Glycine N-Methyltransferase Is an Example of Functional Diversity

Abstract

The cytochrome P-4501A1 (CYP1A1) gene is regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein, which has recently been identified as glycine N-methyltransferase (GNMT) (Raha, A., Wagner, C., Macdonald, R. G., and Bresnick, E. (1994)J. Biol. Chem. 269, 5750–5756). The role of GNMT as a 4 S PAH-binding protein in mediating the induction of cytochrome P-4501A1 has been investigated further. GNMT cDNA, which was cloned into a pMAMneo vector containing the Rous sarcoma virus promoter and the neomycin resistance gene, was stably transfected into D422 Chinese hamster ovary (CHO) cells. Several positive clones were selected by reverse transcription-polymerase chain reaction and assayed for the expression of recombinant protein. Western blot analysis indicated the expression of significant levels of the 4 S protein in the stably transfected CHO cells (CHO-GNMT). Cytosolic preparations from the CHO-GNMT showed high benzo[a]pyrene (B[a]P) binding but no 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity when compared with clones transfected with the pMAMneo vector alone (CHO-neo) or the parental CHO cells. Challanging the CHO-GNMT cells with 4 μm B[a]P resulted in elevated levels of CYP1A1 mRNA. Equally effective in inducing CYP1A1 mRNA were benzo[e]pyrene and 3-methylcholanthrene. On the other hand, TCDD did not induce CYP1A1 gene expression in these cells. B[a]P-treated CHO-GNMT, expressing the 4 S protein, also showed CYP1A1 protein by Western blotting and exhibited ethoxyresorufin-O-deethylase activity; neither the CHO-neo or parental CHO cells were positive for any of these measures. No Ah receptor message or protein was detectable in the parental CHO, CHO-neo, or CHO-GNMT cells. Furthermore, no XRE binding activity was observed in TCDD-treated cytosolic preparations or nuclear extracts from CHO-GNMT cells that were treated with TCDD. These studies unequivocally establish that GNMT is a PAH-binding protein that can mediate the induction of CYP1A1 by PAHs such as B[a]P through an Ah receptor-independent pathway. The cytochrome P-4501A1 (CYP1A1) gene is regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein, which has recently been identified as glycine N-methyltransferase (GNMT) (Raha, A., Wagner, C., Macdonald, R. G., and Bresnick, E. (1994)J. Biol. Chem. 269, 5750–5756). The role of GNMT as a 4 S PAH-binding protein in mediating the induction of cytochrome P-4501A1 has been investigated further. GNMT cDNA, which was cloned into a pMAMneo vector containing the Rous sarcoma virus promoter and the neomycin resistance gene, was stably transfected into D422 Chinese hamster ovary (CHO) cells. Several positive clones were selected by reverse transcription-polymerase chain reaction and assayed for the expression of recombinant protein. Western blot analysis indicated the expression of significant levels of the 4 S protein in the stably transfected CHO cells (CHO-GNMT). Cytosolic preparations from the CHO-GNMT showed high benzo[a]pyrene (B[a]P) binding but no 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity when compared with clones transfected with the pMAMneo vector alone (CHO-neo) or the parental CHO cells. Challanging the CHO-GNMT cells with 4 μm B[a]P resulted in elevated levels of CYP1A1 mRNA. Equally effective in inducing CYP1A1 mRNA were benzo[e]pyrene and 3-methylcholanthrene. On the other hand, TCDD did not induce CYP1A1 gene expression in these cells. B[a]P-treated CHO-GNMT, expressing the 4 S protein, also showed CYP1A1 protein by Western blotting and exhibited ethoxyresorufin-O-deethylase activity; neither the CHO-neo or parental CHO cells were positive for any of these measures. No Ah receptor message or protein was detectable in the parental CHO, CHO-neo, or CHO-GNMT cells. Furthermore, no XRE binding activity was observed in TCDD-treated cytosolic preparations or nuclear extracts from CHO-GNMT cells that were treated with TCDD. These studies unequivocally establish that GNMT is a PAH-binding protein that can mediate the induction of CYP1A1 by PAHs such as B[a]P through an Ah receptor-independent pathway. Polycyclic aromatic hydrocarbons such as B[a]P 1The abbreviations used are: B[a]P, benzo[a]pyrene; TCDD, 2,3,7,8-tetrachloro-p-dioxin; CYP1A1, cytochrome P-4501A1; PAH, polycyclic aromatic hydrocarbon; B[e]P, benzo[e]pyrene; CHO, Chinese hamster ovary; GNMT, glycineN-methyltransferase; RT, reverse