Abnormal Akt signalling in bladder epithelial cell explants from patients with interstitial cystitis/bladder pain syndrome can be induced by antiproliferative factor treatment of normal bladder cells

Abstract

To determine whether protein kinase B (Akt) signalling and secretion of specific downstream effector proteins are abnormal in specific cell fractions of bladder epithelial cells from patients with interstitial cystitis/bladder pain syndrome (IC/BPS), as explanted bladder epithelial cells from patients with IC/BPS produce a frizzled 8-related glycopeptide antiproliferative factor (APF) that inhibits normal bladder epithelial cell proliferation and expression of several proteins known to be regulated by Akt signalling. A related secondary objective was to determine whether treatment of normal bladder epithelial cells with active synthetic asialo-antiproliferative factor (as-APF) induces similar changes in Akt signalling and specific downstream effector proteins/mRNAs. Cell proteins were extracted into four subcellular fractions from primary bladder epithelial explants of six patients who fulfilled modified National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) criteria for IC/BPS and six age- and gender-matched controls. Total and/or phosphorylated cellular Akt, glycogen synthase kinase 3β (GSK3β), and β-catenin; total cellular JunB; and secreted matrix metalloproteinase 2 (MMP2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) levels were determined by Western blot. MMP2, JunB, p53, uroplakin 3 (UPK3), and β-actin mRNAs were quantified by quantitative reverse transcriptase-polymerase chain reaction. Akt activity was determined by nonradioactive assay. IC/BPS cells had lower Akt activity, along with lower Akt ser473- and GSK3β ser9-phosphorylation and higher β-catenin ser33,37/thr41-phosphorylation in specific fractions as compared with matched control cells. IC/BPS explants also had evidence of additional downstream abnormalities compared with control cells, including lower nuclear JunB; lower secreted MMP2 and HB-EGF; plus lower MMP2, JunB, and UPK3 mRNAs but higher p53 mRNA relative to β-actin. Each of these IC/BPS cell abnormalities was also induced in normal cells by as-APF. These findings indicate that IC/BPS cells have abnormal Akt activity with downstream protein expression abnormalities including decreased MMP2 and HB-EGF secretion. They also support the hypothesis that APF plays a role in the pathogenesis of IC/BPS via its effects on cell Akt signalling and HB-EGF production.