Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in thecpkmouse, a model of autosomal recessive polycystic kidney disease

Abstract

Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-β/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-β/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-β, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in mice. pSmad3L expression was increased in mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in mice. Smad3 knockout/ double-mutant mice revealed amelioration of abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-β/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention.

Cystin gene mutations cause autosomal recessive polycystic kidney disease associated with alteredMycexpression

“…64 Similarly, Myc is down-regulated in Cys1 cpk/cpk ;Smad3 +/mice and such double mutants have a milder phenotype than cpk mice. 25 Our findings that complementation of mutant cpk with cystin-GFP rescues the cystic phenotype and restores normal Myc expression confirms that cystin acts in vivo as a negative regulator of Myc. 10.…”

“…19,23 In previous work, we have demonstrated that in renal collecting duct epithelia, cystin physically interacts with necdin in a regulatory complex that modulates Myc expression. 24 Cystin deficiency-associated disruption of ciliary signaling and/or overexpression of Myc is associated with aberrant SMAD3 phosphorylation, 25 overexpression of Fos and Kras protooncogenes, [19][20][21] elevated levels of growth factors, 26 aberrant localization and abundance of the epidermal growth factor receptor (EGFR) on the apical surface of collecting duct cells 27 , abnormal levels of basement membrane components, [28][29][30] and epithelial cell adhesion molecules. 31,32 Until now, the relevance of these effects of cystin deficiency for human disease was unclear in the absence of ARPKD patients with mutations in human CYS.…”

Cystin genetic variants cause autosomal recessive polycystic kidney disease associated with altered Myc expression

“…Fourth, pharmacological inhibition of glucogen synthase kinase 3 beta (GSK3beta), which accelerates cyst formation in cpk mice, leads to decreased My c expression and amelioration of the cystic phenotype 68 . Similarly, Myc is down-regulated in Cys1 cpk/cpk ; Smad3 +/− mice and these double mutants have a milder phenotype than cpk mice 26 . Our findings that complementation of mutant cpk with cystin-GFP rescues the cystic phenotype and restores normal Myc expression provides further evidence that cystin acts in vivo as a negative regulator of Myc .…”

“…Cystin deficiency-associated disruption of ciliary signaling and/or overexpression of Myc is associated with aberrant SMAD3 phosphorylation 26 , overexpression of Fos and Kras proto-oncogenes 20 – 22 , elevated levels of growth factors 27 , aberrant localization and abundance of the epidermal growth factor receptor (EGFR) on the apical surface of collecting duct cells 28 and altered levels of basement membrane components 29 – 31 and epithelial cell adhesion molecules 32 , 33 . Until now, the relevance of these effects of cystin deficiency for human disease was unclear in the absence of ARPKD patients with variants in human CYS1 .…”

Protein Kinase A Downregulation Delays the Development and Progression of Polycystic Kidney Disease

“…Strategies directly targeting PKA, rather than targeting cAMP through inhibition of adenylyl cyclases or activation of phosphodiesterases may be more effective in conditions where PKA activation occurs independently from cAMP. Many of these conditions may occur in ADPKD, for example 115 – 118 : ( 1 ) activation of NF-kB signaling dissociates the NF-κB:IκB complex, which under basal conditions interacts and holds PKA C in its inactive state 119 , 120 . ( 2 ) Activation of TGF- β signaling results in the formation of heterodimeric complexes of Smad2 and Smad3 with Smad4, which bind to the PKA R subunits and activate PKA C activity 121 , 122 .…”