SMAD2/3 and TGF-ß1 suppress expression from the megalin promoter.

(A) LLC-PK1 cells were seeded in triplicate in 24-well plates. The cells were transfected 48 h later with the megalin promoter construct (-1500PromBasic) and a ß-galactosidase vector (pCMVß). Co-transfections were performed in the presence or absence of plasmids encoding for SMAD2 and SMAD3. After 24 h, the transfected cells were lysed, and luciferase activity was measured. Cells expressing the megalin promoter construct showed a 50% decreased in luciferase activity when the SMAD transcription factors were overexpressed. (B) The overexpression of SMAD factors increased luciferase expression from the p800luc plasmid (positive control). Data in (A) and (B) are expressed as the means ± SD, and significant differences with the control (pCMV5) are indicated by *P≤ 0.001. (C) LLC-PK1 cells were transfected with the megalin promoter construct (1500PromBasic) and a ß-galactosidase vector (pCMVß). After 24h the cells were incubated in the presence or absence of TGF-ß1 2.5 ng/ml for another 24h and luciferase activity was measured in the cell lysates. Cells expressing the megalin promoter construct in the presence of TGF-ß1 show decreased luciferase activity. (D) Positive control with the p800luc plasmid in which TGF-ß1 increases luciferase expression. Data in (C) and (D) are expressed as the means ± SD, and significant differences with the control are indicated by #P≤0.05. (E) LLC-PK1 cells were transfected with pCMV5 or with SMAD2 and SMAD3 cDNAs. After 48 h of expression, the cells were lysed and megalin, SMAD2 and SMAD3 were analyzed by immunoblot; tubulin was used as a loading control. (F) Quantification of megalin/tubulin ratio in cells transfected with SMAD2 and SMAD3. (G) LLC-PK1 cells, expressing pCMV5 or SMAD2 and SMAD3 cDNAs were permeabilized with 0.1% saponin and incubated with anti-megalin and anti-FLAG. Cells were then incubated with anti-mouse Alexa-647 and anti-rabbit Alexa -488 antibodies and analyzed by flow cytometry. The results are plotted as the % of the total megalin observed in SMAD2/3 positives cells. Data in (F) and (G) are expressed as the means ± SE, and significant differences with the control are indicated by #P≤0.05.