Modulation of expression in BEAS-2B airway epithelial cells of α-L-fucosidase A1 and A2 by Th1 and Th2 cytokines, and overexpression of α-L-fucosidase 2.
Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-γ, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/and IFN-γ. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise α-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p < 0.01) in response to IFN-γ, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-γ stimulation (>twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-γ addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-γ-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active α-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-γ, a Th1 cytokine. Active α-L-fucosidase 2 was overexpressed in BEAS-2B cells.
Veterinary Sciences Centre, School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
Anna D
AD
Sobkowicz
Mary E
ME
Gallagher
Colm J
CJ
Reid
Daniel
D
Crean
Stephen D
SD
Carrington
Jane A
JA
Irwin
D001249:Asthma / Q000235:genetics* / Q000473:pathology; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007249:Inflammation / Q000235:genetics* / Q000473:pathology; D007371:Interferon-gamma / Q000235:genetics; D018417:Th1 Cells / Q000378:metabolism; D018418:Th2 Cells / Q000378:metabolism; D005644:alpha-L-Fucosidase / Q000235:genetics*
D016207:Cytokines; C000593239:FUCA1 protein, human; D007371:Interferon-gamma; C000593240:FUCA2 protein, human; D005644:alpha-L-Fucosidase