IGF-binding proteins mediate TGF-beta 1-induced apoptosis in bovine mammary epithelial BME-UV1 cells.
TGF-beta 1 is an antiproliferative and apoptogenic factor for mammary epithelial cells (MEC) acting in an auto/paracrine manner and thus considered an important local regulator of mammary tissue involution. However, the apoptogenic signaling pathway induced by this cytokine in bovine MEC remains obscure. The present study was focused on identification of molecules involved in apoptogenic signaling of transforming growth factor-beta 1 (TGF-beta 1) in the model of bovine mammary epithelial cell line (BME-UV1). Laser scanning cytometry (LSC), Western blot and electrophoretic mobility shift assay (EMSA) were used for analysis of expression and activity of TGF-beta 1-related signaling molecules. The earliest response occurring within 1-2 h after TGF-beta 1 administration was an induction and activation of R-Smads (Smad2 and Smad3) and Co-Smad (Smad4). An evident formation of Smad-DNA complexes began from 2nd hour after MEC exposure to TGF-beta 1. Similarly to Smads, proteins of AP1 complex: phosphorylated c-Jun and JunD appeared to be early reactive molecules; however, an increase in their expression was detected only in cytosolic fraction. In the next step, an increase of IGF binding protein-3 (IGFBP-3) and IGFBP-4 expression was observed from 6th hour followed by a decrease in the activity of protein kinase B (PKB/Akt), which occurred after 24 h of MEC exposure to TGF-beta 1. The decrease in PKB/Akt activity coincided in time with the decline of phosphorylated Bad expression (inactive form). Present study supported additional evidence that stimulation of insulin-like growth factor I (IGF-I) was associated with complete abrogation of TGF-beta 1-induced activation of Bad and Bax and in the consequence protection against apoptosis. In conclusion, apoptotic effect of TGF-beta 1 in bovine MEC is mediated by IGFBPs and occurs through IGF-I sequestration, resulting in inhibition of PKB/Akt-dependent survival pathway.
Gajewska
Małgorzata
M
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland.
Motyl
Tomasz
T
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000502:physiology; D005260:Female; D018969:Insulin-Like Growth Factor Binding Proteins / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000187:drug effects* / Q000378:metabolism; D018613:Microscopy, Confocal; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D016755:Proto-Oncogene Proteins c-jun / Q000187:drug effects / Q000378:metabolism; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000187:drug effects / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology* / Q000502:physiology; D053773:Transforming Growth Factor beta1
D004268:DNA-Binding Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D011518:Proto-Oncogene Proteins; D016755:Proto-Oncogene Proteins c-jun; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt