Progress 10/01/02 to 09/30/07
Outputs
The overall objective of this Hatch project was to determine how growth hormone receptor (GHR) gene expression in cattle is regulated by tissue-specific, developmental stage-specific, hormonal and nutritional factors. The following experiments have been conducted to achieve this objective: 1) Using DNase I footprinting analysis, gel-shift assays, yeast one-hybrid system and transient transfection analysis to identify nuclear protein-binding regions in the bovine GHR promoter 1A and the binding proteins; 2) Using ribonuclease protection assays, Western blotting analysis, transient transfection analysis, and gel-shift assays to identify the transcription factor and cis-regulatory region that mediate GH regulation of GHR mRNA expression in the bovine liver.
Impacts
Using candidate transcription factor approach, we have obtained strong evidence supporting a role for a liver-enriched transcription factor called HNF-4a in activating the GHR1A promoter in the adult bovine liver. We have also found that nutritional intake- and parturition-associated changes in GHR1A mRNA expression in the bovine liver are not due to increased HNF-4a mRNA expression. Using yeast one-hybrid system, we have identified chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), HNF-4g (another liver enriched transcription factor), and zinc-finger binding protein 89 (ZBP-89) as additional binding proteins to the GHR1A promoter. These results suggest that GHR gene expression in the bovine liver and hence GH action are under the control of complex mechanism involving multiple transcription factors, and that growth and milk production in cattle, which are traits controlled by GH, may be enhanced by increasing the expression of growth hormone
receptor gene through modulating the expression or activity of transcription factors HNF-4a, HNF-4g, COUP-TFII and ZBP-89 in the liver. In studying the mechanism of GH regulation of GHR expression in the bovine liver, we have found that GH can increase both GHR mRNA and protein expression in the liver. We have further found that the increases in GHR mRNA are mostly due to increased expression of the liver-specific GHR mRNA variant GHR1A. Through transient transfection analyses of serially truncated fragments of the GHR1A promoter, gel-shift assays and mutation analyses, we have obtained data strongly suggesting that GH stimulates the expression of GHR1A mRNA expression in the bovine liver by inducing the binding of a transcription factor called STAT5b to the GHR1A proximal promoter. These results identify another transcription factor in STAT5b that controls GHR gene expression in the bovine liver. Continuous injection of GH is being used to increase milk production in cattle. Our
finding that GH can upregulate GHR gene expression in the bovine liver provides a novel mechanistic link between growth hormone and its effects on milk production in cattle.
Publications
- Jiang H, Lucy MC. 2001. Involvement of hepatocyte nuclear factor-4 in the expression of the growth hormone receptor 1A messenger ribonucleic acid in bovine liver. Molecular Endocrinology 15:1023-1034.
- Xu Q, Walther N, Jiang H. 2004. Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and hepatocyte nuclear factor 4a (HNF-4a) and HNF-4g regulate the bovine growth hormone receptor 1A promoter through a common DNA element. Journal of Molecular Endocrinology. 32:947-961.
- Jiang H, Wang Y, Wu M, Gu Z, Frank SJ, Torres-Diaz R. 2007. Growth hormone stimulates hepatic expression of bovine growth hormone receptor messenger ribonucleic acid through signal transducer and activator of transcription 5 activation of a major growth hormone receptor gene promoter. Endocrinology. 148:3307-3315.
