Nidogen-2 Is A Novel Endogenous Ligand Of LGR4 To Inhibit Vascular Calcification

Vascular calcification is a prevalent pathological phenomenon in chronic kidney disease,diabetes mellitus and atherosclerotic diseases,characterized by ectopic deposition of calcium and phosphorus crystals in the vascular wall.Vascular calcification is considered to be an independent predictor of cardiovascular events,for which there is no effective pharmaceutical treatment.Basement membrane protein nidogen-2 is a key component of the vascular extracellular matrix microenvironment and it’s pivotal for the maintenance of contractile phenotype in vascular smooth muscle cell（VSMCs）.However,whether nidogen-2 is involved in vascular calcification remains unclear.Nidogen-2 protein levels were significantly reduced in high phosphate（Pi）-induced calcifying VSMCs,aortas from 5/6 nephrectomy-induced chronic renal failure mice,aortas from cholecalciferol-overload mice and aortas from mice that were periadventitially administered with Ca Cl₂.Nidogen-2 deficiency exacerbated high Pi-induced VSMC calcification as characterized by calcium deposition,expression of osteogenic markers and Alizarin red staining,whereas the addition of purified nidogen-2 protein markedly alleviated VSMC calcification in vitro.Nidogen-2 knockout mice exhibited aggravated aorta calcification compared to wild-type（WT）mice in response to 5/6 nephrectomy,cholecalciferol-overload and Ca Cl₂ administration.Further unbiased coimmunoprecipitation and interactome analysis of purified nidogen-2 and membrane protein in VSMCs revealed that nidogen-2 directly binds to leucine-rich repeat G-protein-coupled receptor 4（LGR4）through its G1-G2 domain.Nidogen-2 binds to purified LGR4 with K_D value 26.77 nmol/L as evidenced by surface plasmon resonance.LGR4 deficiency in VSMCs in vitro or in vivo abolished the protective effect of nidogen-2 on vascular calcification.Of interest,through a series signaling transduction assays including Top Flash luciferase,BRET,cell-surface ELISA and Glosensor,we found that nidogen-2 biased activated LGR4-Gαq-PKC signaling to counteract VSMCs osteochondrogenic transition and mineralization.In summary,nidogen-2 is a novel endogenous ligand of LGR4 that biased activated Gαq-PKC signaling and inhibited vascular calcification.Our current study not only highlights the importance of nidogen-2 in the modulation of vascular calcification,but also uncovers a novel endogenous biased ligand for LGR4 with high affinity.