Immune-Specific Up-Regulation of Adseverin Gene Expression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin CAMILLA SVENSSON and KATARINA LUNDBERG

To identify genes that are regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and possibly involved in TCDD-induced immunotoxicity, we used the differential display technique to screen for differentially expressed genes in the mouse thymus. Here we show that TCDD increased the expression of adseverin, a Ca-dependent, actin-severing protein. The induction of adseverin is doseand time-dependent in parallel with the induction of CYP1A1, which is currently the most frequently used marker for TCDD exposure. A comparison between mouse strains with different TCDD responsiveness indicated that the induction of adseverin is dependent on the aryl hydrocarbon receptor, a transcription factor known to mediate most of TCDD’s biological effects. Examination of additional organs revealed that the up-regulation of the adseverin gene expression is immune-specific. Using an anti-adseverin antibody, we confirmed the induction of adseverin by TCDD at the protein level and it was confined to the thymic cortex, which harbors immature thymocytes that are known target cells of TCDD. Considering adseverin’s role in actin cytoskeletal reorganization, our observations reveal new mechanistic aspects of how TCDD might exert some of its immunotoxic effects. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other structurally related halogenated aromatic hydrocarbons are persistent environmental pollutants that induce a wide variety of biological and toxic effects in mammals (reviewed in Birnbaum and Tuomisto, 2000). The immune system is one of the most sensitive targets for TCDD; signs of TCDD exposure include thymus atrophy and suppression of cell-mediated and humoral immune responses (reviewed in Kerkvliet, 1995). In fetal and adult mouse thymus, TCDD causes a rapid but transient decrease in cell proliferation followed by a reduction in thymocyte number (Lundberg et al., 1990). In addition, TCDD seems to affect thymocyte differentiation, resulting in a relatively higher number of CD8 thymocytes (Esser and Welzel, 1993). In contrast, the cell number and CD4/CD8 ratio in peripheral lymphoid organs is largely unaffected by TCDD unless the mice have been challenged with an antigen (Lundberg et al., 1991). Thus, within the immune system, TCDD seems to preferentially affect proliferating and/or differentiating cells. Different mechanisms have been proposed to explain TCDD-induced thymus atrophy and both the thymic stroma (Greenlee et al., 1985; Kremer et al., 1994) and the thymocytes (McConkey et al., 1988; Staples et al., 1998b) have been suggested to be direct targets. One of the more recently proposed mechanisms comes from an observation in fetal thymus organ cultures where TCDD induced the cell-cycle inhibitor p27 (Kolluri et al., 1999). This up-regulation of p27 could explain the initial decrease in thymocyte proliferation and number observed after TCDD exposure. However, although the proliferation returns to normal within a few days, the cell number in the thymus remains low for several weeks (Lundberg et al., 1990; Staples et al., 1998a). Thus, to explain atrophy duration other or additional mechanisms must be considered. One such mechanism is the reduced ability of early progenitor cells in the bone marrow and fetal liver to seed the thymus after TCDD-exposure (Fine et al., 1990). Another hypothesis is that TCDD initiates apoptosis in immature thymocytes (McConkey et al., 1988), although that theory has been questioned (Staples et al., 1998a). Thus, to get a clear picture of TCDD-induced thymic atrophy more information is needed. Most effects of TCDD are believed to be mediated through its high-affinity binding to the aryl hydrocarbon receptor (AhR), a ubiquitously expressed transcription factor, and the AhR-nuclear translocator protein. This complex binds to dioxin-responsive elements in the promoter region of the target genes and regulates their transcription (Hankinson, 1995). This work was supported by the Swedish council for work life research, Pnr 95–0437. ABBREVIATIONS: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aryl hydrocarbon receptor; PBS, phosphate-buffered saline; FCS, fetal calf serum; IL, interleukin; RT, reverse transcription; PCR, polymerase chain reaction; HPRT, hypoxanthine phosphoribosyl nucleotide transferase; ab, antibody; differential display, differential display RT-PCR; bp, base pair(s); PIP2, phosphatidylinositol 4,5-bisphosphate; PLCg, phospholipase Cg. 0026-895X/01/6001-135–142$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 1 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 784/911438 Mol Pharmacol 60:135–142, 2001 Printed in U.S.A. 135 at A PE T Jornals on Jne 2, 2017 m oharm .aspeurnals.org D ow nladed from Despite much information on the AhR, its physiological function remains unclear and no