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International Immunology Advance Access originally published online on November 29, 2006
International Immunology 2007 19(2):117-126; doi:10.1093/intimm/dxl128
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

TGF-ß type I receptor kinase inhibitor down-regulates rheumatoid synoviocytes and prevents the arthritis induced by type II collagen antibody

Michitomo Sakuma1,2, Kyosuke Hatsushika1, Kensuke Koyama1, Ryohei Katoh3, Takashi Ando1, Yoshiyuki Watanabe1, Masanori Wako1, Mirei Kanzaki1, Shinichi Takano1, Hajime Sugiyama2, Yoshiki Hamada2, Hideoki Ogawa4, Ko Okumura4 and Atsuhito Nakao1,4

1 Department of Immunology
2 Department of Orthopaedic Surgery
3 Department of Human Pathology, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan
4 Atopy Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan

Correspondence to: A. Nakao; E-mail: anakao{at}yamanashi.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is characterized by hypertrophic synovial tissues comprising excessively proliferating synovial fibroblasts and infiltrating inflammatory cells. Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that regulates cell growth, inflammation and angiogenesis by acting on various cell types. In RA synovial tissues, TGF-ß is expressed at high levels. However, the precise role of TGF-ß in RA remains unclear. We herein demonstrated a causal link between the TGF-ß-induced RA synovial cell proliferation and induction of platelet-derived growth factor (PDGF)-AA. In addition, TGF-ß induced IL-6 and vascular endothelial growth factor (VEGF) production by RA synovial fibroblasts associated with nuclear factor-kappa B activation. These effects of TGF-ß on RA synovial fibroblasts were suppressed by TGF-ß type I receptor kinase inhibitor HTS466284. Furthermore, HTS466284 significantly prevented anti-collagen type II antibody-induced arthritis in mice according to the clinical manifestations, histology, tumor necrosis factor-{alpha}, PDGF and VEGF expression and 5-bromo-2'-deoxyuridine incorporation. These in vitro and in vivo results suggest that TGF-ß plays a role in the development of synovial hyperplasia consisting of synovial cell proliferation, inflammation and angiogenesis. The blockade of TGF-ß signaling may thus become an additional strategy for the treatment of RA.

Keywords: rheumatoid arthritis


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily affects the synovial tissue in multiple joints associated with auto-antibody production such as anti-collagen (CL) type II antibody (OMIM no. 180300 [OMIM] ). RA synovial tissue is characterized by synovial hyperplasia with an increased number of synovial fibroblasts, infiltrating inflammatory cells consisting predominantly of macrophages and lymphocytes and angiogenesis (1, 2). Although the pathogenesis of RA remains unclear, pro-inflammatory cytokines such as tumor necrosis factor-{alpha} (TNF-{alpha}) and IL-1ß produced by synovial fibroblasts and immune cells are known to play a pivotal role in the maintenance of RA based on clinical trials with clearly positive results (1, 3–5). However, as many as 20% of RA patients do not yet respond to anti-TNF-{alpha} or IL-1ß therapy, thus suggesting that other factors also contribute to RA.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that regulates cell growth, differentiation and survival and it is involved in various human disorders including cancer and fibrotic diseases (6). TGF-ß binds to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptor is activated by type II receptor upon ligand binding and transduces signals principally through Smad proteins by inducing the phosphorylation of the proteins with its kinase activity (7). Activin-like kinase (ALK)-5 is a ubiquitously expressed type I receptor for TGF-ß, transmitting signals through Smad2/3. ALK-1 is also a type I receptor for TGF-ß, which is predominantly expressed in vascular endothelial cells, and transmits signals through Smad1/5. The wide-ranging interest in developing therapeutics targeting TGF-ß has been recently raised regarding the inhibition of cancer and fibrotic diseases (8, 9). Several small-molecule inhibitors of TGF-ß type I receptor kinase activity have been recently developed and are thus considered to be one of the promising reagents for the treatment of cancer and fibrotic diseases (10–15). TGF-ß type I receptor kinase inhibitors, which have already been developed, have been demonstrated to efficiently suppress the kinase activity of TGF-ß type I receptor ALK-5, but not ALK-1, probably due to structural differences between ALK-5 and ALK-1 (11–15).

In RA, an analysis of the cytokine mRNA and proteins in the RA synovial tissue revealed that TGF-ß is expressed at high levels in RA patients (16–19) as well as TNF-{alpha} and IL-1ß, thus suggesting its potential role for RA. However, the precise role of TGF-ß in RA appears to remain controversial. Brandes et al. (20) reported that the intra-peritoneal administration of TGF-ß suppressed both the acute and chronic arthritis induced by streptococcal cell wall (SCW) fragments into susceptible rats and arthritis induced by CL type II in DBA/1 mice. Furthermore, the intra-peritoneal injection of neutralizing TGF-ß antibody enhanced the arthritis induced by CL type II in DBA/1 mice (21). These results suggest that exogenous or endogenous TGF-ß functions as anti-arthritic. In contrast, Allen et al. (22) and Fava et al. (23) showed that an injection of TGF-ß into the joint cavity of rats induced synovial erythema, swelling and leukocyte infiltration. In addition, the intra-articular injection of neutralizing TGF-ß antibody suppressed the acute and chronic arthritis induced by SCW in rats (24). These results suggest that exogenous or endogenous TGF-ß functions as pro-arthritic. The differential regulation of inflammation by local and systemic TGF-ß may explain these apparently opposite results (25), but this issue remains to be unresolved. In addition, there have been few studies that have clearly shown the effects of TGF-ß on synovial fibroblasts derived from patients with RA in detailed mechanical analysis. Furthermore, only a few reports have so far compared the responses of RA synovial fibroblasts with other fibroblasts such as dermal fibroblasts (26).

