, MO) were dissolved in corn oil and PBS, respectively. Experimental animals. C57BL/6 mice used for generating FTOCs and for in-vivo treatment with TCDD, DEX, and E2 were bred at the animal facility at Uppsala Biomedical Centre with original breeding pairs obtained from B&K Universal, Solna, Sweden. The strains used for the construction of the chimeric mice were the 129/Sv ⫻ C57BL/6N AhR ⫺/⫺ and AhR ⫹/⫹ mice (B6Ly5.2 phenotype), originally obtained from P. Fernandez-Salguero and F. Gonzalez (National Cancer Institute, National Institutes of Health, Bethesda, MD), and C57BL/6J (B6Ly5.1) mice, originally obtained from Dr. E. A. Boyse (Memorial Sloan-Kettering Cancer Center, New York). All experimental animals were housed and cared for according to The Guide for the Care and Use of Laboratory animals (14). Production and TCDD treatment of chimeric mice. AhR ⫹/⫹ or AhR ⫺/⫺ bone marrow chimeras were constructed and maintained at the SUNY Upstate Medical University, Syracuse, NY, USA, as previously described (9). The different chimeric mice constructed were (1) AhR ⫹/⫹ bone marrow donor into AhR ⫹/⫹ recipient (AhR ⫹/⫹ 3 AhR ⫹/⫹), (2) AhR ⫹/⫹ bone marrow donor into AhR ⫺/⫺ recipient (AhR ⫹/⫹ 3 AhR ⫺/⫺), (3) AhR ⫺/⫺ bone marrow donor into AhR ⫹/⫹ recipient (AhR ⫺/⫺ 3 AhR ⫹/⫹), and (4) AhR ⫺/⫺ bone marrow donor into AhR ⫺/⫺ recipient (AhR ⫺/⫺ 3 AhR ⫺/⫺). Irradiation and bone marrow reconstitution did not alter the thymic response to TCDD as compared to nonmanipulated mice (9). At 8 weeks of age, chimeric male mice were age-matched, randomly divided into 3– 6 animals per treatment group and injected ip with either 30 g/kg of TCDD (in this case from Cambridge Isotopes, Cambridge, MA) in olive oil or olive oil alone (0.1 ml/20 g). All mice were killed 10 days after the injection by CO 2 asphyxiation and cell suspensions of thymocytes prepared as previously described (9). Thymocytes were then subjected to RNA isolation with TRIzol (Invitrogen, Groningen, The Netherlands) according to the manufacturer’s recommendations. Fetal thymus organ culture. FTOCs were prepared as described previously (15). Thymus lobes from C57BL/6 fetuses on day 15 of gestation were placed on cellulose acetate filters (Sartorius AG, Goettingen, Germany), resting on metallic grids in culture plates with 2.5 ml RPMI (SBL, Uppsala, Sweden) supplemented with 10%
FBS, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The lobes were cultured with or without the addition of 135 mM 2-deoxyguanosine (Sigma), a base analogue that eliminates proliferating cells, here mainly thymocytes, while leaving the nonproliferating epithelial cells intact (16), at 37°C and 5% CO 2 in a water-saturated atmosphere. At day 4 a fraction of deoxyguanosinetreated lobes were homogenized and examined in a hemocytometer to check for the absence of thymocytes. No thymocytes could be detected at this time point. Deoxyguanosine-treated and non-treated thymic lobes were then transferred into fresh media, again with or without deoxyguanosine. TCDD was added to half of the cultures to a final concentration of 10 nM TCDD/0.1% 1,4-dioxane and control groups received an equal volume of 1.4-dioxane. Twenty-four hours after addition of TCDD the lobes were harvested and whole thymuses, thymocytes and thymic stroma were subjected to RNA isolation with TRIzol (Invitrogen). Thymocytes were prepared from lobes that had not been treated with deoxyguanosine by gently pressing the lobes through a steel net