transcription; PCR, polymerase chain reaction; GMSA, gel mobility shift assay; EROD, ethoxyresorufin O-de-ethylase.1The abbreviations used are: B[a]P, benzo[a]pyrene; TCDD, 2,3,7,8-tetrachloro-p-dioxin; CYP1A1, cytochrome P-4501A1; PAH, polycyclic aromatic hydrocarbon; B[e]P, benzo[e]pyrene; CHO, Chinese hamster ovary; GNMT, glycineN-methyltransferase; RT, reverse transcription; PCR, polymerase chain reaction; GMSA, gel mobility shift assay; EROD, ethoxyresorufin O-de-ethylase., 3-methylcholanthrene, and TCDD are environmental pollutants that elicit a variety of toxic, teratogenic, and carcinogenic responses in exposed animals (1Poland A. Knutson J. Glover E. Clin. Physiol. 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This has been used by for a of in and The were in an of and in with containing and The hepatoma which was used as a positive in the XRE binding was and as O. 1991; PubMed Scopus Google Scholar). The GNMT was by from a rat liver The and reverse were and The was purified by gel and the was to to published GNMT K. Y. M. J. 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The GNMT was from a and promoter elements and from and CHO cells were in part as a for transformation because of the of endogenous GNMT gene expression and the of with using the clones were and into for was by of the clones for the 4 S PAH-binding protein and gene The with of CHO-GNMT parental and CHO-neo are in A positive from rat is in the CHO-GNMT cells addition to the liver exhibited the 4 S No were observed with the CHO or CHO-neo cells Furthermore, no positive was observed in the from the CHO-GNMT when the reactions were in the of reverse CHO-GNMT cells GNMT protein, protein was by gel and the were to and with to is in CHO-GNMT cells significant levels of GNMT when compared with the levels in parental or CHO-neo cells. B[a]P and TCDD binding were in the CHO cells A and B[a]P binding activity was observed with extracts from the CHO-GNMT cells no binding activity was with extracts from the CHO or CHO-neo cells. The B[a]P interaction, as by a analysis not was with a binding of of protein. These are in with and protein observed with rat liver G.M. Tierney B. Bresnick E. Cancer Res. 1981; 41: 4408-4415PubMed Google Scholar). TCDD binding activity was in cytosolic extracts from the positive hepatoma cells no TCDD binding activity was observed in extracts from the CHO-GNMT CHO-neo not or parental CHO not cells. The CHO-GNMT, CHO-neo, and CHO cells were treated with B[a]P, B[e]P, 3-methylcholanthrene, or TCDD for 8 after which was and that been to was with CYP1A1 and B[a]P, B[e]P, and 3-methylcholanthrene a positive CYP1A1 mRNA and TCDD, which represents the ligand for the Ah receptor, was not effective in inducing CYP1A1 in the CHO-GNMT cells. was also in inducing CYP1A1 in these cells not were from CHO-neo, and CHO-GNMT cells and were for CYP1A1 protein The protein was with CYP1A1 protein. CHO-GNMT cells CYP1A1 protein in to B[a]P treatment The of CYP1A1 in the from and B[a]P-treated is indicated as a positive The of B[a]P treatment the activity of a EROD, was in CHO, CHO-neo, and CHO-GNMT these are in with the PAH for a induction of in CHO-GNMT cells compared with in the parental and CHO-neo of B[a]P treatment (CYP1A1) activity in CHO cells were to and treated with 4 μm B[a]P in for The the from in a The cells were to and treated with 4 μm B[a]P in for The the from The Ah receptor is a characterized protein that in the induction of CYP1A1 by binding to XREs The or the of the Ah receptor in the CHO, CHO-neo, and CHO-GNMT cells was by both and Western No detectable of the protein were in any of these cells not The of Ah receptor activity was by a functional involving nuclear translocation and XRE binding with the CHO-GNMT hepatoma cells were used as the positive The of the receptor complex was demonstrated with cytosolic extracts from cells that been treated in with TCDD No such complex was with extracts from CHO-GNMT cells that been treated The of a receptor complex was also with nuclear extracts from cells that been with TCDD no such complex was observed with TCDD-treated CHO-GNMT nuclear extracts These that CHO-GNMT cells any endogenous expression of Ah receptor and that the PAH-induced through a Ah receptor-independent pathway. The 4 S PAH-binding protein was identified as GNMT the of several including of PAH binding activity by to GNMT, and of the in and A. Wagner C. R.G. Bresnick E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The