Reagents

Dulbecco’s modified Eagle’s medium (DMEM), Minimum Essential Medium alpha (alpha-MEM), L-glutamine, penicillin/streptomycin (P/S) and trypsin were acquired from Gibco (Gibco Life Technologies, Inc). MEM vitamins, fatty acid-free BSA, Trichostatin A (TSA) and the protease inhibitors (phenylmethylsulfonyl fluoride (PMSF), antipain, leupeptin, pepstatin, orthovanadate and NaF) were acquired from Sigma (Sigma-Aldrich). Foetal bovine serum (FBS) was acquired from HyClone (HyClone Laboratories, Inc). Lipofectamine 2000, random primers, primers and RNase were acquired from Invitrogen. DNAse I, reverse transcriptase M-MuLV Reverse, SYBR Green master mix, Pfu DNA Polymerase and Taq polymerase were acquired from Fermentas. Other reagents included bicinchoninic acid (BCA) assays (Pierce Thermo Fisher Scientific Inc.), luciferase assays (Promega), recombinant TGF-β1 (R&D Systems), and RNASolv reagent (Omega Bio-Tek). The following antibodies were used: anti-E-cadherin antibody (BD Bioscience), anti-ß-tubulin antibody (Millipore), phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425), SMAD2 #3122 and SMAD3 #9523 antibodies (Cell Signalling Technology) and anti-FLAG antibody (Clontech). Horseradish peroxidase-conjugated secondary antibodies and anti-rabbit and anti-mouse antibodies were acquired from Molecular Probes. Alexa Fluor 488 Rabbit, Alexa Fluor 555 Mouse and Alexa Fluor 488 Mouse secondary antibodies were obtained from Invitrogen. The polyclonal antiserum to the recombinant human megalin cytoplasmic domain (anti-megT) has been previously described [62]. The ALK inhibitor SB-431542 (Abcam) was kindly donated by Dr. Enrique Brandan, Department of Cellular and Molecular Biology, Faculty of Biological Sciences, P. Catholic University of Chile.

Plasmids

pCMV5, pCMV5-SMAD2-FLAG, and pCMV5-SMAD3-FLAG were provided by Dr. Seong-Jin Kim (CHA University, Korea). The 1500 pGL3-basic megalin promoter (-1500PromBasic) [63] was a gift from Dr. F. Lammert (Homburg, Germany). p800LUC, which contains the PAI-1 promoter [64], was kindly provided by Dr. Rifkin; and the pCMVß vector was obtained from Clontech.

Cell culture and treatments

We used the LLC-PK1 epithelial cell line derived from porcine kidneys (provided by Dr. Carlos P Vio, Departamento de Fisiologia, Pontifícia Universidade Católica). Cells were cultured in alfa-MEM supplemented with 7.5% FBS containing 100 U/ml P/S, 2 mM glutamine and 2.5 μg/ml Plasmocin. These cells have been previously used in studies on proximal tubule function and megalin expression and function [27, 35]. The dog gallbladder epithelial cell line dGBECs (provided by Dr. Sum Lee, Department of Medicine, Veterans Affairs Medical Center, Seattle) are polarized cells that express several proteins present in the gallbladder, including megalin and cubilin [35]. These cells were cultured in low-glucose DMEM supplemented with 2 mM glutamine, 1% MEM non-essential amino acids solution, 1% MEM vitamin solution, 7.5% FBS and P/S. The cells were seeded at approximately 70–80% confluence in complete media for 24 h and maintained at 37°C in 5% CO₂. HEK293 cells were grown and maintained in high-glucose DMEM supplemented with 10% FBS containing 100 U/ml P/S and 2.5 μg/ml plasmocin. All the cultures were maintained in humidified chambers. For bovine serum albumin (BSA) treatments, LLC-PK1 cells were plated at 3 x 10⁴ cells/cm² for 48 h, subsequently grown overnight in low-serum medium (alfa-MEM supplemented with 0.5% FBS containing 100 U/ml P/S, 2 mM glutamine and 2.5 mg/ml Plasmocin) and treated with fatty acid-free BSA or vehicle (water) in serum-free medium at different time points (0.5, 1, 6 or 12 h) and concentrations (0.01, 0.1, 10, or 20 ng/ml).

For TGF-ß1 treatments, LLC-PK1 cells or dGBECs were plated at 3 x 10⁴ cells/cm² for 48 h and subsequently grown overnight in low-serum medium. For dGBEC we used low-glucose alfa-MEM supplemented with 2 mM glutamine, 1% MEM non-essential amino acids solution, 1% MEM vitamin solution, 0.5% FSB and P/S. Cells were treated with recombinant TGF-β1 (1, 2.5, 5, or 10 ng/ml) or vehicle (4 mM HCL + 1 mg/ml BSA) in low-serum medium for different times (6, 12, 30 or 48 h). For TSA treatment, cells were plated at 3 x 10⁴ cells/cm², incubated for 48 h and subsequently treated for 6 h with 10, 50, 100 or 500 nM with TSA in the presence or absence of TGF-β1 (2.5 ng/ml). For experiments aimed at inhibiting the TGF-ß1-RI receptor kinase activity, LLC-PK1 cells were plated and treated with 10 μM SB-431542 for 20 h for qPCR and 48 h for immunoblot experiments.

