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Intrinsic sex differences in the early growth hormone responsiveness of sex-specific genes in mouse liver.

ABSTRACT: Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic patho-physiologies.

SUBMITTER: Wauthier V

PROVIDER: S-EPMC2840812 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Intrinsic sex differences in the early growth hormone responsiveness of sex-specific genes in mouse liver.

Wauthier Valerie V Sugathan Aarathi A Meyer Rosana D RD Dombkowski Alan A AA Waxman David J DJ

Molecular endocrinology (Baltimore, Md.) 20100211 3

Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct cla ...[more]

PMID: 20150183

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Project description:Pituitary GH-secretory profiles are sex dependent and regulate the sexually dimorphic expression of a large number of genes in the liver. The slow response of many sex-specific liver genes to changes in plasma GH status suggests that GH acts in the liver via both direct and indirect mechanisms organized in a hierarchical regulatory network. Presently, genome-wide liver transcription profiling was conducted to elucidate the global impact of pituitary hormone ablation on the sex specificity of rat liver gene expression and to identify sex-specific genes that respond rapidly to GH as candidates for direct targets of GH action. Hypophysectomy abolished the sex specificity of approximately 90% of 1032 sex-dependent genes, consistent with the dominant role of pituitary GH in regulating liver sexual dimorphism. Two major classes of sex-specific genes were identified: genes that were down-regulated after hypophysectomy and may be subject to positive GH regulation (461 class I genes), and genes that were up-regulated after hypophysectomy and may be subject to negative GH regulation (224 class II genes). Fifty class I sex-specific genes were induced, and 38 class II sex-specific genes were suppressed within 90 min of a physiological GH pulse, suggesting they are primary GH response genes. A further 71 sex-specific genes responded after a second GH treatment and may correspond to secondary response genes. Twenty four DNA-binding proteins were identified as early GH response genes, of which 15 were induced and nine were suppressed by GH. Five of these 24 genes displayed sex-specific expression, consistent with a hierarchical transcriptional network controlling sex-specific liver gene expression. Class II male-specific genes, such as Cyp2a2 and Cyp2c13, were down-regulated within 30 min of GH pulse treatment, as determined by heterogeneous nuclear RNA analysis, suggesting that transcription of these genes is restricted to the GH-free interpulse period in adult male rat liver. We conclude that GH acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression.

Project description:A series of two color gene expression profiles obtained using Agilent 44K expression microarrays was used to examine sex-dependent and growth hormone-dependent differences in gene expression in rat liver. This series is comprised of pools of RNA prepared from untreated male and female rat liver, hypophysectomized (‘Hypox’) male and female rat liver, and from livers of Hypox male rats treated with either a single injection of growth hormone and then killed 30, 60, or 90 min later, or from livers of Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart and killed 30 min after the second injection. The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. A comparison of untreated male liver and untreated female liver liver gene expression profiles showed that of the genes that showed significant expression differences in at least one of the 6 data sets, 25% were sex-specific. Moreover, sex specificity was lost for 88% of the male-specific genes and 94% of the female-specific genes following hypophysectomy. 25-31% of the sex-specific genes whose expression is altered by hypophysectomy responded to short-term growth hormone treatment in hypox male liver. 18-19% of the sex-specific genes whose expression decreased following hypophysectomy were up-regulated after either one or two growth hormone injections. Finally, growth hormone suppressed 24-36% of the sex-specific genes whose expression was up-regulated following hypophysectomy, indicating that growth hormone acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression. For full details, see V. Wauthier and D.J. Waxman, Molecular Endocrinology (2008) This series is comprised of pools of liver RNA prepared from untreated male, hypophysectomized (‘Hypox’) male, untreated female and Hypox female rats (3-4 livers/pool), as well as liver RNA prepared from Hypox male rats treated with a single growth hormone injection and killed either 30, 60, or 90 minutes later (pool of n = 4 livers) or from Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart (pool of n = 5 livers). The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver. Dye swapping experiments were carried out for each of the six hybridization experiments, as follows. The Alexa 555-labeled cDNA from one of the two untreated male pools was mixed with the Alexa 647-labeled cDNA from one of the two untreated female pools. Similarly, Alexa 647-labeled cDNA from the second untreated male pool was mixed with the Alexa 555-labeled cDNA from the second untreated female pool. Together, these two mixed cDNA samples comprise a fluorescent reverse pair (dye swap). Dye swaps were similarly carried out for each of the five other competitive hybridization experiments, except that for experiments 5 and 6, a single pool of M-Hypox + GH liver cDNA, or a single pool of M-Hypox + 2GH liver cDNA, was used in each half of the fluorescent reverse pair. Two microarrays, one for each mixed cDNA sample, were hybridized for each of the six fluorescent reverse pairs, giving a total of 12 microarrays.

Project description:The importance of gonadal hormones in the regulation of vascular function has been documented. An alternate and essential contribution of the sex chromosomes to sex differences in vascular function is poorly understood. We reported previously sex differences in microvessel permeability (P(s)) responses to adenosine that were mediated by the cAMP signaling pathway (Wang J, PhD thesis, 2005; Wang J and Huxley V, Proceedings of the VIII World Congress of Microcirculation, 2007; Wang J and Huxley VH, Am J Physiol Heart Circ Physiol 291: H3094-H3105, 2006). The two cyclic nucleotides, cAMP and cGMP, central to the regulation of vascular barrier integrity, are hydrolyzed by phosphodiesterases (PDE). We hypothesized that microvascular endothelial cells (EC) would retain intrinsic and inheritable sexually dimorphic genes with respect to the PDEs modulating EC barrier function. Primary cultured microvascular EC from skeletal muscles isolated from male and female rats, respectively, were used. SRY (a sex-determining region Y gene) mRNA expression was observed exclusively in male, not female, cells. The predominant isoform among PDE1-5, present in both XY and XX EC, was PDE4. Expression mRNA levels of PDE1A (male > female) and PDE3B (male < female) were sex dependent; PDE2A, PDE4D, and PDE5A were sex independent. Barrier function, P(s), was determined from measures of albumin flux across confluent primary cultured microvessel XY and XX EC monolayers. Consistent with intact in situ microvessels, basal monolayer P(s) did not differ between XY (1.7 +/- 0.2 x 10(-6) cm/s; n = 8) and XX (1.8 +/- 0.1 x 10(-6) cm/s; n = 10) EC. Cilostazol, a PDE3 inhibitor, reduced (11%, P < 0.05) P(s) in XX, not XY, cells. These findings demonstrate the presence and maintenance of intrinsic sex-related differences in gene expression and cellular phenotype by microvascular EC in a gonadal-hormone-free environment. Furthermore, intrinsic cell-sex likely contributes significantly to sexual dimorphism in cardiovascular function.

Dataset Information

Intrinsic sex differences in the early growth hormone responsiveness of sex-specific genes in mouse liver.

Publications

Intrinsic sex differences in the early growth hormone responsiveness of sex-specific genes in mouse liver.

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