Article Text

PDF

Anti-apoptogenic function of TGFβ1 for human synovial cells: TGFβ1 protects cultured synovial cells from mitochondrial perturbation induced by several apoptogenic stimuli
  1. A Kawakami1,
  2. S Urayama3,
  3. S Yamasaki1,
  4. A Hida1,
  5. T Miyashita1,
  6. M Kamachi1,
  7. K Nakashima1,
  8. F Tanaka1,
  9. H Ida1,
  10. Y Kawabe3,
  11. T Aoyagi4,
  12. I Furuichi4,
  13. K Migita1,
  14. T Origuchi2,
  15. K Eguchi1
  1. 1The First Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
  2. 2Department of Physical Therapy, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
  3. 3Department of Internal Medicine, National Ureshino Hospital, Saga, Japan
  4. 4Department of Orthopaedics, National Ureshino Hospital, Saga, Japan
  1. Correspondence to:
    Dr A Kawakami
    The First Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan; atsushiknet.nagasaki-u.ac.jp

Abstract

Objective: To investigate anti-apoptogenic mechanism of transforming growth factor β1 (TGFβ1) towards synovial cells.

Methods: Isolated synovial cells, treated or not with TGFβ1, were cultured in the presence or absence of anti-Fas IgM, proteasome inhibitor Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or C2-ceramide. After cultivation, apoptosis of synovial cells was examined by the presence of hypodiploid DNA+ cells, the presence of terminal deoxy (d)-UTP nick end labelling+ cells (TUNEL+ cells), activation of caspases, and disruption of mitochondrial transmembrane potential (ΔΨm).

Results: Activation of caspase-9 and ΔΨm was found in anti-Fas IgM treated synovial cells. The increment of both hypodiploid DNA+ cells and TUNEL+ cells accompanied by the activation of caspase-8 and caspase-3 was also determined in anti-Fas IgM treated synovial cells. These hallmarks for apoptosis induced by anti-Fas IgM were significantly suppressed in TGFβ1 treated synovial cells. LLL-CHO, etoposide, and C2-ceramide also caused ΔΨm, the increment of both hypodiploid DNA+ cells and TUNEL+ cells, and the activation of both Leu-Glu-His-Asp ase (LEHDase; caspase-9 like activity) and Asp-Glu-Val-Asp ase (DEVDase; caspase-3 like activity) in synovial cells. As determined in anti-Fas IgM treatment, TGFβ1 significantly reduced apoptotic cell death of synovial cells induced by the above chemicals.

Conclusions: The protective effect of TGFβ1 for mitochondrial homoeostasis may be important in the anti-apoptogenic function of TGFβ1 for synovial cells.

  • rheumatoid arthritis
  • transforming growth factor β1
  • apoptosis
  • caspase
  • DEVDase, Asp-Glu-Val-Asp ase
  • DiOC6, 3, 3′-dihexyloxacarbocyamine iodide
  • Δψm, disruption of mitochondrial transmembrane potential
  • IETDase, Ile-Glu-Thr-Asp ase
  • LEHDase, Leu-Glu-His-Asp ase
  • LLL-CHO, Z-Leu-Leu-Leu-aldehyde
  • RA, rheumatoid arthritis
  • TGFβ1, transforming growth factor β1
  • TUNEL, terminal deoxy (d)-UTP nick end labelling

Statistics from Altmetric.com

Cytokines and growth factors present in rheumatoid synovial tissues are important factors which regulate an apoptotic process of synovial cells.1–5 Transforming growth factor β1 (TGFβ1) is highly expressed in rheumatoid synovial tissues,6 and found to possess an anti-apoptogenic effect for synovial cells; this was demonstrated by the experimental results showing that TGFβ1 inhibits Fas mediated apoptosis as well as proteasome inhibitor induced apoptosis in cultured synovial cells.2,3 TGFβ1 not only suppresses Fas expression, but increases the expression of Bcl-2 and Bcl-xL in cultured synovial cells.2,3 the latter finding implies that TGFβ1 protects synovial cells from apoptogenic stimuli through a mitochondria dependent mechanism.

