NFkappaB activation is essential for miR-21 induction by TGFβ1 in high glucose conditions

Transforming growth factor betal (TGF beta 1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGF beta 1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGF beta 1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGF beta 1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGF beta 1 in high glucose conditions. The induction by TGF beta 1 was dependent on NF kappa B activation and subsequent ROS generation. TGF beta 1 was instrumental in degrading the NF kappa B inhibitor I kappa B alpha and facilitating the nuclear translocation of NF kappa B p65 subunit. EMSA studies showed enhanced DNA binding activity of NF kappa B in the presence of TGF beta 1. ChIP assay revealed binding of p65 to miR-21 promoter. NF kappa B activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGF beta 1-NF kappa B-miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers. (C) 2014 Elsevier Inc. All rights reserved.