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.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/38476 |
| Date | 06 June 2008 |
| Creators | Romagnolo, Donato |
| Contributors | Animal Science, Akers, Robert Michael, Wong, Eric A., Gwazdauskas, Francis, Johnson, J.L., Turner, J.D., Vinson, William E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation, Text |
| Format | xi, 206 leaves, BTD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | OCLC# 29179597, LD5655.V856_1993.R652.pdf |
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