IRS4, a novel modulator of BMP/Smad and Akt signalling during early muscle differentiation

Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration.

was applied as vehicle control. Lysate were subjected to Western blotting using indicated antibodies. Quantification depicts total FLAG-Smad5 levels (see red indication on the blot) normalised to β-actin relative to β-galactosidase. (e) (f) Impact of IRS proteins on transcriptional activity of BMP Smads. C2C12 cells transfected with BRE-luc, RL-TK and IRS1-4-myc or β-galactosidase were stimulated with 5 nM BMP2 for 6 h (a) or 24 h (b), respectively. Bar chart depicts means ± SD of RLU from triplicate measurements relative to β-galactosidase. (g) IRS4 reduces BMP2-induced ID1 protein expression. Transfected C2C12 cells were stimulated with 5 nM BMP2 for 6 h. Lysates were subjected to Western blotting using indicated antibodies. (h) C2C12 cells transfected with BRE-luc, RL-TK and IRS4-myc, truncations thereof or β-galactosidase were stimulated with 5 nM BMP2 for 6 h. Bar chart depicts means ± SD of RLU from triplicate measurements relative to β-galactosidase. RLU= relative luciferase units (BRE-luc/RLTK-luc) Supplementary Figure S3: Expression of IRS4 and myogenic markers during mouse limb development (a-d) IRS4 is expressed during mouse limb development. RNA from mouse limbs of indicated embryonic developmental stages was isolated, reverse-transcribed and subjected to gene expression analysis via qRT-PCR. Bar charts summarise triplicate measurements and depict MNE ± SEM representative for 2 independent experiments. Data corresponds to these combined in Fig 5a. Supplementary Figure S4: Characterisation of BMP signalling in immortalised human myoblasts (a) RNA from cultured human myoblasts was isolated, reverse-transcribed and used for gene expression analysis via qRT-PCR. Bar chart depicts means ± SEM from triplicate measurements. (b) Human myoblasts were stimulated with 10 nM BMP2 for indicated times; lysates were subjected to Western blotting using indicated antibodies. (c) Human myoblasts were differentiated with or without 10 nM BMP2 for indicated times; gene expression was analysed via qRT-PCR. Bar chart depicts MNE ± SEM from triplicate measurements. (d) Concomitantly, images were acquired at indicated times. (e) IRS4 is expressed in human myoblasts. IRS4 expression was verified via flow cytometry using a specific antibody (black line; control IgG: grey shaded curve). (f) siRNA-mediated knockdown of IRS4. Knockdown efficiency was analysed via qRT PCR (left) and Western blotting (right) (g) Control samples for the In situ proximity ligation assay (PLA) of IRS4 and BMPRII or Smad1. Human myoblasts were subjected to in situ PLA (green signal) to visualize the endogenous association of IRS4 with BMPRII or Smad1; nuclei and the actin cytoskeleton were stained using DAPI and Phalloidin594. The association of Smad1 with Smad4 served as positive control. Figure S5: Targeted IRS4 knockdown decreases myogenic differentiation of primary foetal myoblasts and primary postnatal satellite cells (a) (b) IRS4 knockdown affects myogenesis in primary mouse cells. Primary mouse myoblasts isolated from E18.5 limb muscles (a) or primary mouse satellite cells isolated from P7 limb muscles (b) were transfected with siRNA targeting either nonspecific sequences (scr si) or mouse IRS4 (mIRS4 si) and differentiated for three (a) or two (b) days, respectively. Myosin heavy chain (MHC) and/or the actin cytoskeleton were stained using a specific antibody or Phalloidin594; nuclei were stained using DAPI. Images were quantified using ImageJ. Bar charts depict means ± SD of at least 16 images per condition.  Human

Isolation of primary foetal mouse myoblasts and cytospin
For the isolation of primary myoblasts, limbs derived from E18.5 foetuses were used. First, the muscle tissue was dissected from the skin and bones and homogenised. Homogenates were transferred into Hank's buffered Salt Solution (HBSS; Biochrom AG/Thermo Scientific) supplemented with 2 mM MgSO4 and 2 mM CaCl2. Then, 50 µl 15% collagenase (Sigma-Aldrich) was added and incubated at 37°C and 1000 rpm for 40 min with rigorous vortexing in between. Each sample was supplemented with growth medium (DMEM (inc. 4,5 g/l glucose) BioWhittaker Cambrex), pipetted through a 40 µM cell strainer into 15 ml tubes and centrifuged at room temperature at 2000 rpm for 5 min. The cell pellet was resuspended in growth medium, added onto poly-D-lysine (1 mg/ml; Millipore) coated glass cover slips, incubated for 1 h at room temperature and subjected to cytospin-preparation at 200 rpm for 5 min followed by fixation and immunofluorescence staining. For RNA isolation, cDNA synthesis and semiquantitative PCR analysis, pure primary myoblast population was used.

Isolation of primary postnatal mouse satellite cells
Forelimb and hindlimb muscles harvested from day 7 postnatal mice (P7) were minced and digested using 20 µl collagenase A (100 µg/µl; Sigma-Aldrich) in DMEM in a shaking water bath at 37°C and 1400 rpm for 30 min followed by addition of 2 U/ml dispase (Sigma-Aldrich) for another 30 min. Next, the sample was passed through a 20G syringe followed by a 70 µm cell strainer to obtain a single cell suspension.  Differentiation was induced using DMEM supplemented with 5% horse serum, 2 mM L-glutamine and penicillin (100 units/ml)/streptomycin (10 µg/ml).
For siRNA-mediated knockdown of mouse IRS4 in primary myoblasts or satellite cells, Lipofectamine RNAiMAX (Invitrogen) was used according to manufacturer's instructions; as control non-targeting siRNA was used. In short, cells were transfected with 25-75 nM siRNA and differentiation was induced 48 h post-transfection.

Semi-quantitative PCR analysis
RNA isolated from freshly isolated, pure primary myoblasts or isolated and cultured primary satellite cells was subjected to cDNA synthesis and amplified by PCR using 2 µl cDNA template, 2 µl dNTPs (1.25 mM; Fermentas), 1 µl of each specific primer (10 µM, see Supplementary Table S2) and 0.5 µl Taq polymerase (5-10 U/µl; homemade) in a final reaction volume of 20 µl. The amplification reaction consisted of an initial denaturation step at 95°C for 10 min followed by 30 cycles of 15 sec at 95°C and 60 sec at 60°C. PCR products were separated on 1.5% agarose gels and visualised via ethidium bromide staining.