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J Am Soc Nephrol 15: 2383-2390, 2004
© 2004 American Society of Nephrology
doi: 10.1097/01.ASN.0000136426.01160.2F
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J Am Soc Nephrol 15:2383-2390, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

CCAAT/Enhancer-Binding Protein {delta} Contributes to Myofibroblast Transdifferentiation and Renal Disease Progression

Masanobu Takeji*,{dagger}, Noritaka Kawada*,{dagger}, Toshiki Moriyama*,{ddagger}, Katsuyuki Nagatoya*, Susumu Oseto*, Shizuo Akira§, Masatsugu Hori*, Enyu Imai* and Takeshi Miwa{dagger}

*Department of Internal Medicine and Therapeutics, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan; {dagger}Genome Information Research Center, Osaka University, Suita, Osaka, Japan; {ddagger}School of Health and Sport Sciences, Osaka University, Toyonaka, Osaka, Japan; and §Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan

Correspondence to Dr. Toshiki Moriyama, Department of Internal Medicine and Therapeutics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan. Phone: +81-6-6879-3632; Fax: +81-6-6879-3639; E-mail: moriyama{at}medone.med.osaka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Myofibroblasts are pivotal participants in pathologic processes in a wide variety of organs, such as lung, liver, and kidney, by producing several inflammatory cytokines and extracellular matrices. The mechanism by which transdifferentiation from original cell to myofibroblast occurs, however, is still unclear. The expression of smooth muscle {alpha}-actin (SM{alpha}A) is the most characteristic feature of myofibroblasts; therefore, it was speculated that any factors that promote SM{alpha}A expression might be the key to transdifferentiation to myofibroblasts and disease exacerbation. A transcription factor CCAAT/enhancer-binding protein {delta} (C/EBP{delta}) was identified and demonstrated to bind to sequences including the CArG motif from SM{alpha}A intron 1 and to increase transcriptional activity of this promoter. Expression of SM{alpha}A and C/EBP{delta} in the glomerular area was upregulated in rat anti-Thy1 glomerulonephritis and mouse Habu-venom glomerulonephritis, both of which are models of mesangioproliferative glomerulonephritis. In the latter model, C/EBP{delta} knockout mice demonstrated significantly less SM{alpha}A expression in the glomerular area on day 8 and less renal functional deterioration on day 14, compared with wild-type mice. These data suggest an important role for C/EBP{delta} in myofibroblast transdifferentiation and glomerulonephritis exacerbation.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tissue injury and sustained abnormal repair processes after initial insults are the major causes of tissue fibrosis leading to end-stage organ failure in many tissues, including lung, liver, and kidney (1,2). One of the common histopathologic features of fibrosis is an early emergence of myofibroblasts, a unique mesenchymal cell population with ultrastructural properties of both muscle and nonmuscle cells. Myofibroblasts express smooth muscle cytoskeletal markers, such as smooth muscle {alpha}-actin (SM{alpha}A), caldesmon, and desmin, and actively produce inflammatory cytokines and extracellular matrices. They are thought to be central participants in wound healing, presumably as an extension or accentuation of their role in normal growth and differentiation. In contrast, uncontrolled generation or activation of myofibroblasts results in excessive formation of granulation tissue and accumulation of extracellular matrices, leading to tissue fibrosis and eventual organ function loss (2,3).

Numerous types of cells have been characterized as the source of myofibroblasts, including pericytes, hepatic stellate cells, mesangial cells, interstitial cells, and granulation tissue fibroblasts. However, the molecular mechanism of myofibroblast formation and of expression of smooth muscle cytoskeletal markers and matrix proteins in these cells have not been well characterized. We have been investigating the pathophysiologic significance of myofibroblasts in renal disease. Both caldesmon and SM{alpha}A are sensitive and useful molecular markers for myofibroblasts in progressive renal disease (4,5). Emergence of SM{alpha}A-expressing myofibroblasts has been documented, focusing on prognostic value in IgA nephropathy (6) and other types of glomerulonephritis (7–9), diabetic nephropathy (10), and chronic allograft nephropathy in posttransplant patients (11). These observations in human renal disease highlight the significance of myofibroblasts in progressive renal disease; therefore, many experimental investigations use myofibroblast expansion as a marker of disease progression and its suppression as a marker of therapeutic efficacy.

