Effect of constitutive expression of porcine IGFBP-3 on proliferation and differentiation of L6 myogenic cells

G. Xi, E. Kamanga-Sollo, M. R. Hathaway, W. R. Dayton, M. E. White

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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 1α (hEF-1α) 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.

Original languageEnglish (US)
Pages (from-to)35-51
Number of pages17
JournalDomestic Animal Endocrinology
Volume31
Issue number1
DOIs
StatePublished - Jul 2006

Bibliographical note

Funding Information:
This research was supported by a USDA National Research Initiative Competitive Grant (Grant #00–10204) from the USDA Cooperative State Research, Education and Extension Service Program and by the Minnesota Agricultural Experiment Station.

Keywords

  • Differentiation
  • IGFBP-3
  • L6 myogenic cells
  • Proliferation
  • Stable transfection

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