Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is associated with a number of cellular defects such as hyperproliferation, apoptosis, and dedifferentiation. Mutations in polycystin-1 (PC1) account for ∼85% of ADPKD. Here, we showed that wild-type (WT) or mutant PC1 composed of the last five transmembrane (TM) domains and the C-terminus (termed PC1-5TMC) inhibits cell proliferation and protein translation, as well as the downstream effectors of mTOR, consistent with previous reports. Knockdown of B56α, a subunit of the protein phosphatase 2A (PP2A) complex, or application of PP2A inhibitor okadaic acid or calyculin A, abolished the inhibitory effect of PC1 and PC1-5TMC on proliferation, indicating that PP2A/B56α mediates the regulation of cell proliferation by PC1. In addition to the phosphorylated S6 and 4EBP1, B56α was also downregulated by PC1 and PC1-5TMC. Furthermore, the downregulation of B56α, which may be mediated by mTOR but not AKT, can account for the dependence of PC1-inhibited proliferation on PP2A.
Highlights
Autosomal dominant polycystic kidney disease (ADPKD) results from mutations in genes coding for polycystin 1 (PC1) and polycystin 2 (PC2), which account for about 85% and 15% of ADPKD, respectively [1]
In order to clarify the effect of PC1 on cell proliferation, we used Alarma-Blue to label HeLa, human embryonic kidney (HEK293T), Madin-Darby canine kidney (MDCK), and mouse inner medullary collecting duct (IMCD3) cells after transfection of PC1-5TMC, a truncation mutant consisting of the last five TM domains (S7-S11), and the C-terminus of human PC1 and found that PC1-5TMC in HeLa, MDCK, and IMCD cells, as well as WT PC1 in HEK293T cells, suppresses proliferation (Figures 1(a) and 1(b)), consistent with previous reports [4,5,6,7,8,9]. e difference in magnitude of the responses can be explained mainly by the fact that different cell types can lead to different cell transfection efficiencies and different inhibition rates of cell proliferation
We found that knockdown of B56α abolished the effect of PC1 on proliferation when the expression of B56α was completely inhibited (Figure 2(c)), which further supported and verified that the PC1-inhibited cell proliferation is through phosphatase 2A (PP2A) (B56α)
Summary
Autosomal dominant polycystic kidney disease (ADPKD) results from mutations in genes coding for polycystin 1 (PC1) and polycystin 2 (PC2), which account for about 85% and 15% of ADPKD, respectively [1]. BioMed Research International patients and mouse models, which may result from loss of PC1 binding with tuberin, suggesting that PC1 inhibits cell proliferation by downregulating the activity of mTOR and its interaction with tuberin [8]. It was later verified PC1 reduces cell size by negatively regulating mTOR and downstream effectors S6K1 and 4EBP1 in a tuberin-dependent manner [9]. PP2A is known to dephosphorylate over 300 types of substrates involved in almost all major cellular signaling pathways including the Wnt, mTOR, and MAPK pathways [13]. We employed culture cells transfected with PC1 or PC1 mutants to investigate the relationship between PC1regulating cell proliferation/translation and PP2A (B56α)
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