We aim to elucidate the molecular mechanisms of SM{alpha}A gene regulation during myofibroblast formation. Intronic CArG motif is essential for the transcriptional activation of SM{alpha}A gene in both smooth muscle cells (12) and renal myofibroblasts (13). Although serum response factor (SRF) is a well-known binding factor for the CArG motif, it is distributed ubiquitously in various cells. Therefore, it is likely that any other transcription factors bind to this region to enhance SM{alpha}A expression and might contribute to production of inflammatory cytokines and extracellular matrices. We report here the molecular identification and characterization of transcription factor CCAAT/enhancer-binding protein {delta} (C/EBP{delta}) that binds to sequences that include the CArG motif from intron 1 of the SM{alpha}A gene. C/EBP{delta} enhances SM{alpha}A expression and also contributes to the production of proinflammatory chemokine leading to renal disease progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Cultured Cells
C/EBP{delta}-deficient (C/EBP{delta}[–/–]) mice and wild-type (C/EBP{delta}[+/+]) mice were generated by mating C/EBP{delta} heterozygous (C/EBP{delta}[+/–]) mice (background strain 129) (14). Sprague-Dawley rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Primary cultured mesangial cells were obtained from C/EBP{delta}(–/–) mice and C/EBP{delta}(+/+) mice as described previously (15). Mesangial cells were cultured in RPMI 1640 medium (Life Technologies BRL, Rockville, MD) with 20% FCS (Life Technologies BRL). Mouse NIH-3T3 fibroblasts were cultured in DMEM with 10% FCS, penicillin G (100 U/ml), and streptomycin (100 µg/ml).

Yeast One-Hybrid Analysis
To identify factors that bind to DNA sequences including the CArG motif from SM{alpha}A intron 1, we used a yeast one-hybrid system. Two oligonucleotides, 5'-AATTCGTTTTACCTAATTATGAAATGTTTTACCTAATTATGAAATGTTTTACCTAATTATGAAATGA-3' and 5'-AATTTCATTTCATAATTAGGTAAAACATTTCATAATTAGGTAAAACATTTCATAATTAGGTAAAACG-3', that contained three tandem repeats of the 20-bp CArG motif at bp + 1098 of SM{alpha}A intron 1, with one point mutation (G to T and C to A; italics) to avoid SRF binding were synthesized, annealed, and inserted upstream of the E1b minimal promoter in pHISi-1 (named pHISi-3CArGM) and the CYC1 minimal promoter in pLacZi (named pLacZ-3CArGM). cDNA cloning by the yeast one-hybrid system was performed as described previously using the MATCHMAKER One-Hybrid System (Clontech, Palo Alto, CA) (16). pLacZ-3CArGM and pHISi-3CArGM were integrated into the yeast (YM4271) genome. Next, the yeast was transformed with a human adult kidney cDNA library (Clontech), which contains a human kidney cDNA library cloned into pACT2 and produces the yeast GAL4 activation domain-cDNA fusion protein. Transformants were selected on uracil-, histidine-, and leucine-deficient plates that contained 20 mM 3-aminotriazole. Large colonies (His+) were assayed for {beta}-galactosidase activity by incubating at 30°C with a buffer that contained 0.8 mM 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside. Plasmids were isolated from blue colonies (LacZ+), and their insert cDNA was analyzed by DNA sequencing.

Transfection Analysis of C/EBP{delta}
cDNA that contained the full coding region of human C/EBP{delta} was obtained by yeast one-hybrid analysis and inserted into the EcoRI site of pCAGGS (named pCAGGS-C/EBP{delta}). The 180-bp sequence of SM{alpha}A intron1 region from bp + 972, which contains an intronic CArG motif, or three tandem copies of the 20-bp intronic CArG motif (bp + 1093 to bp + 1112) were inserted into pSV0-CAT plasmid to construct reporter plasmids for transfection assay, designated as pSV-int180-CAT and pSV-3CArG-CAT, respectively. NIH-3T3 fibroblasts were seeded for transfection assay into 6-cm dishes at a density of 4 x 104 cells/cm2. For each dish, 0.5 µg of reporter plasmid, 0 to 4 µg of expression plasmid, and 0.5 µg of luciferase reporter plasmid were transiently transfected with Lipofectamine Reagent (Life Technologies BRL). After 72 h, cells were harvested by scraping in lysis buffer. Promoter activities were evaluated by CAT and luciferase assays.

