The role of CRIP1 and its interaction with Kinectin in the immune mechanisms of hypertension

Background

Hypertension is a complex multifactorial disease and the molecular mechanisms underlying this process are not fully understood. Cysteine-rich protein 1 (CRIP1) has been identified in human monocytes in association with changes in blood pressure (BP), and its expression was significantly altered in circulating monocytes in an angiotensin II-induced mouse model of hypertension. This suggests that CRIP1 may play a role in immune-related mechanisms of hypertension, and the identification of its interaction partners and signaling pathways may elucidate its pathophysiological impact in this condition.

Aim

This study aimed to identify novel CRIP1 interaction partners potentially involved in the immune-related pathogenesis of hypertension.

Material and Methods

A Yeast Two-Hybrid assay was conducted to identify potential protein interaction partners for CRIP1. CRIP1 from monocytic THP-1 cells was immunoprecipitated using a CRIP1-specific antibody, and the predicted interacting proteins were validated by immunoblotting. For protein-protein interactions, recombinantly produced MBP (maltose binding protein) fused CRIP1 protein, 6x-His tagged Kinectin 1 protein, and DDK tagged NF-kB protein were used. CRIP1 silencing was achieved in THP-1 cells via a vector-based shRNA system.

Results

Kinectin and Beta-tropomyosin were first identified as potential CRIP1 protein interaction partners via a Yeast Two-Hybrid Assay, with confidence scores B (high) and D (moderate), respectively. Previous studies have demonstrated that Kinectin, a protein involved in cytoskeletal interactions, and CRIP1 interact with NF-kB/p65 in cancer cells via co-immunoprecipitation, affecting its activation and transcriptional activity. In this study, we confirmed the functional interactions of Kinectin and NF-kB with CRIP1 in monocytes using immunoblot analysis of immunoprecipitated CRIP1. Interestingly, in an in vitro experiment using the human monocytic cell line THP-1, downregulation of CRIP1 (shCRIP1) significantly reduced the gene expression and protein levels of Kinectin, while NF-kB/p65 remained unaffected. However, less phosphorylation of NF-kB/p65 was observed in shCRIP1 cells, indicating a reduced activation of the NF-kB signaling pathway after priming with lipopolysaccharide (LPS). Additionally, a protein-protein interaction assay with recombinantly produced proteins demonstrated that Kinectin, but not NF-kB/p65, directly interacts with CRIP1.

Conclusion

Given that CRIP1 expression in human monocytes has been linked to hypertension, our results suggest that CRIP1 may contribute to the pathogenesis of this disease by interacting with Kinectin, thereby influencing NF-kB/p65 activation and the regulation of pro-inflammatory genes. Ongoing experiments aim to translate these findings into clinical applications.