Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling

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Abstract

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereomicroscopically and processed to paraffin sections. TCDD exposure impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling.

Introduction

Dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are ubiquitous environmental pollutants with a wide range of adverse effects on different species. They are resistant to biological degradation, accumulate in tissue lipid and enrich in the food chain. Dioxins pass the placenta and are lactationally transferred to the offspring, including humans (Furst et al., 1994). Most vertebrate species are responsive to developmental toxicity of TCDD but their sensitivities to various toxic endpoints differ (Peterson et al., 1993). Developmental effects of TCDD range from, e.g., cardiovascular toxicity in fish (Cantrell et al., 1998, Hornung et al., 1999, Teraoka et al., 2002) and birds (Walker and Cantron, 2000) to cleft palate and hydronephrosis in mice (Abbott and Birnbaum, 1990, Abbott et al., 1987a, Pratt et al., 1984) and disturbed development of reproductive tract in rats (Hurst et al., 2000). Among hormonally responsive organs that are sensitive to gestational, lactational and/or pregnancy-related exposure of rats and mice are the mammary gland (Fenton et al., 2002, Vorderstrasse et al., 2004) and prostate (Ko et al., 2002, Roman et al., 1998). Clinical findings suggest that children's developing teeth can be sensitive to dioxins even at environmental levels (Alaluusua et al., 1999). Experimental studies show that TCDD is toxic to developing rodent teeth as well and that morphological consequences of exposure essentially depend on not only the dose but also the stage of tooth development (Gao et al., 2004, Kattainen et al., 2001, Miettinen et al., 2002, Partanen et al., 2004).

Development of the salivary glands starts as an intrusion of embryonic oral epithelium into the underlying mesenchyme and continues under the regulation of epithelial–mesenchymal interactions through clearly distinguishable morphological stages (Hoffman et al., 2002, Jaskoll and Melnick, 1999). As the first step of branching morphogenesis, clefts are formed in the globular initial bud. This then undergoes division to daughter buds, the basal portion of which lengthens to form cords, i.e., future ducts. Repeated cleft formation, budding and branching of the proliferating gland anlage epithelium is accompanied by differentiation of cells along ductal and acinar cell lines. The epithelium, forming the functional portion of a fully developed salivary gland, does not undergo branching in the absence of mesenchyme or its substitutes (Nogawa and Takahashi, 1991). Salivary gland branching morphogenesis has been found to involve a variety of factors, including extracellular matrix constituents (Nakanishi et al., 1988), regulators of their degradation (Nakanishi et al., 1986), basement membrane components (Kadoya et al., 2003, Nogawa and Takahashi, 1991) and growth factors such as members of the epidermal growth factor (EGF) (Jaskoll and Melnick, 1999, Kashimata et al., 2000, Koyama et al., 2003, Morita and Nogawa, 1999, Nogawa and Takahashi, 1991), fibroblast growth factor (FGF) (Hoffman et al., 2002, Morita and Nogawa, 1999) and tumor necrosis factor (Melnick et al., 2001) families and their receptors. Many of the signaling pathways are developmentally stage and/or site specific (Jaskoll and Melnick, 1999, Koyama et al., 2003, Morita and Nogawa, 1999). Synthesis of the extracellular matrix glycoprotein fibronectin (FN) by epithelial cells at the sites of cleft formation, facilitating conversion of cadherin-mediated cell–cell interactions (Menko et al., 2002, Sakai et al., 2003) to cell–matrix interactions, has been shown to be of crucial importance to salivary gland branching morphogenesis (Sakai et al., 2003).

The majority of TCDD effects, including developmental effects, are thought to be mediated by the aryl hydrocarbon receptor (AhR) (Gonzalez and Fernandez-Salguero, 1998, Gu et al., 2000, Mimura et al., 1997, Peters et al., 1999). AhR is a transcription factor possessing a subunit for high-affinity binding of TCDD and related compounds (Schmidt and Bradfield, 1996). At the early phases of response, AhR dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. Subsequent binding of the heterodimer to dioxin-responsive elements of DNA initiates a cascade of events eventually leading to the transcription of mRNAs for xenobiotic-metabolizing enzymes such as cytochrome P4501Al (CYP1Al) and probably a number of other proteins (Schmidt and Bradfield, 1996). A high-affinity endogenous ligand of AhR is currently unknown but the receptor plays a central role in the regulation of cell cycle and apoptosis (Nebert et al., 2000). Interference by TCDD with mouse tooth morphogenesis involves enhanced apoptosis of dental epithelial cells but only of those that are predetermined to undergo apoptosis even during normal development (Partanen et al., 2004).

The concept that the mediation of TCDD effects could involve epidermal growth factor receptor (EGFR) signaling goes back to the finding that effects of TCDD in newborn mice, such as precocious eyelid opening and incisor tooth eruption, and retardation of hair growth (Madhukar et al., 1984, Madhukar et al., 1988) resemble the classical effects of EGF. EGFR is a transmembrane receptor protein tyrosine kinase, the ligands of which are several different EGF-related peptides including the EGF and transforming growth factor α (TGFα). Knowledge of the functional spectrum of EGFR has increasingly extended from a promoter of cell proliferation to a downstream element in various signaling pathways (Hackel et al., 1999), acting in co-operation with, e.g., integrin-dependent adhesion (Cabodi et al., 2004, Moro et al., 1998). While TCDD can modulate EGFR signaling by modifying its density or binding capacity (Sewall et al., 1995, Tuomisto et al., 1996) or the expression levels of its main ligands, it does not bind to the receptor directly (Madhukar et al., 1984).

TCDD is a potent modulator of epithelial cell growth and differentiation (Loertscher et al., 2002). Accordingly, development of many organs formed as a result of epithelial–mesenchymal interactions is sensitive to TCDD. Here we show that TCDD impairs mouse submandibular gland branching morphogenesis and that the impairment is associated with activation of AhR and reduced EGFR signaling.

Section snippets

Organ culture

The use of animals was approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Science of the University of Helsinki. Pregnant mice (NMRI × NMRI) were anesthetized with CO2 and killed by cervical dislocation. Submandibular and sublingual glands from E13 mice (the day of vaginal plug designated E0) were dissected under a stereomicroscope and moved onto polycarbonate Nuclepore filters (pore size 0.1 μm; Corning Inc., New York, USA) supported by a stainless steel

Stages of submandibular and sublingual gland development at the start of culture

Submandibular and sublingual glands from E13 mouse embryos were cultured for 2 or 4 days in the presence of TCDD, EGF, FN or TCDD in combination with EGF or FN. Control explants were cultured in DMEM or with DMSO (vehicle control).

Stereomicroscopic examination of the explants at the start of culture showed that the submandibular gland was formed of an elongated epithelial stalk starting from the oral epithelium and ending in four to six buds. The sublingual gland appeared as a single epithelial

Discussion

This study showed that TCDD impairs epithelial branching morphogenesis of cultured mouse salivary gland and that the impairment is associated with induction of CYP1A1. Consistent with the enlarged buds, TCDD did not markedly affect cell proliferation. Increased epithelial apoptosis after TCDD exposure showed no definite distribution pattern and could not be correlated with reduced branching. Exogenous EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but

Acknowledgments

We thank Marjatta Kivekäs for expert technical assistance. This study was supported by EU (project QLK4-CT-1999-01446), Academy of Finland (Contract no. 206689), the Finnish Dental Society Apollonia and the Finnish Cultural Foundation.

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