Preparation of C/EBP{delta} Protein and Electrophoretic Gel Mobility Shift Assay
Glutathione S-transferase (GST)-C/EBP{delta} was constructed in a pGEX 6P-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ). GST-C/EBP{delta} was transformed into Escherichia coli BL21 and induced by 1 mM isopropyl-D-thiogalactopyranoside. Recombinant proteins were purified using glutathione-Sepharose 4B gels. The probe for the CArG motif (CArG #0) consists of 5'-GTTTTACCTAATTAGGAAATGCTT and 5'-AAGCATTTCCTAATTAGGTAAAAC annealed to each other. The probe for C/EBP{delta} consensus consists of 5'-TGCAGATTGCGCAATCTGCA annealed to itself. Both oligonucleotides were 5'-end-labeled by T4 polynucleotide kinase with [{gamma}-32P]ATP (Amersham Biosciences, Tokyo, Japan). Binding reactions were performed in a 20-µl reaction mixture that contained binding buffer, 0.1 µg of C/EBP{delta} protein, 1 µg of poly-dIdC, and probe. Samples were incubated at room temperature for 30 min and fractionated on 5% polyacrylamide native gels in 0.5x Tris-borate EDTA buffer. After drying, gels were analyzed using the BAS system (Fujifilm, Tokyo, Japan). Competition assays were performed by adding nonlabeled C/EBP consensus probe or nonlabeled CArG #0 probe.

Renal Disease Models of Rat and Mouse
Acute mesangioproliferative glomerulonephritis was produced in rats by a single injection of anti-Thy1 monoclonal antibody (OX-7). On day 0, anti-Thy1 antibody was injected into the tail vein of 6-wk-old male rats at a dose of 1.5 mg/kg body wt (17). On day 7, kidneys were perfused with ice-cold PBS and removed. Habu-venom glomerulonephritis (HVGN), a murine model for acute mesangioproliferative glomerulonephritis, was induced in 6- to 8-wk-old male C/EBP{delta}(–/–) and C/EBP{delta}(+/+) mice. For inducing HVGN, heminephrectomized mice received an injection of lyophilized venom from Habu snake Trimeresurus flavoviridis (Wako, Osaka, Japan) dissolved in saline at 1.5 mg/kg body wt, and their urine was collected twice a week. Eight and 14 d after disease induction, mice were killed, and their kidneys and blood samples were collected (13).

Glomeruli Isolation, RNA Extraction, and Reverse Transcription–PCR
Total RNA of cultured cells and glomeruli were isolated using TRIzol reagent (Life Technologies BRL). Glomeruli were isolated from kidney by a differential sieving method (15). Kidneys were removed from killed animals, and renal cortices were dissected from the kidney with a scalpel, then minced and passed through stainless steel mesh of different pore sizes (120, 75, and 53 µm). Glomeruli were retained on the last mesh with a purity of >95%, as indicated by microscopic evaluation (the remaining few percent consisted of tubular fragments). cDNA was prepared from 1 µg of each RNA sample, using MuLV reverse transcriptase (Applied Biosystems, Foster City, CA), random hexamers, RNase inhibitor, and dNTP mixture in a final volume 20 µl. Semiquantitative PCR was performed with 1 µl of template cDNA, PCR primers (10 pmol each), and AmpliTaq DNA Polymerase (Applied Biosystems) in a final volume of 20 µl. PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. Primers used were murine SM{alpha}A 5'-ATCGTCCACCGCAAATGC (forward) and 5'-AAGGAACTGGAGGCGCTG (reverse); glyceraldehyde-3-phosphate dehydrogenase 5'-AGTATGACTCCACTCACGGCAA (forward) and 5'-TCTCGCTCCTGGAAGATGGT (reverse); C/EBP{delta} 5'-GCAGACAGTGGTGAGCTTGG (forward) and 5'-AAGCATGCGCAGTCTCTTCC (reverse); and monocyte chemoattractant protein-1 (MCP-1) 5'-AGCCAACTCTCACTGAAGCC (forward) and 5'-CATTCAAAGGTGCTGAAGACC (reverse).