|
Progress 10/01/05 to 09/30/06
Outputs
The overall objective of this Hatch project was to determine how growth hormone receptor gene expression in cattle is regulated by tissue-specific, developmental stage-specific, hormonal and nutritional factors. During the period of 10/1/2005 to 09/30/2006, we continued our focus on the molecular mechanism by which growth hormone (GH) increases GH receptor (GHR) gene expression in the liver of cattle. In cattle as well as many other mammals, hepatic GHR mRNA is expressed as variants that differ in the 5'-untranslated region, due to the use of different promoters in transcription and alternative splicing of pre-mRNA. Ribonuclease protection assays and Western blotting analysis revealed that injection of recombinant bovine GH in a slow-release formula caused a nearly equivalent increase (P less than 0.05) in both GHR mRNA and GHR protein, respectively, in the liver of cattle one week after the injection. The increases in GHR mRNA were highly correlated (P less than
0.01) with increases in liver insulin-like growth factor-I (IGF-I) mRNA. Ribonuclease protection assays also revealed that the GH injection increased (P less than 0.05) increased liver expression of the liver-specific GHR mRNA variant GHR1A mRNA without affecting the expression of GHR1B and GHR1C mRNAs, the other two major GHR mRNA variants. Transient transfection analyses of a ca. 3 kb GHR1A promoter, the promoter that directs GHR1A mRNA transcription, in reconstituted GH-responsive cells showed that the GHR1A promoter could be robustly activated by GH, suggesting that GH augmentation of GHR1A mRNA expression is at least in part mediated at the transcriptional level. Further transfection analyses of serially 5'-truncated fragments of this GHR1A promoter narrowed the GH-responsive region down to a ca. 200 bp DNA fragment that contained a putative transducer and activator of transcription-5 (STAT5) binding site. Electrophoretic mobility shift assays demonstrated that this putative
STAT5 binding site was able to bind to STAT5b protein. Deletion of this putative STAT5-binding site also abolished (P less than 0.01) most of the response of the GHR1A promoter to GH. These observations together suggest that chronic action of GH stimulates the expression of the liver-specific GHR1A mRNA variant through binding STAT5b to its promoter, thereby increasing GHR protein expression in the bovine liver.
Impacts
Growth hormone is being used to increase milk production in cattle. The underlying mechanism is not clear. This stud was conducted towards understanding this mechanism (Response). The data from the study indicate that growth hormone injection upregulates growth hormone receptor gene expression in the liver of cattle (Results). The increase in growth hormone receptor expression will likely amplify the action of growth hormone in the tissue, including its action on milk production. Therefore, the finding that growth hormone can upregulate growth hormone receptor gene expression provides a novel explanation for why additional growth hormone to cattle can increase their milk production (Relevance).
Publications
- Xu, Q., Springer, L., Merchant, J. L. and Jiang, H., 2006. Identification of zinc finger binding protein 89 (ZBP-89) as a transcriptional activator for a major bovine growth hormone receptor promoter. Molecular and Cellular Endocrinology. 251:88-95
- Zhou, Y. and Jiang, H., 2006. A milk trait-associated polymorphism in the bovine growth hormone receptor gene does not affect receptor signaling. Journal of Dairy Science. 89:1761-1764.
- Wu, M., Akers, R. M., Torres-Diaz, R., Frank, S. J., Hall, J. B., Beal, W. E. and Jiang, H., 2006. Food deprivation-induced decrease in blood insulin-like growth factor-I is associated with decreased liver growth hormone receptor mRNA and protein in steers. Journal of Animal Science 84 (Supplement 1):451.
- Zhou, Y., Akers, R. M. and Jiang, H., 2006. Growth hormone stimulates the expression of milk protein genes in bovine mammary epithelial cells overexpressing growth hormone receptor. Journal of Animal Science 84 (Supplement 1):427.
- Jiang, H. and Eleswarapu, S., 2006. Identification of growth hormone-regulated genes in the bovine liver by a microarray analysis. Journal of Animal Science 84 (Supplement 1):354.
|
Progress 10/01/04 to 09/30/05
Outputs
The overall objective of this Hatch project was to determine how growth hormone receptor gene expression in cattle is regulated by tissue-specific, developmental stage-specific, hormonal and nutritional factors. During the period of 10/1/2004 to 09/30/2005, we focused on the molecular mechanism by which growth hormone (GH) increases growth hormone receptor (GHR) gene expression in the liver. Ribonuclease protection assays revealed that continuous growth hormone administration caused a significant increase in the expression of GHR1A mRNA, a major GHR mRNA variant, whereas it had no effect on the expression of other GHR mRNA variants. Transient transfection analyses of an approximately 3 kb GHR1A promoter, the promoter that directs GHR1A mRNA transcription, in reconstituted GH-responsive cells showed that the GHR1A promoter could be robustly activated by GH, suggesting that GH augmentation of GHR1A mRNA expression is at least in part mediated at the transcriptional
level. Further transfection analyses of serially truncated fragments of this GHR1A promoter narrowed the GH-responsive region down to an approximately 200 bp DNA fragment that contained a consensus signal transducer and activator of transcription-5 (STAT5) binding site. We are currently in the process of confirming the ability of this candidate STAT5 binding site to bind STAT5 protein.