natural ligand has been identified. The list of TCDD-regulated genes is continuously growing and includes genes coding for xenobiotic metabolizing enzymes (CYP1A1, CYP1B1, glutathione-S-transferase), cytokines (IL-1b, transforming growth factor-b, plasminogen activator inhibitor-2), and others (major histocompatibility complex Q1b, Ecto-ATPase, p27) (Whitlock, 1993; Okey et al., 1994; Dong et al., 1997; Gao et al., 1998; Kolluri et al., 1999). CYP1A1 is the most characterized of these AhR-regulated genes and has become a useful marker for TCDD exposure. However, none of the above-mentioned genes, except perhaps Kip1 (Kolluri et al., 1999), has shown a direct causal relationship with the adverse effects of TCDD. To identify genes that might be involved in TCDD-induced immunotoxicity, we used the differential display reverse transcription-polymerase chain reaction (differential display) technique (Liang and Pardee, 1992) to screen for differentially expressed genes in mouse thymus. The thymus is a critical target organ because of its central role in T cell development, which has activation steps similar to those in mature T cells (Zuniga-Pflucker et al., 1993). Therefore, studies of TCDD-induced gene regulation in the thymus may give mechanistic explanations of immunotoxicity in both the thymus and in peripheral lymphoid organs. Here we show that TCDD induces the expression of adseverin, an actin-binding protein important for normal cell functions. We also examine the induction of adseverin in other organs and in two different mouse strains and conclude that the up-regulation of adseverin gene expression is immune-specific and dependent on the AhR. Materials and Methods Experimental Animals. Female C57BL/6J (B6) AhR (TCDD high responding) mice were bred in our own animal facilities. Original breeding pairs were purchased from B&K Universal (Solna, Sweden). Four-week-old female DBA/2J AhR (TCDD low responding) mice were obtained from Møllegaard og Bomhultgård, Ry, Denmark, and allowed to acclimatize for a minimum of 1 week before use. All mice were housed and cared for in accordance with the National Research Council guidelines. Briefly, they were housed in a pathogen-free environment on a 12-h light/dark cycle and given a standard pellet diet and tap water ad libitum. Treatments. TCDD (98.4% pure; Larodan Fine Chemicals, Malmö, Sweden) was dissolved in 1,4-dioxan and subsequently diluted with corn oil. At 5 to 6 weeks of age the mice were weightmatched, randomly allocated into treatment groups with 3–5 mice in each group and then injected i.p. with different concentrations of TCDD in a total volume of 10 ml/mouse. Control mice were administered the vehicle only. For the differential display experiments, B6 mice were exposed to 50 mg of TCDD/kg for 24 h. In the dose-response experiments, which were performed after 24 h of exposure, B6 mice received 0.5, 2.5, 5, 10, or 50 mg of TCDD/kg and DBA mice received 0.5, 5, 10, or 50 mg of TCDD/kg. In the time-response studies, B6 mice were injected a dose of 10 mg of TCDD/kg and killed 3 h, 6 h, 24 h, 1 week, 2 weeks, or 7 weeks later. For prenatal studies, pregnant B6 mice were administered 10 mg of TCDD/kg at day 12 or 17 of gestation and sacrificed 24 h later by cervical dislocation, after which thymus [gestational day (gd) 18] or liver (gd 13) were dissected from their fetuses. RNA Preparation. In all experiments, the mice were killed by CO2 asphyxiation and the thymuses were removed, weighed, and immediately dissolved in guanidinium isothiocyanate. Total RNA was isolated by phenol-chloroform extraction (Chomczynski and Sacchi, 1987) and treated with DNase I. From B6 mice exposed to 10 mg of TCDD/kg for 24 h, RNA samples were also prepared from lymph nodes, spleen, liver, adrenal glands, kidney, thymocytes, and bone marrow cells. Thymocytes were isolated by gently pressing the thymus through a steel mesh in sterile PBS with 3% FCS and passing the cell suspension over cotton wool followed by three rounds of washing in PBS with 3% FCS. Bone marrow cells were collected by cutting of the ends of the femur and flushing the cavity with sterile PBS with 3% FCS using a 25-gauge needle. RNA samples were also prepared from fetal liver and thymus. Differential Display. Differential display was performed on thymus samples from B6 mice exposed to 50 mg of TCDD/kg for 24 h using the Delta Differential Display Kit (CLONTECH, Palo Alto, CA). Briefly, reverse transcription (RT) of control and TCDD samples were done in a total volume of 10 ml/sample containing 2 mg of denatured total RNA, 0.1 mM oligo dT primer, 2 ml of 53 RT buffer (Promega, Madison, WI), 1 mM dNTP, and 200 U of Moloney murine leukemia virus reverse transcriptase. The reactions were incubated at 42°C for 60 min and then stopped by heating at 75°C for 10 min. The cDNA was then amplified in the presence of 1 mM arbitrary 59 primer and 1 mM oligo dT primer according to the manufacturer’s