We show in this study that mitochondrial perturbation as well as both DNA fragmentation and the activation of caspases in cultured synovial cells, induced by several apoptogenic stimuli, are significantly suppressed by TGFβ1 treatment, which may be closely associated with the anti-apoptogenic function of TGFβ1.

MATERIALS AND METHODS

Synovial cell culture

Synovial cells were isolated from synovial tissues obtained from 18 patients with rheumatoid arthritis (RA) who met the American College of Rheumatology criteria for RA7 at the time of orthopaedic surgery, as we previously described.2–5 In some experiments, synovial cells isolated from patients with osteoarthritis were also used in this study. The adherent synovial cells used in this study at third to fifth passages were less than 1% reactive with monoclonal antibodies, including CD3, CD68, CD20, and von Willebrand factor, which are defined as fibroblast-like synovial cells.

Induction of synovial cell apoptosis by several apoptogenic stimuli

Synovial cells were cultured with or without recombinant human TGFβ1 (5 ng/ml; R&D Systems Inc, Minneapolis, MN) for 48 hours in RPMI 1640 containing 2% bovine serum albumin. After incubation, apoptosis sensitivity in untreated or TGFβ1 treated synovial cells was examined by further incubation with anti-Fas IgM (1 μg/ml for 12 hours; MBL, Nagoya, Japan), Z-Leu-Leu-Leu-aldehyde (LLL-CHO, 10 μM for 24 hours, Peptide Institute, Osaka, Japan), etoposide (50 μM for 24 hours; TopoGen, Inc, Columbus, Ohio), or C2-ceramide (50 μM for 24 hours; Sigma). Apoptosis of synovial cells was quantified by the presence of hypodiploid DNA+ cells, the presence of terminal deoxy (d)-UTP nick end labelling+ cells (TUNEL+ cells), activation of caspases, and disruption of mitochondrial transmembrane potential (ΔΨm) as previously described.3,5,8,9

DNA fragmentation was estimated by the presence of hypodiploid DNA+ cells and TUNEL+ cells, determined by flow cytometry (Epics XL, Beckman Coulter, Hialeah, FL). Detection of hypodiploid DNA+ cells was done by propidium iodide staining (100 μg/ml; Sigma Chemical Co, St Louis, MO), and TUNEL was examined by Mebstain Apoptosis Kit (MBL, Nagoya, Japan).

Activation of caspases in synovial cells was studied by western blot analysis, colorimetric protease assay and flow cytometry. Western blot analysis was done by enhanced chemiluminescence system (Amersham, Arlington Heights, IL, anti-caspase-3; Transduction Laboratories, Lexington, KY, anti-caspase-8; MBL, anti-caspase-9; MBL). Decrement of procaspase expression and/or the appearance of cleaved products indicate the activation of each caspase.10 In addition to western blotting, increment of an enzymatic activity of Asp-Glu-Val-Asp ase (DEVDase: caspase-3 like activity), Ile-Glu-Thr-Asp ase (IETDase: caspase-8 like activity), and Leu-Glu-His-Asp ase (LEHDase: caspase-9 like activity) was used for detection of activation in each caspase.

Enzymatic activity of DEVDase (intracellular DEVDase+ cells) was detected by flow cytometry (Epics XL) by the use of DEVD substrate (OncoImmunin, Inc, College Park, MD) as previously described.8 Enzymatic activity of both IETDase and LEHDase was examined by colorimetric protease assay kit (MBL), and the activity of IETDase and LEHDase was evaluated by a spectrophotometer at an optical density of 405 nm (Multiskan JX, LABSYSTEMS, Tokyo, Japan), according to the manufacturer’s protocol.

Expression of Bcl-2 (anti-Bcl-2; Dako Japan, Kyoto, Japan), Bcl-xL (anti-Bcl-xL; Trevigen, Gaithersburg, CA), and Bax (anti-Bax; Santa Cruz Biotechnology, Santa Cruz, CA) in synovial cells was studied by western blotting, and a relative expression ratio of Bcl-2 to Bax and of Bcl-xL to Bax was calculated by the software NIH Image (1.61) as follows: density of Bcl-2 or Bcl-xL/density of β-actin to density of Bax/density of β-actin. β-Actin (anti-β-actin; Sigma) was used as an internal control protein in western blotting.

