Insulin-like growth factor (IGF) binding protein-5 blocks skeletal muscle differentiation by inhibiting IGF actions

Abstract

Signaling through the IGF-I receptor by locally produced IGF-I or -II is critical for normal skeletal muscle development and repair after injury. In most tissues, IGF action is modulated by IGF binding proteins (IGFBPs). IGFBP-5 is produced by muscle cells, and previous studies have suggested that when overexpressed it may either facilitate or inhibit IGF actions, and thus potentially enhance or diminish IGF-mediated myoblast differentiation or survival. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5, we studied its effects in cultured muscle cells. Purified wild-type (WT) mouse IGFBP-5 or a variant with diminished extracellular matrix binding (C domain mutant) each prevented differentiation at final concentrations as low as 3.5 nm, whereas analogs with reduced IGF binding (N domain mutant) were ineffective even at 100 nm. None of the IGFBP-5 variants altered cell number. An IGF-I analog (R(3)IGF-I) with diminished affinity for IGFBPs promoted full muscle differentiation in the presence of IGFBP-5(WT), showing that IGFBP-5 interferes with IGF-dependent signaling pathways in myoblasts. When IGFBP-5(WT) or variants were overexpressed by adenovirus-mediated gene transfer, concentrations in muscle culture medium reached 500 nm, and differentiation was inhibited, even by IGFBP-5(N). As 200 nm of purified IGFBP-5(N) prevented activation of the IGF-I receptor by 10 nm IGF-II as effectively as 2 nm of IGFBP-5(WT), our results not only demonstrate that IGFBP-5 variants with reduced IGF binding affinity impair muscle differentiation by blocking IGF actions, but underscore the need for caution when labeling effects of IGFBPs as IGF independent because even low-affinity analogs may potently inhibit IGF-I or -II if present at high enough concentrations in biological fluids.

Figure 1

Purification and Characterization of Recombinant Mouse IGFBP-5 and Variants A, Diagram of mouse IGFBP-5, showing N-, L-, and C-domains. Amino acid substitutions to create N and C mutants are indicated. B, Purification scheme using heparin-affinity chromatography. C, Purification of IGFBP-5^WT, showing elution pattern from heparin-agarose column (CM, conditioned culture medium; FT, flow through) by silver staining. D, Detection of purified IGFBP-5 and variants after staining with Gelcode blue. E, Detection of purified IGFBP-5 and variants (N, C, and N/C) by immunoblotting (top panel), IGF-II ligand blotting (middle), and heparin ligand blotting (bottom).

Figure 2

IGFBP-5 Inhibits MyoD-Mediated Muscle Differentiation Results are shown of experiments using C3H10T1/2 mesenchymal stem cells infected with Ad-MyoD, followed by incubation in DM for 24 or 48 h with added purified IGFBP-5 (WT), or variants (N, C, or N/C). A, Experimental scheme. B, Immunoblots of whole cell protein lysates for myogenin, troponin T, Akt phosphorylated at serine 473 (pAkt ^S473), Akt, MyoD, α-tubulin, and immunoblots of conditioned DM for IGFBP-5. The final IGFBP-5 concentrations in DM were 35 nm. C, Immunocytochemistry for troponin T (red) and myogenin (green) after incubation for 48 h with 3.5, 10.5, or 35 nm of IGFBP-5 or variants as indicated. Magnification, ×200.

Figure 3

IGFBP-5 Inhibits Muscle Differentiation C3H10T1/2 cells were infected with Ad-MyoD, followed by incubation in DM for 48 h with purified IGFBP-5 (WT), or variants (N, C, or N/C) at 3.5, 10.5, or 35 nm, as in Fig. 2. A, Percentage of myogenin-positive cells (*, P < 0.05; **, P < 0.01 vs. cells incubated with IGFBP-5 WT or C). B, Fusion Index (*, P < 0.002; **, P < 0.003 vs. cells incubated with IGFBP-5 WT or C). C, Myotube area (*, P < 0.01; **, P < 0.005 vs. cells incubated with IGFBP-5 WT or C). Con, Cells incubated without IGFBP-5.

