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Am J Physiol Renal Physiol 291: F1070-F1077, 2006. First published May 23, 2006; doi:10.1152/ajprenal.00013.2006
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Transforming growth factor-beta1 differentially mediates fibronectin and inflammatory cytokine expression in kidney tubular cells

Weier Qi,1,* Xinming Chen,1,* John Holian,1 Ellein Mreich,1 Stephen Twigg,2 Richard E. Gilbert,3,4 and Carol A. Pollock1

1Department of Medicine, Kolling Institute, University of Sydney, Royal North Shore Hospital, Sydney; 2Department of Medicine, University of Sydney, and Royal Prince Alfred Hospital, Sydney; 3Department of Medicine, St. Vincent’s Hospital, Melbourne, Australia; and 4Department of Medicine, University of Toronto, St. Michael’s Hospital, Toronto, Ontario, Canada

Submitted 17 January 2006 ; accepted in final form 15 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transforming growth factor-beta1 (TGF-beta1) is not only an important fibrogenic but also immunomodulatory cytokine in the human kidney. We have recently demonstrated that TGF-beta1 induces interleukin-8 (IL-8), macrophage chemoattractant protein-1 (MCP-1), and fibronectin production in renal proximal tubular (HK-2) cells. However, the unique dependence of IL-8, MCP-1, and fibronectin on TGF-beta1 expression is unknown. The TGF-beta1 gene was effectively silenced in HK-2 cells using small-interference (si) RNA. Basal secretion of IL-8 and MCP-1 decreased (both P < 0.05) but, paradoxically, fibronectin increased (P < 0.05) in TGF-beta1-silenced cells compared with cells transfected with nonspecific siRNA. Significant increases were observed in mRNA for the TGF-beta2 (P < 0.05), TGF-beta3 (P < 0.05) isoforms and pSmad2 (P < 0.05), which were reflected in protein expression. Concurrent exposure to pan-specific TGF-beta antibody reversed the observed increase in fibronectin expression, suggesting that TGF-beta2 and TGF-beta3 isoforms mediate the increased fibronectin expression in TGF-beta1-silenced cells. An increase in the DNA binding activity of activator protein-1 (AP-1; P < 0.05) was also observed in TGF-beta1-silenced cells. In contrast, nuclear factor-{kappa}B (NF-{kappa}B) DNA binding activity was significantly decreased (P < 0.0005). These studies demonstrate that TGF-beta1 is a key regulator of IL-8 and MCP-1, whereas fibronectin expression is regulated by a complex interaction between the TGF-beta isoforms in the HK-2 proximal tubular cell line. Decreased expression of TGF-beta1 reduces chemokine production in association with reduced NF-{kappa}B DNA binding activity, suggesting that immunomodulatory pathways in the kidney are specifically dependent on TGF-beta1. Conversely, decreased expression of TGF-beta1 results in increased TGF-beta2, TGF-beta3, AP-1, and pSmad2 that potentially mediates the observed increase in fibronectin.

small interference RNA; chemokine; pSmad; activator protein-1; nuclear factor-{kappa}B

TRANSFORMING GROWTH FACTOR (TGF)-beta is known to be a multifunctional peptide. The functions of TGF-beta can be grouped into three broad areas: modulation of inflammatory cell function, growth inhibition and differentiation, and control of extracellular matrix production. Within the kidney, TGF-beta1 is recognized among the TGF-beta isoforms to play the most important role in renal fibrogenesis and progressive kidney disease. However, the protean and important biological functions of TGF-beta1 are appreciated in TGF-beta1 knockout mice, which develop diffuse mononuclear cell infiltrates that prove lethal within a few weeks of birth (19, 32). The other TGF-beta isoforms present in the kidney, TGF-beta2 and TGF-beta3, have also been shown to be fibrogenic in in vitro and in vivo models of fibrotic disease (10, 12, 13, 28, 39). In contrast, TGF-beta2 and TGF-beta3 knockout mice display nonlethal developmental defects (24, 30). Plasma levels of TGF-beta2 and TGF-beta3 are undetectable, suggesting that they are restricted to more localized autocrine and paracrine modes of action (2). We have recently demonstrated that TGF-beta1 is not only an important fibrogenic cytokine but also an important regulator of chemokine production in renal tubular cells, with differential dependence on downstream production of connective tissue growth factor (27).

It is well known that TGF-beta signals through its receptors, TGF-beta type I and type II receptors, with downstream activation of the Smad signaling pathway. TGF-beta is known to mediate its fibrotic effects by activating the receptor-associated Smads (Smad-2 and -3). The pSmad-2 and pSmad-3 associate to form a heteromultimer with Smad-4 (Co-Smad). This complex is then translocated to the nucleus, where it can regulate target gene expression (31).