Transient plasmid transfection and siRNA silencing of SMAD2/3

LLC-PK1 or HEK293 cells were plated at 3 x 10⁴ cells/cm² for 48 h and subsequently transfected with FLAG-tagged SMAD2 and FLAG-tagged SMAD3 expression vectors and -1500PromBasic or p800Luc-reporter plasmids, using Lipofectamine Reagent for 6 h, according to the manufacturer’s instructions. Finally, cells were maintained for 24–48 h in complete media before cell lysis or crosslinking. The empty pCMV5 vector was used as control. SMAD2/3 silencing in LLC-PK1 cells was performed using DsiRNA synthesized by Integrated DNA Technologies (IDT). The nucleotide target sequences for pig were 5´-GGUGAAGAAGCUAAAGAAAACAGGA-3´ and 5´-UCCUGUUUUCUUUAGCUUCUUCACCAA-3´ (siSMAD2 #1) 5´-ACAGUCAUCAUGAACUCAAAGCAAT-3´ and 5´-AUUGCUUUGAGUUCAUGAUGACUGUGA-3´ (SiSMAD2 #2), 5´-GAUAUAUCAGUGAAGAUGGAGAAAC-3´and 5´-GUUUCUCCAUCUUCACUGAUAUAUCCA-3´ (siSMAD2 #3), 5´-GGAUGCAACCUGAAGAUCUUCAACA-3´ and 5´- UGUUGAAGAUCUUCAGGUUGCAUCCUG-3´ (siSMAD3 #1), 5´- ACUGUGGUCAAGGAAGAAGACAUTT-3´ and 5´- AAAUGUCUUCUUCCUUGACCACAGUGA-3´ (siSMAD3 #2), 5´- GGUCAAGGAAGAAGACAUUUUUCTC-3´ and 5´-GAGAAAAAUGUCUUCUUCCUUGACCAC-3´ (siSMAD3 #3).

LLCPK1 cells were transfected with a mix of SMAD2 siRNAs (100 nM) and a mix of SMAD3 siRNAs (100 nM) or control siRNA and Lipofectamine 2000 for 6h. The cells were used 48h post transfection.

RNA isolation, reverse transcription and qPCR

Total RNA was extracted using an RNA-Solv isolation system following the manufacturer's instructions. First, genomic DNA was degraded in DNAse I for 15 min at 25°C. The RNAs were then quantified using NanoDrop equipment. The first strand of cDNA was synthesized by reverse transcription using RevertAid H Minus M Mulv 200 U/μl, 1 μg of RNA in 20 μl of reaction buffer and random primers, and incubation at 42°C for 60 min. PCR was performed to verify reverse transcription using Taq polymerase and a 0.4 μl aliquot of cDNA with specific primer pairs. The sequences of the primers used for qPCR were as follows: pig megalin 5’-CTGCTCTTGTAGACCTGGGTTC-3’ (sense) and 5’- TCGGCACAGCTACACTCATAAC-3’ (antisense); pig PAI-1 5’- CCGTCAGCAAGAGGCAGCAAGAG-3’ (sense) and 5’-TGGTGATGGGAGGAGGCAGAGC-3’ (antisense); pig actin 5’- CCAGATCATGTTCGAGACCTTC-3’ (sense) and 5’- TCTTCATGAGGTAGTCGGTCAG-3’ (antisense); dog megalin 5’-CCTGCCCAAGCTACCGAGCT-3’ (sense) and 5’-CGGGCCCAAAACCAGATACTC-3 (antisense); dog GADPH 5’- GCATCGGAGGGACCCCTCAAA-3’ (sense) and 5’-CACCCGGTTGCTGTAGCCAAA-3’ (antisense). The real-time PCR reactions were conducted using SYBR Green master mix according to the manufacturer's instructions. A 7500 real-time PCR system (Applied Biosystems) was used to measure the relative RNA levels and the data were analysed using 7500 system SDS software.

Immunoblot analysis

Cells were lysed in lysis buffer (phosphate-buffered saline (PBS) containing 1% Triton X-100, 2 mM PMSF, 1 mM pepstatin, 2 μM antipain, 1 μM leupeptin, and 0.3 μM aprotinin) for 30 min at 4°C. The cells were then removed using a cell scraper, and the nuclei eliminated by centrifugation at 20,000x g for 5 min at 4°C. Protein concentrations were measured using a BCA protein assay kit. Equal amounts of protein from each extract were subjected to reducing conditions, and megalin, E-cadherin or ß-tubulin were immunodetected as previously described [35, 62]. The immunoblots were visualized using the Pierce enhanced chemiluminescence (ECL) system, and densitometric analysis was performed using the Gel Doc 2000 Gel Documentation System and Quantity One version 4 software (BioRad, CA, USA). The β-tubulin signal was used to normalize protein levels.

Immunofluorescence

LLC-PK1 cells and dGBECs were washed twice with Ca²⁺/Mg²⁺ PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO_4, 1 mM MgCl₂ and 0.1 mM CaCl₂), fixed with 4% paraformaldehyde in PBS for 30 min at 25°C and then immediately permeabilized with 0.2% Triton X-100 in PBS for 10 min at 25°C. Nonspecific binding sites were blocked with 0.2% gelatin in PBS for 5 min, and the slides were then incubated with primary antibodies (anti-megT, 1:500; anti-E-cadherin, 1:500; or anti-pSMAD2/pSMAD3, 1:100) overnight at 4°C. The coverslips were then washed with blocking buffer and incubated with secondary antibodies (Alexa Fluor 488 Rabbit, Alexa Fluor 555 Mouse and Alexa Fluor 488 Mouse; all 1:500) for 2 h at 25°C. The coverslips were then washed with PBS and mounted on a slide with a drop of Fluoromount-G. Finally, the slides were examined using a Leica DM 2000 fluorescence microscope.

Determination of megalin levels by flow cytometry

For flow cytometry analyses of total megalin, LLC-PK1 cells were plated on 6 well plates and transfected with the plasmids pCMV5 or FLAG-tagged SMAD2 and FLAG-tagged SMAD3 using Lipofectamine. The SMAD2/3-expressing cells were washed with PBS and incubated with PBS containing 1 mM EDTA for 5 min, mechanically detached, and collected by centrifugation at 700xg for 5 min. The cells were resuspended in PBS, 1% heat-inactivated FBS, saponin 0.05% and gently mixed at 4°C for 30 min to permeabilize them. Permeabilized cells were then incubated with anti-megalin and anti-FLAG antibodies at 4°C for 1h in the presence of 0.05% saponin followed by incubation with Alexa 488 or Alexa 647 secondary antibodies (Molecular Probes) at 4°C. Cells were analyzed in a FACSCalibur flow cytometer (Beckton & Dickinson). Total megalin fluorescence was determined in SMAD2/3 positive cells after subtracting the corresponding blank controls.

