Tissue-Specific Gene Expression in Mouse Hepatocytes Cultured in Growth-Restricting Medium

Abstract

Culture conditions which maintain hepatocytes in their in vivo state are not known. This hampers the study of liver gene expression and of direct responses of liver genes to hormonal stimulation. We argued that hepatocytes that were unable to divide might retain in vivo characteristics. We therefore plated mouse (BALB/c) hepatocytes on plastic dishes in medium lacking arginine and measured the levels and transcription rates of six tissue-specific mRNAs over a period of days. Alpha-fetoprotein mRNA began to accumulate at about 48 h of culture, and transcription could sometimes be detected after 72 h. The levels and transcription rates of four mRNAs (albumin, alpha-1-antitrypsin, apolipoprotein A1, and major urinary protein [MUP]) fell sharply. The rate of transcription of transferrin mRNA fell less rapidly, and its level remained high, partly due to its longer half-life. The overall pattern of gene expression in the plated cells did not exactly parallel that of either fetal or regenerating liver. The hepatocytes remained responsive to hormonal stimulation. Insulin and dexamethasone each tended to counteract changes in mRNA levels, for example, preventing the accumulation of alpha-fetoprotein mRNA. The effects of insulin were primarily due to changes in transcription rates. Bovine growth hormone and thyroxine elevated the levels of most of the mRNAs. Many of the effects of these hormones, when added singly, could not be ascribed to changes in transcription. The level of MUP mRNA was strongly affected by added hormones. The mRNA level at 5 days was increased by added insulin, dexamethasone, growth hormone, and thyroxine. In the presence of these three hormones, the decay in the transcription rate of the MUP genes was reduced about 10-fold. We conclude that hepatocytes plated under these nongrowing conditions can provide insights into the hormonal responsiveness of tissue-specific genes.

Differential Expression in Male and Female Mouse Liver of Very Similar mRNAs Specified by Two Group 1 Major Urinary Protein Genes

“…The experimental lanes (1 to 13) contain the following total tissue RNA samples: 1, testosterone-induced female liver (3 ,ug); 2 to 7, male liver (3, 1, 0.3, and 0.1 ,ug and 30 and 10 ng, respectively); 8, female liver (30 pLg); 9, mammary gland (30 ,ug); 10, male submaxillary gland (30 1Lg); 11, female submaxillary gland (30 g.g); 12, male lachrymal gland (30 F.g); 13, female lachrymal gland (30 p,g). The remaining lanes (14 to 17) contain the following controls: 14, group 1, pl7.G1 (3 ng); 15, group 1 (30 ng); 16, group 3, pT7.G3 (30 These were cloned downstream of a T7 RNA polymerase promoter, and the in vitro transcripts (see Materials and Methods) were run on denaturing gels in parallel with mouse RNA samples to give positive and negative hybridization controls.…”

“…Control lanes 14 to 16 in each panel contain different positive control samples, as follows: (a) BLI, pTZ.BL1 (3 ng,.1 ng, and 30 pg, respectively); (b) BSJ, pTZ.BS1 (3 ng, 1 ng, and 30 pg, respectively). Control lanes 17 and 18 contain the same samples in both panels: lane 17, group 1 negative control for both probes, pTZ.MUP11 (30 ng); lane 18, male kidney RNA (30 p.g). BS].…”

“…The group 1 liver MUPs are secreted into the plasma and when excreted make up most if not all of the urinary MUP. The expression of the group 1 genes is strongly dependent on testosterone, thyroxine, and growth hormone (9,17,23,29,30). The dependency on testosterone leads to sexual dimorphism in Mup expression, and female expression can be induced to male levels by testosterone induction (9).…”

“…Variation among individual female mice in levels of liver Mup mRNA. Panel a was probed with the cDNA clone Mup-l I and exposed for 3 h; panel b was probed with the universal group 1 oligonucleotide and exposed for 16 h. The expected positions of group 2 and Mup-15 negative control transcripts are shown by arrows.Experimental lanes (1 to 13) contain the following total cellular RNA samples: 1, testosterone-induced female liver (3 ,ug); 2 to 4, male liver (3, 1, and 0.3 ,ug, respectively); 5 and 6, 7-week-old female liver (30 and 10 ,ug, respectively); 7 and 8, 8-week-old female liver (30 and 10 ,ug, respectively); 9 and 10, female liver (8 to 10 weeks old; 30 and 10 ,ug, respectively); 11 and 12, female liver (8 to 10 weeks old; 30 and 10 ,ug, respectively); 13, mammary gland(30…”

A Mup Promoter-Thymidine Kinase Reporter Gene Shows Relaxed Tissue-Specific Expression and Confers Male Sterility upon Transgenic Mice

“…By analogy with the mechanism of action of other steroid hormones (61), the data suggest that testosterone, through its receptor, is acting on sequences within the 2.2-kb BS6 promoter region to increase transcription. As yet it is not known whether the cis-acting signals responsible for the effects of thyroxine and growth hormone on MUP RNA synthesis in the liver (33,54) are also present.…”