Tristetraprolin regulates IL-8 mRNA stability in cystic fibrosis lung epithelial cells

Abstract

Cystic fibrosis (CF) is due to mutations in the CFTR gene and is characterized by hypersecretion of the proinflammatory chemokine IL-8 into the airway lumen. Consequently, this induces the highly inflammatory cellular phenotype typical of CF. Our initial studies revealed that IL-8 mRNA is relatively stable in CF cells compared with those that had been repaired with [WT]CFTR (wild-type CFTR). Relevantly, the 3′-UTR of IL-8 mRNA contains AU-rich sequences (AREs) that have been shown to mediate posttranscriptional regulation of proinflammatory genes upon binding to ARE-binding proteins including Tristetraprolin (TTP). We therefore hypothesized that very low endogenous levels of TTP in CF cells might be responsible for the relative stability of IL-8 mRNA. As predicted, increased expression of TTP in CF cells resulted in reduced stability of IL-8 mRNA. An in vitro analysis of IL-8 mRNA stability in CF cells also revealed a TTP-induced enhancement of deadenylation causing reduction of IL-8 mRNA stability. We conclude that enhanced stability of IL-8 mRNA in TTP-deficient CF lung epithelial cells serve to drive the proinflammatory cellular phenotype in the CF lung.

Elevated miR-155 Promotes Inflammation in Cystic Fibrosis by Driving Hyperexpression of Interleukin-8

“…7, the effect of raising miR-155, lowering SHIP1, and activating PI3K/ Akt is to activate MAPKs, thereby stabilizing IL-8 mRNA and increasing the levels of IL-8 protein expression. These data thus lend further support to the concept that the proinflammatory phenotype of the CF airway is due to increased stability of the IL-8 mRNA and subsequent increased expression of IL-8 protein (12,46). SHIP1 and PI3K/Akt Signaling-The only predicted and validated microRNA to target SHIP1 is miR-155 (44), and the connection of SHIP1 to activation of PI3K/AKT signaling has been validated using both pharmacologic and molecular methods.…”

“…For example, the stability of mRNAs encoding many inflammatory genes, including IL-8, is regulated by interactions between AUrich elements (AREs) in the 3Ј-untranslated region (3Ј-UTR) and specific ARE-binding proteins. In the case of CF lung epithelial cells in culture, which constitutively secrete high levels of IL-8, we have recently reported that high levels of IL-8 mRNA are sustained by mutation-dependent reduction in the ARE-binding protein tristetraprolin (TTP) (12). We also found a similarly low level of TTP in primary CF lung cells, which had been obtained acutely by brush biopsy of CF patients.…”

“…The primary bronchial epithelial cells were obtained from lung brush biopsies of CF patients as described earlier (12), and the blood was collected from CF patients and controls under a Uniformed Services University of the Health Sciences Institutional Review Board-approved protocol. The cells were stored in RNA later or TRIzol, and total RNA was isolated using the miRVana kit.…”

“…In previous work, we have shown that IL-8 mRNA is greatly stabilized in CF cells, both in vitro and in vivo, and that IL-8 protein expression rates were also elevated as a consequence (12,46). We therefore hypothesized that if SHIP1 levels were exogenously elevated in CF cells, then the half-life of IL-8 mRNA would be reduced, and the expression levels of IL-8 protein would also be reduced.…”

“…miR-155 Stabilizes IL-8 mRNA by Activation of MAPKs-In CF cells, both in vitro and in vivo, we have previously identified intrinsically reduced levels of the ARE-interacting protein TTP (12) and increased levels of MAPK signaling (46) as being independently responsible for IL-8 mRNA stabilization. Nonetheless, in these previous studies, the CF-specific drivers for either of these specific activities had yet to be identified.…”