A counter-regulatory rise in GC levels is important to the inflammatory response, that when impaired is associated with high mortality in inflammatory states. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regenerates intracellular glucocorticoids (GC), amplifying their actions, and has been postulated to play a role in the development of inflammation-associated arthritis, obesity and myopathy. Pro-inflammatory cytokines (e.g. TNF-α) increase expression and activity of 11β-HSD1 in some cell types, but the molecular mechanisms of this regulation is unclear. C2C12 murine muscle cells express 11β-HSD1 and we hypothesised that its activity would be responsive to TNF-α, possibly through an NF-κB dependent mechanism. We identified 2 putative p65 binding sites in the P1 (exon1a) and P2 (exon1b-23kb downstream of P1) promoters and used ChIP analysis to identify whether binding occurred under TNF-α stimulation (10 mg/ml for 1, 2 and 24 h) in C2C12 myotubes differentiated for 5 days. We did not detect binding in the P2 promoter; however exposure to TNF-α produced an NF-κB dependent P65 translocation to the nucleus and binding at position -156bp of the P1 promoter within 1 hour, still detectable after 24 h. Interaction of P65 with HSD11B1 reduced the overall levels of transcript by 34 fold and a similar decrease in protein abundance was also observed. Combined treatment of TNF-α with the protein synthesis inhibitor anisomycin showed the level of HSD11B1 mRNA to be unchanged compared to TNF-α alone, confirming HSD11B1 as a primary P65 target. Finally, TNF-α exposure attenuated the 11β-HSD1 protein half-life and significantly reduced enzyme activity and thereby the ability to generate GC by approximately 50%. Previous work has shown local 11β-HSD1 activity to be stimulated at sites of inflammation. These data suggest that acute inflammatory stimuli signalling through the NF-κB pathway specifically in muscle could act to temper GC regeneration by 11β-HSD1, permitting the maintenance of an acute inflammatory.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.