Inhibition of connexin43 gap junction channels by the endocrine disruptor ioxynil
Introduction
Gap junctions are specialized plasma membrane domains enriched in intercellular channels that mediate transport of ions, metabolites and small signaling molecules (less than 1 kDa) between neighbouring cells (Saez et al., 2003). Gap junctions enable cells to directly cooperate metabolically and play important roles in the control of cell growth and differentiation, maintenance of tissue homeostasis and in embryogenesis (Wei et al., 2004). Gap junctions are expressed in nearly all cell types of the human body. Their extensive expression is reflected in the diversity of their physiological functions. For instance, gap junctions play important roles in the cardiovascular, digestive, reproductive and immune systems (Saez et al., 2003). Dysfunctional intercellular communication via gap junctions has been causally linked to a number of pathological conditions, including cataracts, deafness, skin disorders, heart failure, neuropathies and cancer (Chadjichristos et al., 2006, Mesnil, 2002, Wei et al., 2004).
The gap junction channels are made of transmembrane proteins called connexins (Willecke et al., 2002). The connexin protein family consists of 21 known members in humans, of which the best-studied isoform is connexin43 (Cx43). Connexins are synthesized in the endoplasmic reticulum and transported to the Golgi apparatus where they oligomerize into structures called hemichannels (Segretain and Falk, 2004). Each hemichannel consists of six connexin molecules. The hemichannels are transported to the plasma membrane and may there dock with hemichannels in the adjacent cell, thereby forming intercellular channels. Gap junctions have a high turnover rate, and have a half-life of 1.5–5 h in most tissue types. Formation and degradation of gap junctions are dynamic processes, and regulation of gap junction assembly and turnover is considered critical in the control of intercellular communication (Segretain and Falk, 2004).
Gap junction channels may be acutely regulated in response to various stimuli, including changes in voltage, pH, and connexin phosphorylation. Phosphorylation of connexins regulates several steps of the connexin life cycle, including its trafficking to the plasma membrane, assembly into gap junctions, channel gating, internalization, and degradation. Various kinases have been associated with connexin phosphorylation, including mitogen-activated protein (MAP) kinase, protein kinase C (PKC), and Src (Rivedal and Opsahl, 2001, Sirnes et al., 2008, Lampe and Lau, 2004).
Several lines of evidence indicate that dysfunction of gap junctions plays an important role in cancer development. Firstly, cancer cells usually have completely or partly reduced levels of gap junction intercellular communication (Leithe et al., 2009, Mesnil, 2002, Yamasaki and Naus, 1996). Secondly, re-establishing gap junction intercellular communication by transfection of connexin genes into tumorigenic cells has been shown to normalize growth and differentiation properties in vitro and in vivo (Langlois et al., 2010, Qin et al., 2002, Eghbali et al., 1991, Goldberg et al., 2000, Omori and Yamasaki, 1999). Thirdly, knockout of connexin genes in mice has been shown to result in higher susceptibility to radiation- or chemical-induced tumor formation (Avanzo et al., 2004, Dagli et al., 2004, King and Lampe, 2004, Temme et al., 1997). Finally, many growth factors, oncogenes and tumor promoters are potent inhibitors of gap junction intercellular communication (Trosko and Ruch, 1998, Warn-Cramer and Lau, 2004). Connexins are also involved in the cellular resistance to several types of toxic stress, potentially in a gap junction independent manner (Lin et al., 2003). At the same time, such stress can affect the degradation rate and life cycle of connexins (VanSlyke and Musil, 2005). Some studies also suggest that gap junctions mediate both cell survival and cell death signals (Krysko et al., 2005). However, the molecular mechanisms by which connexins regulate cell growth and protect against toxic insults are still incompletely understood. To clarify their roles in carcinogenesis and toxicity, more information on how gap junctions are affected by different chemical compounds is needed. Furthermore, knowledge on the molecular mechanisms underlying inhibition and degradation of gap junctions in response to chemicals may have important implications for better understanding of their toxicity.