Quantification of megalin levels at the cell surface and degradation assay

The levels of megalin at the cell surface were measured by cell surface biotinylation. LLC-PK1 cells were grown in 6-well plates. After treatment with TGF-β1 the cells were washed three times with ice-cold PBS and then biotinylated with sulfo-NHS-LC-biotin (Pierce) to a final concentration of 0.5 mg/mL for 1 h. After biotinylation, the cells were washed three times with ice-cold PBS, and free biotin was quenched by incubation with 50 mM NH4Cl in PBS for 10 min. Cells were lysed in lysis buffer (150 mM NaCl, 20 mM Tris, pH 8.0, 5 mM EDTA, 1% Triton-X-100, 0.2% BSA and protease inhibitors) at 4°C. Biotinylated cell-surface proteins were then adsorbed to streptavidin agarose beads for 16 h at 4°C. Beads were washed and then the bound proteins were analyzed by SDS-PAGE followed by immunoblotting. Megalin half-life was measured in LLC-PK1 cells, cultured on 6-well plates at 5x10⁶cells/cm². Cells were treated for the indicated times with cycloheximide (100 mg/ml) with or without 2.5 ng/ml of TGF-β1. After cell lysis, samples were processed for western blot analysis. Quantifications were done in Image J, by measuring the Megalin/Tubulin ratio and normalizing to time 0.

Quantification of carboxy-terminal fragment levels

LLC-PK1 cells were plated on 6-well plates in serum-free medium for 10 h. Then, the medium was replaced by serum-free medium containing 10 μM of gamma-secretase inhibitor DAPT (or vehicle) for 14 h. The cells were then treated with 2.5 or 20 ng/ml TGF-β1 for 10 h in the presence of DAPT. Cells were lysed in RIPA (50 mM Tris pH 8.0, 150 mM NaCl, 150 mM EDTA pH 8.0, 1% NP40, 0.5% Deoxycholate, 0.1% SDS) and both carboxy-terminal fragments (CTF) and full-length megalin were detected after SDS-PAGE by western blot analyses.

Promoter and reporter assays

LLC-PK1 cells were co-transfected using Lipofectamine 2000 with -1500PromBasic (300 ng), mutants or the p800Luc (300 ng) luciferase reporter plus FLAG-tagged SMAD2 and SMAD3 vectors or with an empty pCMV5 vector as a control. The p800Luc luciferase reporter plasmid containing the PAI-1 promoter was used as a positive control. A fixed amount (100 ng) of internal control (pCMV-ß) was also co-transfected to allow for normalization of transfection efficiency. Luciferase activity was assayed in a luminometer using a Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Luciferase activity was normalized to determine the transfection efficiency using ß-galactosidase expression. The results are expressed as fold increase of activity compared to the control vector.

Site-directed mutagenesis

To generate single mutations in the SBEs identified in the megalin promoter (-1500PromBasic), the following primers were used: Site 1 (MutSBE1): 5´ TCTTTCCTTCATTTTTACTctTaaGGTTGTGCTTTCCTTTTC 3´ (sense) and 5´ GAAAAGGAAAGCACAACCttAagAGTAAAAATGAAGGAAAGA 3´ (antisense); Site 2 (MutSBE2): 5´ TCTAAAGGGCTTTATGCACctTaaGGAGGGTGGGGACTGGCG 3´ (sense) and 5´ CGCCAGTCCCCACCCTCCttAagGTGCATAAAGCCCTTTAGA 3´ (antisense). Mutants were generated by PCR using Pfu DNA Polymerase according to the manufacturer’s protocol. Mutations were confirmed by DNA sequencing.

Chromatin immunoprecipitation assays

Transfected HEK293 cells (10⁶ per chromatin immunoprecipitation (ChIP) assay) were cross-linked in suspension for 8 min at 25°C in PBS containing 1% formaldehyde before quenching with 1.25 mM glycine. Cells were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF (a serine protease inhibitor), a broad-range protease inhibitor cocktail (see Reagents section) and 20 mM butyrate) for 30 min at 4°C on a rotator, and sonicated 5 times for 30 sec on ice at 30% power (cycle 0.5) to generate chromatin fragments of ~200–500 bp. After sedimentation, chromatin was diluted 10-fold in RIPA buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% Na-deoxycholate, and 140 mM NaCl) containing a protease inhibitor cocktail, 1 mM PMSF and 20 mM butyrate and then incubated on a rotator overnight at 4°C with antibodies pre-coupled to beads in A/G protein agarose (50 μl A/G protein plus 10 μg FLAG antibody incubated at 4°C for 3 h). An unrelated HA antibody was used as control. ChIP material was collected and washed 3 times in 1 ml ice-cold RIPA buffer. Crosslinking was reversed, and DNA was eluted for 6 h on a shaker at 37°C in elution buffer (50 mM NaCl, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 20 mM butyrate) containing 1% SDS and 50 μg/ml proteinase K. The DNA was isolated by phenol-chloroform extraction and ethanol precipitation. The purified DNA was amplified by qPCR to detect the SBE promoter sequences.

The primers used were as follows: for the megalin SBE promoter, (-605 FW) 5’-CGCCTCTTTCCCTTTCTTTC-3’ and (-605 RV) 5’-ACGAGGAGGAAAGTCAAGAAT-3’ and (-57 FW) 5’-GATTCCCGCATGCTTGTT-3’ and (-57 RV) 5’-GAGGCATCCCGTTTTCTACC-3’; and for the PAI-1 SBEs promoter, (-580 FW) 5’-GCAGGGATGAGGGAAAGACC-3’ and (-580 RV) 5’-AACAGCCACAGGCATGCAG-3’ and (-280 FW) 5’-AGCTGAACACTAGGGGTCCT-3’ and (-280 RV) 5’-GCCTGAGAACCTCCCTTGAC-3’. These latter PCR primers were designed according to the reported sequence data for the PAI-1 promoter [65].

Nuclear extracts

Cells were washed twice with PBS and centrifuged for 10 min at 300x g. The pellet was resuspended in 1 ml of hypotonic lysis buffer and incubated for 30 min at 4°C. Nuclei were centrifuged at 10,000xg for 10 min at 4°C, and histones were extracted with 400 μl of 0.2 M sulphuric acid (H₂SO₄) and incubated in a rotator for 30 min at 4°C. Eluted immunoprecipitated samples were precipitated for 30 min on ice in 25% (v/v) trichloroacetic acid (TCA) followed by centrifugation for 10 min at 16,000xg and two washes with ice-cold acetone. Samples were air dried and dissolved in sample buffer, and the protein concentration was then measured by BCA assay. The nuclear proteins were frozen in liquid nitrogen and stored in aliquots at -80°C.