Immunohistochemistry
Mouse monoclonal anti-SM{alpha}A (1A4; peroxidase conjugate DAKO, Glostrup, Denmark) and rabbit polyclonal anti-C/EBP{delta} antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used for immunohistochemical detection of SM{alpha}A and C/EBP{delta}. Paraffin-embedded sections (2-µm thickness) obtained from tissues fixed in 4% paraformaldehyde were blocked and incubated overnight with primary antibodies at 4°C. Sections for C/EBP{delta} immunostaining were incubated with the second biotinylated goat anti-rabbit antibody at 1:150 (Vector ABC Kit; Vector Laboratories, Burlingame, CA) for 1 h at room temperature, then incubated in an avidin-biotinylated horseradish peroxidase complex (Vector) for 40 min at room temperature. Peroxidase activity was visualized with p-dimethyl aminobenzaldehyde (DAB). For evaluating the quantitative level of SM{alpha}A expression in glomeruli, the outline of the glomeruli was encircled on the computer display, and the SM{alpha}A-positive (DAB-positive) area was determined by color density. The SM{alpha}A-positive area percentage (DAB-positive area/encircled area) was calculated from 30 glomeruli in each group using MacSCOPE software (Mitani-Corp, Fukui, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of Intronic CArG Motif Binding Factor
To identify factors that bind to the CArG motif in intron 1 of SM{alpha}A gene, we used a yeast one-hybrid screening system. Approximately 5 x 106 human kidney cDNA clones were screened, and 38 double-positive clones were obtained. After sequence analysis, 20 of the 38 clones represented the same binding factor, identified as C/EBP{delta} (Figure 1), containing the sequence of the DNA-binding domain. These results indicate that C/EBP{delta} is a candidate for the intronic CArG motif binding.



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  		Figure 1. Domain structure of human CCAAT/enhancer-binding protein {delta} (C/EBP{delta}) gene (GenBank accession no. NM_005195) (top) and cDNA clones obtained from yeast one-hybrid analysis (bottom). Each cloned cDNA was produced from the 3'-polyA region. In the coding region, shown by a thick line, gray box (bp + 41 to bp + 583), filled box (bp + 583 to bp + 715), and hatched box (bp + 715 to bp + 850) represent transcription activating domain, DNA-binding domain, and leucine-zipper domain, respectively. Numbers of obtained clones are indicated on the right of each bar.

 
Confirmation of Specific Binding of C/EBP{delta} to Intronic CArG Motif
As we assumed that the intronic CArG motif overlaps with a sequence compatible with the consensus sequence for the C/EBP family (T[T/G]NNGNAA[T/G]) (18) as shown by the underline in Figure 2a, and to exclude the possibility that the point-mutated CArG motif used in yeast one-hybrid cloning might have affected the binding reactivity, we performed electromobility shift assay to confirm binding of C/EBP{delta} to the original intronic CArG motif in SM{alpha}A. C/EBP{delta} protein produced in E. coli showed binding not only to C/EBP consensus sequence (Figure 2b) but also to the intronic CArG sequence (Figure 2c). Complex formation was inhibited after preincubation with unlabeled C/EBP consensus sequence and also, although less competitive, with unlabeled intronic CArG sequence. These results demonstrate the sequence-specific binding of C/EBP{delta} to the intronic CArG sequence.



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  		Figure 2. Electromobility shift assay (EMSA) detecting complex formation of in vitro translated C/EBP{delta} protein and probes. (a) The intronic CArG motif (CArG#0) and its flanking sequence (top) and sequences of the probes used for EMSA (bottom). The boxed sequence (from bp + 1098 to bp + 1107) represents the CArG motif (CC[A/T]6GG), and the underlined sequence represents the C/EBP consensus (T[T/G]NNGNAA[T/G]); N, any nucleotide. (b and c) EMSA of C/EBP{delta} protein with C/EBP consensus probe (b) or the CArG#0 probe (c). Lane 1 is without competitor DNA, and lanes 2 and 3 are incubated with nonlabeled CArG#0 (x200) and C/EBP consensus (x200) oligonucleotides, respectively.

