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The transforming growth factor (TGF-β1) is an important factor in renal chronic inflammatory reactions.［1］ In normal physiological metabolism TGF-β1 regulates the production and degradation of excellular matrix (laminin, fibronectin, collagen etc.) mainly by Smads, activator protein-1 (AP-1).［2］ Different stimuli can induce overexpression of TGF-β1 by the same and different pathways. Overexpression of TGF-β1 results in the deposition of excellular matrix. Now most researches about therapy focus on the lower events, but the results were not exciting as expected. If the activity of TGF-β1 was inhibited, normal metabolism will be affected. So we explored its function at the transcriptional level and tried to find some clues for therapy from the upper events.
The glomerular mesangial cells (MsC) are the specific cells with some characters of smooth muscle cells in the glomerulus. The MsC injury was considered to be central to glomerular sclerosis. Hyperglucose and some cytokines, such as tumour necrosis factor-α (TNF-α) and interleukin-1 (IL-1), could induce NF-κB activity in MsC.［3,4］ It has been reported that oxidized low density lipoprotein (Ox-LDL) could injure MsC and induce matrix deposition.［5］ So we investigated the nuclear factor-κB (NF-κB) activity and expression of TGF-β1 in MsC exposed to Ox-LDL.
METHODS
Cell culture
Mesangial cells were obtained from 7-10 week old SD rats and passed 5-8 generations. MsC were appraised by immunocytochemical methods. Cells were cultured in RPMI1640 with 10% fetal bull serum (GIBCO BRL Company, USA) and then divided into three groups: control group; Ox-LDL group; Ox-LDL+inhibitor of protein kinase group ［H-89: inhibitor of protein kinase A (PKA); PD 98059: inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK-1); SB203580: inhibitor of p38 MAP kinase (p38 MAPK); Staurosporine: inhibitor of protein kinase C (PKC); Proteasome Inhibitor Ⅰ; all were obtained from Sant Cruz Company］.
Oxidation of LDL
LDL was obtained from Sigma Company and was oxidized by CuSO4 as described previously.［6,7］ The oxidation was stopped by adding butylated hydroxytoluene (2, 6-di-t-butyl-p-creso-1) to a final concentration of 0.1 mmol/L. Oxidized LDL was separated from CuSO4 by dialysis against phosphate-buffered saline (PBS) and equilibrated into the cell culture medium. The protein content of LDL was determined according to Lowry.
Mutation
This was performed using the directions of the QuickChangTM Kit of Stratagene Company. The rat TGF-β1 promoters were constructed in pGL3-Basic plasmid with a luciferase-reporting gene.［8］ The sequence from -1550 to +57 bp was analyzed. There was a binding site of NF-κB (－715-－707, AGGGACTT). We replaced it with ATTGACTT. Mutation primer sequences: 5'- GGT-GTGGAGTGTTGAGTAACTTCACTGCCACC-3' (sense), 5'-GGTGGCAGTGAAGTTACTCAACACTC-CACACC-3' (antisense). The plasmids with wild and mutated promoters were differently co-transfected with SV40 into MsC stimulated by Ox-LDL. After 48 hours, the cells were harvested and the luciferase activity was determined.
NF-κB Decoy-oligodeoxynucleotide (ODN) technique
The synthesized ODN with binding sequence of NF-κB are transfected into cells and competed with the promoter of target genes. Double-stranded ODN was prepared from complementary single-stranded phosphorothioate-bonded ODN obtained from Shanghai Biochemistry Technology Company, China, by melting it at 100℃ for 5 minutes, followed by a cool-down phase of 4 hours at an ambient temperature. The efficiency of the hybridization reaction was verified with 2.5% agarose gel electrophoresis. NF-κB-decoy sequence was 5'-TAGTTGAGGGGA-CTTTCCCAGGCA-3', NF-κB-mutated sequence was 5'-TAGTTGAGGTTAAGGTCCCAGGCA-3'. The ODNs were co-transfected into MsC with the wild and mutated promoters differently.
Electrophoretic mobility shift analysis
Nuclear extracts and reaction mixtures were prepared as described previously.［8］ The double stranded gel shift oligonucleotides (Promega Company, USA) for NF-κB was end-labeled with (γ-32P) adenosine triphosphate by using the 5'-end-labeling kit from Promega Company. Gel supershift assay: added 2 μg anti-p50, p52, p65, RelB or c-Rel to the mixture differently and placed it on ice for 3 hours before adding the probe.