present was to the GNMT gene into cells that the to protein as as the Ah These cells a model to the induction by PAHs such as B[a]P and 3-methylcholanthrene. The of the functional role of GNMT as a PAH-binding protein that may mediate the induction In the present GNMT was stably into CHO which the endogenous expression of protein as as the Ah receptor, by of a that a virus as the promoter and exhibited neomycin resistance as the the of the 4 S PAH-binding protein, as a of PAH-induced expression of CYP1A1 the present to B[a]P in the that the of GNMT expression into these CHO cells in the PAH-induced expression of Furthermore, is by the of detectable levels of Ah receptor in have also been to any mRNA for the Ah receptor in the or cells not have any TCDD binding and XRE binding in these cells. B[e]P been demonstrated to a ligand for the 4 S PAH-binding protein (17Tierney B. Weaver D. Heintz N. Schaeffer W.I. Bresnick E. Arch. Biochem. Biophys. 1980; 200: 513-523Crossref PubMed Scopus (74) Google Scholar, 18Zytkovicz T.H. Cancer Res. 1982; 42: 4387-4393PubMed Google Scholar, 19Collins S. Marletta M.A. Mol. Pharmacol. 1984; 26: 353-359PubMed Google Scholar, 20Houser W.H. Hines R.N. Bresnick E. Biochemistry. 1985; 24: 7839-7845Crossref PubMed Scopus (46) Google Scholar, 21Houser W.H. Cunningham C.K. Hines R.N. Schaeffer W.I. Bresnick E. Arch. Biochem. BIophys. 1987; 259: 215-223Crossref PubMed Scopus (29) Google Scholar, 22Gibbons J.A. Babish J.G. Toxicologist. 1990; 10: 193-195Google Scholar, 23Gibbons J.A. Babish J.G. Chem. Biol. Interact. 1992; 83: 203-220Crossref PubMed Scopus (8) Google Scholar). In the present have that PAH is also to induce the expression of CYP1A1 in the CHO TCDD, a ligand for the Ah receptor, to These the role of GNMT as a of CYP1A1 expression induced by PAHs such as B[a]P, B[e]P, and 3-methylcholanthrene through an Ah receptor-independent pathway. GNMT has a binding K. Y. M. J. Biochem. 1987; PubMed Scopus Google Scholar) and has been in rat liver by Wagner C. Proc. Acad. Natl. Sci. U. S. A. PubMed Scopus Google Scholar). GNMT has also been as a of gene expression by of substrates that have not been identified K. Y. M. J. Biochem. 1987; PubMed Scopus Google Scholar). is that GNMT may act in expression of CYP1A1 by substrates that gene, because has been by to gene activity Cell. 1980; 20: Full Text PDF PubMed Scopus Google Scholar, A. Science. 1980; PubMed Scopus Google Scholar, Science. 1981; PubMed Scopus Google Scholar, R. A. Proc. Natl. Acad. Sci. U. S. A. 1982; PubMed Scopus Google Scholar, A. W. Proc. Natl. Acad. Sci. U. S. A. 1982; PubMed Scopus Google Scholar, M. J. R.A. Cell. Full Text PDF PubMed Scopus Google Scholar). The present that GNMT as an and the other as a transcriptional This of is not to GNMT but is by several other that have as both and Several of the in the pathway have been to J. Biochem. PubMed Scopus Google Scholar). the of also in a as K. J. J.K. M.A. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). of the that the activity of in has been identified as H.K. Chaney W.G. Anderson C.W. Davis R.G. Vishwanatha J.K. J. Biol. Chem. 1991; 266: 5169-5176Abstract Full Text PDF PubMed Google Scholar). and by mechanism GNMT is to in GNMT of protein in rat and liver J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, Wagner C. Arch. Biochem. Biophys. 1981; PubMed Scopus Google Scholar). GNMT present as a which represents the Wagner C. Proc. Natl. Acad. Sci. U. S. A. 1984; PubMed Scopus Google Scholar). On the other hand, is the that as a PAH-binding and a transcriptional A. Wagner C. R.G. Bresnick E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). have also recently R. Weaver J, A. Wagner C. Bodwell J.E. Bresnick E. J. Biol. Chem. 1996; 271: 32551-32556Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) that the of GNMT the binding of B[a]P and nuclear is that the of and localization of GNMT are by as has been for other trans-acting factors K. J. J.K. M.A. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). studies that is in the PAH-binding of the protein. the factors in the of functional transcriptional are the of the of GNMT and the intracellular of the The of the of the GNMT, is In have demonstrated that GNMT is a PAH-binding protein that the induction of CYP1A1 by an Ah receptor-independent pathway. are to the factors that the of GNMT and the expression of Wagner of and of the of for the of specific GNMT and the Ah receptor,