 
Transcriptional Activation of Intronic CArG Motif by C/EBP{delta}
To investigate whether C/EBP{delta} affects SM{alpha}A gene expression, we tested the transcriptional activity of reporter plasmid (pSV-int180-CAT), which contains 180 bp of SM{alpha}A intron 1 including the CArG motif as enhancer and CAT gene as reporter, by co-transfecting to NIH-3T3 fibroblasts with C/EBP{delta} expression vector (pCAGGS-C/EBP{delta}). CAT activity of pSV-int180-CAT vector was increased by the co-transfection of pCAGGS-C/EBP{delta} vector in a dose-dependent manner up to 0.5 µg/dish with a 220% increase (Figure 3a). NIH-3T3 fibroblasts do not express SM{alpha}A in the basal condition, and this result suggests that C/EBP{delta} itself has positive activity through SM{alpha}A intron 1. Next, pSV-3CArG-CAT, which contains three copies of the 20-bp intronic CArG motif, was co-transfected with pCAGGS-C/EBP{delta} vector into NIH3T3 fibroblasts, showing a significant increase in CAT activity (180% with 0.5 µg/dish) compared with absence of pCAGGS-C/EBP{delta} (Figure 3b). As it has been reported that the sequence in intron 1 is essential for SM{alpha}A gene transcriptional activation (12,13), these results demonstrate involvement of C/EBP{delta} in transcriptional activation of SM{alpha}A gene via the intronic CArG motif.



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  		Figure 3. Co-transfection analysis of the interaction of C/EBP{delta} with sequence including CArG#0. pSV-int180-CAT (a) or pSV-3CArG-CAT (b) was co-transfected with pCAGGS-C/EBP{delta} into NIH-3T3 fibroblasts. Data are obtained from five repetitive experiments. CAT activity was corrected by luciferase activity in each transfection. Relative CAT activity was determined as the ratio of activity when co-transfected with pCAGGS only. Values are expressed as mean ± SD; *P < 0.01.

 
Upregulation of SM{alpha}A and C/EBP{delta} in Rat Anti-Thy1 Glomerulonephritis
To investigate the in vivo relevance of C/EBP{delta} expression to myofibroblasts, we examined expression of C/EBP{delta} and SM{alpha}A in rat experimental mesangioproliferative glomerulonephritis. Reverse transcriptase–PCR analysis of SM{alpha}A and C/EBP{delta} mRNA was performed in isolated glomeruli obtained from untreated and anti-Thy1 glomerulonephritis rats on day 7. Both SM{alpha}A and C/EBP{delta} mRNA were upregulated in anti-Thy1 glomerulonephritis (Figure 4a). In immunohistochemical examination with consecutive sections from untreated rats, staining of SM{alpha}A was observed only in smooth muscle of the arteriolar wall (Figure 4b), and positive staining of C/EBP{delta} was minute in both glomerulus and arteriolar wall (Figure 4d). In contrast, anti-Thy1 glomerulonephritis rat showed SM{alpha}A and C/EBP{delta} immunostaining in glomerulus with similar distribution (Figure 4, c and e).



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  		Figure 4. Assessment of smooth muscle {alpha}-actin (SM{alpha}A) and C/EBP{delta} expression in anti-Thy1 glomerulonephritis. (a) Reverse transcriptase–PCR (RT-PCR) detection of SM{alpha}A (top), C/EBP{delta} (middle), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; bottom) mRNA in isolated glomeruli from untreated rats (left three lanes) and from anti-Thy1 glomerulonephritis rats on day 7 (right three lanes). Each lane represents an individual rat. (b through e) Immunostaining for SM{alpha}A (b and c) and C/EBP{delta} (d and e) in consecutive sections of the same glomerulus from untreated rat (b and d) and anti-Thy1 glomerulonephritis rat (c and e). Arrows in b and d indicate arteriolar walls. Magnification, x400.