Many lines of evidence suggest nuclear transcription factors AP-1 and NF-{kappa}B play regulatory roles in TGF-beta1-dependent extracellular matrix and chemokine production (1, 3, 7, 20, 34, 37). IL-8, MCP-1, and fibronectin promoters contain both AP-1 and NF-{kappa}B binding sites and, in turn, TGF-beta1 regulates the activation of AP-1 and NF-{kappa}B (21, 33, 35, 40, 41).

The present study aimed to determine whether the secretion of chemokines (IL-8, MCP-1) and the extracellular matrix protein fibronectin is uniquely dependent on TGF-beta1 by using a renal proximal tubular cell model in which TGF-beta1 is selectively silenced.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The immortalized human kidney proximal tubular cell line (HK-2, ATCC) was used as the proximal tubular cell model. Cells were grown to subconfluence in keratinocyte serum-free media (Invitrogen) under conditions previously described (27).

Materials. TGF-beta1 was selectively silenced in the HK-2 cells using small interference (si) RNA methodology. TGF-beta1 and nonspecific double-stranded siRNA (21-mer RNA molecules) were chemically synthesized (Ambion). Human IL-8, TGF-beta2 ELISA kits, pan-specific TGF-beta antibody, and rabbit IgG were all purchased from R&D Systems (Minneapolis, MN), a human MCP-1 immunoassay kit was from BioSource International, and a TGF-beta1 ELISA kit was from Promega (Madison, WI). Antibodies to fibronectin, TGF-beta3, and pSmad2 were purchased from Neomarkers (Santa Cruz, CA) and Cell Signaling (Danvers, NY), respectively.

Experimental protocol. TGF-beta1 siRNA (30 nM/l) was introduced into HK-2 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In parallel, cells were transfected with 30 nM/l nonspecific siRNA, which served as the control data set. RNA was extracted for verification of TGF-beta1 gene silencing in the cells.

The basal levels of chemokine (IL-8 and MCP-1) and fibronectin protein in the TGF-beta1 gene-silenced and control cells were determined. Media was replaced 24 h after transfection with either TGF-beta1-specific or nonspecific siRNA, and conditioned media was collected at 48 h for measurement of IL-8, MCP-1, and fibronectin. The downstream Smad-2 signaling pathway, TGF-beta2 and TGF-beta3 isoforms, and the TGF-beta-responsive transcription factors AP-1 and NF-{kappa}B were assayed.

To determine whether TGF-beta2 and TGF-beta3 account for the increased fibronectin in TGF-beta1- silenced cells, HK-2 cells were transfected with TGF-beta1 siRNA, and 5 µg/ml pan-specific TGF-beta antibody was added 6 h after transfection. Treatment media was replaced 24 h after transfection, and the supernatant was collected at 48 h for measurement of fibronectin protein.

siRNA. RNA molecules (21 mer) were chemically synthesized (Ambion). The complementary oligonucleotides were 2'-deprotected, annealed, and purified by the manufacturer. The sequence targeting TGF-beta1 (accession no. NM_000660) was 5'-AAGGGCTACCATGCCAACTTC-3'.

RT-PCR. RNA was extracted using a RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA was treated with DNase I (Invitrogen) and then reverse transcribed using Superscript II RT (Invitrogen). Sequence-specific primers for human TGF-beta1, TGF-beta2, TGF-beta3, Smad2, beta-actin, and fibronectin are shown in Table 1. Amplified products were electrophoresed through 1.5% (wt/vol) agarose gels and visualized by ethidium bromide staining. Bands were scanned using Gel Documentation (Bio-Rad) and quantitated by densitometry using Quantity One software (Bio-Rad). beta-Actin was used as an internal control for sample normalization. To confirm that DNase I-treated RNA samples have no genomic DNA contamination, one negative control was always included without any Superscript II RT in reverse transcription, and subsequently this sample was used for PCR. There was no band observed on agarose gels in the negative control samples.


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  		Table 1. Sequence-specific primers for human TGF-beta1, -beta2, -beta3, Smad 2, beta-actin, and fibronectin

 
IL-8, MCP-1, TGF-beta1, and TGF-beta2 immunoassays. Conditioned media was collected and centrifuged at 3,000 rpm and 4°C for 10 min to remove cell debris and then stored at –80°C. IL-8, MCP-1, TGF-beta1, and TGF-beta2 were quantified using an ELISA according to the manufacturer’s instructions.

Western blotting. Conditioned media samples were subjected to SDS-PAGE under reducing conditions. Proteins were then transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech). Nonspecific binding sites were blocked overnight (5% nonfat milk and 0.1% Tween 20 in PBS) after which the membranes were incubated with primary fibronectin antibody for 2 h at room temperature, followed by washing four times, after which they were incubated with peroxidase-labeled secondary antibodies (Amersham Pharmacia Biotech) for 1 h and again washed four times. The blots were then detected using ECL (Amersham Pharmacia Biotech). The bands corresponding to fibronectin (220 kDa) were quantitated using Quantity One software (Bio-Rad). Coomassie brilliant blue staining was used to confirm that an equal amount of protein was loaded in each lane.

