Cell-type specificity of ectonucleotidase expression and upregulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

doi:10.1016/S0003-9861(02)00465-4

Archives of Biochemistry and Biophysics

Volume 407, Issue 1, 1 November 2002, Pages 49-62

https://doi.org/10.1016/S0003-9861(02)00465-4 Get rights and content

Abstract

We report here that induction of ectoATPase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is cell-type specific and not a generalized response to aryl hydrocarbon (Ah) receptor activation. TCDD increased [¹⁴C]-ATP and -ADP metabolism in two mouse hepatoma lines, Hepa1c1c7 and Hepa1-6 cells, but not in human hepatoma HepG2 or HuH-7 cells, human umbilical vein endothelial cells (HUVEC), chick hepatoma (LMH) cells, or chick primary hepatocytes or cardiac myocytes, even though all of those cell types were Ah receptor-responsive, as evidenced by cytochrome P4501A induction. To determine whether the differences in ectonucleotidase responsiveness to TCDD might be related to differences in cell-type ectonucleotidase expression, ATP and ADP metabolite patterns, the products of several classes of ectonucleotidases including ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), ectophosphodiesterase/pyrophosphatases (E-NPP enzymes) and ectoalkaline phosphatase activities were examined. Those patterns, together with results of enzyme assays, Western blotting, or semiquantitative RT–PCR show that NTPDase2 is the main ectonucleotidase for murine and human hepatoma cells, NTPDase3 for chick hepatocytes and LMH cells, and an E-NPP enzyme for chick cardiac myocytes. Evidence for NTPDase2 expression was lacking in all cells except the mouse and human hepatoma cells. TCDD increased expression of the NTPDase2 gene but only in the mouse and not in the human hepatoma cells. TCDD did not increase NTPDase3, NTPDase1, E-NPP, or alkaline phosphatase in any of the cell types examined. The failure of TCDD to increase ATP metabolism in HUVEC, chick LMH cells, hepatocytes, and cardiac myocytes can be attributed to their lack of NTPDase2 expression, while the increase in ATP metabolism by TCDD in the mouse but not the human hepatoma cells can be explained by differences in TCDD effects on mouse and human hepatoma NTPDase2 gene expression. In addition to characterizing effects of TCDD on ectonucleotidases, these studies reveal major differences in the complements of ectonucleotidases present in different cell types. It is likely that such differences are important for cell-specific susceptibility to extracellular nucleotide toxicity and responses to purinergic signaling.

Section snippets

Cell culture

Murine (Hepa1c1c7 and Hepa1-6) and chicken hepatoma (LMH) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human hepatoma cells (HuH-7 and HepG2) were gifts from Dr. Hugh Robertson (Weill Cornell Medical College, New York, NY). Hepa1c1c7 cells were grown in minimal essential medium α (MEMα), HuH-7 and Hepa1-6 in Dulbecco’s modified Eagle medium (DMEM), HepG2 cells in MEM, and LMH cells in Waymouth’s MB 752/1 medium, each supplemented with 10% fetal bovine serum

TCDD increases ATP metabolism in mouse hepatoma cells

TCDD treatment increased the rate of metabolism of extracellular ATP by mouse Hepa1c1c7 and Hepa1-6 cells (Fig. 2). TCDD increased the rates for ATP metabolism (Table 1) 2.3-fold for Hepa1c1c7 and 3-fold for Hepa1-6 cells (p<0.001). ADP metabolism was below the limits of detection for both mouse hepatoma cell lines, indicating that neither of these cell lines metabolized extracellular ADP to any appreciable extent.

TCDD does not increase ATP or ADP metabolism in other cell types

To determine whether the increase in ectoATPase activity induced by TCDD is a

Discussion

Our results demonstrate that induction of ectoATPase by TCDD is cell-type specific and limited to a single member of the ectonucleotidase family, NTPDase2. In addition, the induction by TCDD of ectoATPase is limited to mouse hepatoma cells among the eight cell types studied here. We confirm the reported increase in ectoATPase activity by TCDD in mouse Hepa1c1c7 cells [5] and show that the induction extends to mouse Hepa1-6 cells. On the other hand, our data indicate that TCDD does not increase

Acknowledgements

Supported by NIH Grant ES 03606 (to A.B.R.), by Merit Review grants from the Department of Veterans Affairs (A.J.M., M.J.B., J.H.F.D.), by NIH Grants HL 47073, HL 46403, and NS 41462 (A.J.M., M.J.B., J.H.F.D.), and by NIH Grant HL59915 (T.L.K.). We thank Alexander V. Birk, Ph.D. for helpful discussions, Dr. James Whitlock, Jr., for guidance in growing Hepa1c1c7 cells, and Dr. Hugh Robertson for providing HepG2 cells.

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Cell-type specificity of ectonucleotidase expression and upregulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Abstract

Section snippets

Cell culture

TCDD increases ATP metabolism in mouse hepatoma cells

TCDD does not increase ATP or ADP metabolism in other cell types

Discussion

Acknowledgements

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Dev. Biol.

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Biochem. Biophys. Res. Commun.

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