Isolation and Characterization of the α 1 -Antitrypsin Gene of Mice

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-l-antitrypsin, apolipoprotein Al, 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. A greater increase occurred when the medium was simultaneously supplemented with insulin, 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.

Section: Resultsmentioning

confidence: 99%

Tissue-specific gene expression in mouse hepatocytes cultured in growth-restricting medium.

Spiegelberg¹,

Bishop²

1988

“…Northern blots were hybridized to riboprobes corresponding to a partial cDNA clone of mouse P3-450 which readily hybridizes to P-450c and P-450d (inset, row A) (18,19), a mouse a1-AT cDNA clone (inset, row B) (3,11), and a mouse TAT cDNA clone (inset, row C) (17). The control P-450 panel was exposed for 72 h; the MCA P450 panel was exposed for 5 h. All other autoradiograms were equally exposed.…”

mentioning

confidence: 99%

Aryl hydrocarbon induction of rat cytochrome P-450d results from increased precursor RNA processing.

Silver

Krauter

1990

We have previously demonstrated that cytochrome P45Od mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.Exposure of most organisms to environmental pollutants results in the induction of sets of genes whose products act to metabolize these compounds. In rat liver exposed to aryl hydrocarbons such as 3-methylcholanthrene (MCA), a-napthaflovone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin, the hydrocarbon-metabolizing enzymes cytochromes P-450c and P-450d are the major induced proteins (12, 13). It has been determined that transcriptional regulation mediated by the cytosolic Ah receptor is primarily responsible for the hydrocarbon-induced accumulation of P-450c and its mouse homolog cytochrome P1-450 in intact liver (14, 26). However, it has been demonstrated that the Ah receptor-dependent 30-fold induction of P-450d mRNA and its mouse homolog P3-450 is mediated primarily at a posttranscriptional level (10,18,19). We and others (15,18,19) have also examined the MCA induction of the P-450c and P-450d genes in primary rat hepatocyte cultures grown in defined medium and have shown that both genes appear to be mainly posttranscriptionally regulated. Thus, the regulation of P-450d in cultured cells is identical with its regulation in liver cells, whereas P-450c loses much of its transcriptional inducibility and becomes regulated at a posttranscriptional step.To define the mechanism responsible for the hydrocarbon induction of P-450d mRNA, we have analyzed (i) the degradation rate, (ii) the relationship between protein synthesis and MCA induction, and (iii) the accumulation of nuclear mRNA processing intermediates in the presence and absence of MCA. These results show that the Ah receptormediated induction of P-450d mRNA is effected at an early step of processing of the nuclear precursor mRNA, perhaps at the process-versus-discard decision point, and not at the level of mature cytoplasmic mRNA turnover. Moreover, the inhibition of protein synthesis has no significant effect on the induction process, suggesting that the Ah receptor, which has been shown to enter the nucleus after hydrocarbon binding (22, 26), may act directly on an early event in the mRNA synthesis of P-450d.Degradati...

“…The locations of exons within the clones were determined by hybridization to cDNA fragments and by sequencing. Exon numbers, sizes, and positions are conserved between M. caroli and M. domesticus (17) (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

“…One occurs at the P1 site, which is critical in defining inhibitor specificity (4,29). Several AT genes within M. domesticus show 2% divergence, including a change in the P1 amino acid residue (17). It is not clear whether the alterations that have been found within the reactive center of various AT polypeptides from M. domesticus and M. caroli affect the function of the inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…In the present paper, we describe more detailed analysis of the structure and expression of the AT gene in M. caroli and compare it with that in M. domesticus (17). Of major interest is the finding that although the 5'-flanking regions of the genes in the two species are quite similar in sequence, they behave very differently with regard to their functions in governing liver-specific AT expression.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Highly conserved upstream regions of the alpha 1-antitrypsin gene in two mouse species govern liver-specific expression by different mechanisms.

Latimer

Berger

1990

ax-Antitrypsin (AT), the major elastase inhibitor in mammalian serum, is produced primarily in the liver. We have characterized AT gene structure and expression in the mouse species Mus caroli, which expresses high levels of AT in the kidneys as well as in the liver. Analysis of cDNA and genomic clones showed that the AT gene in M. caroli exhibits high sequence homology (>90%) to the gene in laboratory mice (M. domesticus) throughout the coding and 5'-flanking regions. Despite this extensive sequence conservation, the functional organization of cis-acting regulatory elements governing liver-specific expression is strikingly different between these species. Transient-transfection assays showed that the proximal region of the M. caroli promoter (i.e., between -120 and -2 relative to the transcriptional start site) is 10-fold more active than the analogous region of M. domesticus in driving the expression of an indicator gene in cultured liver cells. The increased activity of the proximal region of the M. caroli AT promoter appears to be the result of one or both of the two base substitutions at positions -46 and -48. The weak proximal promoter in M. domesticus is compensated for by the presence of upstream, liver-specific enhancers between -199 and -520; the analogous region in M. caroli is inactive. Thus, during the course of evolution, the modest 7% sequence divergence that has occurred between the 5'-flanking regions of the AT genes in these two species has generated distinct, yet equally effective, modes of hepatocyte-specific expression.The molecular basis for the liver-specific expression of atl-antitrypsin (AT), a major seine protease inhibitor that functions in the control of neutrophil elastase activity (4, 29), has been intensely investigated in humans and mice. For the human gene, maximal liver-specific expression requires 261 nucleotides of the 5'-flanking region, which can be subdivided into a distal element located between -261 and -210 and two proximal elements located between -137 and -37 (6,8,26). Although the proximal elements function only in hepatic cells, the distal element acts as a nonspecific enhancer (8). In striking contrast, the mouse gene, which is structurally similar to the human gene, requires 500 bases of the upstream region for full activity (12); several domains that function specifically in hepatic cells and that interact with hepatocyte-enriched DNA binding proteins have been identified within this region (7, 12). Thus, liver-specific expression of the AT gene is maintained by different mechanisms in the two species, reflecting the accumulation of regulatory mutations since the two species diverged.With one known exception, all mammals express AT predominantly in the liver. The wild-derived mouse species Mus caroli, in sharp contrast to laboratory mice (M. domesticus), transcribes the AT gene in the kidneys as well as the liver (2,19,24). Renal AT expression in M. caroli is specific to tubule cells, where it is regulated by androgens during development (19). This species differe...