All-trans retinoic acid inhibits mesangial cell proliferation by up-regulating p21Waf1/Cip1 and p27Kip1 and down-regulating Skp2

J Nephrol. Nov-Dec 2012;25(6):1031-40. doi: 10.5301/jn.5000090.

Abstract

Background: The aim of this study was to examine effects of all-trans retinoic acid (ATRA) on rat mesangial cell proliferation, apoptosis and underlying mechanisms.

Methods: Cultured HBZY-1 rat mesangial cells received the following treatments: group 1: controls (DMSO); group 2: TGF-beta(1) (10 µg/L); groups 3-5: ATRA (0.1, 1.0 and 10 µmol/L) + TGF-beta(1) (10 µg/L). After treatments, the cells were studied by CCK-8 assay, cell cycle assay and TUNEL staining. p21(Waf1/Cip1), p27(Kip1) and Skp2 mRNA levels were measured by real-time PCR. p21(Waf1/Cip1), p27(Kip1) and Skp2 protein levels were detected by Western blot. The localization of p21(Waf1/Cip1) and p27(Kip1) proteins was observed by immunofluorescence and confocal microscopy.

Results: Compared with controls, TGF-beta(1) significantly enhanced proliferation and inhibited apoptosis of mesangial cells. ATRA dose-dependently reversed both TGF-beta(1)-induced decreases in p21(Waf1/Cip1) mRNA and protein levels and p27(Kip1) protein level and increases in Skp2 mRNA and protein levels, and inhibited TGF-beta(1)-induced proliferation through G(1) arrest. Meanwhile, after ATRA treatments, p21(Waf1/Cip1) and p27(Kip1) proteins mainly localized in the nucleus, and their concentrations in cytoplasm decreased.

Conclusion: ATRA inhibited TGF-beta(1)-induced mesangial cell proliferation by up-regulating p21(Waf1/Cip1) mRNA and protein levels, and p27(Kip1) protein level, and down-regulating Skp2 mRNA and protein levels, but it had no significant effect on apoptosis in rat mesangial cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Blotting, Western
  • Cell Line
  • Cell Proliferation / drug effects*
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism*
  • Cyclin-Dependent Kinase Inhibitor p27 / genetics
  • Cyclin-Dependent Kinase Inhibitor p27 / metabolism*
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • G1 Phase Cell Cycle Checkpoints / drug effects
  • In Situ Nick-End Labeling
  • Mesangial Cells / drug effects*
  • Mesangial Cells / metabolism
  • Mesangial Cells / pathology
  • Microscopy, Confocal
  • RNA, Messenger / metabolism
  • Rats
  • Real-Time Polymerase Chain Reaction
  • Resting Phase, Cell Cycle / drug effects
  • Reverse Transcriptase Polymerase Chain Reaction
  • S-Phase Kinase-Associated Proteins / genetics
  • S-Phase Kinase-Associated Proteins / metabolism*
  • Signal Transduction / drug effects
  • Time Factors
  • Transforming Growth Factor beta1 / metabolism
  • Tretinoin / pharmacology*
  • Up-Regulation

Substances

  • Cdkn1a protein, rat
  • Cdkn1b protein, rat
  • Cyclin-Dependent Kinase Inhibitor p21
  • RNA, Messenger
  • S-Phase Kinase-Associated Proteins
  • Transforming Growth Factor beta1
  • Cyclin-Dependent Kinase Inhibitor p27
  • Tretinoin