Differential Effect of TGF-β1 and Eotaxin on Novel CLC3 Ion-Channel Variants in Human Peripheral Blood Eosinophils
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Authors
Gaurav, Rohit
Kim, Min-Jung
Agrawal, Devendra K.
Issue Date
2012-03-28
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Generic
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Abstract
Rationale: Chloride channels play an important role in various cellular processes including differentiation, proliferation, apoptosis and migration of cells. Previously, we reported the involvement of CLC3 in TGF-β-induced migration, presence of CLC3b and CLC3e transcript variants and membrane expression of the CLC3 in human peripheral blood eosinophils. In this study, we examined the effect of eotaxin in CLC3 expression in eosinophils.
Methods: Eosinophils were isolated and purified (>99% pure >98% viable) from venous blood of healthy donors with negative selection. Cells were incubated with either TGF-β1 (10 ng/ml) or eotaxin (10 ng/ml) for 24 hours. Total RNA was isolated using mirVana kit and 500 ng RNA was reverse transcribed using Improm RT II procedure to quantify CLC3 mRNA transcripts. The qPCR data was analyzed using GAPDH as the reference gene with 2-ΔΔCq as fold increase.
Results: There was a 2-fold increase in CLC3 mRNA transcripts (exon 10-exon 11) with TGF-β1 (n=4, p<0.05) relative to GAPDH. Eotaxin has no statistically significant effect on CLC3 mRNA transcripts (exon 10-exon 11). CLC3b mRNA (exon 12-exon 14) was increased 3-4-fold with TGF-β1 (n=4, p<0.05) compared to 2-fold increase with eotaxin. Eotaxin increased the mRNA transcripts CLC3e (exon 13-exon 14) 3-4-fold (n=4, p<0.05) compared to 2-2.5-fold increase with TGF-β1. However, CLC3e mRNA transcripts decreased in combination of TGF-β1 and eotaxin. Conclusions: mRNA transcript levels of CLC3 with different primer sets suggest the presence of more transcript variants in human blood eosinophils than the known variants, CLC3b and CLC3e. The significant increase in the transcript level of CLC3e with eotaxin suggests the role of CLC3e ion-channel in eotaxin-induced migration of eosinophils in allergic asthma.
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Creighton University