Steroid-enhancing casein kinase inhibitors: a new class of anti-inflammatory anti-fibrotic agents?

M. Li, C. Keenan, C. Xia, S. Y. Langenbach, J. Mangum, T. Harris, A. G. Stewart. Pharmacology and Therapeutics, University of Melbourne, Parkville, AUSTRALIA.

Introduction Chronic obstructive respiratory diseases are commonly treated with inhaled glucocorticoids (GC). However, in severe asthma and in chronic obstructive pulmonary disease GC have insufficient impact on inflammation and remodelling. Transforming growth factor-beta1 (TGF-beta1) is a mediator of airway wall remodelling that induces resistance to GCs in airway epithelium (1,2). However, the mechanisms have proved to be elusive. A role for nuclear canonical Smad signalling was excluded using siRNA against Smad4. Roles for multiple non-canonical kinase pathways including ERK, Phospho-inositide-3-kinase (PI3K), Jun Kinase (JNK) and p38 Mitogen-activated protein kinase (p38MAPK) were also excluded by the use of selective kinase inhibitors (2). We undertook an operational approach to identify candidate pathways of the GC insensitivity.

Methods The effects of TGF-β1 on GC transactivation were measured using GC response element (GRE)-controlled reporter and by RT-qPCR of GC-inducible gene expression. The TGF-beta1 proteome of BEAS-2B cells was identified using differential in-gel electrophoresis (2D DIGE). Data are presented as mean ± SEM for n experiments.

ResultsTGF-beta1 (1-100 pM) and TGF-beta3 (1-100 pM) impaired GRE transactivation and GC-induced gene expression, whereas TGF-beta2 (up to 400pM) did not. Concurrent inhibition of well-characterised non-canonical TGFβ1-activated kinase pathways (ERK, JNK, p38MAPK, PI3K) did not prevent TGF-beta1-suppression of GC activity. Of the 748 (±35, n=5 separate proteomics analyses) protein changes induced by TGF-beta1 (2D-DIGE) only 24 were not inhibited by the cocktail of kinase inhibitors (UO126, 1 microM; SP600125 1 microM; SB202190 1 microM; LY294002 10 microM). Of these 24 candidates only 4 (3 up, 1 down) were common to TGF-beta 1 and 3, with phospho-cofilin being prioritised based on prior evidence of induction of GC insensitivity. TGF-beta1-induced phospho-cofilin was inhibited by the LIM kinase (LIMK) inhibitor, LIMKi3 (10 microM) as was the TGF-beta1 GC insensitivity (% control dexamethasone 30 nM response: vehicle 54±6%, cf LIMKi3 112±8%, n=3). In a separate investigation, supported by an association of casein kinase1 (CK1) with the TGFβ receptor (3), the use of the selective CK1 delta/epsilon inhibitor PF670462 (1-10 microM) concentration-dependently reduced TGF-beta1-induced phospho-cofilin, prevented the GC insensitivity and reduced expression of fibrogens, including plasminogen-activator inhibitor-1.

Conclusions Casein kinase inhibitors present a potential new class of steroid-enhancing TGF-beta modulatory agents for chronic respiratory diseases.

References (1) Salem S et al. (2012). Br J Pharmacol 166:2036-2048 (2) Keenan C et al. (2014) Resp Res e15:55 (3) Hall et al. (1996) Biochem J 316: 303-310.