Immunocytochemical staining. Cells were fixed on coverslips using ice-cold 4% formalin for 30 min followed by washing three times in PBS. Cells were then permeabilized with 0.2% Triton X-100 for 20 min followed by three washings in PBS. Cells were treated with 4% H2O2 in PBS to quench the endogenous peroxidase activity, and then the nonspecific background was blocked using serum-free protein block (Dako). Cells were incubated with TGF-beta3 and pSmad2 primary antibodies diluted in 1:200 at room temperature for 1 h followed by three washings in PBS. LSAB2 system-horseradish peroxidase was used for detection of TGF-beta3 and pSmad2 according to the manufacturer’s instructions (Dako). Mayer’s hematoxylin was used to counterstain the cells. A negative control (no primary antibody) was performed. Coverslips were mounted on glass slides using Dako glycerol mounting media. Slides were blinded, and three random fields were digitized using a Nikon microscope attached to a digital camera.

EMSA. Nuclear extract was prepared using NucBuster Protein Extraction Kit (Novagen) according to the manufacturer’s instructions. AP-1 wild-type oligonucleotide sequences were 5'-CGCTTGATGAGTCAGCCGGAA-3' and 5'-TTCCGGCTGACTCATCAAGCG-3'; and NF-{kappa}B wild-type oligonucleotide sequences were 5'-CGCTTGATGACTCAGCCGGAA-3' and 5'-GCGAACTACTGAGTCGGCCTT-3'. A DIG Gel Shift Kit (Roche) was used to label AP-1 and NF-{kappa}B oligonucleotide 3'-ends with DIG-11-dUTP. In brief, 40 µg of nuclear extract were used in the binding reaction and incubated at room temperature for 1 h. The binding reactions were then electrophoresed through 6% nondenaturing polyacrylamide gels and transferred to a positively charged nylon membrane (Roche). The membrane was then cross linked using a UV transilluminator for 3 min. The membrane was subjected to immunological detection using DIG-AP conjugate provided in the DIG Gel Shift Kit. Chemiluminescence was detected using ECL hyperfilm (Amersham). Both shift bands and free probes were scanned and quantitated using Gel Documentation and Quantity One (Bio-Rad). Signal percentage [shift band vs. (shift band + free probe)·100%] was calculated, and results were expressed as a percentage of control values.

Statistical analysis. All results are expressed as a percentage of the control values (100%). Each experiment was performed independently a minimum of three times. Results are expressed as means ± SE. Statistical comparisons between groups were made by ANOVA, with pairwise multiple comparisons made by Fisher’s protected least-significant difference test. Analyses were performed using the software package Statview version 4.5 (Abacus Concepts, Berkeley, CA). P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TGF-beta1 siRNA-mediated gene silencing. TGF-beta1 siRNA (30 nM/l) decreased TGF-beta1 mRNA expression by 89 ± 2.8%. A RT-PCR representative gel is shown in Fig. 1A. Total and active TGF-beta1 secretion was also decreased by 66 ± 7.5 and 60 ± 0.5%, respectively (Fig. 1, B and C). Nonspecific siRNA is used as a control to exclude any effect caused by TGF-beta1 siRNA not specifically due to silencing of TGF-beta1. Transfection efficiency, estimated by transfecting HK-2 cells with green fluorescent protein plasmids (data not shown), was 85%, hence paralleled the reduction in expression of TGF-beta1.


Figure 1
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  		Fig. 1. Effectiveness of transforming growth factor (TGF)-beta1 small-interference (si) RNA-mediated gene silencing. Nonspecific or TGF-beta1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Forty-eight hours after transfection, RNA was extracted. TGF-beta1 mRNA levels were determined by RT-PCR. A representative RT-PCR gel is shown (A). Media was replaced at 24 h after transfection, and conditioned media was collected at 48 h for measurement of total (B) and active TGF-beta1 (C) using ELISA. Results are means ± SE and are standardized as a percentage of control. ***P < 0.0005 vs. control. **P < 0.005 vs. control; n = 3.

 
Basal secretion of IL-8 and MCP-1 decreased in TGF-beta1-silenced cells. The basal levels of IL-8 and MCP-1 in TGF-beta1-silenced cells decreased to 62.7 ± 5.4 and 74.3 ± 6.2% (both P < 0.05), respectively, compared with nonspecific control (Fig. 2, A and B). We have recently demonstrated that TGF-beta1 upregulates IL-8 and MCP-1 through a connective tissue growth factor-independent pathway (27). These data further confirm that TGF-beta1 plays a positive role in the regulation of IL-8 and MCP-1 in HK-2 cells.


