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

Störung des BMP / Noggin-Gleichgewichts stört die Integrität der Myofaser und führt zu einer Plastizität der Säugetiermuskulatur

Myogenesis is a highly co-ordinated process driven by paracrine signalling in conjunction with an intricate biomechanical niche. Bone morphogenetic proteins (BMPs) and the antagonist Noggin balance proliferation and differentiation of muscle progenitors and adult muscle stem cells. However, there is a lack of systematic understanding of how BMP/Noggin signalling co-ordinates with the microenvironment during fetal myogenesis in mammals. Understanding the mechanisms that drive the myogenic program is essential to determine the molecular basis of muscle disease and regeneration. This study investigated the role of BMP/Noggin signalling during limb myogenesis. A comprehensive phenotypic analysis of a Noggin knockout mouse model (Nog KO) showed altered proliferation, differentiation and fusion of myogenic progenitor cells altogether decreasing myofiber formation. This was re-capitulated by a CRISPR/Cas9 generated Noggin-deficient C2C12 myoblast cell line. Furthermore, a striking disappearance of myofibers was observed in fetal stages which was accompanied by loss of the basal lamina and ectopic expression of the extracellular matrix component Tenascin-C (TnC). This altered the biomechanical niche by reducing the stiffness of the muscle tissue. Myotubes in these muscles were characterized by exacerbated BMP/SMAD signalling and displayed hallmarks of de-differentiation including cell cycle re)entry in otherwise post-mitotic myonuclei. Furthermore, in vivo genetic lineage tracing analysis in the Nog KO fetuses revealed trans-differentiation of the myogenic cells into chondrogenic, osteogenic and connective tissue cells. Analysis of mouse myotubes in vitro demonstrated that myonuclear cell cycle re-entry can be initiated by BMPs with superb potency, such as generated upon protease cleavage, in combination with TnC and substrate stiffness below the endogenous range for muscle. This study reveals a previously unseen capacity of muscle fragmentation and cell- fate switch in a mammalian organism. It was demonstrated that a combination of exacerbated BMP signalling and the biomechanical niche influences muscle-cell plasticity. This work highlights the importance of maintaining the intricate balance between BMP and Noggin during developmental myogenesis. This knowledge can be extrapolated to gain a novel perspective on muscle diseases like muscular atrophy and to develop new tools to foster muscle regeneration

Cooperation of BMP and IHH signaling in interdigital cell fate determination

<div><p>The elaborate anatomy of hands and feet is shaped by coordinated formation of digits and regression of the interdigital mesenchyme (IM). A failure of this process causes persistence of interdigital webbing and consequently cutaneous syndactyly. Bone morphogenetic proteins (BMPs) are key inductive factors for interdigital cell death (ICD) in vivo. NOGGIN (NOG) is a major BMP antagonist that can interfere with BMP-induced ICD when applied exogenously, but its in vivo role in this process is unknown. We investigated the physiological role of NOG in ICD and found that <i>Noggin</i> null mice display cutaneous syndactyly and impaired interdigital mesenchyme specification. Failure of webbing regression was caused by lack of cell cycle exit and interdigital apoptosis. Unexpectedly, <i>Noggin</i> null mutants also exhibit increased <i>Indian hedgehog</i> (<i>Ihh</i>) expression within cartilage condensations that leads to aberrant extension of IHH downstream signaling into the interdigital mesenchyme. A converse phenotype with increased apoptosis and reduced cell proliferation was found in the interdigital mesenchyme of <i>Ihh</i> mutant embryos. Our data point towards a novel role for NOG in balancing <i>Ihh</i> expression in the digits impinging on digit-interdigit cross talk. This suggests a so far unrecognized physiological role for IHH in interdigital webbing biology.</p></div

Primer sequences for real-time RT-qPCR.

<p>Primer sequences for real-time RT-qPCR.</p

Noggin mutants display impaired interdigit marker expression.

