Abstract
Necroptosis, a form of programmed cell death, is characterized by the loss of membrane integrity and release of intracellular contents, the execution of which depends on the membrane-disrupting activity of the Mixed Lineage Kinase Domain-Like protein (MLKL) upon its phosphorylation. Here we found myofibers committed MLKL-dependent necroptosis after muscle injury. Either pharmacological inhibition of the necroptosis upstream kinase Receptor Interacting Protein Kinases 1 (RIPK1) or genetic ablation of MLKL expression in myofibers led to significant muscle regeneration defects. By releasing factors into the muscle stem cell (MuSC) microenvironment, necroptotic myofibers facilitated muscle regeneration. Tenascin-C (TNC), released by necroptotic myofibers, was found to be critical for MuSC proliferation. The temporary expression of TNC in myofibers is tightly controlled by necroptosis; the extracellular release of TNC depends on necroptotic membrane rupture. TNC directly activated EGF receptor (EGFR) signaling pathway in MuSCs through its N-terminus assembly domain together with the EGF-like domain. These findings indicate that necroptosis plays a key role in promoting MuSC proliferation to facilitate muscle regeneration.
Highlights
Necroptosis is tightly regulated by the kinase activities of Receptor Interacting Protein Kinases 1 (RIPK1) and Receptor Interacting Protein Kinase 3 (RIPK3).[1,2,3,4] Upon necroptosis induction, RIPK1 binds with RIPK3 and form amyloidal death complex through their RIP homotypic interaction motif (RHIM) domains to activate their kinase activities.[5,6]The amyloidal RIPK1-RIPK3 complex propagates death signal to a downstream effector protein Mixed Lineage Kinase Domain-Like protein (MLKL)
Necroptosis-deficient mice exhibit muscle regeneration defects To explore the function of RIPK1/RIPK3/MLKL-axis dependent necroptosis in muscle regeneration, we generated necroptosisdeficient mice by knocking out the gene of MLKL using the CRISPR/Cas[9] system (Supplementary information, Fig. S1a–c)
No obvious defects of skeletal muscle were observed in Mlkl−/− mice, as their body weight and average myofiber size are the same as WT mice (Supplementary information, Fig. S1d, e), suggesting that necroptosis is not required for muscle development
Summary
Necroptosis is tightly regulated by the kinase activities of RIPK1 and Receptor Interacting Protein Kinase 3 (RIPK3).[1,2,3,4] Upon necroptosis induction, RIPK1 binds with RIPK3 and form amyloidal death complex through their RIP homotypic interaction motif (RHIM) domains to activate their kinase activities.[5,6]The amyloidal RIPK1-RIPK3 complex propagates death signal to a downstream effector protein MLKL. MLKL; on Thr357/Ser[358] of human MLKL), which releases the auto-inhibition of MLKL and enable its oligomerization through its brace region.[9,10] The anti-phospho-MLKL antibody was widely used on sections as the biomarker to detect necroptosis happening in vivo.[11,12,13,14,15,16,17] Following phosphorylation and oligomerization, the N-terminus MLKL directly binds with phosphatidylinositol phosphates (PIPs), which enables it to translocate to membrane compartments, which leads to membrane rupture and cellular contents release.[7,8,9,10,18,19,20] Both Mlkl- and Ripk3deficient mice were demonstrated to be developmentally normal.[21,22,23] It has been widely reported that MLKL-mediated necroptosis plays an essential role in driving inflammation by releasing damage-associated molecular patterns (DAMPs).[24,25,26,27] necroptosis has been considered to be detrimental in vivo due to its pro-inflammatory features. Whether necroptotic cell-released factors play beneficial roles under pathophysiological conditions is not clear
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