Differential regulation of glutathione S-transferases in cultured hepatocytes

Abstract

Specific cDNA probes were used to determine steady-state mRNA levels for the multiple glutathione S-transferases in primary hepatocyte cultures. In the first 24 hr of culture, gene transcripts for the Ya family decreased sharply, Yb3 disappeared completely, but changes in levels of mRNA for Yb1 and Yb2 were smaller. These results suggest that the isoenzymes are regulated independently. Yp mRNA, which is present at greatly elevated levels in hyperplastic nodules and hepatocellular carcinomas but not in normal adult livers, was hardly detectable in freshly isolated hepatocytes, but Yp transcripts rapidly accumulated in the first 24 hr in culture and continued to increase for 72 hr. Decreased levels in Ya and Yc and increases in Yp were detected by immunoblotting methods, indicating that translation products changed together with mRNA levels in the cultured cells. The appearance of Yp transcripts in hepatocytes was effectively blocked by addition of dexamethasone to the culture medium. Elevations of Yp levels are characteristic of the cell culture system and factors regulating Yp transcription in nodules and carcinomas may also be operative in cultured hepatocytes.

Steroid 5.ALPHA. Reductase mRNA Type I is Differentially Regulated by Androgens and Glucocorticoids in the Rat Liver

“…3). The expression of several liver specific and non-specific mRNAs have been shown to vary temporally in primary hepatocyte cultures [29,30]. In our study supplementing culture medium with Dex (1 mM) resulted in a five-and two-fold reduction in the mRNA expression of steried 5aR type I after 24 h and 48 h, respectively, compared with 5aR1 mRNA levels in cells cultured in steroid free medium.…”

“…Steroid 5a reductase type I mRNA is expressed and hormonally regulated in rat primary hepatocyte culture: To determine whether rat primary hepatocyte cultures represent in vitro systems capable of supporting the regulation of steroid 5aR type I mRNA, steady state levels of this mRNA in hepatocytes grown in cultures were analyzed. Control mRNA for GST-Yb was essentially unchanged throughout all culture conditions [29]. Under similar culture conditions steroid 5aR type I mRNA abundance declined to 25% and 4% of fresh cell levels after 24 h and 48 h, respectively (Fig.…”

“…First, differential effects of these two steroids on several gene products and cellular functions have been demonstrated, such as androgen receptor concentrations [24,31], mouse mammary tumor virus (MMTV)-LTR related transcripts [21,[32][33][34] and growth rates of tumor cell lines [31][32][33][34]. Secondly, there is substantial homology between sequences mediating androgen effects and DNA sequences known to be essential for glucocorticoid responses as in the case of MMTV-LTR [21,[22][23][24][25][26][27][28][29][30][31][32][33][34][35]. Finally, defective formation of 5a-reduced androgens and glucocorticoids has been demonstrated in patients with a genetic deficiency of steroid 5aR [23,24] suggesting that both steroids can serve as substrates for a single enzyme.…”

Insulin and Glucagon Regulation of GlutathioneS-Transferase Expression in Primary Cultured Rat Hepatocytes

“…The cytosolic GSTs are homo or heterodimeric enzymes, and the major GST subunits expressed in the adult liver are alpha-class subunits A1, A2, and A3 and the mu-class subunits M1 and M2 (Mannervik et al, 1985). GST subunit P1, a member of the pi class, is not expressed in adult rat liver but is expressed in fetal liver, primary cultured rat hepatocytes, and during the early stages of hepatocellular carcinoma (Abramovitz et al, 1989;Tee et al, 1992;Tsuchida and Sato, 1992).Diabetes mellitus leads to a series of metabolic disturbances; they are found not only in the metabolism of carbohydrates, lipids, and proteins, but also in xenobiotic metabolism. Cytochrome P450 (CYP) 2B1, 2E1, 3A, and 4A protein and activity levels have been reported to be increased in experimental animals and humans with diabetes (Bellward et al, 1988;Barnett et al, 1990;Song et al, 1990 GSTs, H89,ethyl]-5-isoquinoline sulfonamide; PI3-kinase, phosphatidylinositol 3-kinase; Br-cAMP, 8-bromo-cAMP; DB-cAMP, dibutyryl-cAMP; PKA, protein kinase A; LDH, lactate dehydrogenase; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; NBD, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; EA, ethacrynic acid; ARE, antioxidant response element; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; PBS-T, 0.05% Tween 20 in PBS.…”

“…The cytosolic GSTs are homo or heterodimeric enzymes, and the major GST subunits expressed in the adult liver are alpha-class subunits A1, A2, and A3 and the mu-class subunits M1 and M2 (Mannervik et al, 1985). GST subunit P1, a member of the pi class, is not expressed in adult rat liver but is expressed in fetal liver, primary cultured rat hepatocytes, and during the early stages of hepatocellular carcinoma (Abramovitz et al, 1989;Tee et al, 1992;Tsuchida and Sato, 1992).…”