2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes an Ah receptor-dependent and ARNT-independent increase in membrane levels and activity of p60Src

doi:10.1016/S1382-6689(97)00016-1

Environmental Toxicology and Pharmacology

Volume 3, Issue 3, July 1997, Pages 211-220

https://doi.org/10.1016/S1382-6689(97)00016-1 Get rights and content

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to affect various cellular activities including growth factor signal transduction, hormone responses, and cell differentiation. The purpose of this study was to examine more closely the very early effects of TCDD on protein tyrosine kinase activity, specifically p60^Src. We found that TCDD causes rapid changes in the plasma-microsomal membrane levels and activity of p60^Src in Hepa 1c1c7, Hepa c4 cells, and SR3Y1 cells, a p60^v-Src overexpressing cell line. Such cellular changes occur within 30 minutes after 10 nM TCDD treatment, as measured by Western blot analysis. TCDD's ability to increase p60^Src levels was found to be: (1) dose-dependent, with an estimated EC₅₀ between 10⁻¹⁰ and 10⁻¹¹ M TCDD; (2) Ah receptor (AhR)-dependent, since TCDD's effect was blocked by co-administration with 1 μM α-naphthoflavone, an AhR antagonist; and interestingly (3) ARNT-independent, since TCDD's effect was observed in Hepa c4 cells, an ARNT⁻ mutant cell line. Since ARNT is a heterodimerization partner of the AhR required for binding of the ligand-activated AhR to dioxin-responsive elements on DNA in the nucleus to transactivate genes controlled by the AhR, an alternative mechanism for TCDD's action is discussed which does not require ARNT. Along with increased membrane levels of p60^Src, we observed a corresponding increase in the activity of a 60 kDa protein tyrosine kinase using two different kinase detection assays. This effect of TCDD was also found to be AhR-dependent, ARNT-independent, and independent of de novo protein synthesis since cycloheximide was unable to completely abolish TCDD's effect. The present findings provide a potentially important mechanism by which TCDD can alter cell growth and differentiation.

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent member of a large class of structurally related chemicals called halogenated aromatic hydrocarbons (HAHs) that occur as environmental contaminants (Safe, 1990). There is considerable evidence that TCDD's biological activity is mediated through binding to a cytosolic receptor, termed the Ah receptor (AhR; Okey et al., 1994). Some of TCDD's effects in animals include body weight loss, immune suppression, teratogenicity, carcinogenicity, and death (Poland and Knutson, 1982, Gasiewicz, 1991). Additionally, TCDD causes hormone or growth factor-like effects. For example, mouse neonates treated with either TCDD or exogenously added epidermal growth factor exhibit premature incisor eruption, early eyelid opening, and body weight loss (Madhukar et al., 1988). TCDD has been termed a growth dysregulator because many of TCDD's effects appear to result from changes in cell growth and differentiation, such as cleft palate (Couture et al., 1990), XB cell hyperkeratinization (Knutson and Poland, 1980, Blankenship and Matsumura, 1994), accelerated differentiation of preimplantation embryos (Blankenship et al., 1993), and carcinogenesis (Sewall et al., 1993).

Because TCDD and related chemicals have been clearly shown to increase protein tyrosine kinase (PTK) activity, some researchers have suggested that the effects of TCDD and related chemicals on cell growth and differentiation are at least partially mediated through protein kinases, particularly protein tyrosine kinases (Madhukar et al., 1988, Clark et al., 1991, Archuleta et al., 1993, Ma and Babish, 1993). While the mechanism by which TCDD increases PTK activity remains unknown, it is clear that TCDD's effects on PTKs potentially represent a very important event in TCDD's toxicity for several reasons. First, many PTKs are very important regulators of cellular functions such as cell division and differentiation (Hunter and Cooper, 1985). Secondly, the activity of PTKs is tightly regulated within cells and any alteration in the regulation of PTKs may play a role in altered gene expression and carcinogenesis (Hunter, 1991, Velu, 1990). Finally, co-administration of protein kinase inhibitors with TCDD has been shown to prevent some of TCDD's toxic effects (Bombick et al., 1988, Muralidhara et al., 1994).