Western blot analysis
Protein extracts (30 μg per lane) were separated by denaturing 10% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate according to standard protocols, and then transferred to a polyvinylidene membrane. Protein was probed by a polyclonal anti-IκBα antibody (1∶2000 dilution; Promega Company, USA). The transferred protein was re-probed by an anti-rabbit IgG antibody conjugated with horseradish peroxidase for 1 hour. The proteins were then detected using enhanced chemiluminescent (ECL) detecting reagents and autoradiography.
DNA transfection and luciferase assay
Plasmid DNA was transfected into the cultured rat MsC with GeneJammerTM according to the instructions when cells reached 60% confluence. pRL-SV40 was used as an internal control to normalize for transfection efficiency. After different treatments, the transfected MsC were washed three times with ice-cold PBS, then incubated with 100 μl of 1×passive lysis buffer for 30 minutes, and harvested. After 2 minutes of centrifugation, the supernatants were stored at -70℃ then used for the measurements. Luciferase activities were determined with the Dual-luciferase reporter assay system according to the instructions of the manufacturers. All transfection experiments were repeated at least three times.
RT-PCR analysis
Total RNA was isolated from cultured MsC by solid-phase extraction with RNeasy kit from the Qiagen company. RT-PCR analyses were also performed to determine the effects of NF-κB decoy-ODN on TGF-β1. Amplification of GAPDH cDNA served as an internal standard. The following primers were used for amplification. TGF-β1: 5'-GCCAGATCCTGTCCAAACTAA-3'(sense) and 5'-TTGGTATCCAGGGCTCTC-3'(antisense); GAPDH:5'-ATCACCATCTTCCAGGAG-3'(sense) and 5'-GAACACGGAAGGCCATGC-3'(antisense).
Statistical analysis
The bands of results were quantified by densitometry. The results of 3 differential experiences were expressed as the mean±standard deviation (SD). Statistical analysis using SPSS(version 8.0) by the Student's t test, with a P value <0.05 was considered as statistically significant.
RESULTS
MsC appraisal
The cells were stained with anti-keratin, anti-CD34, anti-desmin. Anti-keratin and anti-CD34 were negative and anti-desmin was positive. So we can remove the chances of epithelial cells and endotheliocytes and approved they were MsC.
The effect of mutation and NF-κB decoy-ODN on TGF-β1 promoter
The relative luciferase activity displayed the transcription and the promoter activity. The activity of luciferase declined after the promoter mutated. The transcription was affected. To determine the effect of NF-κB decoy-ODN on the transcription of the promoters, NF-κB decoy-ODN was co-transfected into MsC with the wild or mutated promoter. Then the cells were exposed to 100 μg/ml Ox-LDL for 36 hours. The NF-κB decoy-ODN inhibited the activity of the wild promoter. But it had no effect on the mutated promoter. The NF-κB control mutated-ODN was co-tranfected into MsC with the wild or mutated promoter, too. It failed to compete with the wild and mutated promoter. The activity of luciferase did not decrease ( Fig. 1 ).
Activity of NF-κB in MsC induced by Ox-LDL
The NF-κB binding activity was enhanced in a time and concentration dependent manner in MsC. After exposure to Ox-LDL (100 μg/ml) for 6 hours, NF-κB binding activity increased and reached a peak at 36 hours ( Fig. 2 ). NF-κB binding activity elevated gradually with an increase of Ox-LDL after exposure for 36 hours ( Fig. 3 ). The non-special probe could not form the retarded band. To further address the question of which members of NF-κB complex are present in the nuclear extract from MsC by Ox-LDL, anti-p65, p50, p52, RelB, c-Rel antibodies were used in supershift assay. Gel supershift assay proved that enhancement of NF-κB activity was mainly from p50 and p65 ( Fig. 4 ).
Effects of protein kinase on NF-κB binding activity induced by Ox-LDL
After exposure to 100 μg/ml Ox-LDL for 36 hours, MsC were treated with the protein kinases for 60 minutes.H-89, PD98059 and SB203580 could not inhibit the activity of NF-κB induced by Ox-LDL in MsC. It was inhibited by Staurosporine partially. Proteasome Inhibitor Ⅰ almost inhibited it completely ( Fig. 5 ). These effects of NF-κB were not attributed to general toxicity because morphological changes were not found after MsC were stained with trypan blue.
Expression of IκBα induced by Ox-LDL
Ox-LDL induced degradation of IκB nearly on adding to MsC. IκBα expression came to the minimum after 30 minutes and recovered to the normal level after 1 hour ( Fig. 6 ).