In this study, we thus sought to define the roles of TGF-ß in the development of RA by examining the effects of TGF-ß on RA synovial fibroblasts and on a different animal model of arthritis in more detail. Our results suggest that TGF-ß is pro-arthritic in the development of RA and the intervention of endogenous TGF-ß signaling may thus have a therapeutic potential for RA.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents
Recombinant human TGF-ß1 was purchased from R&D Inc. (Minneapolis, MN, USA). A selective small-molecule inhibitor of TGF-ß type I receptor kinase HTS466284 (11), a platelet-derived growth factor (PDGF) receptor kinase inhibitor AG1296 and a nuclear factor-kappa B (NF-{kappa}B) inhibitor BAY11-7082 were purchased from Calbiochem (San Diego, CA, USA). An NF-{kappa}B inhibitor Helenalin was purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, USA).

Cell culture
Human synovial fibroblasts were obtained as previously described (27). In brief, after enzymatic digestion, human synovial cells were isolated from synovial tissues of the knee joints of RA at the time of total knee arthroplasty operations. The cells were suspended in DMEM (Invitrogen–GIBCO, Carlsbad, CA, USA) containing 10% FCS and 100 µg ml–1 streptomycin, and then were cultured in monolayers. After three to five passages, the subcultured cells were composed of morphologically uniform fibroblastic cells (synovial fibroblasts) that were free of macrophages. A human bronchial epithelial cell line, BEAS2B (28), was maintained in F-12 Nutrient Mixture medium (HAM) with L-glutamine (GIBCO/Invitrogen) supplemented with 10% FCS and 100 µg ml–1 streptomycin.

Cell viability assay
RA synovial fibroblasts (5 x 103 cells per well) were cultured in DMEM containing 0.1% FCS with or without indicated doses of TGF-ß1 for 48 h in a flat-bottom 96-well microtiter plate. In some experiments, the indicated doses of HTS466284 or AG1296 were added to the culture. Cell viability was then determined by measuring the metabolic activity using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)[2H]tetrazolium monosodium salt (WST) using Tetra Color ONE kit (Seikagaku Corporation, Tokyo, Japan) according to the manufacturer's instructions. In our preliminary studies, the metabolic activity measured in this assay was proportional to the cell number which was directly counted by a trypan blue exclusion assay.

Reverse transcription–PCR
Total RNA was extracted from cells (1 x 106) using the Isogen solution (Nippon Gene, Toyama, Japan) as recommended by the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using the first-strand cDNA synthesis kit (Ready To Go) (Amersham Biosciences Corp., Piscataway, NJ, USA). PCR amplification [PDGF-AA and PDGF-BB: 94°C for 45 s, 55°C for 45 s and 72°C for 45 s, 35 cycles; Smad7 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 94°C for 60 s, 55°C for 60 s and 72°C for 60 s, 35 cycles] was performed in a DNA engine cycler (MJ Research, Inc., Waltham, MA, USA). The PCR products were separated by 2.0% agarose gel electrophoresis and stained with 0.5 µg ml–1 ethidium bromide. The primer pairs (PDGF-AA, PDGF-BB) used were purchased from R&D Inc. Human Smad7 and GAPDH primers are as follows—Smad7: forward, 5'-CATCACCTTAGCCGACTCTG-3' and reverse, 5'-GTCTTCTCCTCCCAGTATGC-3' and GAPDH: forward, 5'-CCACCCATGGCAAATTCCATGGCA-3' and reverse, 5'-TCAAGACGGCAGGTCAGGTCCACC-3'.

Quantitative real-time PCR
Total RNA was extracted from cells (1 x 106) as described above and cDNA was synthesized from 2 µg of total RNA using Reverse Transcriptase System (Applied Biosystems). A quantitative PCR analysis was performed using the ABI7500 real-time PCR system (Applied Biosystems) according to the manufacturer's instructions. The primers and probes for c-Myc and GAPDH were purchased from Applied Biosystems. The ratio of each gene to that of GAPDH was calculated, and the value of 1.0 was assigned to cells that were incubated without TGF-ß1.

Western blotting
Cells (1 x 106) cultured in standard medium were lysed and suspended in RIPA buffer [20 mM Tris–HCl (pH 8.0), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, 0.5% deoxycholate and 0.1% SDS] supplemented with 0.1 mM phenylmethylsulfonylfluoride, 100 mM Na3VO4 and 20 mM ß-glycerophosphate. The extracts were cleared by centrifugation. Whole-cell extracts (20 µg protein) were separated on 9–15% SDS–polyacrylamide gels and then were transferred onto Immobilon-P membrane (Millipore, Billerica, MA, USA). The detection of specific proteins was performed by using antibodies for c-Myc (Calbiochem), phosphorylated Smad2, Smad2/3 (Upstate Biotechnology Inc., Lake Placid, NY, USA), TGF-ß type I receptor and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive bands were detected by enhanced chemiluminescence (ECL-plus; Amersham Life Science, Piscataway, NJ, USA) using HRP-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology Inc.). The density of detected bands was evaluated in a Chemi Doc XRS-J (version 1; Bio-Rad Laboratories, Hercules, CA, USA) and Quantity One software (version 4.5; Bio-Rad Laboratories).

Cytokine array
The amounts of several cytokines in the culture supernatants were determined using a cytokine protein array (TranSignal Human Cytokine Antibody Array 1.0, Panomics, Inc., Redwood City, CA, USA) according to the manufacturer's instructions.

ELISA
The amounts of IL-6 or vascular endothelial growth factor (VEGF) in the culture supernatants and the amounts of serum TNF-{alpha} were determined by using the human IL-6, VEGF and mouse TNF-{alpha} ELISA kits (R&D Inc.) according to the manufacturer's instructions.