in PBS with 3% FCS and passing the cell suspension through a sterile cotton mesh. The viability and purity of the cells was found to be ⬎95% by eosin exclusion. We used ten to twenty thymus lobes, depending on treatment, to generate one RNA sample and three RNA samples were collected from each treatment group. In vivo treatment of C57BL/6 with TCDD, E2 or DEX. At 5 to 6 weeks of age, female C57BL/6J mice were weight-matched and randomly allocated into treatments groups of 3– 4 mice per group. The mice were injected with either TCDD (10 g/kg, ip), E2 (5 mg/kg, sc) or DEX (5 mg/kg, ip). Control mice were injected with an equal volume of the pertinent vehicle. At the indicated times of exposure (Figs. 3 and 4) the mice were killed by CO 2 asphyxiation and their thymuses were removed. Thymocytes were isolated as described in the FTOC section, counted and subjected to RNA isolation with TRIzol (Invitrogen). Reverse transcription. One microgram of total RNA isolated from thymocytes of AhR chimeras, from thymocytes of in vivo treated C57BL/6 mice and from whole thymic lobes, isolated thymocytes and stroma from FTOCs, were treated with 1 U DNase I Amp grade (Life Technologies) according to the manufacturer’s instruction and subjected to reverse transcription (RT) to produce cDNA. The RT was performed in a total volume of 40 l containing 1 g oligo(dT) primer (Amersham Pharmacia Biotech, Uppsala, Sweden), 8 l 5 ⫻ RTbuffer (Promega, Madison, WI), 1 mM dNTP, 20 U RNase inhibitor (Amersham Pharmacia Biotech) and 400 U of Moloney murine leukemia virus reverse transcriptase (Promega). The samples were incubated at 37°C for 60 min and then heated to 70°C for 10 min to stop the reactions. Polymerase chain reaction (PCR). Five microliters of the cDNA samples were amplified in a total volume of 50 l containing 0.5 M of 5⬘ and 3⬘ primers, 0.2 mM dNTPs, 1⫻ PCR Buffer (Clontech, Palo Alto, CA) and 1 l Advantage polymerase mix (Clontech). The samples were incubated at 94°C for 4 min and then amplified with 25 and 30 cycles for adseverin and CYP1A1 respectively: 94°C for 15 s, 62°C for 30 s and 68°C for 30 s with a final extension at 68°C for 7 min. As an internal control of the amount of cDNA used, the housekeeping gene hypoxanthine phosphoribosyl nucleotide phosphate (HPRT) was amplified for 25 cycles along with the target gene. The following primers were used: 5⬘-TGGCTTTGGGAAGGTGTACATCA-3⬘ and 5⬘-GCCATTGTTTCGTGGCAGTTTTA-3⬘ for adseverin; 5⬘-GGCATTCATCCTTCGTCCCCTT-3⬘ and 5⬘-TCACAGCGGGCGTGTTTTAAAGT-3⬘ for CYP1A1; and 5⬘-CACAGGACTAGAACACCTGC-3⬘ and 5⬘-GCTGGTGAAAAGGACTCT-3⬘ for HPRT. After amplification, 15 l of each PCR sample was run on a 1.5% agarose gel containing 0.4 g ethidium bromide (EtBr)/ml. The gel was photographed in a gel documentation system (GDS 5000 from UVP Ltd, Cambridge, England) and the bands were analyzed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov).
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FIG. 1. Effects of TCDD on the mRNA levels of adseverin (Ads) (A and B) and CYP1A1 (CYP) (C and D) in thymocytes from AhR chimeric mice. The EtBr-stained gel images (A and C) where each lane represents one out of three to five individual samples are shown. The data shown (B and D) are the mean of three to five independent experiments expressed as target gene/HPRT ratios in vehicle (䊐) or TCDD (■)-exposed thymocytes. Error bars indicate SE. *P ⬍ 0.05; **P ⬍ 0.01, with respect to vehicle-treated, matched control.
