Mammalian target of rapamycin regulates vascular endothelial growth factor-dependent liver cyst growth in polycystin-2-defective mice.
Polycystic liver disease may complicate autosomal dominant polycystic kidney disease (ADPKD), a disease caused by mutations in polycystins, which are proteins that regulate signaling, morphogenesis, and differentiation in epithelial cells. The cystic biliary epithelium [liver cystic epithelium (LCE)] secretes vascular endothelial growth factor (VEGF), which promotes liver cyst growth via autocrine and paracrine mechanisms. The expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), and phosphorylated mammalian target of rapamycin (p-mTOR) and the protein kinase A (PKA)-dependent phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) are also up-regulated in LCE. We have hypothesized that mammalian target of rapamycin (mTOR) represents a common pathway for the regulation of hypoxia-inducible factor 1 alpha (HIF1alpha)-dependent VEGF secretion by IGF1 and ERK1/2. Conditional polycystin-2-knockout (Pkd2KO) mice were used for in vivo studies and to isolate cystic cholangiocytes [liver cystic epithelial cells (LCECs)]. The expression of p-mTOR, VEGF, cleaved caspase 3 (CC3), proliferating cell nuclear antigen (PCNA), IGF1, IGF1R, phosphorylated extracellular signal-regulated kinase, p-P70S6K, HIF1alpha, and VEGF in LCE, LCECs, and wild-type cholangiocytes was studied with immunohistochemistry, western blotting, or enzyme-linked immunosorbent assays. The cystic area was measured by computer-assisted morphometry of pancytokeratin-stained sections. Cell proliferation in vitro was studied with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assays. The treatment of Pkd2KO mice with the mTOR inhibitor rapamycin significantly reduced the liver cyst area, liver/body weight ratio, pericystic microvascular density, and PCNA expression while increasing expression of CC3. Rapamycin inhibited IGF1-stimulated HIF1alpha accumulation and VEGF secretion in LCECs. IGF1-stimulated LCEC proliferation was inhibited by rapamycin and SU5416 (a vascular endothelial growth factor receptor 2 inhibitor). Phosphorylation of the mTOR-dependent kinase P70S6K was significantly reduced by PKA inhibitor 14-22 amide and by the mitogen signal-regulated kinase inhibitor U1026.


CONCLUSIONS
These data demonstrate that PKA-dependent up-regulation of mTOR has a central role in the proliferative, antiapoptotic, and pro-angiogenic effects of IGF1 and VEGF in polycystin-2-defective mice. This study also highlights a mechanistic link between PKA, ERK, mTOR, and HIF1alpha-mediated VEGF secretion and provides a proof of concept for the potential use of mTOR inhibitors in ADPKD and conditions with aberrant cholangiocyte proliferation.
Spirli
Carlo
C
Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, CT 06520, USA.
Okolicsanyi
Stefano
S
Fiorotto
Romina
R
Fabris
Luca
L
Cadamuro
Massimiliano
M
Lecchi
Silvia
S
Tian
Xin
X
Somlo
Stefan
S
Strazzabosco
Mario
M
D000818:Animals; D003560:Cysts / Q000209:etiology* / Q000473:pathology; D004195:Disease Models, Animal; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D008107:Liver Diseases / Q000209:etiology* / Q000473:pathology; D051379:Mice; D016891:Polycystic Kidney, Autosomal Dominant / Q000503:physiopathology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D050396:TRPP Cation Channels / Q000172:deficiency*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000502:physiology*
D048049:Extracellular Signal-Regulated MAP Kinases; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007334:Insulin-Like Growth Factor I; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D050396:TRPP Cation Channels; D042461:Vascular Endothelial Growth Factor A; C497443:Hif1a protein, mouse; C519530:insulin-like growth factor-1, mouse; C546843:mTOR protein, mouse