Toxicological risk evaluation is based on results from several biological assays. To evaluate a substance for carcinogenic risk, rodent cancer bioassays in combination with assays for genotoxicity are often used. However, since many tumor-inducing chemicals do not induce direct DNA damage, it is crucial to obtain mechanistic knowledge of their biological effects in order to obtain reliable predictions on their possible relevance in human disease induction. The ability of chemicals to inhibit gap junction channels is associated with carcinogenicity, but not with direct genotoxicity (Rosenkranz et al., 2000). This makes knowledge on the ability of chemicals to block gap junctions an important supplement in the evaluation of chemicals with regard to human health effects.
The pesticide ioxynil (4-hydroxy-3,5-diiodobenzonitrile) has previously been reported to act as an endocrine disrupting chemical and has a range of actions on the thyroid axis (Ishihara et al., 2003, Sugiyama et al., 2005, Morgado et al., 2007, McKinlay et al., 2008). Furthermore, both ioxynil and its derivative ioxynil octanoate (4-cyano-2,6-diiodophenyl octanoate) have been reported to induce tumors in animal bioassays (Report Directorate, 2004). However, the molecular mechanisms underlying the possible tumorigenic potential of these compounds are unknown. In the present study we provide evidence that phenolic and octanoic ioxynil are potent inhibitors of Cx43 gap junction channels and that both compounds induce degradation of Cx43. The data further indicate that the two chemicals differ in their ability to activate MAP kinase and phosphorylate Cx43. The findings may have implications for risk assessment of ioxynil and its derivates, and better understanding of the mechanisms underlying dysregulation of Cx43 gap junctions in carcinogenesis.
Section snippets
Cells
The non-tumorigenic rat liver epithelial cell line IAR20 was obtained from International Agency for Research on Cancer, Lyon, France. The cells were originally isolated from the liver of normal inbred BD-VI rats (Montesano et al., 1975), and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco BRL Life Technologies, Inchinnan, UK). IAR20 cells were plated onto 60 mm (1 × 106) Petri dishes (Costar, Cambridge, MA, USA) 48 h before
Results
The chemical structures of ioxynil and ioxynil octanoate are shown in Fig. 1A. Ioxynil octanoate contains an eight carbon long chain attached to the phenolic ioxynil by an ester bond, resulting in significant change in chemical properties such as solubility in water and organic solvents. As a cellular model system for analyzing the effect of ioxynil and ioxynil octanoate on gap junction intercellular communication, the rat liver epithelial cell line IAR20 was used. These cells endogenously
Discussion
Toxicological risk evaluation is based on results from a large number of biological tests. The evaluation is often complex and it is important that it is based on the best and most relevant assays. To evaluate a substance for carcinogenic risk, rodent cancer bioassays in combination with several assays for genotoxicity are often used. This is because accumulation of genetic changes in somatic cells is essential for the genesis of cancer, and that the ability of a substance to directly induce
Acknowledgments
We thank Astri Nordahl and Zeremariam Yohannes for excellent technical assistance. The work was supported by the Norwegian Cancer Society and the Research Council of Norway.
References (50)
- et al.
The effect of endocrine disrupting chemicals on thyroid hormone binding to Japanese quail transthyretin and thyroid hormone receptor
Gen. Comp Endocrinol.
(2003) - et al.
The effects of connexin phosphorylation on gap junctional communication
Int. J. Biochem. Cell Biol.
(2004) - et al.
Ubiquitination and down-regulation of gap junction protein connexin-43 in response to 12-O-tetradecanoylphorbol 13-acetate treatment
Journal of Biological Chemistry
(2004) - et al.
Effect of selected pesticides and their ozonation by-products on gap junctional intercellular communication using rat liver epithelial cell lines
Chemosphere
(2001) - et al.
Endocrine disrupting pesticides: implications for risk assessment
Environ. Int.
