The Mechanism Of TGF-? Repressing TIMAP And Its Functional Significance In Macrophage M2 Phenotypic Phagocytosis

BackgroundTIMAP(TGF-? inhibited membrane-associated protein)is a novel TGF-?downstream protein we cloned from glomerular endothelial cells years ago.TIMAP is a member of the myosin phosphatase targeting subunit(MYPT)family and contains the interaction site for catalytic subunit of protein phosphatase 1(PP1c)and 5 ankryn repeats at N terminus and a C-terminal CAAX box that directs its expression on the cell membrane through prenylation.But so far the mechanism of TGF-?repressing TIMAP and its functional relevance to TGF-? bioactivity remain largely unknown.TGF-P is an important cell growth factor that regulates many cellular processes such as tissue development,inflammation,tissue fibrosis and tumor.Although we originally identified TIMAP from glomerular endothelial cells,which share with hematopoietic cells the common precursor-hemantioblast,our preliminary study found that TIMAP is also highly expressed in mouse macrophages,which are professional inflammatory and phagocytic cells regulated by TGF-?.Macrophages can differentiate into pro-inflammatory M1 subtype via classical pathway,or alternatively to an anti-inflammatory M2 subtype,depending on the micro-environmental stimulations.Early in inflammatory diseases,macrophages are attracted to the inflammatory site and release a large amount of pro-inflammatory cytokines to promote inflammation;but later on the disease progression,macrophages polarize to M2 phenotype,accompanied by expression of M2 specific cytokines such as TGF-? and IL-10,migrate to the injured site to clean harmful cell debris through phagocytosis and repair injury by depositing extracellular matrix proteins.However,macrophages often become hyper-responsive,resulting in uncontrolled or deregulated release of TGF-? that exacerbates acute tissue injury and promotes the development of chronic diseases,such as tissues fibrosis,cancer cell metastasis.Macrophage migration and phagocytosis involve dynamic cytoskeleton rearrangement and cell membrane movement,which are driven by myosin and F-actin contractivity that is positively regulated by myosin light chain(MLC)phosphorylation.The level of MLC phosphorylation is controlled by myosin light chain kinase(MLCK)and myosin phosphatase(MP)whose holoenzyme is composed of catalytic subunit of protein phosphatase PP1c,a small protein M20 of unknown function and one of the MP family members including TIMAP.Recent study indicated that TIMAP assisted PP1c in dephosphorylating MLC in vitro,suggesting that TIMAP might affect TGF-?-associated macrophage M2 tissue repairing process via its modulation on MLC phosphorylation.ObjectiveThe aims of this study were to explore:a)the molecular mechanism of TGF-?repressing TIMAP;b)The functional relevance of TGF-? repressing TIMAP in macrophage M2 phenotypic activities;c)whether TIMAP affects macrophage M2 polarization and migration/phagocytosis via its regulation on MLC phosphorylation.Methods and ResultsWe found that TIMAP is highly expressed in macrophages,the primary inflammatory cells regulated by TGF-?.By using a TIMAP promoter reporter construct,we demonstrated that TIMAP transcription repression by TGF-? is inhibited by HDAC inhibition.TGF-? treatment caused selective HDAC3 protein upregulation in cells and ChIP assay indicated that HDAC3 and Smad2/3 were recruited to the Smad binding element(SBE)sites on the TIMAP promoter.Meanwhile the levels of acetylated histone3(Ac-H3)in the same locus were decreased,suggesting TGF-? represses TIMAP transcription through HDAC3-dependent Smad signaling.TIMAP seems to antagonize TGF-P induction of macrophage M2 polarization;TIMAP negatively affected the induction of macrophage M2 maker Arg-1 and IL-10,suggesting that TIMAP participates in TGF-?-associated wound remodeling and tissue repairing processes.TIMAP is physically associated with and dephosphorylated myosin light chain in macrophage,which is an essential cytoskeleton component in promoting cell motility.In addition,TIMAP levels negatively correlate with TGF-?-induced macrophage migration and phagocytosis in HDAC3 and MLC phosphorylation-dependent manners,suggesting that TIMAP dephosphorylation of MLC constitutes a critical component of TGF-?-mediated M2 macrophage activities.ConclusionsIn summary,our studies have revealed a novel regulatory pathway that mediates TGF-?-associated wound repair/tissue remodeling processes.TIMAP is repressed by TGF-P via HDAC3-Associated Smad signaling,TIMAP negatively regulates macrophage M2 phenotypic phagocytosis by dephosphorylating MLC.Given the fact that hyperactive TGF-p activities are frequently associated with various chronic disorders and HDAC inhibitor or TGF-? signaling blockade protect against tissue fibrosis,inflammatory disease and tumor.The strategies targeting HDAC3/TIMAP axis might potentially counteract TGF-?-associated pathological processes.