Figure 2
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  		Fig. 2. Basal secretion of IL-8 and MCP-1 decreased in TGF-beta1-silenced cells. Nonspecific or TGF-beta1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Media was replaced at 24 h after transfection, and conditioned media was collected at 48 h for measurement of IL-8 and MCP-1 using ELISA. Results are means ± SE and are standardized as a percentage of control. *P < 0.05 vs. control; n = 6.

 
Enhanced basal secretion of fibronectin in TGF-beta1-silenced cells. Fibronectin mRNA expression paradoxically increased (299 ± 45% vs. control; P < 0.05; Fig. 3A), paralleled by an increase in fibronectin protein expression (161 ± 9.6% vs. control; P < 0.05; Fig. 3B) in TGF-beta1-silenced cells compared with cells transfected with nonspecific siRNA.


Figure 3
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  		Fig. 3. Enhanced basal secretion of fibronectin in the TGF-beta1-silenced cells. Nonspecific or TGF-beta1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Twenty-four hours after transfection, cells were replaced with HK-2 growth media for another 24 h, and then RNA was extracted to verify that the TGF-beta1 gene was effectively silenced. A: RT-PCR for fibronectin was performed. B: conditioned media samples were used to measure fibronectin production. Results are means ± SE and are standardized as a percentage of control. *P < 0.05 vs. control; n = 6.

 
TGF-beta1-induced fibronectin secretion in HK-2 cells has been previously demonstrated in our laboratory (27). The enhanced mRNA and protein levels of fibronectin in TGF-beta1-silenced cells was unexpected, and the mechanisms underlying the increased matrix production were further explored.

Increased TGF-beta2, TGF-beta3, and pSmad2 occurs in TGF-beta1-silenced cells. mRNA levels of TGF-beta2, TGF-beta3, and Smad2 significantly increased to 289 ± 76, 164 ± 31, and 195 ± 32% respectively, in TGF-beta1-silenced cells compared with cells transfected with nonspecific siRNA (all P < 0.05; Fig. 4, A, C, and E). TGF-beta2 protein was significantly increased in TGF-beta1-silenced cells to 149 ± 5.7% of that observed in cells transfected with nonspecific siRNA (P < 0.05; Fig. 4B). TGF-beta3 and pSmad2 expression was shown to be significantly increased when assessed immunocytochemically (Fig. 4, D and F, respectively). This supports the view that TGF-beta1 plays a critical role in regulating the other TGF-beta isoforms in the human kidney and suggests that TGF-beta1 endogenously functions to limit TGF-beta2 and TGF-beta3, which can also induce fibronectin production through activation of Smad2.


Figure 4
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  		Fig. 4. mRNA and protein levels of TGF-beta2, TGF-beta3, and Smad2 in the TGF-beta1-silenced cells. Nonspecific or TGF-beta1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Twenty-four hours after transfection, cells were replaced with HK-2 growth media for another 24 h, and then RNA was extracted to verify that the TGF-beta1 gene was effectively silenced. RT-PCR was used to measure mRNA levels of TGF-beta2 (A), TGF-beta3 (C), and Smad2 (E); ELISA was used to measure protein level of TGF-beta2 (B). Immunocytochemistry staining was used to measure protein levels of TGF-beta3 (D) and pSmad2 (F). TGF-beta1-silenced cells were either exposed to 5 µg/ml pan-specific TGF-beta antibody or 5 µg/ml rabbit IgG (negative control) 6 h after transfection. Treatment media was replaced at 24 h after transfection. A supernatant was collected at 48 h after transfection for measurement of fibronectin protein (G). ***P < 0.0005 vs. nonspecific siRNA. *P < 0.05 vs. nonspecific siRNA. ###P < 0.0005 vs. TGF-beta1 siRNA plus rabbit IgG; n = 3.

 
Increased fibronectin returned to the control level (cells transfected with nonspecific siRNA) when TGF-beta1-silenced cells were exposed to 5 µg/ml pan-specific TGF-beta antibody (Fig. 4G). These data further suggest that elevated levels of TGF-beta2 and TGF-beta3 account for the increased fibronectin in TGF-beta1-silenced cells,

DNA binding activity of AP-1 and NF-{kappa}B in TGF-beta1-silenced cells. AP-1 DNA binding activity increased to 162 ± 14% (P < 0.05) compared with cells transfected with nonspecific siRNA (Fig. 5A). In contrast, NF-{kappa}B DNA binding activity decreased to 18 ± 2% (P < 0.0005) compared with cells transfected with nonspecific siRNA (Fig. 5B). This suggests differential roles of these transcription factors in mediating fibrogenic and immunomodulatory responses.