Reporter assay
RA synovial fibroblasts (2.5 x 104) were plated in 24-well plate in 0.5 ml medium. At 12 h after plating, the cells were transfected with 200 ng of NF-{kappa}B reporter plasmid (NF-{kappa}B TransLucent Reporter Vector, Panomics, Inc.), which detected the activation of NF-{kappa}B, and 5 ng pRL-CMV (Promega, Madison, WI, USA) using FuGENE6 transfection reagent (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. At 8 h after transfection, 10 ng ml–1 TGF-ß was added into the culture with or without 10 µM HTS466284. After 72 h of stimulation, the cells were harvested and treated with a PikaGene dual SeaPansy Luminescence detection kit (Toyo Ink, Tokyo, Japan) in order to measure the luciferase activity. The luminescence was measured with Gene Light 55 (Microtec Nition, Chiba, Japan). The pRL-CMV plasmid was used as an internal control of transfection efficiency.

Induction of arthritis
BALB/c mice were purchased from SLC (Tokyo, Japan) and kept under specific pathogen-free conditions. The arthritogenic anti-CL type II mAb cocktails obtained from Chondrex (Seattle, WA, USA) contains four mAbs (F10, A2, D8 and D1) in equal amounts. To establish anti-CL type II antibody-induced arthritis (29), the mice were intra-peritoneally injected with 2 mg per mouse of anti-CL type II mAb cocktail (day 1) and 3 days later (day 4) with 50 µg per mouse of LPS (Chondrex). HTS466284 (11) or a control vehicle [dimethyl sulfoxide (DMSO)] was intra-peritoneally administered on day 5 and thereafter every day until sacrifice. The animal experiments were approved by the Institutional Review Board of the University of Yamanashi.

Clinical evaluation of arthritis
Starting on day 1 after mAb injection, the mice were blindly inspected for disease progression. The clinical severity of the disease was scored using a scoring system based on the number of inflamed joints in forepaws, hind paws and ankles, inflammation being defined by swelling: 0, normal; 1, slight swelling; 2, mild swelling; 3, moderate swelling and 4, severe swelling. All paws and ankles were graded, thus resulting in a maximal clinical score of 24 per mouse, and then were expressed as the mean arthritic index on a given day.

Histology
The hind paws (tarsocrural joint) were removed post-mortem on day 11 after mAb injection, fixed and decalcified. The decalcified paws were embedded in paraffin, sectioned and stained with hematoxylin and eosin. For immunohistochemistry, the paws were immediately dissected and frozen in OCT compound in liquid nitrogen. The samples were stored at –80°C until cryosectioned. The sections were stained with anti-PDGF-A and anti-VEGF antibodies (Santa Cruz Biotechnology Inc.) through the use of peroxidase-based VECTASTAIN ABC kits with diaminobenzidene substrate (Vector Laboratories, Burlingame, CA, USA).

5-Bromo-2'-deoxyuridine incorporation
The mice were given 5-bromo-2'-deoxyuridine (BrdU) on day 8 after the injection of anti-CL type II antibody at 4 h before sacrifice to mark proliferating cells. The synovial tissue sections were incubated with anti-BrdU mAb (Dako Cytomation, Hamburg, Germany) followed by HRP-conjugated goat anti-mouse Ig (Dako Cytomation). BrdU-positive cells with synovial cell-like morphology were counted at x400 and are expressed as a percentage of the number of the BrdU-positive cells in the total of 100 synovial fibroblasts.

Data analysis
The data are summarized as the mean ± SD. The unpaired Student's t-test was used for the statistical analysis of the results. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
TGF-ß stimulates proliferation of RA synovial fibroblasts via PDGF induction
To investigate the roles of TGF-ß in the development of RA, we first examined the effects of TGF-ß on proliferation of synovial fibroblasts derived from patients with RA. TGF-ß significantly stimulated the proliferation of RA synovial fibroblasts in a dose-dependent manner based on the assay by detecting the amounts of cellular metabolites (WST assay) (Fig. 1A). The direct counting of the cell number at 72 h after TGF-ß stimulation also showed a significant increase in the cell number at the dose of 10 ng ml–1 TGF-ß (Fig. 1B). Since PDGF or fibroblast growth factor up-regulation by TGF-ß has been proposed to be a mechanism leading to the growth stimulation by TGF-ß in fibroblasts (30, 31), we examined the effects of PDGF receptor kinase inhibitor AG1296 on TGF-ß-induced proliferation of RA synovial fibroblasts. TGF-ß-induced proliferation was abolished by the addition of 5 µM AG1296 in RA synovial fibroblasts (Fig. 1C). In line with these findings, a reverse transcription–PCR analysis showed that TGF-ß induced the mRNA expression of PDGF-AA, but not PDGF-BB, in RA synovial fibroblasts (Fig. 2A). Furthermore, western blot and real-time PCR analyses showed that TGF-ß induced the mRNA and protein expression of a cell cycle regulator, c-Myc (32), in RA synovial fibroblasts (Fig. 2B and C). Interestingly, the TGF-ß-induced c-Myc mRNA and protein expressions were not inhibited by AG1296 (Fig. 2B and C), thus suggesting that the TGF-ß-induced c-Myc expression was independent of PDGF receptor signaling.


Figure 1
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  		Fig. 1. TGF-ß stimulates the proliferation of RA synovial fibroblasts. (A) RA synovial fibroblasts were cultured in DMEM containing 0.1% FCS in the presence or absence of the indicated doses of TGF-ß for 48 h (left panel) or cultured in the presence of 10 ng ml–1 TGF-ß with or without the indicated doses of TGF-ß type I receptor kinase inhibitor HTS466284 for 48 h (right panel). Cellular viability was then estimated by a WST assay. The data were represented as OD450. (B) RA synovial fibroblasts (3.0 x 104 cells) were cultured in DMEM containing 0.1% FCS in the presence or absence of the indicated doses of TGF-ß for 72 h and then direct cell counting was performed by a trypan blue exclusion assay. (C) RA synovial fibroblasts were cultured in the presence or absence of 10 ng ml–1TGF-ß with or without 5 µM AG1296, an inhibitor of PDGF receptor kinase, for 48 h. Cellular viability was then estimated by a WST assay. The data were represented as OD450. Values represent the mean ± SD. *P < 0.05 compared with the corresponding control. Representative results of four independent experiments using synovial fibroblasts derived from four RA patients are shown (n = 4).

