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-B in induced and noninduced HAS activation. Transforming growth factor (TGF) ␤1 as well as interleukin (IL)-1␤ are both strong inducers of HAS1 transcription. Stimulation of fibroblast-like synoviocytes with IL-1␤ resulted in rapid degradation of IB␣, an event that was preceded by IB␣ phosphorylation. Interestingly, TGF␤1 neither affected IB␣ levels, nor did it cause phosphorylation of IB␣. In addition, TGF␤1 had no effect on IB␤ and IB⑀ levels. Electrophorectic mobility shift assays demonstrate that IL-1␤ is a potent inducer of NF-B translocation; however, TGF␤1 treatment did not result in shifting bands. Two adenovirus constructs were used to further clarify differences in TGF␤1-and IL-1␤-induced HAS1 activation. Overexpressing IB␣ completely abolished the IL-1␤ effect on HAS1 but did not interfere with TGF␤1-induced HAS1 mRNA accumulation. Identical results were obtained when a dominant negative IKK was overexpressed. Interestingly, neither overexpression of IB␣ nor of IKK had any effect on HAS2 and HAS3 mRNA levels. Taken together, HAS1 can be activated by distinct pathways; IL-1␤ utilizes NF-B, and TGF␤1 does not. Furthermore, HAS2 and HAS3 are activated without the involvement of NF-B.

The Anti-rheumatic Gold Salt Aurothiomalate Suppresses Interleukin-1β-induced Hyaluronan Accumulation by Blocking HAS1 Transcription and by Acting as a COX-2 Transcriptional Repressor

“…However, this gene is readily up-regulated in response to a series of cytokines (18). We demonstrated that IL-1␤-induced HAS1 activation depends on the activation and translocation of the transcription factor NFB (23). Within the hyaluronan synthase family, HAS1 is therefore the only gene that shares a dependence on NFB activation for its up-regulation with a large group of other molecules that are characterized by their pro-inflammatory properties (31).…”

“…AuTM Does Not Prevent IL-1␤-induced IB␣ Degradation-NFB activation is of relevance for IL-1␤-induced COX-2 and HAS1 activation (23). Enzymatic degradation of IB is in most cases a prerequisite for subsequent NFB translocation, and a series of anti-inflammatory drugs have been shown to exert their beneficial effects by blocking IB degradation.…”

“…That COX-2 is relevant in this context is evidenced by the demonstration that PGE2 is one of the most potent activators of HAS1 transcription (33). Secondly, in FLS, AuTM interferes with the translocation of a series of transcription factors, among them NFB, a transcription factor that has been shown to be essential for the activation of IL-1␤-induced HAS1 transcription and many other pro-inflammatory genes (23,31,34,35). Although the inhibitory action of AuTM on NFB activation might be attributed to its effect on PGE2, other mechanisms must be at work as well.…”

“…Aliquots were used for PCR. Primer sequences and experimental conditions for conventional block cycler RT-PCR as well as for real time RT-PCR have been described in detail elsewhere (22,23). Each RT-PCR experiment included a dissociation curve to verify the specificity of the amplicon as well as no-template controls.…”

Hyaluronan Production in Synoviocytes as a Consequence of Viral Infections

“…Basal mRNA expression levels in unstimulated FLS were chosen to represent 1ϫ expression of a given gene. Amplification curves and equations for calculations have been reported elsewhere (17). Primers used in real time PCR experiments were selected based on published data (14,17,18) or generated with the help of software provided by the supplier of the primers MWG Biotech AG.…”