The integrated band intensities were normalized to the mRNA expression of the HPRT gene (target gene/HPRT) and the fold of induction calculated:((target gene treated ⫻ HPRT control)/(target gene control ⫻ HPRT treated)) (17). Statistics. A two-tailed Student’s t test for paired variables, in the MicrosoftExcel ’98 program, was used to evaluate differences between treatment and control groups. Results were considered significant at P ⬍ 0.05.
RESULTS Thymocytes are direct targets of TCDD. Using RTPCR we determined the mRNA level of adseverin in thymocytes isolated from different AhR chimeras treated with a single injection of either 30 g TCDD/kg or vehicle 10 days earlier. There was a low constitutive level of adseverin mRNA in thymocytes from all vehicle treated mice. Upon TCDD treatment the level of adseverin mRNA was increased, but only in thymocytes with the AhR ⫹/⫹ genotype and irrespective of the genotype of the thymic stroma (Figs. 1A and 1B). Thymocytes from AhR ⫹/⫹ 3 AhR ⫹/⫹ mice showed a 22-fold increase of adseverin mRNA after TCDD treatment and in thymocytes from AhR ⫹/⫹ 3 AhR ⫺/⫺ mice the increase of adseverin mRNA was 9-fold. The lower relative increase in thymocytes from AhR ⫹/⫹ 3 AhR ⫺/⫺ mice was due to a higher constitutive level of adseverin in the controls of these mice. This difference between the controls was however not significant (P ⫽ 0.11). In contrast, no induction of adseverin was observed in
AhR ⫺/⫺ thymocytes isolated from AhR ⫺/⫺ 3 AhR ⫹/⫹ or AhR ⫺/⫺ 3 AhR ⫺/⫺ chimeras treated with TCDD (Figs. 1A and 1B). In parallel, we also measured the induction of CYP1A1, another AhR regulated gene, and it followed the pattern of adseverin (Figs. 1C and 1D). Adseverin gene expression can be regulated by TCDD in thymocytes but not in the thymic stroma. To investigate if adseverin is also induced in the thymic stroma upon TCDD treatment we used the FTOC technique to determine the levels of adseverin and CYP1A1 mRNA in whole thymus lobes, thymocytes and thymic stroma after TCDD-exposure. There was a constitutive level of adseverin mRNA in both thymocytes and stroma (Figs. 2A and 2B). TCDD treatment increased the levels of adseverin mRNA in whole thymus 2.6-fold and in isolated thymocytes 2.9-fold (Figs. 2A and 2B). However, TCDD failed to induce adseverin in thymic stroma (Figs. 2A and 2B). This is in contrast to CYP1A1 gene expression, which was induced in both thymocytes and stroma (Figs. 2C and 2D). Induction of adseverin is not a common effect of agents causing thymus atrophy. Although the TCDDregulated induction of adseverin seems to be mediated via the AhR, we considered the possibility that increased levels of adseverin in thymocytes could be a common early effect of different agents that cause thymus atrophy. This was investigated by using RT-PCR and comparing the effects of TCDD, E2, and DEX on
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FIG. 2. Effects of TCDD on the mRNA levels of adseverin (Ads) (A and B) and CYP1A1 (CYP) (C and D) in isolated thymocytes, whole thymus and thymic stroma from FTOCs. The EtBr-stained gel images (A and C) where each lane represents one out of three individual samples are shown. The data shown (B and D) are the mean of three independent experiments expressed as target gene/HPRT ratios in vehicle (䊐) or TCDD-exposed (■) thymocytes, whole thymus, and stroma. Error bars indicate SE. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001, with respect to vehicle-treated, matched control.
the adseverin mRNA levels in thymocytes. There was a marked, 8.3-fold, increase in adseverin mRNA levels in thymocytes 24 h after exposure to 10 g TCDD/kg (Fig. 3). At this time-point there was no effect on cellularity in the thymus by TCDD (Fig. 4). However, at 1 week of exposure the cell number was reduced to 40% of control (Fig. 4). E2 is known to induce thymus atrophy with
FIG. 3. Effects of TCDD, E2, and DEX on the level of adseverin mRNA in thymocytes revealed by RT-PCR. Gel images of the separated PCR products were quantified by densitometric analysis and the data shown are the mean of three independent experiments expressed as the fold of induction of adseverin above control. The ratio of adseverin/HPRT in the controls was defined as 1 (broken line). Error bars indicate SE *P ⬍ 0.05, with respect to vehicletreated, matched control.