 

Figure 2
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  		Fig. 2. TGF-ß induces PDGF-AA and c-Myc expression in RA synovial fibroblasts. (A) RA synovial fibroblasts cultured in DMEM containing 0.1% FCS were stimulated with 10 ng ml–1 TGF-ß for the indicated hours with or without 10 µM HTS466284. RNA was then extracted from the cells and reverse transcription–PCR using specific primers for PDGF-AA and -BB, Smad7 and GAPDH was performed. (B) RA synovial fibroblasts cultured in DMEM containing 0.1% FCS were stimulated with 10 ng ml–1 TGF-ß for 24 h in the presence or absence of 10 µM HTS466284 or 5 µM AG1296. The cells were then lysed and a western blot analysis with specific antibodies against human c-Myc and ß-actin was performed. The density of the detected bands (c-Myc and ß-actin) was quantified in a Chemi Doc XRS-J, and the ratio (c-Myc/ß-actin) was calculated and presented below the western blot panel. (C) RA synovial fibroblasts cultured in DMEM containing 0.1% FCS were stimulated with 10 ng ml–1 TGF-ß for the indicated hours with or without 10 µM HTS466284 or 5 µM AG1296. RNA was then extracted from the cells and real-time PCR was performed using specific primers and probes for c-Myc and GAPDH. The ratio of each gene to that of GAPDH was calculated, and the value of 1.0 was assigned to cells that were incubated without TGF-ß1. (D) The cell lysates of cultured RA synovial fibroblasts or human bronchial epithelial cell line BEAS2B (as a positive control) were subjected to a western blot analysis with a specific antibody against TGF-ß type I receptor. (E) RA synovial fibroblasts cultured in DMEM containing 0.1% FCS were stimulated with 10 ng ml–1 TGF-ß for the indicated times in the presence or absence of 10 µM HTS466284. The cells were then lysed and a western blot analysis with specific antibodies against phosphorylated Smad2 and Smad2/3 was performed (Smad2, upper band and Smad3, lower band). Values represent the mean ± SD. *P < 0.05 compared with corresponding control. Representative results of four independent experiments using synovial fibroblasts derived from four RA patients are shown (n = 4).

 
To determine whether the TGF-ß-induced proliferation depends on the kinase activity of TGF-ß type I receptor ALK-5, we examined the effects of TGF-ß type I receptor kinase inhibitor HTS466284 on TGF-ß-induced activation of RA synovial fibroblasts. We confirmed that RA synovial fibroblasts expressed a significant amount of TGF-ß type I receptor (Fig. 2D) and 10 µM HTS466284 significantly inhibited TGF-ß-induced mRNA expression of Smad7, which is a well-known direct target gene of TGF-ßR-/Smad-mediated signaling (33) (Fig. 2A). HTS466284 (10 µM) also suppressed TGF-ß-induced phosphorylation of Smad2 in RA synovial fibroblasts (Fig. 2E). These results confirmed the efficacy of HTS466284 for suppressing TGF-ß signaling on a cell-based assay. As shown in Figs 1(A) and 2(A–C), 10 µM HTS466284 significantly suppressed the TGF-ß-induced proliferation, PDGF-AA expression and c-Myc expression in RA synovial fibroblasts.

TGF-ß induces IL-6 and VEGF production in RA synovial fibroblasts via NF-{kappa}B activation
We next examined the effects of TGF-ß on cytokine productions by RA synovial fibroblasts using a cytokine protein array. TGF-ß strongly induced IL-6 and VEGF and, less matrix metalloproteinase 3 (MMP3), production by RA synovial fibroblasts (Fig. 3A). Interestingly, the RA synovial fibroblasts constitutively produced IL-4, which was suppressed by TGF-ß (Fig. 3A). Helenalin (5 µM) and BAY11-7082, which are inhibitors of NF-{kappa}B, suppressed the TGF-ß-induced IL-6 and VEGF production in RA synovial fibroblasts without affecting cell viability (Fig. 3B and data not shown), thus suggesting the NF-{kappa}B pathway to be involved in the cytokine production. Interestingly, the inhibition of TGF-ß-induced IL-6, but not VEGF, production by Helenalin was partial, thus suggesting that other pathways are involved in the IL-6 production. Consistent with these findings, TGF-ß increased the luciferase activity of NF-{kappa}B reporter plasmid, thereby detecting the activation of NF-{kappa}B, after the transfection of the construct into RA synovial fibroblasts (Fig. 3C). Again, 10 µM HTS466284 suppressed TGF-ß-induced IL-6, VEGF and MMP3 production and the activation of NF-{kappa}B reporter construct in RA synovial fibroblasts (Fig. 3A–C). HTS466284 alone did not affect the luciferase activity of the NF-{kappa}B reporter plasmid (data not shown).