In silico analysis

To locate the transcription factor binding sites in the human megalin promoter, tools from MatInspector software (Genomatix) were used as described above [66, 67].

Statistical analysis

Immunoblots were quantified using ImageJ 1.45s software, and qPCR analysis was performed using a relative quantification mathematical model, as previously described [68]. Data are expressed as the means ± SD from at least three independent experiments. Comparisons were performed using analysis of variance (ANOVA) followed by Bonferroni correction. All of the statistical analyses were performed using GraphPad Prism 5.

Decrease megalin mRNA and protein levels induced by TGF-β1 in epithelial cell lines is dependent on the activation of TGF- ß type I receptor kinase

Although it has been established that TGF-ß1 reduces megalin protein levels [47, 52], it is not known whether TGF-ß1 directly regulates megalin transcription. Therefore, megalin mRNA levels were measured by qPCR in two cell lines that express megalin endogenously: LLC-PK1 cells, a cell line derived from pig kidney proximal tubules and dGBECs derived from dog gallbladder epithelial cells. The cells were treated with TGF-ß1 for different times and at different concentrations (Fig 1). TGF-ß1 concentrations of 2.5 ng/ml and 10 ng/ml significantly decreased megalin mRNA levels by 40% in LLC-PK1 cells, while treatment with 10 ng/ml TGF-ß1 decreased megalin mRNA levels by 60% in dGBECs (Fig 1A and 1C). These results indicate that both cell types exhibit a reduction in megalin mRNA expression following TGF-ß1 stimulation. At lower TGF-ß1 concentrations, only LLC-PK1 cells showed decreased megalin mRNA levels. However, megalin mRNA levels show the largest decrease (as percentage) in dGBECs when exposed to higher TGF-ß1 concentrations. Next, we studied the effects of TGF-ß1 exposure over time in LLC-PK1 cells and dGBECs by incubating them with 2.5 ng/ml and 10 ng/ml TGF-ß1 respectively. In both cell types, megalin mRNA levels significantly decreased after 6h of TGF-β1 treatment (Fig 1B and 1D). mRNA expression of plasminogen-activator inhibitor (PAI-1), a known TGF-ß1 target gene, was measured as positive control (Fig 1E and 1F) to show that the cells had an active, operative TGF-ß1 signalling pathway.

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Fig 1. Effect of TGF-ß1 treatment on megalin mRNA levels.

(A) LLC-PK1 cells and (C) dGBECs were treated with the indicated TGF-ß1 concentrations for 12 h, and megalin mRNA levels were analysed by real-time PCR. Megalin mRNA levels analysed by real-time PCR after treating LLC-PK1 cells (B) and (D) dGBECs with 2.5 or 10 ng/ml TGF-ß1 respectively for different times (6, 12, 30 and 48 h). As control, we measured PAI-1 mRNA levels in LLC-PK1 cells (E) that had been treated with TGF-ß1 at the indicated concentrations for 12 h or (F) treated with 2.5 ng/ml TGF-ß1 for different times (as shown in B and D). Values were normalized using either actin or GADPH as a housekeeping gene (n = 3 independent experiments). The results are expressed as the means ± standard deviation (SD). Significant differences compared with the control (vehicle) are indicated by *P≤0.05, **P≤0.01, ***P≤0.001.

https://doi.org/10.1371/journal.pone.0213127.g001

Given the effect of TGF-ß1 on megalin mRNA levels, we evaluated changes in megalin protein levels by treating LLC-PK1 cells and dGBECs with 2.5 ng/ml or 10 ng/ml TGF-ß1, respectively, for 48 h. Immunoblot and immunofluorescence analyses of megalin expression in LLC-PK1 cells (Fig 2A and 2B) and dGBECs (Fig 2C and 2D) showed that megalin expression was decreased. These results are also concordant with previous studies performed in OK cells [52]. Interestingly, the distribution of megalin in LLC-PK1 and dGBECs differs in confluent cells. In dGBECs (Fig 2D) it is clearly perinuclear and vesicular, whereas in LLC-PK1 (Fig 2B) besides some vesicular localization, megalin is also found in clusters close to the cell periphery. As control, we measured the expression of the epithelial marker E-cadherin (Fig 2A and 2C), a known TGF-ß1 target downregulated by this cytokine [69]. Taken together, these results indicate that TGF-ß1 decreases megalin mRNA levels, causing a concomitant reduction in megalin protein levels.

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Fig 2. Effect of TGF-ß1 treatment on megalin protein levels.

LLC-PK1 cells and dGBECs were treated with 2.5 or 10 ng/ml TGF-ß1 1, respectively, for 48 h, and total cell lysates (40 μg) from LLC-PK1 cells (A) and dGBECs (C) were subjected to SDS-PAGE and immunoblotted with antisera against megalin (top panel) or with E-cadherin antibodies (middle panel). The graphs below the immunoblots show the densitometric analyses of megalin and E-cadherin levels normalized with the housekeeping gene ß-tubulin (bottom panel). The results are expressed as the mean ± standard deviation (SD). Significant differences are indicated by *P≤0.05. Scale bars, 20 μm. (n = 3 independent experiments). (B and D) Immunofluorescence for megalin and E-cadherin in confluent LLC-PK1 cells and dGBEC treated with TGF-ß1 for 48 h in the presence or absence of the inhibitor SB-431542 (10 μM). Cells were grown on coverslips, fixed in 4% paraformaldehyde and double-stained using antibodies specific for megalin (B and D; green) and E-cadherin (B and D; red). Overlapping images with Hoechst-stained nuclei (blue) are shown.

