Role of CRACR2A in the development of pulmonary arterial hypertension - 17/02/23

Pulmonary arterial hypertension (PAH) is a rare, progressive, and devastating disease resulting from the obstruction of distal pulmonary arteries. Pulmonary vascular remodeling in PAH involves pulmonary arterial endothelial cells (hPAECs) and pulmonary arterial smooth muscle cells (hPASMCs) dysfunctions. An alteration of intracellular Ca²⁺ could be a significant cause of pulmonary vascular cell dysfunctions (proliferation/contraction/migration) [1Srikanth S., Jung H.J., Kim K.D., Souda P., Whitelegge J., Gwack Y. A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells Nat Cell Biol 2010 ;  12 : 436-446 [cross-ref]

Cliquez ici pour aller à la section Références]. Recently, store-operated channels (SOCs), made up of two essential components: stromal interaction molecules (STIM1) as endo/sarcoplasmic reticulum (ER/SR) Ca²⁺ sensor proteins and Orai1 Ca²⁺ channels at the plasma membrane have been shown to play essential roles in vascular cells. In a previous study, store-operated-Ca²⁺ entry is increased in PAH-hPASMC in association with a two-fold increase of Orai1 protein expression (Masson B et al., in revision). Importantly, Orai1 requires STIM1 molecules to be functional and several associated regulatory partners such as calcium release activated channel regulator 2A (CRACR2A). Indeed, CRACR2A, a cytosolic Ca²⁺ sensor, plays a crucial and essential role in SOCE by regulating STIM1-Orai1 interaction [1Srikanth S., Jung H.J., Kim K.D., Souda P., Whitelegge J., Gwack Y. A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells Nat Cell Biol 2010 ;  12 : 436-446 [cross-ref]

Cliquez ici pour aller à la section Références]. Recently, loss of function mutations in the CRACR2A gene has been identified in an Italian cohort of PAH patients [2Barozzi C., Galletti M., Tomasi L., De Fanti S., Palazzini M., Manes A., et al. A combined targeted and whole exome sequencing approach identified novel candidate genes involved in heritable pulmonary arterial hypertension Sci Rep 2019 ;  9 : 753

Cliquez ici pour aller à la section Références]. However, the role of CRACR2A in PAH pathogenesis is unknown.

Our goal was to study the role of CRACR2A in vitro as a SOCE modulator, as an Orai1 expression regulator, and its contribution to PAH phenotype in hPASMCs. In the present study, we used control and PAH-hPASMCs in which we modulated CRACR2A level of expression by siRNA strategy or overexpression strategy. We measured the consequences of CRACR2A knockdown on SOCE by Ca²⁺ imaging measurement (FURA2/AM probes), on hPASMCs proliferation (BrdU assay), and migration capacity (Wound healing assay).

Using immunofluorescence staining on human control and PAH lungs, we found that CRACR2A is located and expressed in control and PAH (hPAECs and hPASMCs). CRACR2A protein expression is unchanged in control and PAH-hPAECs, whereas its expression was reduced in PAH-hPASMCs. Using 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, Orai1 expression, and proliferation. In contrast, CRACR2A overexpression in PAH-hPASMCs reduced SOCE.

We found that CRACR2A plays an essential role in SOCE regulation in both control and PAH-hPASMCs, and could partly explain why Orai1 expression/function is increased in PAH-hPASMCs. These results suggest that CRACR2A is involved in PAH pathogenesis.