Figure 3
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  		Fig. 3. TGF-ß induces IL-6 and VEGF production in RA synovial fibroblasts through the activation of NF-{kappa}B. (A) RA synovial fibroblasts were cultured in DMEM containing 0.1% FCS in the presence or absence of 10 ng ml–1 TGF-ß with or without 10 µM TGF-ß type I receptor kinase inhibitor HTS466284 for 48 h. The supernatants were then collected and subjected to a cytokine protein array. The table presented in the bottom indicates the corresponding cytokines on the protein array membrane. (B) RA synovial fibroblasts were cultured in DMEM containing 0.1% FCS in the presence or absence of 10 ng ml–1 TGF-ß with or without 5 µM Helenalin for 48 h. The supernatants were then collected and IL-6 and VEGF concentrations were measured by ELISA. (C) RA synovial fibroblasts cultured in DMEM containing 10% FCS were transfected with NF-{kappa}B-Luc reporter plasmid. Eight hours after the transfection, the culture medium was changed to DMEM containing 0.1% FCS and cells were stimulated with or without 10 ng ml–1 TGF-ß in the presence or absence of 10 µM HTS466284. Seventy-two hours after the stimulation, the cells were harvested and the luciferase activity was measured with a luminometer. Values represent the mean ± SD. *P < 0.05 compared with corresponding control. Representative results of four independent experiments using synovial fibroblasts derived from four RA patients are shown (n = 4).

 
TGF-ß type I receptor kinase inhibitor HTS466284 prevents the arthritis induced by anti-CL type II antibody in mice
Because TGF-ß type I receptor kinase inhibitor HTS466284 efficiently down-regulated TGF-ß-induced activation of RA synovial fibroblasts, we investigated whether the inhibitor is also effective in vivo as well as in vitro. For this purpose, we used a mouse model of arthritis, which was developed by the injection of mAbs against type II CL followed by the subsequent injection of bacterial LPS to reduce the threshold of the arthritogenic dose of mAbs and the required number of mAb clone (CL type II antibody-induced arthritis) (29). One advantage of this model is that it can easily induce arthritis regardless of the genetic background of the mice and share distal-stage effector mechanisms with RA, as judged by a massive recruitment of leukocytes, the involvement of pro-inflammatory cytokines such as TNF-{alpha} and synovial hyperplasia. In addition, we can focus on the effector phase of RA because the development of this arthritis model is independent of T cells and B cells (34). Wild-type mice receiving control vehicle (DMSO) developed the arthritis after the injection of anti-type II CL mAbs plus LPS, beginning on day 5 after the administration of the mAbs, with subsequent increasing severity of inflammation until day 11 (Fig. 4A and B). The mice receiving HTS466284 (1 or 10 mg kg–1 per mouse) on day 5 and thereafter every day developed limited clinical manifestations of arthritis as judged by the clinical scoring (Fig. 4A and B). There was a tendency to show a dose-dependent response although the data were not statistically significant (Fig. 4B). A histological examination of the hind paws on day 11 showed a significantly reduced cellular infiltration and synovial hyperplasia in the mice receiving HTS466284 (Fig. 4C). Immunohistochemical staining with anti-VEGF and anti-PDGF-A antibodies showed the expression of VEGF and PDGF-A in the synovial tissue to be up-regulated after the induction of arthritis, which was reduced by treatment with HTS466284 (Fig. 4D). Serum TNF-{alpha} levels were up-regulated by the induction of arthritis, which was also reduced by treatment with HTS466284 (Fig. 4E). In addition, as a measure of DNA synthesis in synovial fibroblasts, we studied a BrdU incorporation assay. On day 8 after the administration of the mAbs, the number of BrdU-positive synovial fibroblasts was up-regulated in the inflamed synovial tissue, which was suppressed by the treatment with HTS466284 (Fig. 4F). Treatment with HTS466284 alone did not affect the gross appearance, histology of the hind paws, VEGF and PDGF expression and BrdU incorporation in mice (data not shown).


Figure 4
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  		Fig. 4. TGF-ß type I receptor kinase inhibitor HTS466284 prevents CL type II antibody-induced arthritis in mice. The mice were intra-peritoneally injected with 2 mg per mouse of anti-CL type II mAb cocktail (day 1) and 3 days later (day 4) with 50 µg per mouse of LPS. The indicated doses of TGF-ß type I receptor kinase HTS466284 or a control vehicle (DMSO) were intra-peritoneally administered on day 5 and thereafter every day. (A) Representative photographs showing the hind paws of the mice treated or untreated with HTS466183 (1 and 10 mg kg–1 per mouse) on days 1 and 11. (B) Clinical scoring measured during the course of study. Values represent the mean ± SD of three mice per group. *P < 0.05 compared with corresponding control. Similar results were obtained from at least three independent experiments. (C) A histological examination of the hind paws (tarsocrural joint) of the mice treated or untreated with HTS466284 (3 mg kg–1 per mouse) on day 11. Representative photographs of hematoxilin and eosin staining are shown. (D). Immunohistochemical examination of the hind paws (tarsocrural joint) of the mice treated or untreated with HTS466284 (3 mg kg–1 per mouse) on day 11. Representative photographs of immunohistochemical staining with anti-VEGF (left panels) and anti-PDGF-A (right panels) antibodies are shown. Positive staining is indicated as brown. (E) Serum TNF-{alpha} levels before or after the induction of arthritis. Sera were obtained from HTS466284-treated (3 mg kg–1 per mouse) or untreated mice before (day 1: white bars) or after the induction of arthritis (day 5: black bars) and then TNF-{alpha} levels were measured by ELISA. (F) BrdU incorporation of synovial fibroblasts in the hind paws (tarsocrural joint) of the mice treated or untreated with HTS466284 (3 mg kg–1 per mouse) (on day 8). BrdU-positive synovial fibroblasts were counted as described in Methods. P < 0.05 compared with the corresponding control (n = 5).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, we demonstrated that TGF-ß stimulated the proliferation of synovial fibroblasts derived from RA patients through PDGF-AA associated with the c-Myc induction (Figs 1 and 2). In addition, TGF-ß induced IL-6 and VEGF production by RA synovial fibroblasts, at least in part, through NF-{kappa}B activation (Fig. 3). All these effects of TGF-ß on RA synovial fibroblasts were efficiently suppressed by TGF-ß type I receptor kinase inhibitor HTS466284 (Figs 1–3). Since the proliferation of synovial fibroblasts and the production of IL-6 and VEGF are critical events for the development of RA (2, 35–40), the in vitro results taken together with the in vivo findings (Fig. 4) suggest that TGF-ß has a pro-arthritic effect, at least, in the distal-stage effector phase (the development of synovial hyperplasia) of RA.