https://doi.org/10.1371/journal.pone.0213127.g002

The canonical TGF-ß1 pathway includes the SMAD proteins, which are transcriptional modulators [19]. Therefore, we tested whether the SMAD2/3 signalling pathway is directly involved in the down-regulation of megalin expression. To inhibit SMAD2/3 phosphorylation (and therefore activation of the pathway), we used the specific inhibitor SB-431542 (SB), which blocks the activity of TGF-ßRI (ALK5) serine/threonine kinase. SB does not interfere with the enzymatic function of other receptors of the same family such as ALK 2,3 and 6 and does not affect the activation of other kinases (ERK, JNK or p38) associated with TGF-ß pathways [69, 70]. As a result of SB treatment, both megalin and E-cadherin levels recovered from the effects of TGF-β1, as shown in the lower immunofluorescence panels (Fig 2B and 2D). When LLC-PK1 cells were treated with TGF-β1 (2.5 ng/ml) and 10 μM SB for 20 h, the effect of TGF-ß1 on megalin mRNA levels was completely reversed, and even increased compared to the control condition (Fig 3A, fourth bar). Similarly, western blot analyses (Fig 3C and 3D) show that SB counteracted the effect of TGF-ß1 restoring megalin protein levels. Furthermore, the addition of 10 μM SB alone resulted in a nearly 2-fold increase in basal megalin mRNA and protein levels compared with the control (Fig 3A, 3C and 3D). PAI-1 mRNA levels were measured as a response-control (Fig 3B). Interestingly, treatment with SB alone induced a significant decrease in PAI-1 mRNA levels by 30% (Fig 3B, third bar), suggesting that under steady-state conditions, LLC-PK1 epithelial cells have a basal activity of the TGF-ßRI/SMAD2/3 signal transduction pathway. In addition, to determine whether SMAD2/3 signalling responds to treatments, we used immunofluorescence to assess nuclear localization of pSMAD2/3. TGF-ß1 induced nuclear translocation of pSMAD2/3, and this effect was inhibited by SB (Fig 3E). As is shown in S1 Fig, under basal conditions the cells show a diffuse SMAD2/3 staining pattern that includes cytoplasm and nucleus. TGF-ß1 treatment for 6 h clearly defines a nuclear SMAD2/3 localization, whereas SB treatment completely abrogates the nuclear staining observed under basal control conditions, again supporting the idea that there is basal activity of the TGF-ßRI/SMAD2/3 pathway in LLC-PK1 cells. Therefore, these results support the hypothesis that in epithelial cells, TGF-ß1 transcriptionally represses megalin expression by activating the TGF-ßRI/SMAD2/3 signal transduction pathway.

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Fig 3. Effect of the TGF-βRI kinase activity inhibitor SB-431542 on megalin levels.

LLC-PK1 cells were exposed to TGF-ß1 (2.5 ng/ml) in the presence or absence of SB-431542 (10 μM) for 20 h and megalin (A) and PAI-1 (B) mRNA levels were measured by real-time PCR. (C) LLC-PK1 cell extracts from cells treated with TGF-ß1 (2.5 ng/ml) in the presence or absence of SB-431542 (10 μM) for 48 h were used to determine megalin protein levels by immunoblotting; β-tubulin was used as the loading control. The bands in the blots were quantified by densitometry, and the results were plotted as the ratio of megalin/β-tubulin for each condition (D) Significant differences compared with the control (vehicle) are indicated by *P≤0.01, **P≤0.001. Significant differences with TGF-β1 alone are indicated by #P≤0.001 (n = 4 experiments). (E) Immunofluorescence for the pSMAD2/3 transcription factors in LLC-PK1 cells. The cells were treated under each condition for 6 h (using the same concentrations described above) and stained using anti-pSMAD2/3 antibodies (green). Overlapping images with Hoechst-stained nuclei (blue) are shown. Scale bars, 20 μm.

https://doi.org/10.1371/journal.pone.0213127.g003

As megalin protein levels can also be reduced by degradation, we determined if megalin half-life [71] was modified by TGF-ß1 treatment. LLC-PK1 cells were treated with TGF-ß1 2.5 ng/ml for up to 12 h in the presence of the translational inhibitor cycloheximide, in order to interrupt protein synthesis and therefore follow the disappearance of megalin attributed to its degradation. We found that megalin protein levels decreased with very similar kinetics in the control and under TGF-ß1 treatment conditions, (Fig 4A and 4B). Previous studies showed that treating lung epithelial cells with a high concentration (20 ng/ml) of TGF-ß1 induces shedding of the megalin ectodomain and a reduction of megalin at the cell surface. These effects can be first detected 4h after the start of treatment and they reach a peak at 10 h [72]. Therefore, we tested if our cells respond similarly, using two different concentrations of TGF-ß1 (2.5 and 20 ng/ml) (Fig 4C). Neither cell surface megalin levels nor total levels of megalin were modified after 10 h of TGF-ß1 treatment. This result suggests that TGF-ß1 (low or high concentrations) does not induce degradation or cell surface reduction of the receptor in this cell type during this period of time (Fig 4D and 4E). Similarly, cell surface and total levels of E-cadherin were not decreased (Fig 4F and 4G).

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Fig 4. Megalin half-life and cell surface levels are not modified by TGF-ß1.

(A) LLC-PK1 cells were treated with cycloheximide and either vehicle or 2.5 ng/ml TGF-ß1 for up to 12 h. At different time points, cells were lysed and the presence of megalin and β-tubulin determined by western blot. (B) Quantification of the megalin/ß-tubulin ratio (n = 3 experiments). (C) LLC-PK1 cells were treated with 2.5 or 20 ng/ml TGF-ß1 for 10 h and then subjected to cell surface biotinylation at 4°C. Cell lysates were precipitated with streptavidin-Sepharose, resolved by SDS-PAGE and probed with anti-megalin or anti-E-cadherin antibodies. The graphs show the densitometric analyses of megalin and E-cadherin levels (surface/total) (D, F) as well as total protein levels (E, G). The results are expressed as the mean ± SEM (n = 4).

https://doi.org/10.1371/journal.pone.0213127.g004

In order to complement this result, we also determined the levels of the megalin carboxy-terminal fragment (CTF), which is generated after ectodomain shedding. We found that CTF levels were not increased after a 10 h treatment with TGF-ß1 2.5 or 20 ng/ml. However, treatment with the γamma-secretase inhibitor DAPT stabilized CTF levels (Fig 5A and 5B). The product of CTF processing is the intracellular cytoplasmic domain of megalin (MICD) which, in the case of this receptor, has been suggested to negatively control its own transcription [72]. We were able to detect the CTF but the MICD is extremely unstable and its detection erratic even in the presence of proteasome inhibitors. However, since TGF-ß1 did not increase CTF levels, we discarded the hypothesis of an increased production of the MICD. According to the results shown in Fig 4, total megalin levels were not modified after 10 h of treatment with TGF-ß1 at the two concentrations tested (Fig 5C and 5D). Overall, these results do not support a role of TGF-ß1 in the degradation or shedding of megalin, at least in the first 10–12 h of treatment.