(2008) Connexins Cancer. Biol. Cell
(2002)- et al.
Disruption of thyroid hormone binding to sea bream recombinant transthyretin by ioxinyl and polybrominated diphenyl ethers
Chemosphere
(2007) - et al.
Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication
Journal of Biological Chemistry
(2002) - et al.
Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells
Experimental Cell Research
(2005) - et al.
Regulation of connexin biosynthesis, assembly, gap junction formation, and removal
Biochimica et Biophysica Acta
(2004)
The detergent resistance of Connexin43 is lost upon TPA or EGF treatment and is an early step in gap junction endocytosis
Biochemical and Biophysical Research Communications
Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication
Biochemical and Biophysical Research Communications
High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32
Curr. Biol.
Regulation of gap junctions by tyrosine protein kinases
Biochimica et Biophysica Acta
Increased susceptibility to urethane-induced lung tumors in mice with decreased expression of connexin43
Carcinogenesis
Connexins in atherosclerosis
Advances in Cardiology
Delayed liver regeneration and increased susceptibility to chemical hepatocarcinogenesis in transgenic mice expressing a dominant-negative mutant of connexin32 only in the liver
Carcinogenesis
Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin 32 cDNA retards growth in vivo
Proceedings of the National Academy of Sciences of the United States of America
Connexin43 suppresses MFG-E8 while inducing contact growth inhibition of glioma cells
Cancer Research
Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate
Journal of Cellular Physiology
Mice deficient for the gap junction protein Connexin32 exhibit increased radiation-induced tumorigenesis associated with elevated mitogen-activated protein kinase (p44/Erk1, p42/Erk2) activation
Carcinogenesis
Regulation of gap junction intercellular communication by the ubiquitin system
Cell Signal.
Gap junctions and the propagation of cell survival and cell death signals
Apoptosis
The Tumor-Suppressive Function of Connexin43 in Keratinocytes Is Mediated in Part via Interaction with Caveolin-1
Cancer Research
Epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43
Journal of Cell Science
Cited by (15)
Endocytic trafficking of connexins in cancer pathogenesis
2023, Biochimica et Biophysica Acta - Molecular Basis of DiseaseWaterborne exposure of zebrafish embryos to micromole concentrations of ioxynil and diethylstilbestrol disrupts thyrocyte development
2013, Aquatic ToxicologyCitation Excerpt :Interestingly, zebrafish exposed to waterborne (μm) clofibrate (cholesterol reducing drug) have severe heart deformities and altered thyroid development and only the most ventral (primordial) thyroid follicles develop (Raldúa et al., 2008). The well established disruptive effect of IOX on tight junction formation and function (Leithe et al., 2011) may certainly explain how IOX can affect cardiac development (and consequently thyrocyte development), since these cellular structures are needed to ensure proper mechanical cellular resistance, tissue integrity and organ function (Auman et al., 2007; Holtzman et al., 2007; Schoenebeck and Yelon, 2007). DES is an estrogen-like compound and it is highly probable that some of the effects observed in the present study were mediated by the estrogen signaling pathway on which it has been demonstrated to act (Henley and Korach, 2010; le Maire et al., 2010; Ma, 2009).
Connexin-dependent signaling in neuro-hormonal systems
2012, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :Recent evidence indicates that these chemicals also affect connexin expression and function. Thus, the pesticide ioxynil, and its derivative ioxynil octanoate, decrease the expression of Cx43, its phosphorylation and the function of the cell-to-cell channels this connexin makes [269]. It is yet not established whether these effects account for the effects of these endocrine disruptors on tumorigenesis and thyroid alterations.
The E3 ubiquitin ligase ITCH negatively regulates intercellular communication via gap junctions by targeting connexin43 for lysosomal degradation
2024, Cellular and Molecular Life SciencesBrain disorders and chemical pollutants: A gap junction link?
2021, Biomolecules