similar kinetics to TCDD (18) and was accordingly also examined at 24 h of exposure. A dose of 5 mg E2/kg had no effect on the level of adseverin mRNA or the thymocyte number at 24 h, but still reduced the cell number to 26% of control, at 1 w of exposure (Figs. 3 and 4). In contrast to TCDD and E2, DEX induces a very rapid but transient thymic atrophy (19) and its effect was therefore analyzed at 3, 6, and 24 h of exposure. At the
FIG. 4. Effects of TCDD, E2 and DEX on thymic cell number. Data shown are the mean thymic cell number based on three individual mice, expressed as % of control (broken line) with the cell number in matched, vehicle-treated controls defined as 100%. Error bars indicate SE. *P ⬍ 0.05, with respect to vehicle-treated, matched control.
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dose of 5 mg DEX/kg no effect on the adseverin mRNA level was observed at 3 or 6 h of exposure (Fig. 3) while a reduction in the cell number was observed after 6 h (Fig. 4). At 24 h of exposure to DEX, there was a slight but not significant increase, 1.7-fold, in adseverin levels (Fig. 3). At this time-point there was marked thymus atrophy with the cell number being 40% of that in control thymi (Fig. 4). DISCUSSION Reduced proliferation and increased differentiation and apoptosis are effects that all have been associated with TCDD-induced thymic atrophy (15, 20, 21). Similar effects have also been demonstrated in megakaryoblastic leukemia cells by reintroduction of the adseverin expression (8). Considering that all stages in lymphocyte life—lymphocyte development, migration and activation—are associated with profound changes in cell morphology and cell signaling both of which require actin modulation (22–24), we believe that adseverin might be a mediator of some of the immunotoxic effects of TCDD. Here, we have further characterized the TCDD-induced adseverin expression in the thymus, reported previously by us (3), to strengthen the evidence for the role of adseverin in TCDD-induced thymic atrophy and precisely define the cellular target cells. By analyzing thymocytes from different AhRchimeric mice, we have shown that adseverin is induced in AhR-expressing thymocytes upon TCDD exposure, regardless of the AhR genotype of the thymic stroma. A similar observation was made for CYP1A1, the most frequently used marker for TCDD activation. The gene expression of adseverin was increased by TCDD to roughly the same level in thymocytes from AhR ⫹/⫹ 3 AhR ⫹/⫹ and AhR ⫹/⫹ 3 AhR ⫺/⫺ chimeras. However, for unknown reasons there was a higher variability in the levels of adseverin mRNA in thymocytes from both vehicle and TCDD-treated AhR ⫹/⫹ 3 AhR ⫺/⫺ chimeras, compared to the AhR ⫹/⫹ 3 AhR ⫹/⫹ thymocytes and therefore the fold of induction was lower in these mice. The constitutive level of adseverin in AhR ⫺/⫺ thymocytes was not significantly different from the levels in AhR ⫹/⫹ thymocytes (Fig. 1B). This could indicate that the AhR is not involved in the basal transcriptional regulation of adseverin in thymocytes under normal conditions. However, another interpretation is that the expression of adseverin is critical for normal cell development, as indicated in the study by Zunino and co-workers (8), and therefore alternative pathways to regulate adseverin gene expression have evolved. It is clear however, that presence of a functional AhR in thymocytes, is pivotal for the regulation of adseverin by TCDD, since no adseverin induction was observed in AhR ⫺/⫺ thymocytes after TCDD exposure. The adseverin induction observed here also coin-
cides with the TCDD-induced thymic effects previously reported by Staples and co-workers (9). In these chimeras they observed that TCDD induces thymus atrophy in mice with AhR ⫹/⫹ hematopoietic cells but not in mice with AhR ⫺/⫺ hematopoietic cells, irrespective of the AhR genotype of the stroma. Although the induction of adseverin in thymocytes and the reduction in thymic weight and cellularity is due to a direct effect on thymocytes it is also possible that TCDD can induce adseverin in the thymic stroma. To investigate this effect we used FTOCs in combination with deoxyguanosine. In controls the constitutive level of adseverin was similar for whole thymus, stroma and thymocytes. However, no adseverin induction could be