Figure 5
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  		Fig. 5. Differential DNA binding activity of AP-1 and NF-{kappa}B in the TGF-beta1-silenced cells. Nonspecific or TGF-beta1 siRNAs (30 nM/l) were introduced into HK-2 cells, respectively, at subconfluence using Lipofectamine 2000. In parallel wells, to act as negative controls, cells were transfected with transfection reagent alone (Mock). Twenty-four hours after transfection, a nuclear extract was collected for measurement of AP-1 (A) and NF-{kappa}B (B) DNA binding activity using EMSA. Signal quantification of shift bands and free probes was performed and the ratio [shift band vs. (shift band + free probe)] was calculated. Results are means ± SE and are standardized as a percentage of control (nonspecific siRNA). ***P < 0.0005 vs. control. *P < 0.05 vs. control; n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The findings of the present studies reinforce the key role that TGF-beta plays in both profibrotic and inflammatory renal disease. However, our findings have clearly demonstrated that the inflammatory response in the kidney, leading to an upregulation of IL-8 and MCP-1, is specifically dependent on the TGF-beta1 isoform and its target transcription factor NF-{kappa}B. Unexpectedly, effective silencing of the TGF-beta1 isoform enhanced fibronectin expression in the proximal tubule, with further investigation suggesting that this was due to an associated upregulation in TGF-beta2 and -beta3 isoforms with targeting of the AP-1 transcription factor and downstream activation of the Smad2 signaling pathway. To our knowledge, this is the first report to demonstrate that upregulation of IL-8 and MCP-1 is uniquely dependent on TGF-beta1 and that silencing of the TGF-beta1 gene results in the increased expression of TGF-beta2, TGF-beta3, and AP-1 DNA binding activity.

All three TGF-beta isoforms have been previously reported to have fibrogenic effects on renal cells. Although TGF-beta1 is generally considered to be the major or predominant isoform involved in renal fibrosis, Wilson and colleagues (39) have demonstrated that all isoforms of TGF-beta induce fibronectin expression in rat mesangial cells and can contribute to pathological matrix accumulation in renal fibrosis repair. TGF-beta2 has been reported to be dominant in the acute phase of nephropathy in streptozotocin-induced type I diabetes in the rat (12), and treatment with a specific neutralizing antibody significantly reduced renal fibrosis in this model (13). In vitro, TGF-beta2 has been reported to induce extracellular matrix (fibronectin, collagen type I, collagen type IV) and play a fibrogenic role in both a human retinal pigment epithelial cell line and cultured human optic nerve head astrocytes (10, 28). TGF-beta3 has also been reported to decrease matrix metalloproteinase-2 and increase tissue inhibitor of metalloproteinase-1 and collagen type I in primary lung fibroblasts (26).

Prior studies have demonstrated the interdependence of the TGF-beta isoforms in different models of fibrogenesis. TGF-beta2 and TGF-beta3 have been shown to stimulate TGF-beta1 production in human fetal lung fibroblasts, rat mesangial cells, and untransformed lung and intestinal epithelial cells (38, 42, 43). This would be consistent with the present studies, as it is likely that the absence of TGF-beta1 production could lead to an unregulated upregulation of TGF-beta2 and -beta3 driving the increase in fibronectin secretion. TGF-beta1 and TGF-beta2 are equally potent in the activation of Smad2 (11, 23), whereas TGF-beta3 is also reported to increase the activation of Smad2 in folliculostellate cells and murine medial edge epithelium cells (5, 8). As Smad2 is known to mediate the downstream fibrogenic response of TGF-beta, an upregulation in the setting of enhanced fibronectin expression was not unexpected.

Our findings are consistent with the phenotypic observations in the rare clinical disorder Camurati-Engelmann disease, characterized by unregulated excessive fibrosis of skin, subcutaneous tissue, and organs and progressive diaphyseal dysplasia characterized by hyperosteosis and sclerosis of the diaphyses of long bones (16). Both activating and inactivating mutations of several components of the TGF-beta signaling pathway have been described, resulting in the same clinical phenotype (4, 14, 29). For example, Kinoshita et al. (16) hypothesize that domain-specific heterozygous missense mutations in TGF-beta1, with resultant inactive protein, is a likely cause of Camurati-Engelmann disease.

Conversely, in experimental models, Chen et al. (6) reported that fibronectin expression decreased in cultured tubular cells derived from TGF-beta1 knockout mice. Interestingly, in these studies a significant upregulation of TGF-beta3 expression was also observed, as was the expression of the type II receptor. However, constitutive expression of fibronectin was reduced. The explanation for these somewhat divergent results remains unclear.

In contrast to the paradoxical increase in fibronectin expression in the TGF-beta1-silenced cells, the production of the inflammatory chemokines IL-8 and MCP-1 was reduced. We have recently demonstrated that TGF-beta1 upregulates IL-8 and MCP-1 expression in proximal tubular epithelial cells (27). Hence, these results are consistent with the recognition that TGF-beta1 plays a central role in the modulation of inflammation in the tubular epithelium.