There have been contrasting findings regarding the effect of TGF-ß on synovial fibroblast growth. Allen et al. (22) showed that TGF-ß stimulated growth of synovial fibroblasts derived from SCW-injected rats whereas Lafyatis et al. (41) showed that TGF-ß inhibited the anchorage-independent growth of RA synovial fibroblasts. Our results clearly indicated that TGF-ß stimulated the proliferation of RA synovial fibroblasts through PDGF-AA production (Figs 1 and 2). Synovial cell proliferation induced by TGF-ß injections in the previous in vivo studies (22, 23) occurred at a delayed time after the injections (~72 h), which was consistent with the possibility that a secondary mitogen such as PDGF might have been required. PDGF is known to have a strong mitogenic activity for RA synovial fibroblasts (42, 43) and the induction of PDGF by TGF-ß was also reported in human dermal fibroblasts and osteosarcoma cells (30, 31, 44). We therefore speculate that a common mechanism (PDGF induction by TGF-ß) may mediate the growth stimulatory effects of TGF-ß on cells from parenchymal origin including RA synovial fibroblasts. The induction of c-Myc by TGF-ß has also been reported in osteosarcoma cells which were growth stimulated by TGF-ß (44). However, it remains to be determined whether the induction of c-Myc by TGF-ß in certain cell types directly contributes to TGF-ß-induced PDGF production and growth stimulation.

TGF-ß induced IL-6 and VEGF production by RA synovial fibroblasts (Fig. 3). Both IL-6 and VEGF increased in RA synovial tissues (37, 39) and recent clinical trials with anti-IL-6R antibody, which have shown promising results, also support the finding that IL-6 signaling plays a pivotal role in RA (38). The inhibition of VEGF signaling also suppressed acute arthritis in an animal model (40). Luttun et al. (45) also reported that the inhibition of VEGF receptor-1 (Flt 1) suppressed inflammation and neovascularization in CL type II-induced arthritis. As a result, TGF-ß may be a potent inducer of these important cytokines for RA, thereby contributing to the disease.

NF-{kappa}B dependence of IL-6 and VEGF expression has been well accepted (46). Our results with inhibitors of NF-{kappa}B pathway indicated that TGF-ß-induced IL-6 and VEGF production in RA synovial fibroblast depended on, at least in part, the activation of NF-{kappa}B. Since TGF-ß signals principally through the Smad pathway (7), we think that TGF-ß-induced activation of NF-{kappa}B in RA synovial fibroblasts is a secondary event following Smad activation. This issue is currently under investigation.

TGF-ß type I receptor kinase inhibitor HTS466284 suppressed CL type II antibody-induced arthritis, thus suggesting that endogenous TGF-ß signaling is involved in the development of arthritis (Fig. 4). The inhibition of arthritis by HTS466284 was not complete even at the high dose (10 mg kg–1 per mouse). We therefore speculate that TNF-{alpha} or other inflammatory cytokines may also play an important role in the full development of the arthritis induced by anti-CL type II antibody.

The inhibitory effects of HTS466284 on synovial fibroblast proliferation and cytokine production in vitro might, in part, explain the in vivo results. Indeed, synovial hyperplasia, inflammation, VEGF and PDGF expression and synovial fibroblast proliferation as judged by BrdU incorporation were significantly suppressed in the mice treated with HTS466284 (Fig. 4C–E). However, because TGF-ß can act on synovial residential cells and immune cells in various ways (6, 47), many other mechanisms, e.g. inhibition of leukocytes migration, should thus be involved in the inhibition of arthritis by TGF-ß type I receptor kinase inhibitor HTS466284.

Our in vivo results were consistent with the findings by Wahl et al. (24) using neutralizing antibody against TGF-ß, thus suggesting that endogenous TGF-ß has a pro-arthritic effect. Small molecular inhibitors have an advantage over the neutralizing antibody in the delivery and cell or tissue penetration. For example, antibody therapy could not efficiently block TGF-ß signaling in an autocrine mode which might occur in pathological sites. Indeed, RA synovial fibroblasts spontaneously produce TGF-ß which might function as a mitogenic factor for the cells (48). We therefore think that our results reinforce the findings by Wahl et al (24). However, we cannot, of course, rule out the possibility that different arthritis models induced a different outcome or other possibilities.

Since the use of anti-TNF-{alpha} treatment has now become widespread (1, 3, 4), the number of RA patients in whom this treatment is unsuccessful thus continues to grow and such patients need alternative treatment. Recent studies have highlighted the therapeutic potential of antagonizing the TGF-ß pathway in cancer and fibrotic diseases and small-molecule inhibitors of TGF-ß type I receptor kinase have been extensively developed by several companies (10). The small-molecule inhibitors of TGF-ß type I receptor kinase appear to be promising for the treatment of cancer and fibrotic diseases in pre-clinical animal models (10). Our results suggest that TGF-ß type I receptor kinase inhibitors may be also a useful option for the treatment of RA. However, a further detailed analysis, including studies using other TGF-ß type I receptor kinase inhibitors or other arthritis models, should be done in the future with a careful evaluation of the unfavorable side effects following such treatments.


   Acknowledgements
 
This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan and from the Ministry of Health, Labor and Welfare, Japan. We thank Drs Yutaka Kanamaru, Masao Furuhashi and Kachio Tasaka for their valuable discussion and technical help, Michiyo Matsumoto and Yuko Ohnuma for their secretarial assistance and Mutsuko Hara for her general assistance.


