ERK1/2-Dependent Vascular Endothelial Growth Factor Signaling Sustains Cyst Growth in Polycystin-2 Defective Mice

doi:10.1053/j.gastro.2009.09.005

Gastroenterology

Volume 138, Issue 1, January 2010, Pages 360-371.e7

https://doi.org/10.1053/j.gastro.2009.09.005 Get rights and content

Background & Aims

Severe polycystic liver disease can complicate adult dominant polycystic kidney disease, a genetic disease caused by defects in polycystin-1 (Pkd1) or polycystin-2 (Pkd2). Liver cyst epithelial cells (LCECs) express vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2. We investigated the effects of VEGF on liver cyst growth and autocrine VEGF signaling in mice with Pkd1 and Pkd2 conditional knockouts.

Methods

We studied mice in which Pkd1 or Pkd2 were conditionally inactivated following exposure to tamoxifen; these mice were called Pkd1^flox/−:pCxCreER (Pkd1KO) and Pkd2^flox/−:pCxCreER (Pkd2KO).

Results

Pkd1KO and Pkd2KO mice developed liver defects; their LCECs expressed VEGF, VEGFR-2, hypoxia-inducible factor (HIF)-1α, phosphorylated extracellular signal–regulated kinase 1/2 (pERK1/2), and proliferating cell nuclear antigen (PCNA). In Pkd2KO but not Pkd1KO mice, exposure to the VEGFR-2 inhibitor SU5416 significantly reduced liver cyst development, liver/body weight ratio, and expression of pERK and PCNA. VEGF secretion and phosphorylation of ERK1/2 and VEGFR-2 were significantly increased in cultured LCECs from Pkd2KO compared with Pkd1KO mice. Inhibition of protein kinase A (PKA) reduced VEGF secretion and pERK1/2 expression. Addition of VEGF to LCECs from Pkd2KO mice increased phosphorylated VEGFR-2 and phosphorylated mitogen signal-regulated kinase (MEK) expression and induced phosphorylation of ERK1/2; this was inhibited by SU5416. Expression of HIF-1α increased in parallel with secretion of VEGF following LCEC stimulation. VEGF-induced cell proliferation was inhibited by the MEK inhibitor U1026 and by ERK1/2 small interfering RNA.

Conclusions

The PKA–ERK1/2–VEGF signaling pathway promotes growth of liver cysts in mice. In Pkd2-defective LCECs, PKA-dependent ERK1/2 signaling controls HIF-1α–dependent VEGF secretion and VEGFR-2 signaling. Autocrine and paracrine VEGF signaling promotes the growth of liver cysts in Pkd2KO mice. VEGF inhibitors might be used to treat patients with polycystic liver disease.

Section snippets

Materials, Antibodies, and Immunohistochemistry

All materials, antibodies, reagents, and their providers are listed in the Supplementary Materials and Methods.

Animals and Treatment

We established an inducible model for Pkd1 and Pkd2 inactivation using conditional Pkd1^flox and Pkd2^flox alleles in combination with the tamoxifen inducible pCXCreER line. The Pkd1^flox allele¹⁶ and the pCXCreER line have been reported previously.¹⁷ The pCXCreER transgene has a generalized promoter that achieves robust expression in bile ducts (Supplementary Figure 1A). The novel Pkd2

Characterization of the Liver Phenotype in Conditional Polycystin Knockout Mice

Pkd1KO and Pkd2KO mice had normal-appearing bile ducts before tamoxifen-induced gene inactivation (data not shown) but developed a bile duct cystic liver phenotype similar to human ADPKD after activation of Cre-mediated recombination by tamoxifen (Figure 1). Liver cysts were evident 4 weeks after induction and progressively enlarged until the time the mice were killed (8 weeks after, 15 weeks of age). These findings show that Pkd1 and Pkd2 expression are required to maintain normal bile ducts

Discussion

Progressive enlargement of liver cysts is responsible for severe complications in polycystic liver disease.² The mechanism of cyst enlargement in ADPKD is not well understood, but the consensus is that a major determinant is the excessive proliferation of the cystic epithelium.⁵

Our previous studies have indicated that estrogens, insulin-like growth factor-1, and VEGF are among the factors able to stimulate cholangiocyte proliferation that are overexpressed in the cystic epithelium of the liver

Acknowledgments

The authors thank Corinne Lobe (University of Toronto, Toronto, Ontario, Canada) for the pCX-CreER mice.

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by National Institutes of Health (NIH) grant DK079005, PKD Foundation (to M.S.), Yale University Liver Center (NIH grant K34989 to M.S. and C.S.), and NIH grants DK51041 and DK54053 (to S.S.). C.S. is a recipient of an American Liver Foundation/American Association for the Study of Liver Diseases Liver Scholar Award. The support of Fondazione S. Martino (Bergamo, Italy) is gratefully acknowledged.

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Basic—Liver, Pancreas, and Biliary TractERK1/2-Dependent Vascular Endothelial Growth Factor Signaling Sustains Cyst Growth in Polycystin-2 Defective Mice

Background & Aims

Methods

Results

Conclusions

Section snippets

Materials, Antibodies, and Immunohistochemistry

Animals and Treatment

Characterization of the Liver Phenotype in Conditional Polycystin Knockout Mice

Discussion

Acknowledgments

Gastroenterology

Biochem Biophys Res Commun

J Biol Chem

Gastroenterology

Gastroenterology

Toxicol In Vitro

Am J Pathol

Curr Biol

Gastroenterology

Kidney Int

Biochem Biophys Res Commun

Gastroenterology

Gastroenterology

J Biol Chem

Magnetic resonance imaging evaluation of hepatic cysts in early autosomal-dominant polycystic kidney disease: the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease cohort

Clin J Am Soc Nephrol

Polycystic disease of the liver

Hepatology

Sirolimus reduces polycystic liver volume in ADPKD patients

J Am Soc Nephrol

Mechanisms of disease: autosomal dominant and recessive polycystic kidney diseases

Nat Clin Pract Nephrol

Cellular and molecular function of mucolipins (TRPML) and polycystin 2 (TRPP2)

Pflugers Arch

Regulation of ryanodine receptor-dependent calcium signaling by polycystin-2

Proc Natl Acad Sci U S A

Pathophysiology of cholangiopathies

J Clin Gastroenterol

Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid

Hepatology

Basic—Liver, Pancreas, and Biliary Tract
ERK1/2-Dependent Vascular Endothelial Growth Factor Signaling Sustains Cyst Growth in Polycystin-2 Defective Mice