The differential effects of the effective silencing of TGF-beta1 on cytokines involved in the inflammatory pathways and proteins contributing to extracellular matrix production may be explained by the differential effects on the transcription factors AP-1 and NF-{kappa}B. Although both AP-1 and NF-{kappa}B have been reported to be involved in the regulation of renal inflammatory responses (25, 36), AP-1 activation has been more closely linked with renal fibrosis and NF-{kappa}B with inflammatory responses. TGF-beta1 enhances both AP-1 and NF-{kappa}B DNA binding activity (9, 17, 18). Conversely, TGF-beta2 has been shown to repress NF-{kappa}B activity in granule neurons (15) and TGF-beta3 has been shown to induce AP-1 in untransformed lung and intestinal epithelial cells (43). Although these findings are not uniformly observed (9, 22), our findings are consistent with these observations that AP-1 and NF-{kappa}B DNA binding activity and activation are differentially regulated by different isoforms of TGF-beta, resulting in divergent effects on the expression of potential participants in fibrotic and inflammatory pathways.

In summary, our studies in this in vitro model of tubular cells suggest that TGF-beta1 is specifically involved in the modification of cytokines potentially involved in inflammatory responses, but fibronectin expression is regulated by a complex interaction between the TGF-beta isoforms and their interacting transcription factors and downstream signaling pathways. A key role of TGF-beta1 in the proximal tubular cells of human kidney, identified in the present study, is to limit the production of the extracellular matrix protein fibronectin, induced by TGF-beta2 and -beta3.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by the National Health and Medical Research Council of Australia and the Juvenile Diabetes Research Foundation. W. Qi is supported by a National Health Medical Research Council Scholarship.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. A. Pollock, Dept. of Medicine, Level 3, Wallace Freeborn Professorial Block, Royal North Shore Hospital, St. Leonards, NSW, Australia 2065 (e-mail: carpol{at}med.usyd.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* W. Qi and X. Chen equally contributed to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bian ZM, Elner SG, Yoshida A, and Elner VM. Differential involvement of phosphoinositide 3-kinase/Akt in human RPE MCP-1 and IL-8 expression. Invest Ophthalmol Vis Sci 45: 1887–1896, 2004.[Abstract/Free Full Text]
  2. Bottinger EP, Letterio JJ, and Roberts AB. Biology of TGF-beta in knockout and transgenic mouse models. Kidney Int 51: 1355–1360, 1997.[ISI][Medline]
  3. Brat DJ, Bellail AC, and Van Meir EG. The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro-oncol 7: 122–133, 2005.[Abstract]
  4. Campos-Xavier B, Saraiva JM, Savarirayan R, Verloes A, Feingold J, Faivre L, Munnich A, Le Merrer M, and Cormier-Daire V. Phenotypic variability at the TGF-beta1 locus in Camurati-Engelmann disease. Hum Genet 109: 653–658, 2001.[CrossRef][ISI][Medline]
  5. Chaturvedi K and Sarkar DK. Role of protein kinase C-Ras-MAPK p44/42 in ethanol and transforming growth factor-beta3-induced basic fibroblast growth factor release from folliculostellate cells. J Pharmacol Exp Ther 314: 1346–1352, 2005.[Abstract/Free Full Text]
  6. Chen S, Hoffman BB, Lee JS, Kasama Y, Jim B, Kopp JB, and Ziyadeh FN. Cultured tubule cells from TGF-beta1 null mice exhibit impaired hypertrophy and fibronectin expression in high glucose. Kidney Int 65: 1191–1204, 2004.[CrossRef][ISI][Medline]
  7. Chen S, Mukherjee S, Chakraborty C, and Chakrabarti S. High glucose-induced, endothelin-dependent fibronectin synthesis is mediated via NF-{kappa}B and AP-1. Am J Physiol Cell Physiol 284: C263–C272, 2003.[Abstract/Free Full Text]
  8. Cui XM, Chai Y, Chen J, Yamamoto T, Ito Y, Bringas P, and Shuler CF. TGF-beta3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion. Dev Dyn 227: 387–394, 2003.[CrossRef][ISI][Medline]
  9. Dhandapani KM, Hadman M, De Sevilla L, Wade MF, Mahesh VB, and Brann DW. Astrocyte protection of neurons: role of transforming growth factor-beta signaling via a c-Jun-AP-1 protective pathway. J Biol Chem 278: 43329–43339, 2003.[Abstract/Free Full Text]