<p>(A) Whole-mount ISH for <i>Msx1</i> and <i>Msx2</i> shows impaired expression in proximal interdigital mesenchyme (yellow arrowheads) for both genes; <i>Msx2</i> appears increased at the digit tips in <i>Nog</i>^<i>-/-</i> hand plates (black arrowheads). (B) Section ISH for <i>Msx2</i> and <i>Id3</i> on longitudinal hand plate sections, digit 3 is shown; orientation as indicated di: distal, do: dorsal, pr: proximal, ve: ventral). <i>Msx2</i> and <i>Id3</i> show increased expression at the digit tips (arrowheads). (C) Expression of <i>Msx1</i> and <i>Msx2</i> was analyzed by real-time qPCR on mRNA extracted from microdissected interdigit tissue. (D) BMP canonical signaling was assessed by immunolabeling for phosphorylated SMADs (pSMAD1/5/8). No aberrant pSMAD signal is visible in the interdigital region in <i>Nog</i>^<i>-/-</i> hand plates. Increased signaling is apparent at the circumference of cartilage condensations in <i>Nog</i>^<i>-/-</i> hand plates. (E) <i>Nog</i>^<i>-/-</i> hand plates exhibit impaired <i>Aldh1a2</i> expression, especially in the proximal interdigital mesenchyme (yellow arrowheads), at E13.5 also in the distal interdigital mesenchyme (black arrowheads). (F) Persistent expression of <i>Fgf8</i> in the AER overlying the interdigits (yellow arrowheads) in <i>Nog</i>^<i>-/-</i> hand plates.</p

Apoptosis and proliferation of interdigital cells are altered conversely in <i>Nog</i> and <i>Ihh</i> mutants.

<p>(A) Apoptosis was assessed by immunolabeling for cleaved Caspase 3. Boxed areas showing the distal interdigital mesenchyme are shown as magnifications below. (B) Cell proliferation was assessed by immunolabeling for 5-Bromodesoxyuridine (BrdU) incorporated into the DNA of dividing cells. Boxed areas showing the distal interdigital mesenchyme are shown as magnifications below. (C) Quantification of interdigital apoptosis depicted as cleaved Caspase 3-positive cells / total number of interdigital cells. (D) Quantification of interdigital proliferation depicted as BrdU-positive cells / total cells. Quantification of proliferation was performed in the central interdigit (dashed line in (B)). (E) Quantification of proliferation within digit metacarpal condensations depicted as BrdU-positive cells / total cells. Error bars represent S.E.M. T-test: * = p<0.05; ** = p<0.01; *** = p<0.001 (n = 3).</p

Increased <i>Indian hedgehog</i> (<i>Ihh</i>) expression and downstream signaling in <i>Nog</i>^<i>-/-</i> hand plates.

<p>The expression of <i>Ihh</i> (A), <i>Gli1</i> (B) and <i>Ptc1</i> (C) was assessed by Whole-mount ISH. Note the strong upregulation of <i>Ihh</i> expression in cartilage condensations and the diffuse expression of the IHH targets <i>Gli1</i> and <i>Ptc1</i> in interdigital mesenchyme in <i>Nog</i>^<i>-/-</i> autopodes. (D, E) E13.5 hand plates were microsurgically dissected into interdigit and digit mesenchyme. Quantitative RT-PCR confirms increased <i>Ihh</i> expression in digit condensations (D) and increased expression of <i>Ptc1</i> and <i>Gli1</i> in interdigit mesenchyme (E). Error bars represent S.E.M. T-test: * = p<0.05; ** = p<0.01; *** = p<0.001 (n = 3).</p

Heterobifunctional Ligase Recruiters Enable Pan-Degradation of Inhibitor of Apoptosis Proteins

Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enables the transfer of ubiquitin tags onto their target proteins leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, degraders outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies

Digit and interdigit formation in <i>Nog</i>^<i>-/-</i> embryos.

<p>Cartilage condensation was assessed by whole-mount in-situ hybridization for <i>Sox9</i> (A-C) and X-Gal staining detecting β-Galactosidase expressed from the <i>Noggin</i> locus in <i>Nog</i>^<i>+/-</i> or <i>Nog</i>^<i>-/-</i> embryos (E). (D) Shows unstained autopods. Stages are indicated; FL: forelimb, HL: hindlimb. Indentation between digits is marked by an arrowhead, the interdigital mesenchyme is marked by an asterisk in (C and D).</p

Expanding the PROTAC Toolbox: Targeted Degradation of the Deubiquitinase USP7 in Cancer

Targeting deubiquitinating enzymes (DUBs) has emerged as a promising therapeutic approach in several human cancers and other diseases. DUB inhibitors are exciting pharmacological tools but often exhibit limited cellular potency. Here we report PROTACs based on an ubiquitin-specific protease 7 (USP7) inhibitor scaffold to degrade USP7. The hit compound CST967 caused highly selective degradation of USP7 and inhibited proliferation of USP7-dependent cancer cells. We present the first DUB degrader, which will be a useful tool to deepen our understanding of USP7