Introdution-Celiac disease (CD) is a complex inflammatory disorder triggered by the consumption of cereals containing gliadins that cause a damage at the intestinal level in genetically predisposed individuals. Type 2 transglutaminase (TG2)-induced modifications of gliadin are strongly implied in CD pathogenesis. Recently, an in vitro model of skin-derived fibroblasts has been developed to study constitutive differences between cells from healthy subjects and CD ones. We aimed to investigate whether control and CD fibroblasts displayed constitutive differences regarding TG2 expression level, activity and cellular distribution. Methods-To analyze TG2 levels in total lysates and in subcellular fractions, western blot analyses were performed by using CUB7402 antibody. To visualize TG2 distribution into cells, confocal microscopic images were analyzed. To monitor TG2 transamidating activity, a quantitative microplate assay was performed by using the substrate pentylamin-biotine. Results-We found that TG2 was expressed in all samples from cell lysates, however protein levels were variable in both groups of subjects. We also demonstrated that TG2 activity was inducible in all cell cultures tested and it well correlated with protein expression level. By performing subcellular fractionating experiments, we observed that TG2 was always more abundant in the membrane fractions, but in CD fibroblasts this phenomenon was greatly more evident. Finally, confocal images showed that TG2 differently distributed into vesicular compartment, in particular we found a higher association with the early endosome compartment in celiac fibroblasts with the respect to control ones. Conclusions-These findings let us to affirm that in CD fibroblasts TG2 preferentially associates to membranes, especially to the early endosome compartment, thus contributing to determine a "CD phenotype" which is independent from gliadin exposition and is also evident far from the main site of inflammation.
Subcellular distribution of type 2 transglutaminase is constitutively different in skin derived-fibroblasts from celiac disease patients and from healthy controls
PAOLELLA, GAETANA;LEPRETTI, MARILENA;DI ZENZO, MARINA;DI LAURO, ALESSANDRO;ESPOSITO, Carla;CAPUTO, IVANA
2015
Abstract
Introdution-Celiac disease (CD) is a complex inflammatory disorder triggered by the consumption of cereals containing gliadins that cause a damage at the intestinal level in genetically predisposed individuals. Type 2 transglutaminase (TG2)-induced modifications of gliadin are strongly implied in CD pathogenesis. Recently, an in vitro model of skin-derived fibroblasts has been developed to study constitutive differences between cells from healthy subjects and CD ones. We aimed to investigate whether control and CD fibroblasts displayed constitutive differences regarding TG2 expression level, activity and cellular distribution. Methods-To analyze TG2 levels in total lysates and in subcellular fractions, western blot analyses were performed by using CUB7402 antibody. To visualize TG2 distribution into cells, confocal microscopic images were analyzed. To monitor TG2 transamidating activity, a quantitative microplate assay was performed by using the substrate pentylamin-biotine. Results-We found that TG2 was expressed in all samples from cell lysates, however protein levels were variable in both groups of subjects. We also demonstrated that TG2 activity was inducible in all cell cultures tested and it well correlated with protein expression level. By performing subcellular fractionating experiments, we observed that TG2 was always more abundant in the membrane fractions, but in CD fibroblasts this phenomenon was greatly more evident. Finally, confocal images showed that TG2 differently distributed into vesicular compartment, in particular we found a higher association with the early endosome compartment in celiac fibroblasts with the respect to control ones. Conclusions-These findings let us to affirm that in CD fibroblasts TG2 preferentially associates to membranes, especially to the early endosome compartment, thus contributing to determine a "CD phenotype" which is independent from gliadin exposition and is also evident far from the main site of inflammation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