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Fig 5. Megalin CTF levels are increased by inhibiting gamma-secretase activity but are not modified by TGF-ß1 treatment.

LLC-PK1 cells were treated with DAPT 10 μM or vehicle for 24 h. During the last 10 h, cells we treated with 2.5 or 20 ng/ml TGF-ß1. (A, C) Cell lysates were processed by SDS-PAGE and probed with anti-megalin or anti-tubulin antibodies. (B, D) The graphs show the densitometric analysis of Megalin CTF/tubulin ratio and of full length Megalin/tubulin ratio with and without DAPT. The results are expressed as the mean ± SEM (n = 4).

https://doi.org/10.1371/journal.pone.0213127.g005

SMAD2/3 proteins interact with two SBEs (SMAD-binding elements) identified in the megalin promoter

The SMAD transcription factors act by forming complexes and they simultaneously activate and repress target genes in the same cell, depending on the context of the SBEs and the presence of proteins that induce the formation of the protein complexes that bind to these sites [14, 18]. We analysed the human megalin promoter with the MatInspector software and identified two consensus SBEs located at positions -605 bp and -57 of the transcription start site (Fig 6A, bold and underlined bases). In order to analyse SMAD2/3 protein-DNA interactions at the SBEs of the human megalin promoter, we performed formaldehyde crosslinking and immunoprecipitation experiments. The human megalin promoter or a parental vector was transfected into HEK293 cells in the presence of FLAG-tagged SMAD2/3, and the cells were then treated with TGF-ß1 to activate the signalling pathway and induce SMAD2/3 translocation into the nucleus. The exogenous SMAD factors were then immunoprecipitated, and the regions containing each SBE site were amplified from the complex by qPCR using two primer pairs designed for this purpose (Fig 6B). The results showed that SMAD2/3 interact with the SBEs localized at positions -605 (SBE1) and -57 (SBE2) in the human megalin promoter (Fig 6B). As a negative control for the immunoprecipitation experiments we used an unrelated antibody (anti-HA). The relative occupancy for the factors binding to the two SBE was determined by qPCR signals derived from ChIP and input samples and expressed graphically in Fig 6C (black bars). The -280 and -680 SBEs from the PAI-1 promoter were analysed as positive controls for ChIP (Fig 6B and 6C, white bars). These results support the idea that SMAD2/3 could directly inhibit megalin expression at the transcriptional level by binding to the SBEs in the megalin promoter; however, we cannot discard the presence of other proteins forming a complex with SMADs transcription factors.

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Fig 6. Association of SMAD2/3 with the megalin promoter.

(A) Schematic representation of the human megalin promoter (-1500 bp), highlighting the putative SBEs used to analyse megalin promoter activity. (B) HEK293 cells were seeded onto 10 cm culture dishes and transfected with the megalin promoter construct (-1500PromBasic) or the PAI-1 promoter construct (p800Luc) 48 h later. Co-transfections were performed in the presence or absence of exogenous FLAG-SMAD2 and FLAG-SMAD3 plasmids. After 24 h, the transfected cells were treated with 5 ng/ml TGF-ß1 for 1 hour and subjected to ChIP as described in the experimental section. The immunoprecipitated DNA was queried using negative control primers, a non-specific primer set and specific primers to the SBE-57 or SBE-605 sites of the megalin promoter and the SBE-280 or SBE-680 sites of the PAI-1 promoter (positive control). PCR was performed using DNA that was immunoprecipitated using an HA antibody as a negative control (HA lane). The PCR from the input DNA served as another positive control (input lane). The products were analysed by agarose gel electrophoresis. (C) The qPCR signals derived from the ChIP samples were divided by the qPCR signals derived from the input sample collected earlier during the ChIP procedure. The relative fold change was calculated from the mean ± SD 2(^)ΔΔCT values (n = 2), as described in the experimental section, and normalized with the input.

https://doi.org/10.1371/journal.pone.0213127.g006

TGF-ß1 represses megalin promoter activity via SMAD2/3

To directly test the repressive effect of SMAD2/3 on the human megalin promoter, LLC-PK1 cells were co-transfected with the human megalin promoter coupled to luciferase (-1500PromBasic) in the presence or absence of overexpressed SMAD2/3. The results showed that the expression of SMAD2/3 triggered a significant decrease (50%) in megalin promoter activity compared with the control without SMAD2/3 transfection (Fig 7A). The positive control was the p800Luc plasmid, containing the PAI-1 promoter fragment (-800 to +71) with two consensus SBEs located at positions -280 and -680 bp of the transcription start site [64, 65]. In this case, SMAD2/3 overexpression induced a 4-fold increase in the activation of the PAI-1 promoter (Fig 7B). Interestingly, in a complementary experiment we found that megalin protein levels were significantly decreased by the overexpression of SMAD2/3. Immunoblots showed that megalin levels decreased almost 40% in cells overexpressing SMADs (Fig 7E and 7F), and we obtained similar results when megalin levels were detected by FACS. The cells expressing SMAD2/3 with the FLAG epitope expressed significantly lower levels of megalin than the controls (Fig 7G). On the other hand, the direct effect of TGF-ß1 was also tested with the same promoters coupled to luciferase and, as expected, the cytokine decreased luciferase expression (about 20%) when the luciferase gene was under the control of the megalin promoter (Fig 7C). Also, as expected, TGF-ß1 increased luciferase activity in cells where the luciferase gene was under the control of p800Luc (Fig 7D) by 2-fold. Therefore, for both promoters the effects of TGF-ß1 were less evident that the effects of the overexpression of SMAD2/3.

