NRP1 interaction with AP2M1 promotes vascular calcification by driving endocytosis of PDGFRβ
J Cheng, Z Guan, H Chen, A Huang, J Ma, Z Zhang, M Feng, Y Chen, C Xie, H HuangAbstract
Background
Arterial calcification is the main cause of cardiovascular death in the patient with chronic kidney disease. Vascular smooth muscle cell (VSMC) transdifferentiation into an osteoblastic phenotype is the key pathological process of arterial calcification. However, the mechanisms remain unclear.
Purpose
This study was designed to explore the role of NRP1 in arterial calcification.
Methods
NRP1 expression was detected in in vitro VSMC calcification models, mouse arterial calcification models induced by adenine phosphate (AP) diet or vitamin D3 (VitD3) overload, and human calcified arterial tissue samples. Pharmacological inhibition with NRP1-specific inhibitor EG00229 and VSMC-specific NRP1 knockout mice (NRP1SMKO) were used to confirm the function of NRP1 in arterial calcification in vivo. Immunoprecipitation-mass spectrometry (IP-MS), co-immunoprecipitation (Co-IP) and non-denaturing polyacrylamide gel electrophoresis were adopted to identify the binding mode between NRP1 and AP2M1, a subunit of clathrin adaptor AP2 complex. A competitive binding peptide was designed according to the specific binding sequence of NRP1 and AP2M1.
Results
NRP1 expression was significantly increased in calcified VSMCs, mouse arterial calcification models and human calcified arterial tissues, and its expression was positively correlated with the calcification marker BMP2. Intraperitoneal injection of EG00229 and NRP1SMKO both significantly reduced arterial calcification in mice. NRP1 bound to the C-terminus of AP2M1 through a novel intracellular sequence (amino acids 881-892), and this binding was independent of the classic SEA sequence of NRP1 and the dileucine motif of AP2M1. The competitive binding peptide targeting this sequence significantly inhibited arterial calcification in vivo.
Conclusion
This study first found that the NRP1-AP2M1 axis promotes arterial calcification. A novel binding sequence between NRP1 and AP2M1 was identified in this study, which reveals a new regulatory mode of NRP1 independent of its classic SEA sequence. The NRP1-targeted peptide designed in this study has a significant anti-calcification effect in vivo, which provides a potential candidate drug for the clinical treatment of arterial calcification. This study also provides new experimental evidence and strategies for the membrane protein-targeted intervention of arterial calcification.Figure 1