Swelling-activated Cl^- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells

Background: Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl^- currents (I_Cl,swell) are upregulated in PASMC proliferation and that nonspecific Cl^- current blockers inhibit proliferation. However, the specific role of I_Cl,swell in PASMC proliferation and its molecular underpinning remain unknown. Methods and results: In the present study, we found that the specific I_Cl,swell blocker, DCPIB (4-[(2-butyl-6,7- dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC₅₀=2.7μmol/l) I_Cl,swell and inhibited (IC₅₀=6.9μmol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl^- channel genes underlying I _Cl,swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl^- channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to I_Cl,swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by >45%) compared with control-shRNA, it did not alter I_Cl,swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for I_Cl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution. Conclusion: Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie I_Cl,swell in these cells.