Adenovirus-mediated gene transfer of mutated IκB kinase and IκBα reveal NF-κB-dependent as well as NF-κB-independent pathways of HAS1 activation

Abstract

It has become increasingly clear that hyaluronan is more than the simple matrix molecule it was once thought to be but instead takes part in a multitude of biological functions. Three genes encode for hyaluronan synthases (HAS). We demonstrated earlier that HAS2 and HAS3 are constitutively activated in type-B synoviocytes (fibroblast-like synoviocytes) and, furthermore, that the only gene that readily responds to stimulation with a series of proinflammatory cytokines is HAS1. Here we probe the involvement of the transcription factor NF-κB in induced and noninduced HAS activation. Transforming growth factor (TGF) β1 as well as interleukin (IL)-1β are both strong inducers of HAS1 transcription. Stimulation of fibroblast-like synoviocytes with IL-1β resulted in rapid degradation of IκBα, an event that was preceded by IκBα phosphorylation. Interestingly, TGFβ1 neither affected IκBα levels, nor did it cause phosphorylation of IκBα. In addition, TGFβ1 had no effect on IκBβ and IκBε levels. Electrophorectic mobility shift assays demonstrate that IL-1β is a potent inducer of NF-κB translocation; however, TGFβ1 treatment did not result in shifting bands. Two adenovirus constructs were used to further clarify differences in TGFβ1- and IL-1β-induced HAS1 activation. Overexpressing IκBα completely abolished the IL-1β effect on HAS1 but did not interfere with TGFβ1-induced HAS1 mRNA accumulation. Identical results were obtained when a dominant negative IKK was overexpressed. Interestingly, neither overexpression of IκBα; nor of IKK had any effect on HAS2 and HAS3 mRNA levels. Taken together, HAS1 can be activated by distinct pathways; IL-1β utilizes NF-κB, and TGFβ1 does not. Furthermore, HAS2 and HAS3 are activated without the involvement of NF-κB. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.