title: Effect of constitutive expression of porcine IGFBP-3 on proliferation and differentiation of L6 myogenic cells.
abstract: We have previously shown that exogenous recombinant porcine IGFBP-3 (rpIGFBP-3) suppresses proliferation and differentiation of L6 myogenic cells in an IGF-I-dependent manner and suppresses proliferation of L6 myogenic cells via an IGF-I-independent mechanism. In order to assess the effects of endogenously produced IGFBP-3, we have transfected L6 myogenic cells with a pEF6/V5 vector containing pIGFBP-3 cDNA under the control of the human elongation factor 1alpha (hEF-1alpha) promoter and with the empty vector. We have isolated a cell population that constitutively produces porcine IGFBP-3 (tL6 cells) and a stable mock transfected cell population containing the empty vector (mtL6 cells). Constitutive expression of IGFBP-3 slightly reduced the expression of IGFBP-5 but had no effect on IGFBP-4 production by L6 myogenic cells. Immunoneutralization of IGFBP-3 increased both IGF-I- and Long-R3-IGF-I-stimulated proliferation of tL6 cells (58 and 33%, respectively) (P<0.01). These data indicate endogenous pIGFBP-3, like exogenous rpIGFBP-3, suppresses the proliferation of L6 myogenic cells via both IGF-I-dependent and -independent pathways. Immunoneutralization of IGFBP-3 also increased IGF-I-stimulated differentiation (21%, P<0.05) but had no effect on Long-R3-IGF-I stimulated differentiation of tL6 myogenic cells. Results indicate that exogenous and endogenous IGFBP-3 affect proliferation and differentiation of L6 myogenic cells in a similar way. Immunohistochemical localization data reveal that pre-incubation with anti-pIGFBP-3 dramatically reduces the level of intracellular IGFBP-3 in tL6 myogenic cells indicating that endogenously produced IGFBP-3 must first be secreted before it is internalized and that anti-pIGFBP-3 prevents internalization of IGFBP-3. TL6 and mtL6 cells provide a good system to further investigate the mechanisms by which IGFBP-3 affects proliferation and differentiation of myogenic cells.
authors.affiliation: Animal Growth and Development Laboratory, Department of Animal Science, University of Minnesota, 348 ABLMS, Eckles Avenue, St. Paul, MN 55108, USA.
authors.forename: G
authors.initials: G
authors.lastname: Xi
authors.forename: E
authors.initials: E
authors.lastname: Kamanga-Sollo
authors.forename: M R
authors.initials: MR
authors.lastname: Hathaway
authors.forename: W R
authors.initials: WR
authors.lastname: Dayton
authors.forename: M E
authors.initials: ME
authors.lastname: White
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D048708:Cell Growth Processes / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003402:Creatine Kinase / Q000378:metabolism; D017077:Culture Media, Conditioned; D007150:Immunohistochemistry / Q000662:veterinary; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011506:Proteins / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D017077:Culture Media, Conditioned; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D011506:Proteins; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D003402:Creatine Kinase