  10. Fuchshofer R, Birke M, Welge-Lussen U, Kook D, and Lutjen-Drecoll E. Transforming growth factor-beta 2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes. Invest Ophthalmol Vis Sci 46: 568–578, 2005.[Abstract/Free Full Text]
  11. Hartner A, Hilgers KF, Bitzer M, Veelken R, and Schocklmann HO. Dynamic expression patterns of transforming growth factor-beta2 and transforming growth factor-beta receptors in experimental glomerulonephritis. J Mol Med 81: 32–42, 2003.[ISI][Medline]
  12. Hill C, Flyvbjerg A, Gronbaek H, Petrik J, Hill DJ, Thomas CR, Sheppard MC, and Logan A. The renal expression of transforming growth factor-beta isoforms and their receptors in acute and chronic experimental diabetes in rats. Endocrinology 141: 1196–1208, 2000.[Abstract/Free Full Text]
  13. Hill C, Flyvbjerg A, Rasch R, Bak M, and Logan A. Transforming growth factor-beta2 antibody attenuates fibrosis in the experimental diabetic rat kidney. J Endocrinol 170: 647–651, 2001.[Abstract]
  14. Janssens K, Gershoni-Baruch R, Guanabens N, Migone N, Ralston S, Bonduelle M, Lissens W, Van Maldergem L, Vanhoenacker F, Verbruggen L, and Van Hul W. Mutations in the gene encoding the latency-associated peptide of TGF-beta 1 cause Camurati-Engelmann disease. Nat Genet 26: 273–275, 2000.[CrossRef][ISI][Medline]
  15. Kaltschmidt B and Kaltschmidt C. DNA array analysis of the developing rat cerebellum: transforming growth factor-beta2 inhibits constitutively activated NF-kappaB in granule neurons. Mech Dev 101: 11–19, 2001.[CrossRef][ISI][Medline]
  16. Kinoshita A, Saito T, Tomita H, Makita Y, Yoshida K, Ghadami M, Yamada K, Kondo S, Ikegawa S, Nishimura G, Fukushima Y, Nakagomi T, Saito H, Sugimoto T, Kamegaya M, Hisa K, Murray JC, Taniguchi N, Niikawa N, and Yoshiura K. Domain-specific mutations in TGFB1 result in Camurati-Engelmann disease. Nat Genet 26: 19–20, 2000.[CrossRef][ISI][Medline]
  17. Komura E, Tonetti C, Penard-Lacronique V, Chagraoui H, Lacout C, Lecouedic JP, Rameau P, Debili N, Vainchenker W, and Giraudier S. Role for the nuclear factor kappaB pathway in transforming growth factor-beta1 production in idiopathic myelofibrosis: possible relationship with FK506 binding protein 51 overexpression. Cancer Res 65: 3281–3289, 2005.[Abstract/Free Full Text]
  18. Konig HG, Kogel D, Rami A, and Prehn JH. TGF-beta1 activates two distinct type I receptors in neurons: implications for neuronal NF-kappaB signaling. J Cell Biol 168: 1077–1086, 2005.[Abstract/Free Full Text]
  19. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, Roberts AB, Sporn MB, Ward JM, and Karlsson S. Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci USA 90: 770–774, 1993.[Abstract/Free Full Text]
  20. Kwon D, Fuller AC, Palma JP, Choi IH, and Kim BS. Induction of chemokines in human astrocytes by picornavirus infection requires activation of both AP-1 and NF-kappa B. Glia 45: 287–296, 2004.[CrossRef][ISI][Medline]
  21. Lee BH, Park SY, Kang KB, Park RW, and Kim IS. NF-kappaB activates fibronectin gene expression in rat hepatocytes. Biochem Biophys Res Commun 297: 1218–1224, 2002.[CrossRef][ISI][Medline]
  22. Lu T, Burdelya LG, Swiatkowski SM, Boiko AD, Howe PH, Stark GR, and Gudkov AV. Secreted transforming growth factor beta2 activates NF-kappaB, blocks apoptosis, and is essential for the survival of some tumor cells. Proc Natl Acad Sci USA 101: 7112–7117, 2004.[Abstract/Free Full Text]
  23. Molin DG, Poelmann RE, DeRuiter MC, Azhar M, Doetschman T, and Gittenberger-de Groot AC. Transforming growth factor beta-SMAD2 signaling regulates aortic arch innervation and development. Circ Res 95: 1109–1117, 2004.[Abstract/Free Full Text]
  24. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, and Sheppard D. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96: 319–328, 1999.[CrossRef][ISI][Medline]
  25. Panchapakesan U, Sumual S, Pollock CA, and Chen X. PPAR{gamma} agonists exert antifibrotic effects in renal tubular cells exposed to high glucose. Am J Physiol Renal Physiol 289: F1153–F1158, 2005.[Abstract/Free Full Text]
  26. Papakonstantinou E, Aletras AJ, Roth M, Tamm M, and Karakiulakis G. Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts. Cytokine 24: 25–35, 2003.[CrossRef][ISI][Medline]