 
SM{alpha}A Expression in Cultured Mesangial Cells from C/EBP{delta}(–/–) Mice
Cultured mesangial cells express substantial amounts of SM{alpha}A and are thought to mimic activated mesangial cells, namely myofibroblasts, found in diseased glomeruli (19). As C/EBP{delta}(–/–) mice have been produced before and reported to exhibit little phenotypic modulation under physiologic conditions (14), we investigated expression levels of SM{alpha}A in cultured mesangial cells from these knockout mice. Expression of SM{alpha}A mRNA in C/EBP{delta}(–/–) cells was lower than that in C/EBP{delta}(+/+) cells (Figure 5). Moreover, mRNA expression of MCP-1, which is considered an important chemokine in the progression of glomerulonephritis, was also reduced in C/EBP{delta}(–/–) mesangial cells (Figure 5). However, other proinflammatory cytokines, such as TGF-{beta}1 and IL-1{beta}, did not show a reduction in their mRNA levels, compared with C/EBP{delta}(+/+) cultured mesangial cells (data not shown).



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  		Figure 5. mRNA expression of SM{alpha}A (top) and monocyte chemoattractant protein-1 (MCP-1; middle) in mesangial cells in culture assessed by RT-PCR. mRNA extracted from C/EBP{delta}(+/+) mesangial cells is indicated in the left two lanes, and mRNA from C/EBP{delta}(–/–) mesangial cells is indicated in the right two lanes.

 
Renal Disease Model in C/EBP{delta}(–/–) Mice
Mouse HVGN is a model for acute mesangioproliferative glomerulonephritis, which evokes acute mesangiolysis followed by mesangial cell proliferation with myofibroblastic transdifferentiation and matrix increment. Similar to rat anti-Thy1 glomerulonephritis, C/EBP{delta} expression was only slightly observed in normal glomeruli and increased in HVGN in C/EBP{delta}(+/+) mice but not at all in C/EBP{delta}(–/–) mice (Figure 6).



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  		Figure 6. Immunohistochemistry of C/EBP{delta} in the glomerulus of C/EBP{delta} (+/+) (a and c) and C/EBP{delta} (–/–) (b and d) mice. (a and b) Normal condition. (b and d) Habu-venom glomerulonephritis (HVGN) on day 8. Magnification, x400.

 
Mesangial SM{alpha}A expression in C/EBP{delta}(–/–) mice on day 8 was milder than in C/EBP{delta}(+/+) mice (Figure 7, c and d). Although mesangial expansion was observed to persist in HVGN on day 14, mesangial SM{alpha}A expression was diminished in both C/EBP{delta}(+/+) and C/EBP{delta}(–/–) mice at that time (Figure 7, g and h), possibly suggesting that the disease was going into the recovery phase. The proportion of glomerular SM{alpha}A-positive area in C/EBP{delta}(–/–) mice was significantly less (11.0 ± 6.9 versus 18.2 ± 8.7%; mean ± SD) than in C/EBP{delta}(+/+) mice (Figure 7i). These data suggest involvement of C/EBP{delta} in mesangial transdifferentiation and SM{alpha}A expression in acute mesangioproliferative glomerulonephritis. Urinary protein excretion tended to be decreased along the time course of HVGN in C/EBP{delta}(–/–) mice versus C/EBP{delta}(+/+) mice. The urinary protein/creatinine ratios were 5.0 ± 2.9 versus 7.6 ± 1.6 on day 4 and 3.6 ± 0.8 versus 4.3 ± 0.7 on day 14 for C/EBP{delta}(–/–) mice versus C/EBP{delta}(+/+), respectively. The renal function was preserved in C/EBP{delta}(–/–) mice. Plasma urea nitrogen was 39.3 ± 2.6 versus 56.9 ± 16.6 and plasma creatinine 0.15 ± 0.02 versus 0.20 ± 0.04 on day 14 (mg/dl, mean ± SD; P < 0.05) for C/EBP{delta}(–/–) mice versus C/EBP{delta}(+/+), respectively.