   Abbreviations
 
ALK, activin-like kinase
BrdU, 5-bromo-2'-deoxyuridine
cDNA, complementary DNA
CL, collagen
DMSO, dimethyl sulfoxide
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
MMP3, matrix metalloproteinase 3
NF-{kappa}B, nuclear factor-kappa B
PDGF, platelet-derived growth factor
RA, rheumatoid arthritis
SCW, streptococcal cell wall
TGF-ß, transforming growth factor-ß
TNF-{alpha}, tumor necrosis factor-{alpha}
VEGF, vascular endothelial growth factor

   Notes
 
Transmitting editor: H. Karasuyama Back

Received 21 June 2006, accepted 1 November 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Feldmann M, Brennan FM, Maini RN. (1996) Rheumatoid arthritis. Cell 85:307.[CrossRef][Web of Science][Medline]
  2. Mor A, Abramson SB, Pillinger MH. (2005) The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction. Clin. Immunol. 115:118.[CrossRef][Web of Science][Medline]
  3. Bathon JM, Martin RW, Fleischmann RM, et al. (1999) A comparison of etanercept and methotrexate in patients with early rheumatoid arthritis. N. Eng. J. Med. 343:1586.
  4. Lipsky PE, van der Heijde DM FM, St Clair EW, et al. (2000) Infliximab and methotrexate in the treatment of rheumatoid arthritis. N. Eng. J. Med. 343:1594.[Abstract/Free Full Text]
  5. Breshihan B, Alvaro-Gracia JM, Cobby M, et al. (1998) Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rhuem. 41:2196.
  6. Massague J. (1990) The transforming growth factor-ß family. Annu. Rev. Cell Biol. 6:597.[CrossRef][Web of Science][Medline]
  7. Heldin CH, Miyazono K, ten Dijke P. (1997) TGF-ß signaling from cell membrane to nucleus through SMAD proteins. Nature 390:465.[CrossRef][Medline]
  8. Derynck R, Akhurst RJ, Balmain A. (2001) TGF-ß signaling in tumor suppression and cancer progression. Nat. Genet. 29:117.[CrossRef][Web of Science][Medline]
  9. Leask A and Abraham DJ. (2004) TGF-ß signaling and the fibrotic response. FASEB J. 18:816.[Abstract/Free Full Text]
  10. Yingling JM, Blanchard KL, Sawyer JS. (2004) Development of TGF-ß signalling inhibitors for cancer therapy. Nat. Rev. Drug Discov. 3:1011.[CrossRef][Web of Science][Medline]
  11. Singh J, Chuaqui CE, Boriack-Sjodin PA, et al. (2003) Successful shape-based virtual screening: the discovery of a potent inhibitor of the type I TGF-ß receptor kinase. Bioorg. Med. Chem. Lett. 13:4355.[CrossRef][Medline]
  12. Sawyer JS, Anderson BD, Beight DW, et al. (2003) Synthesis and activity of new aryl- and heteroaryl-substituted pyrazole inhibitors of the transforming growth factor-ß type I receptor kinase domain. J. Med. Chem. 46:3953.[CrossRef][Web of Science][Medline]
  13. Uhl M, Aulwurm S, Wischhusen J, et al. (2004) SD-208, a novel transforming growth factor-ß receptor I kinase inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma cells in vitro and in vivo. Cancer Res. 64:7954.[Abstract/Free Full Text]
  14. Hjelmeland MD, Hjelmeland AB, Sathornsumetee S, et al. (2004) SB-431542, a small molecule transforming growth factor-ß-receptor antagonist, inhibits human glioma cell line proliferation and motility. Mol. Cancer Ther. 3:737.[Abstract/Free Full Text]
  15. DaCosta-Byfield S, Major C, Laping NJ, Roberts AB. (2004) SB-505124 is a selective inhibitor of transforming growth factor-ß type I receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 65:744.[Abstract/Free Full Text]
  16. Fava R, Olsen N, Keski-Oja J, Moses H, Pincus T. (1989) Active and latent forms of transforming growth factor-ß activity in synovial effusions. J. Exp. Med. 169:291.[Abstract/Free Full Text]
  17. Lotz M, Kekow J, Carson DA. (1990) Transforming growth factor-ß and cellular immune responses in synovial fluids. J. Immunol. 144:4189.[Abstract]
  18. Taketazu F, Kato M, Gobl A, et al. (1994) Enhanced expression of transforming growth factor-ßs and transforming growth factor-ß type II receptor in the synovial tissues of patients with rheumatoid arthritis. Lab. Investig. 70:620.[Web of Science][Medline]
  19. Cheon H, Yu SJ, Yoo DH, Chae IJ, Song GG, Sohn J. (2002) Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-ß1 in synovial fibroblasts from rheumatoid arthritis and normal individuals. Clin. Exp. Immunol. 127:547.[CrossRef][Web of Science][Medline]
  20. Brandes ME, Allen JB, Ogawa Y, Wahl SM. (1991) Transforming growth factor ß-1 suppresses acute and chronic arthritis in experimental animals. J. Clin. Invest. 87:1108.[Web of Science][Medline]
  21. Thorbecke GJ, Shah R, Leu CH, Kuruvilla AP, Hardison AM, Palladino MA. (1992) Involvement of endogenous tumor necrosis factor alpha and transforming growth factor-ß during induction of collagen type II arthritis in mice. Proc. Natl Acad. Sci. USA 89:7375.[Abstract/Free Full Text]
  22. Allen JB, Manthey CL, Hand AR, Ohura K, Ellingsworth L, Wahl SM. (1990) Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor-ß. J. Exp. Med. 171:231.[Abstract/Free Full Text]
  23. Fava RA, Olsen NJ, Postlethwaite AE, et al. (1991) Transforming growth factor-ß1 (TGF-ß1) induced neutrophil recruitment to synovial tissues: implication for TGF-ß-driven synovial inflammation and hyperplasia. J. Exp. Med. 173:1121.[Abstract/Free Full Text]
