Keay Susan K, Zhang Chen-Ou
Department of Medicine, Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, MD, USA.
Department of Veterans Affairs Medical Center, Medical Service, Baltimore, MD, USA.
BJU Int. 2016 Jul;118(1):161-72. doi: 10.1111/bju.13457. Epub 2016 Mar 29.
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.
确定间质性膀胱炎/膀胱疼痛综合征（IC/BPS）患者膀胱上皮细胞的特定细胞组分中蛋白激酶B（Akt）信号传导及特定下游效应蛋白的分泌是否异常，因为IC/BPS患者的膀胱上皮细胞外植体可产生一种卷曲蛋白8相关糖肽抗增殖因子（APF），该因子可抑制正常膀胱上皮细胞增殖以及几种已知受Akt信号传导调控的蛋白的表达。一个相关的次要目的是确定用活性合成去唾液酸抗增殖因子（as-APF）处理正常膀胱上皮细胞是否会在Akt信号传导及特定下游效应蛋白/信使核糖核酸中诱导类似变化。
从6例符合美国国立糖尿病、消化和肾脏疾病研究所（NIDDK）修订的IC/BPS标准的患者以及6例年龄和性别匹配的对照者的原发性膀胱上皮外植体中提取细胞蛋白，分为四个亚细胞组分。通过蛋白质印迹法测定细胞总Akt和/或磷酸化Akt、糖原合酶激酶3β（GSK3β）和β-连环蛋白；细胞总JunB；以及分泌的基质金属蛋白酶2（MMP2）和肝素结合表皮生长因子样生长因子（HB-EGF）水平。通过定量逆转录聚合酶链反应对MMP2、JunB、p53、尿路上皮蛋白3（UPK3）和β-肌动蛋白信使核糖核酸进行定量。通过非放射性测定法确定Akt活性。
与匹配的对照细胞相比，IC/BPS细胞在特定组分中的Akt活性较低，同时Akt丝氨酸473和GSK3β丝氨酸9的磷酸化水平较低，而β-连环蛋白丝氨酸33、37/苏氨酸41的磷酸化水平较高。与对照细胞相比，IC/BPS外植体还存在其他下游异常的证据，包括核内JunB水平较低；分泌的MMP2和HB-EGF水平较低；以及相对于β-肌动蛋白，MMP2、JunB和UPK3信使核糖核酸水平较低，但p53信使核糖核酸水平较高。as-APF在正常细胞中也诱导了IC/BPS细胞的每一种异常。
这些发现表明，IC/BPS细胞具有异常的Akt活性以及包括MMP2和HB-EGF分泌减少在内的下游蛋白表达异常。它们还支持以下假设，即APF通过其对细胞Akt信号传导和HB-EGF产生的影响在IC/BPS的发病机制中起作用。
