Sp1 and Smad3 are required for high glucose-induced p21(WAF1) gene transcription in LLC-PK1 cells.
The cyclin-dependent kinase inhibitor p21(WAF1) is required for diabetic glomerular hypertrophy. High glucose-induced hypertrophy in proximal tubule cells is dependent on transforming growth factor-beta (TGF-beta). Many of the TGF-beta-induced effects are dependent on Smad2/3. Thus, the molecular mechanisms of high glucose-induced p21(WAF1) and hypertrophy were studied in high glucose-cultured proximal tubule-like LLC-PK(1) cells. We found that high glucose (30 mM) induced hypertrophy at 72 h. High glucose also increased the expression of p21(WAF1) protein and p21(WAF1) mRNA transcription and abundance at 48 h. The DNA element in the 5' regulatory region of p21(WAF1) gene essential for high glucose-induced p21(WAF1) gene transcription was identified as Sp1 by a series of the 5' regulatory region of p21(WAF1) gene deletion mutants. Moreover, high glucose activated Smad2/3 while increasing the Sp1 DNA-binding activity. High glucose also increased the Sp1-dependent transcriptional activity of p21(WAF1) gene. High glucose-induced hypertrophy was attenuated by p21(WAF1) short interfering RNA and Smad3 dominant-negative plasmid transfection. We concluded that high glucose induced hypertrophy via Sp1-Smad2/3-dependent activation of p21(WAF1) gene transcription in LLC-PK(1) cells.
Chuang
Tsai-Der
TD
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Guh
Jinn-Yuh
JY
Chiou
Shean-Jaw
SJ
Chen
Hung-Chun
HC
Hung
Wen-Chun
WC
Chuang
Lea-Yea
LY
D000818:Animals; D018929:Cell Culture Techniques; D003470:Culture Media; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D019084:Fluorescent Antibody Technique, Indirect; D017353:Gene Deletion; D005947:Glucose / Q000494:pharmacology*; D018374:LLC-PK1 Cells; D008156:Luciferases / Q000378:metabolism; D010957:Plasmids; D034741:RNA, Small Interfering / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D013552:Swine; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D003470:Culture Media; D050759:Cyclin-Dependent Kinase Inhibitor p21; D034741:RNA, Small Interfering; D051900:Smad3 Protein; D016329:Sp1 Transcription Factor; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase; D005947:Glucose