detected in the stroma while induction was observed in both whole thymus and thymocytes. The fold of adseverin induction in TCDD-treated FTOCs is lower than the fold of induction we have observed in TCDD-exposed adult thymocytes. It should also be noted that the ratio of adseverin/HPRT is higher in control thymocytes from FTOC as compared to adult thymocytes. This may be due to in vitro culture conditions or may reflect a real difference in adseverin expression between adult and fetal thymus. Preliminary data indicates that immature thymocytes express higher levels of adseverin than more mature subsets (our own unpublished data). In contrast to adseverin, CYP1A1 was induced in both stroma and thymocytes, and the level of induction was approximately the same in the two compartments of FTOCs. This result shows that with respect to CYP1A1 induction TCDD has an effect on the stroma. Thus, while CYP1A1 seems to be induced in most cell types upon TCDD exposure, the induction of adseverin is not only confined to lymphoid tissues, but more specifically to the hematopoietic compartment. This agrees with previous results from our group (3) and others showing that certain TCDDmediated effects on gene expression are tissue- or cellspecific (25). Although we have shown here that TCDD has an AhR-dependent effect on adseverin gene regulation in thymocytes, we also wanted to know if other agents causing thymic atrophy could induce adseverin gene expression in an AhR–independent way. We used DEX and E2, agents with well documented immunomodulatory properties, which also induce thymic atrophy, although by different mechanisms (19, 26). We have previously shown that the induction of adseverin by TCDD appears before any sign of thymus atrophy is evident (3). Therefore, we found it relevant to examine the effects of the different compounds early in the time course of atrophy. TCDD was used as a positive control and in agreement with our previous study (3), adseverin induction was observed in thymocytes before any signs of atrophy was evident, 24 h after exposure to TCDD. In contrast, E2— known to induce thymus atrophy with similar kinetics to TCDD (18, 26)—and
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DEX— known to induce atrophy faster than TCDD— did not induce adseverin expression significantly within 24 h. A small but not statistically significant adseverin induction was observed after 24 h of DEX exposure, a time when the thymocyte number was already reduced by approximately 60%. It is possible that this slight increase in adseverin level is due to an indirect effect of DEX, which selectively reduces the number of double positive thymocytes (18). Our preliminary data show that the DN subset has a higher constitutive expression of adseverin than DP cells (unpublished data). Thus, if the remaining cells have a higher constitutive level of adseverin, a relative increase would be detected. To summarize our findings, the TCDD-induced adseverin expression in the thymus is restricted to thymocytes and dependent on AhR activation in these cells. In addition, the induction of adseverin is not a common toxic effect of agents causing thymic atrophy but rather seems to be strictly regulated. The cellspecific induction of adseverin by TCDD indicates that although the presence of an AhR is obligatory for TCDD-induced adseverin expression, other factors are also required to mediate this response. Such factors remains to be identified but could be transcription factors specifically expressed in the developing thymocytes. Analysis of the promoter region of adseverin as well as functional studies of adseverin may be helpful in revealing such regulatory factors and hence new mechanistic aspects on TCDD-induced immunotoxicity.
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ACKNOWLEDGMENTS The authors thank Nancy Fiore, J. Erin Staples, and Mike Laiosa (all of the Department of Microbiology and Immunology, SUNY Upstate Medical University) for generating the original samples from chimeras, for organizing samples for shipment to Uppsala and for discussion of the results. We also thank Raili Engdahl and Lena Norgren (Department of Pharmaceutical Biosciences, Div. of toxicology, Uppsala University) for technical assistance.
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