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Fig 7. 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.

https://doi.org/10.1371/journal.pone.0213127.g007

To directly address the role of SMAD2/3 in the repressive effect of TGF-ß1 on megalin, we silenced the expression of SMAD2/3 in LLC-PK1 cells and then we transfected them with the different promoter constructs coupled to luciferase. The silencing was very efficient (84% decrease of SMAD2 and 72% of SMAD3), as seen in the immunoblot for SMAD 2 and SMAD3 (Fig 8A and 8B). Under conditions in which the expression of SMAD2/3 was significantly downregulated, the repressive role of TGF-ß1 on the activation of the megalin promoter was not evident, suggesting that at least in part, TGF-ß1 affects megalin expression through the canonical SMAD2/3 pathway. However, due to the error in the analysis it cannot be completely excluded that some transcriptional repressive effect of the cytokine may still remain (Fig 8C). As control, the TGF-ß1 induced activation of p800Luc was blocked in the SMAD2/3 knock-down cells (Fig 8D). At the protein level, the downregulation of megalin induced by the cytokine was clearly dependent on the expression of SMAD2/3 (Fig 8E and 8F). The reduction of SMAD2/3 expression was associated with a significant decrease in the activation of the p800Luc promoter under non-stimulation conditions (Fig 8D), indicating a basal activation of the system in this cell type, similarly to what was found when TGF-ßRI was inhibited by SB (Fig 3B).

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Fig 8. The effects of TGF-ß1 on megalin expression depend on the expression of SMAD2/3.

(A) LLC-PK1 cells were transfected with control siRNA o SMAD2/SMAD3-specific siRNAs. After 48 h of expression, SMAD2 and SMAD3 were analyzed by immunoblot and tubulin was measured as a loading control (B) Quantification of SMAD2/tubulin or SMAD3/tubulin in cells transfected with siRNA. (C, D) SMAD2/SMAD3 were silenced with SMAD2/SMAD3- siRNAs in LLC-PK1 cells. The cells were then transfected with the megalin promoter construct 1500PromBasic (C) or the p800luc plasmid (D). After 24h the cells were incubated with vehicle or 2.5 ng/ml of TGF-ß1for another 24h. Luciferase activity was determined using a Luciferase Reporter Assay System, and the ß-galactosidase signal was used to normalize the results. Data are expressed as the means ± SD, and significant differences with the control are indicated by *P≤0.05, while significant differences between non silenced and silenced cells are indicated by ### P≤0,001. (E, F) LLC-PK1 cells were transfected with control siRNA o SMAD2/SMAD3-siRNAs. After 24 h of expression, cells were cultured in low serum medium (0.5% FBS) for 16 h and then treated in the presence or absence of 2.5 ng/ml of TGF-ß1 or 24h. After 60h of silencing and 24h of treatment with TGF-ß1, the cells were lysed and the analyzed by immunoblot. In (E) the expression of megalin, SMAD2 and SMAD3 were determined and the detection of tubulin was used as a loading control. (F) Shows the quantification of megalin/tubulin, as % of the control, in cells transfected with siRNA and treated with TGF-ß1. Data are expressed as the means ± SE. Significant differences with the control are indicated by *P≤0.05.

https://doi.org/10.1371/journal.pone.0213127.g008

Taking into account the presence of two SBEs in the megalin promoter, we established the contribution of each one in the repression of the promoter. SBE1 and SBE2 were mutated by base substitution (Fig 9A, asterisks). The plasmids were then transfected into LLC-PK1 cells in the presence or absence of overexpressed SMAD2/3, and their activities were measured. The single-site SBE mutants (Mut-SBE1 or Mut-SBE2) were able to respond to the presence of SMAD2/3 decreasing the expression of luciferase (Fig 9B, black bars compared with white bars). However, the double-mutant SBEs (MutSBE1/2) did not respond to the repressive effect of SMAD2/3 (Fig 9B, black bars compared with white bars). These data indicate that the two SMAD response elements independently repress the megalin promoter. In addition, in the SBE mutant promoters, the basal activity was significantly increased compared with the wild type promoter in the absence of overexpressed SMAD factors (Fig 9B, all white bars), suggesting that endogenous SMAD factors were basally active in LLC-PK1 cells (as suggested in Fig 3A, 3C and 3D and S1 Fig). Finally, the relevance of these two SBEs in the repressor effect of TGF-ß1 was determined by the same type of assays using the wild-type and mutant promoters (Fig 9C). In the SBE1 mutant and in the double SBE1/2 mutant, the repressor role of TGF-ß1 was not significant, although given the error in the analysis, the hypothesis cannot be excluded that a component of TGF beta inhibition remains. The mutant SBE2 responded to the cytokine but to a lesser extent than the wild-type promoter. Overall, these results strongly suggest that both SBEs identified in the human megalin promoter could function as binding sites for a transcriptional repressor complex that is linked by SMAD2/3 and have a role in the repressive effect of TGF-ß1.

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Fig 9. SMAD2/3 suppress megalin promoter activation at SBEs.

(A) Schematic representation of SBEs and site-directed mutations (asterisks) generated in the human megalin promoter constructs. (B) LLC-PK1 cells were transfected with wild type or mutant SBE constructs of the human megalin promoter (1500PromBasic) and with the ß-galactosidase vector (pCMVß). Co-transfections were performed in the presence (black bars) or absence (white bars) of SMAD2 and SMAD3 cDNAs. After 24 h, the transfected cells were lysed, and luciferase activity was measured. Luciferase activity was normalized with ß-galactosidase expression to determine transfection efficiency. The results are expressed as the means ± SD. Significant differences with the respective controls (untransfected SMAD factors) are indicated by *P≤ 0.001. Significant differences with the wild type construct are indicated by #P≤0.001. (C) LLC-PK1 cells expressing wild type or mutant SBE constructs of the human megalin promoter (1500PromBasic) and ß-galactosidase vector (pCMVß) were incubated in the presence or absence of 2.5 ng/ml of TGF-ß1 for 24h. Later, the cells were lysed, and luciferase activity was measured. The results are expressed as the means ± SD. Significant differences with the respective controls are indicated by ∞P≤ 0.05. Significant differences with the wild type construct are indicated by &&P≤0.01, &&&P≤0.001.

https://doi.org/10.1371/journal.pone.0213127.g009

Albumin reduces megalin expression by a mechanism that involves the TGF-βRI/SMAD2/3 pathway

