CRIP1: Role in the pathogenesis of hypertension by regulating interactions with cytoskeletal proteins

Abstract Background Hypertension is a complex polygenic disease and the underlying molecular mechanisms remain unknown. In an interdisciplinary approach, we identified cysteine-rich protein 1 (CRIP1) in human monocytes as being strongly associated with hypertension, suggesting a link between CRIP1 and the pathophysiology of hypertension via the immune system. The identification of CRIP1 protein interaction partners and associated signaling pathways would provide insight into the pathophysiological significance of CRIP1 under hypertensive conditions. Objective The overall aim of our project is to evaluate the functional role of CRIP1 in hypertension and cardiovascular disease (CVD). Here, using systems medicine approaches including experimental settings and human cohort data, we focused on the identification of novel CRIP1 protein interaction partners that may be associated with the pathogenesis of hypertension and cardiovascular disease. Methods Monocytic CRIP1 was immunoprecipitated and co-immunoprecipitated proteins in the eluate were analyzed by LC-MS/MS and Pathway and Process Enrichment Analysis and validated by immunoblot techniques. Silencing of CRIP1 was established in the monocyte-like cell line THP-1 using a vector-based shRNA system. The association of CRIP1 with genes encoding cytoskeletal proteins was accessed in a human cohort by transcriptome-based analysis of human monocytes in the Guttenberg-Gesundheitsstudie (GHS; n = 1527). Results First, a Yeast Two-Hybrid Assay was performed and two potential protein interaction partners were predicted for CRIP1: KTN1 and TPM2. To obtain comprehensive information on interacting proteins, a co-immunoprecipitation assay with a specific antibody against monocytic CRIP1 confirmed these as interaction partners and revealed further protein interactions with proteins that are crucial for monocyte migration and invasion behavior of monocytes by LC-MS/MS and Pathway and Process Enrichment Analysis, and immunoblot analysis. In an in vitro experiment with the human monocytic cell line THP-1, downregulation of CRIP1 by >70% significantly reduce the abundance of these proteins. In addition, in a transcriptome-based analysis of human monocytes, most of the genes encoding proteins of microtubule cytoskeleton organization (GO:0000226), actin filament and cytoskeleton organization (GO:0007015 and GO:0030036), Arp2/3 complex-mediated actin nucleation (GO:0034314), and cytoskeleton-dependent intracellular transport (GO:0030705) were associated with CRIP1 expression. Conclusion As CRIP1 expression in human monocytes has already been linked to hypertension, our results suggest that CRIP1 may play a role in the pathogenesis of this disease by regulating interactions with cytoskeletal proteins that lead to impaired monocyte migration and invasion behaviour. Further experiments are underway to translate our findings into clinical application.