  27. Qi W, Chen X, Polhill TS, Sumual S, Twigg S, Gilbert RE, and Pollock CA. Transforming growth factor-beta1 induces IL-8 and MCP-1 through a connective tissue growth factor-independent pathway. Am J Physiol Renal Physiol 290: F703–F709, 2006.[Abstract/Free Full Text]
  28. Saika S, Yamanaka O, Ikeda K, Kim-Mitsuyama S, Flanders KC, Yoo J, Roberts AB, Nishikawa-Ishida I, Ohnishi Y, Muragaki Y, and Ooshima A. Inhibition of p38MAP kinase suppresses fibrotic reaction of retinal pigment epithelial cells. Lab Invest 85: 838–850, 2005.[CrossRef][ISI][Medline]
  29. Saito T, Kinoshita A, Yoshiura K, Makita Y, Wakui K, Honke K, Niikawa N, and Taniguchi N. Domain-specific mutations of a transforming growth factor (TGF)-beta 1 latency-associated peptide cause Camurati-Engelmann disease because of the formation of a constitutively active form of TGF-beta 1. J Biol Chem 276: 11469–11472, 2001.[Abstract/Free Full Text]
  30. Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, Cardell EL, and Doetschman T. TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development 124: 2659–2670, 1997.[Abstract]
  31. Schnaper HW, Hayashida T, and Poncelet AC. It’s a Smad world: regulation of TGF-beta signaling in the kidney. J Am Soc Nephrol 13: 1126–1128, 2002.[Free Full Text]
  32. Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N, and Doetschman T. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359: 693–699, 1992.[CrossRef][Medline]
  33. Tamura K, Nyui N, Tamura N, Fujita T, Kihara M, Toya Y, Takasaki I, Takagi N, Ishii M, Oda K, Horiuchi M, and Umemura S. Mechanism of angiotensin II-mediated regulation of fibronectin gene in rat vascular smooth muscle cells. J Biol Chem 273: 26487–26496, 1998.[Abstract/Free Full Text]
  34. Tchilibon S, Zhang J, Yang Q, Eidelman O, Kim H, Caohuy H, Jacobson KA, Pollard BS, and Pollard HB. Amphiphilic pyridinium salts block TNF alpha/NF kappa B signaling and constitutive hypersecretion of interleukin-8 (IL-8) from cystic fibrosis lung epithelial cells. Biochem Pharmacol 70: 381–393, 2005.[CrossRef][ISI][Medline]
  35. Tseng L, Tang M, Wang Z, and Mazella J. Progesterone receptor (hPR) upregulates the fibronectin promoter activity in human decidual fibroblasts. DNA Cell Biol 22: 633–640, 2003.[CrossRef][ISI][Medline]
  36. Wang W, Huang XR, Li AG, Liu F, Li JH, Truong LD, Wang XJ, and Lan HY. Signaling mechanism of TGF-beta1 in prevention of renal inflammation: role of Smad7. J Am Soc Nephrol 16: 1371–1383, 2005.[Abstract/Free Full Text]
  37. Weigert C, Brodbeck K, Brosius FC III, Huber M, Lehmann R, Friess U, Facchin S, Aulwurm S, Haring HU, Schleicher ED, and Heilig CW. Evidence for a novel TGF-beta1-independent mechanism of fibronectin production in mesangial cells overexpressing glucose transporters. Diabetes 52: 527–535, 2003.[Abstract/Free Full Text]
  38. Wen FQ, Kohyama T, Skold CM, Zhu YK, Liu X, Romberger DJ, Stoner J, and Rennard SI. Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts. Inflammation 27: 9–19, 2003.[CrossRef][ISI][Medline]
  39. Wilson HM, Minto AW, Brown PA, Erwig LP, and Rees AJ. Transforming growth factor-beta isoforms and glomerular injury in nephrotoxic nephritis. Kidney Int 57: 2434–2444, 2000.[CrossRef][ISI][Medline]
  40. Xiao YQ, Malcolm K, Worthen GS, Gardai S, Schiemann WP, Fadok VA, Bratton DL, and Henson PM. Cross-talk between ERK and p38 MAPK mediates selective suppression of pro-inflammatory cytokines by transforming growth factor-beta. J Biol Chem 277: 14884–14893, 2002.[Abstract/Free Full Text]
  41. Yamada M, Kim S, Egashira K, Takeya M, Ikeda T, Mimura O, and Iwao H. Molecular mechanism and role of endothelial monocyte chemoattractant protein-1 induction by vascular endothelial growth factor. Arterioscler Thromb Vasc Biol 23: 1996–2001, 2003.[Abstract/Free Full Text]
  42. Yu L, Border WA, Huang Y, and Noble NA. TGF-beta isoforms in renal fibrogenesis. Kidney Int 64: 844–856, 2003.[CrossRef][ISI][Medline]
  43. Yue J and Mulder KM. Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a smad-dependent pathway. J Biol Chem 275: 35656, 2000.[Free Full Text]



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