Studies On NLK Mediating The Differentiation Of Muscle Cells By Regulating SRF Signaling Pathway

Skeletal muscle plays an important role in the movement of the body and the maintenance of the normal form of the body,and its growth and development also have a strong impact on the ability of life.Skeletal musle formation requires the fusion of myoblast cells to produce multinucleated fibers,a multi-step process contolled by a variety of myogenic regulators.SRF(serum response factor),a major regulator of early cytoskeleton genes,regulates cell differentiation and reproduction by binding to MKLs(cardiac protein-related transcription factor)or ELKs transcription co-proteins,which have an important effect on skeletal muscle formation,but the specific molecular mechanism is not clear.NLK(nemo-like kinase)is an typical kinase of MAPK family,which can phosphorylate the substrate and play a series of roles.We find the specific deletion of NLK in skeletal muscles of C57 mice will make the muscle microtubules of mice bulky,thus increasing the weight of mice and skeletal muscles such as gastrocnemius.Therefore,we hypothesized whether NLK is crucial to skeletal musle development in mice.In this paper,we firstly used human renal epithelial cell screening to find that NLK can effiectively up-regulate the SRF/ELK1 signaling pathway,so that the transcription and expression levels of its downstream relate genes are enhanced,and down-regulated the SRF/MKL1 signaling pathway,so as to weaken the trancriptiong and expression of its downstream genes.Then we constructed human colerectal cancer cell lines and muscle myoblast cell lines with NLK knockout and knockdown respectively.Through experiments,it was further proved that NLK,whether in human or mouse cells,plays the same role in the above two pathways,and the differentiation of myoblast cells was significantly accelerated after NLK knockdown.Therefore,NLK can inhibit the differentiation of cells.Since NLK can affect the above two signaling pathway,we wondered it could interact with one or more proteins.Therefore,we designed experiements to verify that NLK can only directly bind to SRF but no direct interaction with ELK1 or MKL1.And we also studied the cellular localization of the two proteins and found that they had a good overlap in the nucleus.NLK is a kinase that phosphorylates the substrate protein,so we developed experiement to confirm that NLK indeed phosphorylates SRF.Therefore,it can be concluded that NLK is phosphorylated after binding with SRF to futhuer affect the above two signaling pathways.In order to prove the NLK phosphorylation of SRF specific molecular mechanism,we will SRF gene maybe NLK phosphorylation of serine and threonine all mutations into alanine,design experiments,again to find NLK phosphorylation site of SRF,and found that this site is likely to be S101 and S103,but this part needs more experiment to verify further.Moreover,the molecular mechanism by which NLK makes mice gain weight also needs further exploration and verification.In summart,NLK can regulate the ELK/SRF signaling pathway through phosphorylation of SRF,thereby inhibiting the differentiation process of mouse myoblasts.Moreover,after specific knockout of NLK by skeletal musle,the body weight and skeletal muscle weight of mice increased.