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  		Figure 7. Histologic assessment by periodic acid-Schiff staining (a, b, e, and f) and immunohistochemistry of SM{alpha}A (c, d, g, and h) in HVGN on day 8 (a through d) and day 14 (e and f). (a, c, e, and g) C/EBP{delta}(+/+) mice. (b, d, f, h) C/EBP{delta}(–/–) mice. (i) Quantitative evaluation of glomerular SM{alpha}A expression. Values are mean ± SD, calculated from 30 glomeruli in each group; *P < 0.001. Magnification, x400.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have been investigating the pathophysiologic significance, nature, and molecular mechanism of phenotypic change of renal cells to myofibroblasts in the process of progressive renal diseases (4,5,13). We hypothesized that the molecular mechanisms underlying the induction of SM{alpha}A in myofibroblasts are closely related to the molecular pathophysiology of progressive renal disease leading to renal fibrosis. The intronic CArG motif from the SM{alpha}A gene is essential for in vivo transcriptional activation of the SM{alpha}A gene in both smooth muscle cells (12) and renal myofibroblasts (13). Interaction of the CArG motif and SRF is a candidate mechanism for transcriptional activation of the SM{alpha}A gene (20,21). However, SRF is a ubiquitous factor, and the CArG motifs are present in a wide variety of genes. We identified C/EBP{delta} as the major candidate transcription factor for transactivating the SM{alpha}A gene in myofibroblasts through its intronic CArG sequence. We also found that C/EBP{delta} can bind directly to DNA in contrast to myocardin, a key co-factor of SRF (22) for smooth muscle differentiation co-activating SM{alpha}A and other smooth muscle gene expression in smooth muscle cells (23,24). We observed that there was no expression of myocardin mRNA in both normal and diseased kidney or cultured mesangial cells (unpublished observation), so its involvement in myofibroblast transdifferentiation is unlikely. It is, however, still unknown whether C/EBP{delta} can cooperate with SRF and/or other transcription factors.

C/EBP{delta} belongs to the C/EBP family, which contains a leucine zipper domain that mediates dimerization with the same or other C/EBP isoforms and interaction with the target sequence. In the present study, we found that C/EBP{delta} can bind to the C/EBP family consensus sequence, which coincides with the intronic CArG sequence and can stimulate promoter activity of the SM{alpha}A gene through it (Figures 2 and 3Go). In addition, as shown in Figure 4, C/EBP{delta} and SM{alpha}A proteins were upregulated and distributed similarly in the mesangial area in experimental glomerulonephritis. In contrast, in smooth muscle cells of the arteriolar wall, SM{alpha}A immunostaining is densely positive but C/EBP{delta} staining is not (Figure 4, b and d). This observation indicates that, in normal smooth muscle cells, C/EBP{delta} is not greatly involved in the expression of SM{alpha}A. This result is the first demonstration of upregulation of C/EBP{delta} in myofibroblasts in vivo in experimental glomerulonephritis and strongly suggests a pivotal role for C/EBP{delta} in the positive regulation of SM{alpha}A in glomerular myofibroblasts.

We found that in vitro cultured mesangial cells isolated from C/EBP{delta}(–/–) mice express much less SM{alpha}A compared with those from C/EBP{delta}(+/+) mice (Figure 5), supporting the in vivo observation of possible involvement of C/EBP{delta} in gene regulation of SM{alpha}A in glomerular myofibroblasts. Moreover, the mesangioproliferative glomerulonephritis model (HVGN) in C/EBP{delta}(–/–) mice showed decreased SM{alpha}A expression in the glomeruli in vivo (Figure 7).

Myofibroblasts are also known to play pivotal roles in the progression of tubulointerstitial fibrosis. There arose a possibility that C/EBP{delta} might be involved with myofibroblasts not only in glomerular mesangial lesions but also in tubulointerstitial lesions; thus, we investigated another renal disease model. Unilateral ureteral obstruction (UUO) is a model of tubulointerstitial fibrosis, in which we previously reported that the emergence of myofibroblast was closely related to the degree of fibrosis (25,26). We analyzed UUO in C/EBP{delta}(–/–) mice and found that SM{alpha}A mRNA upregulation in whole kidneys of UUO (on day 7) was significantly attenuated in C/EBP{delta}(–/–) mice compared with C/EBP{delta}(+/+) mice (data not shown). In addition to results from the HVGN model, these results suggest that C/EBP{delta} is engaged in the process of transdifferentiation not only in mesangial cells but also in renal interstitial fibroblasts and might play a role in exacerbation of progressive renal diseases.

