Roles Of TRPC3 In LPS Or ACh-induced Mouse Airway Smooth Muscle Cells Proliferation

Airway remodeling is a major feature of asthma and chronic obstructive pulmonary disease(COPD) which is characterized by increased airway smooth muscle cell(ASMC) mass. Lipopolysaccharide(LPS), a major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is ubiquitously present as a contaminant of cigarette smoke, organic dusts, and environmental pollution, has been proven to be an inducer of airway remodeling, while the mechanisms remains unclear. Acetylcholine(ACh) has been reported to be the primary parasympathetic neurotransmitter in the airways and paracrine or autocrine hormone released from non-neuronal origins, such as ASMCs, inflammatory cells and airway epithelial cell. Recent studies have revealed the pathological and physiological role of ACh in airway remodeling except for its well-known functions in contraction, however, the mechanisms are limited of investigation. Canonical transient receptor potential channel-3(TRPC3)-encoded nonselective cation channels(NSCCs) have been proven to be important native constitutively active channels and play significant roles in contraction and airway hyperresponsiveness by regulating the concentration of intracellular free Ca2+([Ca2+]i) in ASMCs, while its functional role in airway remodeling is unclear. In this study, cultured ASMCs were stimulated with LPS or ACh, then, the possible role of TRPC3 in ASMCs proliferation was investigated to provide new evidence of searching key target for the airway remodeling intervention.Part ?TRPC3-mediated Ca2+ entry contributes to mouse airway smooth muscle cell proliferation induced by lipopolysaccharideObjectives: Investigate whether TRPC3 mediated Ca2+ entry is involved in LPS-induced mouse ASMCs proliferation.Methods:To test this aim, mouse ASMCs were cultured with or without LPS treatment for 48 h, then cell viability was examined by MTT assay, TRPC3 expression was determined with Western blotting and NSCC currents were measured with a patch clamp recording, [Ca2+]i changes were investigated using calcium imaging, and finally, small interfering RNA(si RNA) technology was used to further test the contribution of TRPC3 to LPS-induced ASMCs proliferation.Results:(1) Increasing concentrations of LPS(0.1-10 ?g/ml) treatment for 24 h or 48 h could promote ASMCs proliferation in a time and concentration dependent manner, LPS(10 ?g/ml, 48 h) induced a more significant increase in ASMCs proliferation; Gd3+(3 ?M) or TRPC3 antibodies(1:200) markedly inhibited the proliferative effects of LPS.(2) LPS treatment induced significant up-regulation of TRPC3 expression, enhancement of NSCC currents in ASMCs, which were all surpressed by Gd3+ or TRPC3 antibodies.(3) LPS treatment induced significant increases in resting [Ca2+]i and ACh elicited [Ca2+]i responses, that were inhibited by Gd3+ or TRPC3 antibodies.(4) Using si RNA(30 n M, 72 h) transfection to knockdown TRPC3 largely inhibited ASMCs proliferation.(5) TRPC3 gene silencing significantly inhibited LPS-induced TRPC3 expression up-regulation and enhancement of NSCC currents.(6) TRPC3 gene silencing largely blocked LPS induced increases of resting [Ca2+]i and ACh-elicited [Ca2+]i responses.Summary:Through this study, we found that LPS treatment produced significant increase in ASMCs proliferation with up-regulation of TRPC3 protein expression and NSCC currents in ASMCs, LPS treatment also enhanced resting [Ca2+]i and ACh-elicited [Ca2+]i responses in ASMCs; Gd3+(3 ?M), TRPC3 antibodies or TRPC3 gene silencing significantly inhibited these effects, eventually inhibited LPS-induced ASMCs proliferation. Results above indicated that LPS could up-regulate TRPC3 protein expression and TRPC3 encoded NSCC currents, which resulted in the elevation of [Ca2+]i in ASMCs, thus, induced ASMCs proliferation.Part ?TRPC3-mediated Ca2+ entry contributes to mouse airway smooth muscle cell proliferation induced by AcetylcholineObjectives:Study the role of TRPC3 mediated Ca2+ entry in ACh-induced mouse ASMCs proliferation which occurs with airway remodeling.Methods:To this aim, primary mouse ASMCs were cultured with or without ACh treatment for 24 h, then cell viability was examined by MTT assay, TRPC3 expression was determined using Western blotting and NSCC currents were measured with a patch clamp recording, [Ca2+]i changes were examined by calcium imaging, and finally, small interfering RNA(si RNA) technology was used to confirm the contribution of TRPC3 to ACh-induced ASMCs proliferation.Results:(1) Increasing concentrations of ACh(1n M-m M) treatment for 24 h or 48 h induced a time and concentration dependent increase in ASMCs proliferation; ACh(100 ?M, 24 h) treatment improved ASMCs proliferation more significantly, which was inhibited by Gd3+(3 ?M) or TRPC3 antibodies(1:200) co-incubation.(2) ACh markedly induced up-regulation of TRPC3 expression, enhancement of NSCC currents in ASMCs, and the effects were all inhibited by Gd3+ or TRPC3 antibodies.(3) ACh largely increased resting [Ca2+]i and KCl-elicited [Ca2+]i responses in ASMCs, that were all suppressed by Gd3+ or TRPC3 antibodies.(4) TRPC3 si RNA(30 n M, 72 h) transfection largely inhibited ACh-induced ASMCs proliferation.(5) TRPC3 gene silencing largely inhibited AChinduced up-regulation of TRPC3 expression, increase of NSCC currents and(6) enhancement of resting [Ca2+]i and KCl-elicited [Ca2+]i responses in ASMCs.Summary:The major findings in this part : ACh treatment significantly induced ASMCs proliferation, up-regulation of TRPC3 expression, enhancement of NSCC currents, elevation of resting [Ca2+]i and KCl-elicited [Ca2+]i responses in ASMCs; these effects were all suppressed by Gd3+, TRPC3 antibodies or TRPC3 gene silencing. Collectively, these data indicated that ACh could up-regulate TRPC3 protein expression and TRPC3 encoded NSCC currents in ASMCs, which induced elevation of [Ca2+]i in ASMCs, in turn, contributing to ASMCs proliferation.Conclusion: Collectively, the results indicate that TRPC3 mediated Ca2+ entry contributes to LPS or ACh-induced ASMCs proliferation that occurs with airway remodeling and suggest it may be a key target for the airway remolding intervention.