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Autocrine mechanisms of action of insulin-like growth factor-I (IGF-I) and hormonal regulation of expression of IGF-finding proteins in mammary epithelial cells

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) genes in mammary epithelial cells. To test the hypothesis that IGF-I affects growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, several cell lines were developed expressing an ovine exon-2 containing IGF-I cDNA under the control of the mouse mammary tumor virus-long terminal repeat (pMMTV-IGF-I), early simian virus (pSV40-IGF-I), and herpes simplex thymidine kinase (pTK-IGF-I) promoters. Stably transfected clones were generated by cotransfection of clonal MAC-T cells with the IGF-I expression vectors and a plasmid conferring resistance to hygromycin-B (HYG-B), using a calcium phosphate precipitation procedure. Induction of the MMTV-LTR with the glucocorticoid dexamethasone (DEX) was required for enhanced expression of IGF-I in MD-IGF-I (MD=Mammary Derived) cells, whereas SV40-IGF-I cells constitutively expressed the highest levels of IGF-I, followed by TK-IGF-I cells. Activity of the MMTV promoter in MD-IGF-I cells was coordinately regulated by lactogenic hormones and extracellular matrix. Acute secretion of DEX-induced recombinant IGF-I by MD-IGF-I cells stimulated cell proliferation through an autocrine/paracrine pathway and triggered the expression of IGFBP-3. Neither acute nor constitutive expression of IGF-I affected expression of type 1 IGF receptor mRNAs, but down-regulated cell surface receptor levels, in the order SV40-> TK- > MD-IGF-I. Secretion of IGF-I-induced IGFBP-3 potentiated the mitogenic actions of IGF-I as evidenced by enhancement of [³H]thymidine uptake into DNA of parental MAC-T cells. This study provides evidence that local production of IGF-I can stimulate cell proliferation of bovine mammary epithelial cells through an autocrine/paracrine mode of action. We suggest that secretion of IGF-I-induced IGFBP-3 by bovine mammary epithelial cells enhances cell responsiveness to IGF-I, but does not prevent down-regulation of the IGF-I receptor in cells constitutively expressing IGF-I. / Ph. D.

Identiferoai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/38476
Date06 June 2008
CreatorsRomagnolo, Donato
ContributorsAnimal Science, Akers, Robert Michael, Wong, Eric A., Gwazdauskas, Francis, Johnson, J.L., Turner, J.D., Vinson, William E.
PublisherVirginia Tech
Source SetsVirginia Tech Theses and Dissertation
LanguageEnglish
Detected LanguageEnglish
TypeDissertation, Text
Formatxi, 206 leaves, BTD, application/pdf, application/pdf
RightsIn Copyright, http://rightsstatements.org/vocab/InC/1.0/
RelationOCLC# 29179597, LD5655.V856_1993.R652.pdf

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