cyclin-d1 and Polycystic-Kidney--Autosomal-Dominant

Autosomal dominant polycystic kidney disease (ADPKD) leads to renal failure. The hallmark of ADPKD is increased epithelial proliferation, which has been proposed to be due to atypical signaling including abnormal JAK-STAT activity. However, the relative contribution of JAK-STAT family members in promoting proliferation in ADPKD is unknown. Here, we present siRNA JAK-STAT-focused screens discovering a previously unknown proliferative role for multiple JAK-STAT components (including STAT1, STAT2, STAT4, STAT5a, and STAT5b). Amongst these, we selected to study the growth hormone/growth hormone receptor/STAT5-axis because of its known role as a regulator of growth in nonrenal tissues. Loss of STAT5 function, facilitated by pharmacological inhibition or siRNAs, significantly reduced proliferation with an associated reduction in cyst growth in vitro. To study whether STAT5 is abnormally activated in vivo, we analyzed its expression using two independent mouse models of ADPKD. STAT5 was nuclear, thus activated, in renal epithelial cyst lining cells in both models. To test whether forced activation of STAT5 can modulate proliferation of renal cells in vivo, irrespective of the Pkd1 status, we overexpressed growth hormone. These mice showed increased STAT5 activity in renal epithelial cells, which correlated with de novo expression of cyclin D1, a STAT5 target gene. Chromatin immunoprecipitation experiments revealed that STAT5 transcriptionally activated cyclin D1 in a growth hormone-dependent fashion, thus providing a mechanism into how STAT5 enhances proliferation. Finally, we provide evidence of elevated serum growth hormone in Pkd1 mutant mice. Thus, the growth hormone/STAT5 signaling axis is a novel therapeutic target in ADPKD.

Topics: Animals; Carrier Proteins; Cell Line; Cell Nucleus; Cell Proliferation; Cyclin D1; Disease Models, Animal; Epithelial Cells; Genotype; Growth Hormone; Humans; Janus Kinases; Kidney; Mice, Transgenic; Phenotype; Polycystic Kidney, Autosomal Dominant; Protein Kinase Inhibitors; RNA Interference; Signal Transduction; STAT5 Transcription Factor; Time Factors; Transfection; TRPP Cation Channels; Tumor Suppressor Proteins

Mesangial proliferative glomerulonephritis (MsPGN) is characterized by widespread mesangial cell proliferation and an accumulation of extracellular matrix (ECM) in the mesangial area. In a previous study we developed a polycystin‑1 N‑terminal fragment (PC‑1 NF) fusion protein that inhibits the proliferation of cyst‑lining epithelial cells in autosomal dominant polycystic kidney disease. In addition, the PC‑1 NF fusion protein arrests the cell cycle of cancer cells at the G0/G1 phase, inhibiting their proliferation. In the present study, the effect of the PC‑1 NF fusion protein on MsPGN was investigated. It was found that the PC‑1 NF fusion protein inhibited the proliferation of rat mesangial cells and induced G0/G1 phase arrest and apoptosis in vitro. PC‑1 NF fusion protein treatment also resulted in a decrease in mRNA expression levels of proliferating cell nuclear antigen, cyclin D1 and B‑cell lymphoma‑2 (Bcl‑2) and an increase in mRNA expression levels of Bcl‑2‑associated X protein (Bax) and p21Waf1. Furthermore, a decrease in Bcl‑2, c‑fos, c‑jun and protein kinase C‑α protein levels was observed, whereas Bax protein levels increased. Additionally, PC‑1 NF fusion protein induced ECM degradation and inhibited ECM expansion. The results also demonstrated that PC‑1 NF fusion protein treatment resulted in a decrease in type IV collagen and tissue inhibitor of metalloproteinase mRNA levels but an increase in matrix metalloproteinase 2 mRNA levels. In combination, these results suggest that the PC‑1 NF fusion protein inhibits proliferation, promotes apoptosis and induces ECM degradation in MsPGN rats. This study offers novel perspectives for the treatment of MsPGN.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Cycle; Cell Line; Cell Proliferation; Cyclin D1; Epithelial Cells; Extracellular Matrix; Glomerulonephritis; Matrix Metalloproteinase 2; Mesangial Cells; Polycystic Kidney, Autosomal Dominant; Proliferating Cell Nuclear Antigen; Protein Kinase C-alpha; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Recombinant Fusion Proteins; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; TRPP Cation Channels

Previous studies have shown that the expression and distribution of keratinocyte growth factor (KGF), also known as FGF-7 (fibroblast growth factor-7) or HBGF-7 (heparin-binding growth factor-7), may be implicated in kidney cyst formation and expansion. However, there are no data on KGF expression in human autosomal dominant polycystic kidney disease (ADPKD) tissue, and it is unknown whether it affects ADPKD cyst-lining epithelial cell epithelial cell proliferation.. The expression and distribution of KGF and KGF receptor (KGFR) mRNA in ADPKD cystic and normal kidney tissues were examined using quantitative real-time polymerase chain reaction (PCR) and in situ hybridization. KGF and KGFR protein expression in the above tissues was analysed by immunohistochemistry and western blot. The effect of KGF on cyst-lining epithelial cell proliferation was assessed by MTT assay, and its effect on the cyst-lining epithelial cell cycle was analysed by flow cytometry. The effect of KGF on cyclin D1 and P21(wafl) gene expression in cyst-lining epithelial cells was also determined.. KGF and KGFR mRNA expression in ADPKD cysts was higher than in normal kidney tissues. KGF and KGFR protein expression was also higher in ADPKD cysts and was localized to cyst-lining epithelial cells, tubular and interstitial cells. In vitro experiments revealed that KGF promoted cyst-lining epithelial cell proliferation, and decreased the ratio of G0/G1 phase but increased that of S phase. In response to KGF, the expression of the cyclin D1 gene in cyst-lining epithelial cells increased markedly while P21(wafl) expression decreased.. KGF and KGFR expression was upregulated in ADPKD kidney tissues. KGF stimulated the proliferation of cyst-lining epithelial cell in vitro by regulating the expression of cyclin D1 and P21(wafl) genes. KGF may play a role in pathogenesis of ADPKD.

Topics: Adult; Aged; Blotting, Western; Cell Proliferation; Cyclin D1; Epithelium; Female; Fibroblast Growth Factor 7; Flow Cytometry; Follow-Up Studies; Gene Expression Regulation; Humans; Immunohistochemistry; In Situ Hybridization; In Vitro Techniques; Kidney Tubules, Proximal; Male; Middle Aged; Polycystic Kidney, Autosomal Dominant; Polymerase Chain Reaction; Receptor, Fibroblast Growth Factor, Type 2; Retrospective Studies; RNA, Messenger