  24. Wahl SM, Allen JB, Costa GL, Wong HL, Dasch JR. (1993) Reversal of acute and chronic synovial inflammation by anti-transforming growth factor-ß. J. Exp. Med. 177:225.[Abstract/Free Full Text]
  25. Wahl SM. (1994) Transforming growth factor-ß: the good, the bad, and the ugly. J. Exp. Med. 180:158.
  26. Berse B, Hunt JA, Diegel RJ, et al. (1999) Hypoxia augments cytokine (transforming growth factor-ß and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts. Clin. Exp. Immunol. 115:176.[CrossRef][Web of Science][Medline]
  27. Takayanagi H, Iizuka H, Juji T, et al. (2000) Involvement of receptor activator of nuclear factor {kappa}B ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum. 43:259.[CrossRef][Web of Science][Medline]
  28. Ke R, Reddel R, Gerwin BI, et al. (1998) Human bronchial epithelial cells with integrated SV40 virus T antigen genes retain the ability to undergo squamous differentiation. Differentiation 38:60.[CrossRef]
  29. Nandakumar KS, Svensson L, Holmdahl R. (2003) Collagen type II-specific monoclonal antibody-induced arthritis in mice. Description of the disease and the influence of age, sex, and genes. Am. J. Pathol. 163:1827.[Abstract/Free Full Text]
  30. Leof EB, Proper JA, Goustin AS, Shipley GD, DiCorleto PE, Moses HL. (1986) Induction of c-sis mRNA and activity similar to platelet-growth-factor by transforming growth factor-ß: a proposed model for indirect mitogenesis involving autocrine activity. Proc. Natl Acad. Sci. USA 83:2453.[Abstract/Free Full Text]
  31. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross R. (1990) TGF-ß induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell 63:515.[CrossRef][Web of Science][Medline]
  32. Grandori C, Cowley SM, James LP, Eisenman RN. (2000) The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell Dev. Biol. 16:653.[CrossRef][Web of Science][Medline]
  33. Nakao A, Okumura K, Ogawa H. (2002) Smad7: a new key player in TGF-ß-associated disease. Trends Mol. Med. 8:361.[CrossRef][Web of Science][Medline]
  34. Nandakumar KS, Backlund J, Vestberg M, Holmdahl R. (2004) Collagen type II (CII)-specific antibodies induce arthritis in the absence of T or B cells but the arthritis progression is enhanced by CII-reactive T cells. Arthritis Res. Ther. 6:R544.[CrossRef][Web of Science][Medline]
  35. Taniguchi K, Kohsaka H, Inoue N, et al. (1999) Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis. Nat. Med. 5:760.[CrossRef][Web of Science][Medline]
  36. Nasu K, Kohsaka H, Nonomura Y, et al. (2000) Adenoviral transfer of cyclin-dependent kinase inhibitor genes suppresses collagen-induced arthritis in mice. J. Immunol. 165:7246.[Abstract/Free Full Text]
  37. Houssiau FA, Devogelaer JP, Van Damme J, de Deuxchaisnes CN, Snick JV. (1988) Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum. 31:784.[Web of Science][Medline]
  38. Nishimoto N, Yoshizaki K, Miyasaka N, et al. (2004) Treatment of rheumatoid arthritis with humanized anti-interleukin-6 receptor antibody: a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum. 50:1761.[CrossRef][Web of Science][Medline]
  39. Fava RA, Olsen NJ, Spencer-Green G, et al. (1994) Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J. Exp. Med. 180:341.[Abstract/Free Full Text]
  40. Grosios K, Wood J, Esser R, Raychaudhuri A, Dawson J. (2004) Angiogenesis inhibition by the novel VEGF receptor tyrosine kinase inhibitor, PTK787/ZK222584, causes significant anti-arthritic effects in models of rheumatoid arthritis. Inflamm. Res. 53:133.[CrossRef][Web of Science][Medline]
  41. Lafyatis R, Remmers EF, Roberts AB, Yocum DE, Sporn MB, Wilder RL. (1989) Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous platelet-derived growth factor and inhibition by transforming growth factor-ß and retinoids. J. Clin. Invest. 83:1267.[Web of Science][Medline]
  42. Remmers EF, Lafyatis R, Kumkumian GK, et al. (1990) Cytokines and growth regulation of synoviocytes from patients with rheumatoid arthritis and rats with streptococcal cell wall arthritis. Growth Factors 2:179.[Medline]
  43. Butler DM, Leizer T, Hamilton JA. (1989) Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor. Differences to the activation by IL-1. J. Immunol. 142:3098.[Abstract]
  44. Matsuyama S, Iwadate M, Kondo M, et al. (2003) SB-431542 and gleevec inhibit transforming growth factor-ß-induced proliferation of human osteosarcoma cells. Cancer Res. 63:7791.[Abstract/Free Full Text]
  45. Luttun A, Tjwa M, Moons L, et al. (2002) Revascularization of ischemic tissues by PIGF treatment, and inhibition of tumor angiogenesis, arthritis, and atherosclerosis by anti-Flt1. Nat. Med. 8:831.[CrossRef][Web of Science][Medline]
  46. Karin M and Delhase M. (2000) The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatory signalling. Semin. Immunol. 12:85.[CrossRef][Web of Science][Medline]
  47. Letterio JJ and Roberts AB. (1998) Regulation of immune responses by TGF-ß. Annu. Rev. Immunol. 16:137.[CrossRef][Web of Science][Medline]
  48. Bucala R, Ritchlin C, Winchester R, Cerami A. (1991) Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts. J. Exp. Med. 173:569.[Abstract/Free Full Text]

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