Figure 4

IGFBP-5 Does Not Alter Cell Viability C3H10T1/2 cells were infected with Ad-MyoD, followed by incubation in DM for up to 48 h with purified IGFBP-5 (WT), or variants (N, C, or N/C) at 3.5, 10.5, or 35 nm, as in Fig. 2. A, Cell number after 24 h in DM. B, Cell number after 48 h in DM. Results are presented as percentage of time 0 (mean ± sd) and were calculated as described in Materials and Methods. Con, Cells incubated without IGFBP-5. None of the results were statistically different from the others.

Figure 5

IGFBP-5 Inhibits Differentiation of C2 Myoblasts without Altering Cell Viability Cells were incubated in DM for 48 h with purified IGFBP-5 (WT), or variants (N, C, or N/C) at 35 or 100 nm. A, Immunoblots of whole cell protein lysates for myogenin, troponin T, Akt phosphorylated at serine 473 (pAkt^S473), Akt, and α-tubulin. B, Immunocytochemistry for troponin T (red) and myogenin (green). Magnification, ×200. C, Immunoblots at 0 and 48 h of conditioned culture medium for IGFBP-5 or variants added at time 0. D, Cell number at 48 h after addition of 100 nm of IGFBP-5^WT or variants. Results are presented as percentage of time 0 (mean ± sd) and were calculated as described in Materials and Methods. Con, Cells incubated without IGFBP-5. None of the results were statistically different from the others.

Figure 6

IGF-I Reverses the Inhibitory Effect of IGFBP-5 on MyoD-Mediated Muscle Differentiation C3H10T1/2 cells were infected with Ad-MyoD, followed by incubation in DM for 48 h with purified IGFBP-5 (WT or C) [35 nm] with or without R³IGF-I [5 nm]. A, Immunocytochemistry for myogenin (green) and troponin T (red). Magnification, ×200. B, Fusion index (*, P < 0.003 vs. cells not incubated with R³IGF-I). Results are presented as mean ± sd and were calculated as described in Materials and Methods. Con, Cells incubated without IGFBP-5.

Figure 7

Overexpression of IGFBP-5 or Variants Inhibits MyoD-Mediated Muscle Differentiation A, C3H10T1/2 cells were infected with Ad-MyoD and Ad-tTA, followed by infection with Ad-IGFBP-5^WT or variants, and incubation in DM ± Dox for 48 h. The final concentration of IGFBP-5 in conditioned medium after 48 h in DM ranged from 500–700 nm. B, Quantitative immunoblots for IGFBP-5^WT and variants (N, C, or N/C), using 1–6 μl of conditioned DM and graded amounts of purified IGFBP-5^WT as standards. C, Immunocytochemistry for myogenin (green) and troponin T (red). Magnification, ×200. D, Myogenin-positive nuclei (mean ± sd; *, P < 0.003; **, P < 0.01 vs. no Dox). E, Cell number (mean ± sd). Results for D and E were calculated as described in Materials and Methods.

Figure 8

Dose-Dependent Inhibition of IGF-I Receptor Activation by IGFBP-5 C3H10T1/2 cells were incubated in serum-free medium for 3 h followed by addition of new serum-free medium for 30 min with IGF-II [10 nm] plus IGFBP-5^WT or variants (N, C, or N/C) at the concentrations indicated. A, Immunoblots (IB) of anti-phosphotyrosine (pTyr) immunoprecipitates (IP) and whole cell protein lysates for the IGF-I receptor (IGF-IR). A representative experiment (of two performed) is shown. B and C, Immunoblots of whole cell protein lysates for Akt phosphorylated at serine 473 (pAkt^S473). Relative levels of pAkt^S473 normalized to Akt are indicated graphically at the bottom of the panels. A representative experiment (of four performed) is shown.