Effects of Ox-LDL on TGF-β1 expression
Ox-LDL could induce the expression of TGF-β1 in a time and concentration dependent manner. After exposure to 100 μg Ox-LDL for 12 hours, TGF-β1 mRNA increased significantly in MsC and reached a peak at 48 hours ( Fig. 7 ). With an increase of Ox-LDL, TGF-β1 mRNA elevated gradually in Ox-LDL-stimulated MsC for 36 hours ( Fig. 8 ).
DISCUSSION
The NF-κB family consists of a group of inducible transcription factors which regulate immune and inflammatory responses and protect cells from undergoing apoptosis in response to cellular stress.［9］ A number of signal transduction cascades can activate the NF-κB pathway to result in the translocation of the NF-κB proteins from the cytoplasm to the nucleus where they activate the expression of specific cellular genes. Hyperglucose induced oxidant stress could activate NF-κB binding activitiy. The TNF-α-NF-κB passway exists in MsC and IL-1 could induce the upregulation of NF-κB binding activity, too.［10,11］ Ox-LDL can injure MsC and cause deposition of extracellular matrix. It has been reported that Ox-LDL could induce the activation of NF-κB in other cells. The results demonstrated that Ox-LDL could upregulate the binding activity of NF-κB in MsC.
NF-κB was a member of the mammalian rel gene family comprised of p105/p50, p100/p52, p65 (RelA), RelB and c-Rel.［12］ The inactivated NF-κB is sequestered in the cytoplasm in homo- or hetero-dimmers. In the research, the probe and the nuclear proteins reacted and formed unique retarded band. Gel supershift assay demonstrated that it was the heterodimmer of p65/p50. Its specific inhibitor IκB is a family that comprises of IκBα, IκBβ, IκBε, IκBγ and bcl-3 in mammals. IκB masks the nuclear localization sequence of NF-κB through its 6-7 ankyrin repeats. The release and translocation of NF-κB needs the phosphorylation and ubiquitinylation of IκB by specific IκB kinases (IKK). The inhibitor of heterodimmer of p65/p50 is IκB.［13］ Ox-LDL induced rapid proteolytic degradation of IκBα that resulted in translocation of p65/p50 into the nucleus and binding to its DNA response element. NF-κB activity is regulated by IκB, protein level, and co-activation protein, etc. Activation of NF-κB induced by Ox-LDL could be mediated by IκB in mesangial cells.
IKK is activated by NF-κB-inducing kinase(NIK). The activation of NIK is mediated by MyD88 of Toll-like receptor, IL-1 receptor associated kinase, and TNF receptor-associated factor. TLR2→MyD88→IRAK→TRAF→NIK→IKK→NFκB signal transduction pathway that induces transcription of chemokine genes.［14,15］ The activity of NF-κB induced by Ox-LDL was not affected by H-89, PD98059 and SB203580 in MsC. It suggested that PKA, MEK-1 and p38 MAPK are not involved in the NF-κB activation induced by Ox-LDL in mesangial cells. Though inhibitors could not inhibit activation of NF-κB, it has been reported that PKA protein could bind to the transactivation domain of p65.［16］ So the PKA pathway should be researched further. Staurosporine partially inhibited the activity of NF-κB. PKC could mediate the activation of NF-κB induced by Ox-LDL in MsC.
Emerging evidence suggests that NF-κB may mediate the transcription of inducible nitric oxide synthase (iNOS),［17］ monocyte chemoattractant protein-1 (MCP-1),［18］ and vascular cell adhesion molecule-1 (VCAM-1)［19］ in mesangial cells. The Ox-LDL could induce the increase of TGF-β1 mRNA in MsC confirmed by RT-PCR assay. NF-κB binding activity was elevated in MsC after exposure to Ox-LDL. Analysis of the promoter region of the rat TGF-β1 gene demonstrates a putative binding site for NF-κB. Mutation of that site resulted in the decline of transcription confirmed by transient transfection. NF-κB decoy oligodeoxynucleotides competed with the wild promoter and inhibited its activity. The mutated promoter vector was also co-transfected with NF-κB into MsC and its activity did not decrease. Neither the wild promoter nor the mutated promoter competed with the NF-κB control mutant decoy oligodeoxynuceotides. So the above mentioned site could be a response element of NF-κB. The result suggested that NF-κB involves in the overexpression of TGF-β1 induced by Ox-LDL in mesangial cells. NF-κB activation could be mediated by PKC partially.
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