ADAMTS16 Activates Latent TGF-β1 To Regulate Cardiac Fibrosis And Dysfunction

Heart failure occurs at the end of heart disease,and is correlated with a high rate of morbidity and mortality.The main cause of heart failure is cardiac fibrosis.However,the clinical tools of treating cardiac fibrosis are limited.Cardiac fibrosis is a scar formation process characterized by abnormal proliferation of fibroblasts and excessive deposition of extracellular matrix(ECM)proteins,leading to abnormal cardiac structure and function.Collagen accumulated in ECM can lead to decreased compliance and increased stiffness of the left ventricle,and pathological signal transduction in cardiomyocytes,which ultimately leads to heart failure.Excessive accumulation of ECM also impairs the electrical activity in cardiomyocytes and increases the risk of arrhythmias.Cardiac fibroblasts(CFs)and myofibroblasts(MFs)are the main sources of ECM.Phenotypic transformation of CFs to MFs is strictly regulated,and is an important basis for maintaining cardiac homeostasis.Most heart diseases are related to cardiac fibrosis.TGF-β1(transforming growth factor-β1)signaling plays an important role in the development of cardiac fibrosis and heart failure through autocrine or paracrine pathways.In many tissues,the continuous production of TGF-β1 is the basis of fibrosis.Activation of TGF-β1 signaling depends on the release of mature TGF-β1 from the latent complex including TGF-β1 and LAP(latency-associated peptide).The activity of TGF-β1 signaling is strictly regulated under physiological conditions.Thrombospondin-1(TSP1)is a major activator of TGF-β1 in heart diseases.Previous studies found that binding of the KRFK sequence of TSP1 to the LSKL sequence of LAP alters the conformation of TGF-β1,making it accessible to its receptor TβRⅠ/TβRⅡ.The receptor-activated downstream Smads are then translocated to the nucleus to transmit extracellular signals.In the process of cardiac fibrosis,TGF-β1 signaling is involved in the phenotypic transformation of cardiac fibroblasts.Studies showed that the stimulation of TGF-β1 induces CFs to differentiate into MFs and increases the synthesis of ECM proteins.It also leads to ECM deposition by inhibiting the activity of metalloproteinases(MMPs)and promoting the synthesis of protease inhibitors such as PAI-1 and TIMPs.The ADAMTSs(disintegrin and metalloproteinase with thrombospondin motifs)are a group of multifunctional proteases and structurally related to the ADAMs family.Typically,The ADAMTS metalloproteinases can degrade key proteins in ECM,and play an important role in cartilage matrix remodeling during morphogenesis and tissue repair.ADAMTS16 is a member of ADAMTS which can cleave α2-macroglobulin,suggesting that ADAMTS16 has potential protease activity.In rats with targeted disruption of the Adamts16 gene,the systolic blood pressure(BP)was lower by 36 mm Hg associated with significantly decreased aortic pulse wave velocity and vascular media thickness compared to that of the control rats.It was suggested that ADAMTS16 is an important candidate gene for hypertension.TSP1 was shown to interact with LAP-TGF-β1 through a KRFK motif and a Wxx W motif(WSPW,WSHW,WGPW)to activate LAP-TGF-β1.A similar,although not identical,Wxx W motif was identified in the TSP1 motif of ADAMTS16.Although we did not identify the KRFK motif in ADAMTS16,we found an RRFR motif which shares similar charged amino acid residues with KRFK.We hypothesize that ADAMTS16 may interact with LAP-TGF-β1 via RRFR and Wxx W motif.As TGF-β1 is considered as one of the most important pro-fibrotic growth factor in cardiovascular diseases,ADAMTS16 may be involved in the regulation of cardiac fibrosis by activating LAP-TGF-β1.In this study,ADAMTS16 was used to investigate its regulatory role in myocardial fibrosis at cellular and model animal levels.Transverse aortic constriction(TAC)surgery characterized by aortic stenosis with increasing left ventricular pressure is commonly used as an experimental model for pressure overload-induced cardiac hypertrophy and heart failure.ADAMTS16 was upregulated in a mouse model for cardiac hypertrophy and heart failure with TAC,and its m RNA expression was strongly correlated with markers(MMP2、MMP9、Col1a1 and Col3a1)for cardiac fibrosis.The data suggested that ADAMTS16 is closely related to cardiac fibrosis.In vitro,ADAMTS16 could promote the expression of α-SMA,a molecular marker of MFs,and enhance the contraction and migration ability of CFs,indicating that ADAMTS16 promotes the phenotypic transformation of CFs to MFs.Structural modeling analysis showed that the Wxx W and RRFR motif of ADAMTS16 resemble that of TSP1,which can interact with LAP-TGF-β1.Co-Immunoprecipitation assays showed that ADAMTS16 can interact with LAP-TGF-β1.Molecular biology-based assessment also indicated that overexpression of WT-ADAMTS16 activated LAP-TGF-β1.however,this effect was abolished by mutation of the RFRR sequence into IIFI(Mut ADAMTS16),si RNA-mediated knockdown of ADAMTS16 expression,and TGF-β1 neutralizing antibody(TGF-β1 NAb)incubation in CFs.Because the RRFR motif of ADAMTS16 is required for binding to and activation of TGF-β1 in CFs,we hypothesized that the critical role of ADAMTS16 in TGF-β1 activation may be mimicked by a RRFR tetrapeptide.Co-Immunoprecipitation assays showed that RRFR tetrapeptide inhibited the interaction between ADAMTS16 and LAP-TGF-β1 with a dose-dependent effect.In addition,RRFR tetrapeptide promoted the activation of LAP-TGF-β1,which was independent of ADAMTS16 as similar results were obtained in CFs treated with Adamts16 specific si RNA or negative control si RNA.In vivo,intraperitoneal injection of RRFR tetrapeptide activated the Smad2/Smad3 signaling pathway downstream of TGF-β1,and significantly deteriorated the degree of cardiac dysfunction and fibrosis in pressure overload-induced heart failure,which could be blocked by TGF-β1 NAb therapy.Taken together,our data showed that:(1)ADAMTS16 is a novel determinant of cardiac fibrosis and heart failure.(2)The RRFR motif in ADAMTS16 plays a key role in the activation of TGF-β1 signaling.(3)the RRFR tetrapeptide treatment aggravated TACinduced cardiac fibrosis,hypertrophy and heart failure by regulating TGF-β1 signaling.(4)The ADAMTS16/TGF-β1 signaling pathway is involved in cardiac fibrosis,cardiac hypertrophy and heart failure.Our results identify a new role for ADAMTS16 in the development of cardiovascular diseases,and suggest that ADAMTS16 may be a new target for the treatment of heart failure and cardiac fibrosis.