Impacts
If the DNA region being studied is found to be important for growth hormone stimulation of growth hormone receptor expression, cattle should contain the right sequence for this DNA region. Otherwise, they may be less responsive to growth hormone and thereby produce less meat or milk.
Publications
- Jiang, H., Lucy, M.C., Crooker, B.A. and Beal, W.E. 2005. Expression of growth hormone receptor 1A mRNA is decreased in dairy cows but not in beef cows at parturition. J Dairy Sci. 88:1370-1377.
- Zhou, Y. and Jiang, H. 2005. Trait-associated sequence variation in the bovine growth hormone receptor 1A promoter does not affect promoter activity in vitro. Anim Genet. 36:156-159.
|
Progress 10/01/03 to 09/30/04
Outputs
The overall objective of this Hatch project was to determine how growth hormone receptor gene expression in cattle is regulated by tissue-specific, developmental stage-specific, hormonal and nutritional factors. During the period of 10/1/2003 to 09/30/2004, we focused on the effect of growth hormone on growth hormone receptor gene expression in the liver and the underlying mechanism. We found that continuous administration of growth hormone increased the expression of the liver-specific growth hormone receptor mRNA variant, also a major growth hormone receptor mRNA variant, GHR 1A mRNA, but not other growth hormone receptor mRNA variants, in the liver of beef cows. We are currently studying the role of some candidate transcription factors in mediating growth hormone up-regulation of GHR 1A mRNA expression.
Impacts
The result that growth hormone can stimulate its receptor gene expression provides a novel mechanism by which administration of growth hormone enhances growth or milk production in cattle.
Publications
- Eleswarapu, S. and Jiang, H. 2005. Growth hormone regulates the expression of hepatocyte nuclear factor-3 gamma and other liver-enriched transcription factors in the bovine liver. Journal of Endocrinology. (in press).
|
Progress 10/01/02 to 09/30/03
Outputs
The overall objective of this Hatch project was to determine how growth hormone receptor gene expression in cattle is regulated by tissue-specific, developmental stage-specific, hormonal and nutritional factors. During 10/2000-09/2002, we focused on the nutritional regulation of growth hormone receptor gene expression. We found that food restriction significantly decreased the levels of total GHR mRNA in the livers of cattle and that this decrease was associated with a significant decrease in both serum IGF-I and liver IGF-I mRNA. We also found that the decrease in liver GHR mRNA by food restriction was mainly due to a decrease in the expression of GHR mRNA variants 1C3 and 1A and that food restriction did not affect liver expression of two other major GHR mRNA variants, named 1B and 1C2.
Impacts
The results of this project so far suggest that nutritional levels have differential effects on the expression of different growth hormone receptor mRNA variants in the liver of cattle. Liver expression of insulin-like growth factor I, an important factor for normal growth, is primarily driven by growth hormone action in normally fed animals. The results of this study suggest that undernutrition may decrease liver expression of growth hormone receptor, thereby decreasing liver expression of insulin-like growth factor I and retarding animal growth.
Publications
- Wang Y, Eleswarapu S, Beal WE, Swecker WS, Akers RM, and Jiang H 2003 Reduced serum insulin-like growth factor (IGF) I is associated with reduced liver IGF-I mRNA and liver growth hormone receptor mRNA in food-deprived cattle. Journal of Nutrition. 3:2555-2560.
- Wang Y, Eleswarapu S, Beal WE, Swecker WS, Akers EM, and Jiang H 2003 Effects of fasting on serum insulin-like growth factor I and liver insulin-like growth factor I and growth hormone receptor mRNA in cattle. Journal of Animal Science. 81 (Supplement 1): 312
|
|