Although high concentrations of albumin are known to decrease megalin expression [57], it is not known if this effect is dependent on the activation of the TGF-ß signalling pathway. Albumin induces TGF-ß1 secretion [73] in human proximal tubule HK-2 cells and in cells from the collecting duct that lack megalin [74]. Therefore, the pro-inflammatory effects of albumin could be megalin independent. To determine whether the albumin-induced megalin reduction is connected to the canonical TGF-ß1 pathway, LLC-PK1 cells were incubated with increasing concentrations of albumin for 24 h and the mRNA expression of megalin and PAI-I was measured. As previously reported, albumin significantly reduced megalin expression at a concentration of 10 mg/ml [57] (Fig 10A). A lower concentration was able to induce PAI-1 expression (Fig 10B). Moreover, the albumin-induced decrease in megalin expression and the increase in PAI-1 expression were significantly overcome by SB (P ≤ 0.05 in both cases), suggesting that the effects of albumin involves, directly or indirectly, activation of the TGF-ß1 receptor (Fig 10C and 10D). Again, we observed that the inhibition of the TGF-ßRI by SB significantly increased megalin mRNA levels and reduced PAI-1 expression as shown before (Fig 3A and 3B). However, in the case of PAI-1 the treatment with albumin in the presence of SB431542 seems to upregulate PAI-1 expression compared to SB431542 alone. This could indicate that albumin activates other pathways that modulate PAI-1 gene expression. To complement these results, the effects of albumin were determined directly in the activation of megalin human promoter as described for TGF-ß1 (Fig 7 and Fig 8). Similarly to the effect of the cytokine, albumin significantly decreased megalin expression (Fig 10E). Moreover, the repressing effect of albumin on megalin was dependent on the presence of SMAD2/3 (Fig 10F) since it was abolished in SMAD2/3 knockdown cells.

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Fig 10. Albumin decreases megalin expression and this effect is counteracted by TGF-βRI inhibition and SMAD2/3 silencing.

LLC-PK1 cells were treated with albumin concentrations ranging from 0.01 to 20 mg/ml for 24 h. Megalin (A) and PAI-1 mRNA levels (B) were measured by qPCR. (C) Megalin and (D) PAI-1 mRNA levels in LLC-PK1 cells that were exposed to 20 mg/ml of albumin and/or the inhibitor SB for 20 h. The results are expressed as the average RQ ± standard deviation (SD). (E) LLC-PK1 cells were transfected with the megalin promoter construct (1500PromBasic) and a ß-galactosidase vector (pCMVß) and incubated in the presence or absence of 20 mg/ml of albumin for 24h. Later, the cells were lysed, and luciferase activity was measured. (F) LLC-PK1 cells were transfected with the 1500PromBasic plasmid and SMAD2/SMAD3- siRNAs, after 24h the cells were treated with 20 mg/ml of albumin for another 24h. Luciferase activity was determined using a Luciferase Reporter Assay System, and the ß-galactosidase signal was used to normalize the results. Data are expressed as the means ± SD. Significant differences with controls are indicated by: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Significant differences between treatments are indicated by # P≤0.05, ## P ≤ 0.01.

https://doi.org/10.1371/journal.pone.0213127.g010

The repression of megalin mRNA expression by TGF-ß1 is reverted by inhibition of histone deacetylases

Transcriptional repression involves, among other mechanisms, histone deacetylase (HDAC) activity [75]. We wanted to evaluate the effects of the HDAC inhibitor TSA on megalin mRNA expression in LLC-PK1 cells by qPCR. TSA treatment, starting at a 50 nM concentration, elicited a significant increase in megalin mRNA levels compared with the vehicle (control) (Fig 11A and 11B, third bar). These results are concordant with previous studies that analysed the regulation of megalin expression by TSA in NRK and Caco-2 cells [76]. We next examined the effect of TSA on TGF-ß1 -induced reduction of megalin expression that had been previously observed in LLC-PK1 cells (Figs 1A, 1B, 2A and 2B). The cells were treated with TGF-ß1 and/or TSA for 6 h and mRNA levels were then evaluated by qPCR. Whereas TGF-ß1 decreased mRNA as expected, TSA significantly counteracted TGF-ß1 response resulting in megalin mRNA levels similar to the control (Fig 11B). Interestingly, TSA alone also increases PAI-I expression, however, the induction of the PAI-1 gene by TGF-ß1 was blocked by TSA and was not additive, as has been described in other cell types [78, 79] (Fig 11C). In contrast to megalin mRNA levels, even though TSA alone increases megalin protein levels after 48 h of treatment, its effect over the reduction induced by TGF-ß1 was not evident under this experimental condition (Fig 11D). As a control for TSA treatment, the acetylated form of histone H3 was detected by immunoblotting after a 6 h treatment (Fig 11E). These results indicate that TSA counteracts the repressive effect of TGF-ß1 on megalin mRNA expression, via modulation of HDAC activity. However long term TGF-ß1 treatment may reduce megalin protein levels involving other pathways in addition or independently of the HDACs.

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Fig 11. TSA increases megalin expression and counteracts the effect of TGF-β1 on megalin mRNA levels in LLC-PK1 cells.

(A) LLC-PK1 cells were treated with the indicated concentrations of TSA for 6 h, and megalin mRNA levels were analysed by real-time PCR. The cells were then exposed to TGF-ß1 (2.5 ng/ml) in the presence or absence of TSA (50 nM) for 6 h. Megalin (B) and PAI-1 (C) mRNA levels were then measured by real-time PCR. (D) LLC-PK1 cell extracts treated with TGF-ß1 (2.5 ng/ml) for 48 h in the presence or absence of TSA (50 nM) were used to measure megalin protein levels by immunoblotting; ß-tubulin was used as the loading control. (E) Cells were treated with TSA for 6 h. Total histones were extracted, and acetylated H3 and total H3 histones were detected by immunoblot analysis. The results are expressed as the means ± SD. Significant differences with the control (vehicle) are indicated by *P≤0.01, **P≤0.001. Significant differences with TGF-ß1 alone are indicated by #P≤0.001.

https://doi.org/10.1371/journal.pone.0213127.g011