Beta-hydroxybutyrate-induced growth inhibition and collagen production in HK-2 cells are dependent on TGF-beta and Smad3.
BACKGROUND
Ketonuria is common in diabetes. The major form of ketone body is beta-hydroxybutyrate (beta-HB), which is metabolized by the proximal tubule. Transforming growth factor beta (TGF-beta) and tubulopathy are important in diabetic nephropathy. Thus, the role of TGF-beta and the downstream Smad3 in beta-HB-induced effects in the human proximal tubule (HK-2 cell) was studied.


METHODS
Effects of beta-HB (0.1 to 10 mmol/L) on HK-2 cells were determined for: proliferation, cell cycle distribution, collagen production, tubular transdifferentiation [expression of alpha-smooth muscle actin (alpha-SMA) protein], TGF-beta, Smad2/3, p21WAF1, and p27kip1.


RESULTS
Beta-HB (0.1 to 10 mmol/L) dose dependently decreased proliferation, arrested the cells in G0/G1 phase of the cell cycle, and increased p21WAF1/p27kip1 protein expression at 48 hours (without affecting p21WAF1/p27kip1 mRNA and transcription). beta-HB (1 mmol/L) increased p21WAF1/p27kip1 protein half-lives. Beta-HB (1 mmol/L) increased TGF-beta transcription at 24 hours and TGF-beta1 mRNA/bioactivity at 48 hours. Beta-HB (1 mmol/L) increased nuclear Smad2/3 protein expression and increased collagen production (without affecting tubular transdifferentiation), which were reversed by Smad7, dominant-negative Smad3, and N-acetylcysteine. Dominant-negative Smad3 reversed beta-HB-induced TGF-beta transcription at 24 hours, and reversed TGF-beta1 bioactivity at 48 hours. Dominant-negative Smad3 reversed beta-HB-induced p21WAF1/p27kip1 protein expression at 48 hours. Finally, N-acetylcysteine, TGF-beta antibody, Smad7, and dominant-negative Smad3 reversed beta-HB (1 mmol/L)-induced growth inhibition at 48 hours.


CONCLUSIONS
Beta-HB activated Smad 2/3 by oxidative stress. TGF-beta and Smad3 mediate beta-HB-induced cell cycle-dependent growth inhibition while Smad3 mediate beta-HB-induced collagen production and p21WAF1/p27kip1 protein expression in HK-2 cells. Moreover, beta-HB increased p21WAF1/p27kip1 protein expression by increasing p21WAF1/p27kip1 protein stability.
Guh
Jinn-Yuh
JY
Department of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chuang
Tsai-Der
TD
Chen
Hung-Chun
HC
Hung
Wen-Chun
WC
Lai
Yung-Hsiung
YH
Shin
Shyi-Jang
SJ
Chuang
Lea-Yea
LY
D020155:3-Hydroxybutyric Acid / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003094:Collagen / Q000096:biosynthesis*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D005799:Genes, Dominant; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology; D018384:Oxidative Stress / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051899:Smad2 Protein; D051900:Smad3 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; D012333:RNA, Messenger; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003094:Collagen; D020155:3-Hydroxybutyric Acid