Previous studies in this lab have shown that a single treatment with TCDD results in a rise in the activity and quantity of p60^Src in vivo which lasts for a long time period in the case of liver plasma membranes (e.g. 20 days after the treatment; Bombick and Matsumura, 1987). The activity and the titer of p60^Src was also found to be increased by the action of TCDD in cell culture models (Bombick and Matsumura, 1987, Bombick et al., 1988). In this study, we tested the hypothesis that TCDD interacts with the AhR to trigger a subsequent increase in activity and increase in cellular localization of p60^Src in the plasma-microsomal membrane at an early stage in the sequence of events of TCDD's action. We found that by thirty minutes, TCDD had already increased membrane levels and kinase activity of p60^Src.

Section snippets

Chemicals

γ-³²P-adenosine triphosphate (ATP; 3000 Ci/mmol) was obtained from Amersham. Powdered cell culture media and antibiotics were purchased from GIBCO (Grand Island, NY). All other biochemicals were procured from Sigma Chemical Company (St. Louis, MO). 2,3,7,8-TCDD was a gift from Dow Chemical Company (Midland, Michigan) and was >99.9% pure (by GLC). Stock solutions of chemicals used in this study were prepared in p-dioxane. A Hamilton^® syringe was used to add chemicals directly to cultured cells.

TCDD increases membrane levels of p60^src

Hepa 1c1c7 cells were treated with 10 nM TCDD in p-dioxane or with p-dioxane alone (as a control) for several time periods ranging from 30 min to 48 h. Membrane levels of p60^Src were determined by Western analysis as shown in Fig. 1 by a representative experiment. Only a single protein band was detected on the blot with a molecular weight of 60 kDa, indicating the presence of p60^Src. Relative levels of p60^Src shown in Fig. 1 were quantitated from the developed X-ray film by using a computerized

Discussion

TCDD's ability to affect protein kinase activity, particularly protein tyrosine kinases (PTKs), has been clearly shown to be an important step in TCDD's mechanism of action (Madhukar et al., 1984, Enan and Matsumura, 1993, Ma and Babish, 1993; reviewed in Matsumura, 1994). Previous studies in our laboratory have shown that TCDD increases plasma membrane levels and activity of p60^Src at time points long after TCDD exposure (Bombick and Matsumura, 1987). Furthermore, recent evidence demonstrates

Acknowledgements

Work funded by research grants ES03575, ES02533, and ES05707 (Molecular Biology Core) from the National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes an Ah receptor-dependent and ARNT-independent increase in membrane levels and activity of p60Src

Abstract

Introduction

Section snippets

Chemicals

TCDD increases membrane levels of p60src

Discussion

Acknowledgements

Reprod. Toxicol.

Biochem. Pharmacol.

J. Biol. Chem.

Cell

Methods in Enzymology

Cell

Mol. Cell. Endocrinol.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

Toxicol. Lett.

J. Biol. Chem.

Curr. Opin. Genet. Dev.

Mol. Cell. Endocrinol.

J. Biol. Chem.

7,12-Dimethylbenz[a]anthracene activates protein-tyrosine kinases Fyn and Lck in the HPB-ALL human T-cell line and increases tyrosine phosphorylation of phospholipase C-g1, formation of inositol 1,4,5-trisphosphate, and mobilization of intracellular calcium

Proc. Natl. Acad. Sci. USA

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes elevation of the levels of the protein tyrosine kinase pp60c-src

J. Biochem. Toxicol.

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes increases in expression of c-erb-A and levels of protein-tyrosine kinases in selected tissues of responsive mouse strains

Proc. Natl. Acad. Sci. USA

Tyrosine dephosphorylation of pp60c-src is stimulated by a serine/threonine phosphatase inhibitor

Oncogene

2,3,7,8-Tetrachlorodibenzo-p-dioxin stimulation of tyrosine phosphorylation in B lymphocytes: potential role in immunosuppression

Mol. Pharmacol.

Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets

Mol. Cell. Biol.

A critical review of the developmental toxicity and teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: recent advances toward understanding the mechanism

Teratology

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpressor cells

J. Cell Biol.

Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

Nucleic Acids Res.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes an Ah receptor-dependent and ARNT-independent increase in membrane levels and activity of p60^Src

TCDD increases membrane levels of p60^src

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes elevation of the levels of the protein tyrosine kinase pp60^c-src

Tyrosine dephosphorylation of pp60^c-src is stimulated by a serine/threonine phosphatase inhibitor

Redistribution of activated pp60^c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets