Effects of Growth Hormone and Insulin-Like Growth Factor-I on Milk Protein Gene Expression and Nutrient Uptake and Cell Proliferation in Clonal Bovine Mammary Epithelial Cells
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The overall objective of this research was to further understand the mechanism by which growth hormone (GH) stimulates milk production in cattle. Three studies were conducted toward this objective. In the first study, the effects of GH and insulin-like growth factor-I (IGF-I), a major mediator of GH action in vivo, on cell proliferation, nutrient transport, and milk protein gene expression in bovine mammary epithelial cell line MAC-T cells were determined. GH increased (P < 0.01) expression of four major milk protein genes in MAC-T cells transfected with GHR expression plasmid. Cotransfection analyses indicated that GH also stimulated (P < 0.01) luciferase reporter gene expression from the promoters of the four milk protein genes in MAC-T cells. These findings together with the fact that GHR mRNA and protein are expressed in the epithelial cells of the bovine mammary gland suggest that GH may directly stimulate milk protein gene expression in the mammary gland. This study also showed that IGF-I increased the proliferation (P < 0.01) and amino acid transport (P < 0.05) in MAC-T cells. Because GH is known to stimulate IGF-I production in animals, IGF-I-mediated mammary epithelial cell proliferation and amino acid uptake may be additional mechanisms by which GH increases milk production in cattle. In the second study, the role of connective tissue growth factor (CTGF) on IGF-I-stimulated proliferation of MAC-T cells was investigated. A microarray analysis revealed that IGF-I decreased CTGF mRNA expression in MAC-T cells (P < 0.01). This effect of IGF-I was further found to be mediated through the PI-3 kinase/Akt signaling pathway from the IGF-I receptor (IGF-IR). CTGF alone stimulated MAC-T cell proliferation (P < 0.01). However, together with IGF-I, CTGF attenuated the proliferating effect of IGF-I on MAC-T cells, and this attenuation was reversed by additional IGF-I. Therefore, IGF-I inhibition of CTGF expression may benefit IGF-I stimulation of MAC-T cell proliferation. CTGF had no effect on IGF-I-induced phosphorylation of IGF-IR or total IGF-IR expression in MAC-T cells, suggesting that CTGF may attenuate IGF-I stimulation of MAC-T cell proliferation through a postreceptor inhibition of the IGF-IR signaling pathway. In the third study, whether a milk yield-associated T/A polymorphism in exon 8 of the bovine GHR gene affected GHR signaling was determined. It was found that the two corresponding GHR variants did not differ in mediating GH induction of gene expression, suggesting that the two GHR variants are not functionally different and hence are unlikely to mediate different effects of GH on milk production. In summary, the results of this dissertation research suggest that GH may directly stimulate milk protein gene expression and indirectly stimulate mammary epithelial cell proliferation and amino acid uptake through IGF-I, thereby stimulating milk production in cattle. The results also suggest that IGF-I stimulation of mammary epithelia cell proliferation may involve an inhibition of CTGF expression in the cells.
- Doctoral Dissertations