Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I

Abstract

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

FIG. 1

Postmitotic expression of IGF-I enhances expression of the markers of muscle terminal differentiation. Control L6E9 myoblasts and L6E9 myoblasts stably transfected with the MLC–IGF-I expression vector (L6MLC/IGF-I) were grown to approximately 80% confluence in GM and then switched to DM (day 0). Total RNA was isolated from proliferative myoblasts (lanes GM) and at 0, 1, 2, 3, and 4 days after switching to DM. Northern blots of total RNA samples (15 μg) were analyzed with the indicated ³²P-labeled probes. Ethidium bromide (EtBr) staining was used to verify equal loading of the RNA sample.

FIG. 2

IGF-I enhances myogenin promoter activity. L6E9 and L6MLC/IGF-I myoblasts were transiently transfected with a myogenin–LacZ–β-Gal vector. Cell cultures were shifted 24 h after transfection to serum-free medium and cultured for an additional 72 h. Fifty separate randomly selected fields per each cell line were counted to quantitate LacZ-expressing cells. The numbers of total cells in each field per each cell line were approximately the same. The number of β-Gal-positive cells per field are indicated on the y axis.

FIG. 3

IGF-I promotes the myogenic program by induction of MEF-2C expression. Northern blots of total RNA (15 μg) isolated from L6E9 and L6MLC/IGF-I myogenic cultures at 0, 1, 2, 3, and 4 days after switching to DM and probed with MEF-2C (30), MCK (8), and β-enolase (35) ³²P-labeled cDNA probes are shown. Ethidium bromide (EtBr) staining was used to verify equal loading of the RNA sample.

FIG. 4

Postmitotic expression of IGF-I induces myotube hypertrophy and cytoskeletal reorganization. L6E9 and L6MLC/IGF-I cell lines were grown to approximately 80% confluence in GM and then switched to DM and cultured for 4 days. (A and B) Eosin-Wright’s stain (phase contrast) of L6E9 and L6MLC/IGF-I differentiated muscle cells; (C and D) Hoechst nuclear staining; (E and F) differentiated cultures immunostained with mouse anti-α actin MAb, processed for alkaline phosphatase activity, and then photographed at the same magnification.

FIG. 5

IGF-I induces accumulation of β1D integrin. Biotinylated total cell extracts from L6E9 and L6MLC/IGF-I proliferative myoblasts and differentiated myotubes were immunoprecipitated with anti-β1D integrin antibody. Proteins were resolved by SDS-PAGE, blotted onto nitrocellulose membrane, and probed with ExtrAvidin-peroxidase. Lane 1, L6MLC/IGF-I proliferative myoblasts plus antibody (Ab); lane 2, L6E9 myotubes plus Ab; lane 3, L6MLC/IGF-I myotubes plus Ab; lane 4, L6MLC/IGF-I myotubes without Ab. Arrows labeled β1D and α7 indicate the bands corresponding to β1D and α7 integrins, respectively.

FIG. 6

IGF-I activates expression of the neonatal isoform of MyHC. L6E9 and L6MLC/IGF-I cell lines were grown to approximately 80% confluence in GM and then switched to DM and cultured for 4 days. Differentiated cultures were immunostained with MAb against either the embryonic or neonatal isoform of MyHC (Immunofluor) or were subjected to nuclear staining (Hoechst).

FIG. 7

Morphological effect of PI 3-kinase inhibitor on IGF-I-induced hypertrophy. Cells were grown as described in the legend to Fig. 1 and treated with a 10 μM concentration of the PI 3-kinase inhibitor LY294002 at different times of culture: during growth in GM, at 80% of confluence (d0), and at days 1, 2, and 3 in DM. Cells treated with LY294002 in GM were switched to DM without PI 3-kinase inhibitor when they were at 80% of confluence. After 4 days, the cultures were washed with PBS, fixed with methanol, and stained with eosin–Wright’s stain.

FIG. 8

Late inhibition of PI 3-kinase does not affect myogenic differentiation. Northern blots of total RNA samples (15 μg) isolated from untreated (lanes L6E9 or L6MLC/IGF-I cells (lanes U) or from cells treated with 10 μM LY294002 at different times of differentiation are shown: during growth in GM, at 80% of confluence (day 0), and at days 1, 2, and 3 in DM. RNAs were probed with IGF-I or myogenin ³²P-labeled probes. Ethidium bromide (EtBr) staining was used to verify equal loading of the RNA samples.

FIG. 9

PI 3-kinase is not required in maintenance of muscle phenotype induced by MLC–IGF-I. (Lower panels) Immunofluorescence analysis of the neonatal isoform of MyHC in L6MLC/IGF-I cultures treated with 10 μM LY294002 inhibitor as myoblasts (panels GM) or differentiated in the presence of inhibitor added immediately upon serum withdrawal (panels d0+LY294002) or 2 days after serum withdrawal (panels d2+LY294002) is shown. U, untreated cultures. Upper panels show Hoechst nuclear staining. All cultures were analyzed 4 days after serum withdrawal.