Role of CRACR2A in the development of pulmonary arterial hypertension

Abstract

Introduction

Pulmonary arterial hypertension (PAH) is a rare, progressive, and devastating disease resulting from the obstruction of distal pulmonary arteries. The pathobiology of PAH is multifactorial, involving all the cell spectrum of pulmonary circulation, including pulmonary arterial (PA) smooth muscle cells (PASMCs), endothelial cells (PECs), and PA adventitial fibroblasts (PAAFs). These cellular dysfunctions are characterized by enhanced migration, proliferation and apoptosis resistance. Moreover, in PAH-hPASMCs, calcium signaling is exacerbated contributing to hPASMCs abnormalities. A mutation of CRACR2A, a regulatory partner of the calcium (Ca²⁺) channel Orai1 has been identified in an Italian PAH patients’ cohort suggesting its involvement in PAH development. Orai1, the main Ca²⁺ channel of the Store-Operated Ca²⁺ Entry (SOCE) is increased in expression and function in PAH-hPASMCs and participate to PAH pathogenesis. SOCE is triggered by STIM1, a calcium sensor of the endoplasmic reticulum (ER) that activates Orai1 after ER calcium depletion. CRACR2A is known to stabilize Orai1/STIM1 interaction.

Methods

We used a whole exome sequencing approach to identify CRACR2A variants. We used a siRNA strategy against CRACR2A in hPASMCs, hPECs, and hPAAF isolated from control and PAH patients. We then measured the consequences of the CRACR2A knockdown on SOCE by Ca²⁺ imaging measurement, on cell proliferation, migration and in vitro angiogenesis. Finally, we measured the consequence on SOCE and Orai1 protein expression in PAH-hPASMCs overexpressing CRACR2A.

Results

We identified additional variants of CRACR2A in PAH patients strengthening CRACR2A involvement in PAH development. Using immunofluorescence staining on human control and PAH lungs, we found that CRACR2A is expressed in control and PAH. By immunoblot, we showed that CRACR2A protein expression is unchanged in control and PAH-hPECs and hPAAFs, whereas its expression is reduced in PAH-hPASMCs. By the siRNA strategy, we found that SOCE, migration, and proliferation were decreased in control-hPASMCs transfected with siCRACR2A. Surprisingly, in PAH-hPASMCs, the knockdown of CRACR2A strongly increased SOCE and Orai1 expression. In contrast, CRACR2A overexpression in PAH-hPASMCs reduced SOCE. In PAH-hPAAFs, the knockdown of CRACR2A increases SOCE as in PAH-hPASMCs.

Conclusion

These results indicate that CRACR2A plays an essential role in pulmonary vascular cells from control and PAH patients by its involvement in SOCE, proliferation, and migration. The reduced CRACR2A expression in PAH-hPASMCs could partly explain increased Orai1 expression and function in PAH-hPASMCs. These results suggest that CRACR2A dysfunction is involved in PAH pathogenesis.