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.
Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, CT 06520, USA.
Carlo
C
Spirli
Stefano
S
Okolicsanyi
Romina
R
Fiorotto
Luca
L
Fabris
Massimiliano
M
Cadamuro
Silvia
S
Lecchi
Xin
X
Tian
Stefan
S
Somlo
Mario
M
Strazzabosco
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