Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smoothmuscle 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,I-swell) are upregulated in PASMC proliferation and that nonspecific Cl- current blockers inhibit proliferation. However, the specific role of I-Cl,I-swell in PASMC proliferation and its molecular underpinning remain unknown.Methods and results:In the present study, we found that the specific I-Cl,I-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=2.7mol/l) I-Cl,I-swell and inhibited (IC50=6.9mol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl- channel genes underlying I-Cl,I-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,I-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,I-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,I-swell (i.e.,CLC-2) also showed a mainly intracellular distribution.Conclusion:Our findings support the conclusion that both I-Cl,I-swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie I-Cl,I-swell in these cells.