C/EBP{delta} is expressed in liver, lung, adipose tissue, and intestine under physiologic conditions and is strongly upregulated at the transcriptional level by inflammatory stimuli, such as turpentine oil, bacterial LPS, and cytokines such as IL-6 and TNF-{alpha} (27,28). C/EBP{delta}(–/–) mouse shows only a mild phenotype of slightly disturbed lipid storage under physiologic conditions (14). These findings indicate that C/EBP{delta} is not essential for development and/or maintenance of these tissues, perhaps because of the redundant function of other C/EBP family proteins. Some investigators have reported a role for C/EBP{delta} in pathologic states. For example, C/EBP{delta} enhances PDGF-{alpha} receptor expression in vascular smooth muscle cells, and its contribution to atherosclerosis is suggested (29). The present report is the first to show the relationship between C/EBP{delta} and kidney disease, suggesting the possibility of attenuation of the disease by suppressing C/EBP{delta} activity. In the pathologic state of other organs to which SM{alpha}A-expressing myofibroblasts contribute, such as liver cirrhosis, chronic pancreatitis, and pulmonary fibrosis, C/EBP{delta} may also play an important role.

Myofibroblasts are thought to be a source of extracellular matrix deposition in sclerosing tissues. C/EBP binding sites have been identified in the promoters of several genes that modulate extracellular matrix expansion, such as type I collagen or tissue inhibitor of metalloproteinases (30,31). It is interesting that C/EBP were reported to regulate type I collagen transcription in hepatic stellate cells through a hydrogen peroxide–dependent pathway (32,33). The binding motifs for C/EBP are now known to be present in various genes that encode most inflammation-inducible molecules, such as IL-1{beta}, MCP-1 (34), granulocyte macrophage colony-stimulating factor receptor gene (35), PDGF receptor (29,36), and intercellular adhesion molecule 1 (37). In the present study, MCP-1 mRNA expression was clearly decreased in C/EBP{delta}(–/–) mesangial cells in culture, but we could see no obvious difference for other cytokines, including TGF-{beta}1, a key mediator of myofibroblast transdifferentiation (38,39). This may be due to the degree of replacement of other transcription factor(s), perhaps including C/EBP{beta}, and for MCP-1 transcription, C/EBP{delta} may have a major contribution.

In the present study, C/EBP{delta}(–/–) mice showed less SM{alpha}A expression in HVGN on day 7, and consequent renal function was relatively less deteriorated. These results suggest that suppressing excessive myofibroblast transdifferentiation could be a therapeutic measure for glomerulonephritis. A recent report pointed out that myofibroblasts are necessary in the repair process of kidney tissue injury (40); however, the present results show amelioration of renal disease by partial inhibition of myofibroblast transdifferentiation in the absence of C/EBP{delta}. Certainly, from the present data, we cannot conclude whether it is beneficial to block the transdifferentiation completely, and further investigation is planned.

In conclusion, we have determined that the intronic CArG motif in SM{alpha}A gene is a binding locus of transcriptional activator C/EBP{delta}. C/EBP{delta} was upregulated in rat and mouse experimental glomerulonephritis, and its expression mirrored SM{alpha}A induction. SM{alpha}A expression was lower in cultured mesangial cells and renal disease models in C/EBP{delta}(–/–) mice compared with C/EBP{delta}(+/+) mice. Furthermore, the degree of renal function loss was attenuated in C/EBP{delta}(–/–) mice. Our results demonstrate the involvement of C/EBP{delta} in transcriptional activation of the SM{alpha}A gene in myofibroblasts and in renal diseases. Further analysis of C/EBP{delta}-dependent gene regulation in myofibroblasts is likely to give us new insights into molecular pathophysiology of progressive renal disease and helps in establishing new therapeutic approaches to myofibroblast-related disease.


   Acknowledgments
 
This work has been supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (T.M.), and by a grant from Takeda Science Foundation (T.M.).

We are grateful to Dr. Wataru Nishida (Department of Neuroscience, Osaka University) for help in preparing GST-C/EBP{delta} fusion protein.


   Footnotes
 
M.T. and N.K. contributed equally to this work.

N.K.’s current affiliation is Center for Hypertension and Renal Disease Research, Georgetown University Medical Center, Washington, DC.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication September 8, 2003. Accepted for publication May 27, 2004.




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