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Studies on growth hormone induced female-characteristic gene transcription in rat liver

thesis
posted on 2024-09-02, 18:04 authored by Cissi Gardmo

Growth hormone (GH) secretion is sexually dimorphic, which has an impact on hepatic gene expression. This is particularly pronounced in rats but has also been demonstrated in other species, including humans. The male GH pattern is transduced by the JAK2-Sta5 signalling pathway, but less is known about the molecular mechanisms leading to GH induced female-specific gene expression. Studies on GH regulation of the prototypical female-specific rat liver gene CYP2C12, have suggested the involvement of the transcription factors C/EBPalpha, Stat5, HNF4 and HN176. However, there is no evidence that these factors transduce the sex-specific expression of the gene.

We used the subtractive suppressive hybridization (SSH) technique to identify additional genes expressed in rat liver in response to the female continuous GH secretion pattern, which could then be used to unravel corresponding molecular mechanisms. The Sadenosylmethionine synthetase mRNA was identified among the SSH sequences, but the use of hypophysectomised rats revealed that this gene is not expressed in response to the female GH pattern, but instead down regulated by the male-specific intermittent GH secretion pattern. This emphasises that sex-characteristic gene expression in rat liver, dependent on the sexually dimorphic pattern of GH secretion, is more complex than just activation of gene transcription by the continuous or the intermittent GH pattern.

The most represented sequence among the SSH clones did not correspond to any known rat gene. Northern blots confirmed that this transcript was expressed in response to the female-specific GH pattern, and cloning of the full-length cDNA revealed homology to the human serum protein alpha1IB-glycoprotein. The rat a1bg promoter was cloned by 'genome walking' and a 2.3kb a1bg promoter construct was used in in vivo transfection assays. Sexually dimorphic reporter expression was displayed and the transactivation was dependent on a continuous exposure of the rat liver to GH. Truncation of the 2.3kb a1bg construct showed that sequences sufficient for the sexually dimorphic expression are contained within the 160bp proximal part of the promoter. In vitro footprinting analysis of the 160bp sequence revealed four protected areas.

Binding of Stat5, HNF6 and NF 1 was shown by electromobility shift assays. In vivo transfection of rat livers with different a1bg constructs proved that the Stat5 binding site contributed to the expression, but probably to an higher extent in male than in female rat liver and thereby not to the female-specific expression. Mutation of the HNF6 site in combination with deletion of the NF1 binding region resulted in very low reporter activity, and the sex differentiated expression was abolished. Thus, for the a1bg gene the HNF6 site in combination with the nearby NF1 binding region constitute a regulatory unit for transducing the female GH pattern effect.

Even though sex-specific effects of GH on hepatic gene expression have not been possible to achieve in primary rat hepatocytes in culture, such a system is valuable for studies on other aspects of GH-dependent molecular mechanisms. We have addressed the issues of liposome-mediated transfection and various culture conditions of primary rat hepatocytes, to obtain a simple and consistent system for the study of hepatic gene transcription, using luciferase as a reporter gene. The 160bp a1bg reporter construct was induced by GH, and primary hepatocytes together with in vivo transfections can now be used to fully explore female-specific GH regulation of the a1bg gene.

List of scientific papers

I. Gardmo C, Swerdlow H, Mode A (2002). Growth hormone regulation of rat liver gene expression assessed by SSH and microarray. Mol Cell Endocrinol. 190(1-2): 125-33.
https://doi.org/10.1016/S0303-7207(02)00004-7

II. Oscarsson J, Gardmo C, Eden S, Mode A (2001). Pulsatile growth hormone secretion decreases S-adenosylmethionine synthetase in rat liver. Am J Physiol Endocrinol Metab. 280(2): E280-6.
https://pubmed.ncbi.nlm.nih.gov/11158931

III. Gardmo C, Persson B, Mode A (2001). Cloning of a novel growth hormone-regulated rat complementary deoxyribonucleic acid with homology to the human alpha1B-glycoprotein, characterizing a new protein family. Endocrinology. 142(6): 2695-701.
https://doi.org/10.1210/en.142.6.2695

IV. Gardmo C, Mode A (2005). Mechanistic studies on hepatic gene expression dependent on female-characteristic GH secretion using in vivo transfection of rat liver. [Manuscript]

V. Gardmo C, Kotokorpi P, Helander H, Mode A (2005). Transfection of adult primary rat hepatocytes in culture. Biochem Pharmacy. [Accepted]
https://doi.org/10.1016/j.bcp.2005.03.028

History

Defence date

2005-05-27

Department

  • Department of Medicine, Huddinge

Publication year

2005

Thesis type

  • Doctoral thesis

ISBN-10

91-7140-375-2

Number of supporting papers

5

Language

  • eng

Original publication date

2005-05-06

Author name in thesis

Gardmo, Cissi

Original department name

Department of Medical Nutrition

Place of publication

Stockholm

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