Mitochondrial perturbation in synovial cells was examined by ΔΨm.9 The cells were reacted with saturating amount of DiOC6 (3,3′-dihexyloxacarbocyamine iodide, Fluoreszenztechnologie, Grottenhofstr, Austria) at 37°C for 15 minutes, washed, and analysed by flow cytometry. In some experiments, synovial cells were cultured in the presence of Z-Val-Ala-Asp-CH2DCB (caspase inhibitor, 200 μM; Phoenix Pharmaceuticals, Inc, Mountain View, CA), and apoptosis of these cells was also examined.

Statistical analysis

Data were expressed as mean (SD). Differences between groups were tested for statistical significance using the Student’s t-test. A p value <0.05 was considered significant.

RESULTS

Inhibition of Fas mediated mitochondrial perturbation in cultured synovial cells by TGFβ1

Although we did not find the cleaved products of each caspase, activation of caspase-3/-8/-9 in synovial cells by anti-Fas IgM was strongly suggested by western blotting (fig 1), which was confirmed by an enzymatic activity assay for DEVDase, IETDase, and LEHDase (table 1). Mitochondrial perturbation with DNA fragmentation in synovial cells was also clearly induced by anti-Fas IgM (table 1). Expression of procaspase-3/-8/-9 in synovial cells was not changed by TGFβ1 (fig 1), but TGFβ1 treatment significantly suppressed ΔΨm, activation of caspase-3/-8/-9, and DNA fragmentation of synovial cells induced by anti-Fas IgM (fig 1, table 1).

Table 1

Inhibition of Fas mediated apoptosis of synovial cells by TGFβ1

Figure 1

Western blot analysis for the activation of caspase-3/-8/-9 in synovial cells induced by anti-Fas IgM, which is inhibited by TGFβ1. Synovial cells isolated from the rheumatoid synovial tissues were cultured with or without TGFβ1 for 48 hours, washed, and further incubated with control mouse IgM or anti-Fas IgM for 12 hours. After cultivation, the expression of procaspase-3/-8/-9 in synovial cells was examined by western blotting as described in the text. Note that the disappearance of procaspase-3/-8/-9 in anti-Fas IgM treated synovial cells, which indicates the activation of each caspase, was significantly inhibited by TGFβ1 treatment. Results are representative data from six determinations. anti-Fas IgM (−); addition of control mouse IgM.

Effect of TGFβ1 for synovial cell apoptosis induced by other apoptogenic stimuli

We next examined whether TGFβ1 treatment protects mitochondrial perturbation induced by other apoptogenic stimuli. LLL-CHO, etoposide, and C2-ceramide, the chemicals triggering apoptosis in a mitochondria dependent fashion,11–13 induced ΔΨm with the presence of DNA fragmentation toward synovial cells (table 2). Z-Val-Ala-Asp-CH2DCB did not inhibit ΔΨm of synovial cells in the process (data not shown), which supported the importance of the mitochondrial pathway in synovial cell death induced by LLL-CHO, etoposide, and C2-ceramide. Activation of both LEHDase and DEVDase was also clearly found in synovial cells treated with the chemicals, and TGFβ1 treatment significantly suppressed the above hallmarks for apoptosis (table 2). As we previously described,3 the relative expression ratio of Bcl-2 or Bcl-xL to Bax in synovial cells was increased by TGFβ1 (untreated synovial cells: Bcl-2 to Bax 0.56 (0.05) and Bcl-xL to Bax; 0.12 (0.01); TGFβ1 treated synovial cells: Bcl-2 to Bax 0.95 (0.07)* and Bcl-xL to Bax 0.88 (0.06)*; *p<0.01 v untreated synovial cells. Results are the mean (SD) of five individual experiments. We found no difference in TGFβ1 induced inhibition for synovial cell apoptosis induced by anti-Fas IgM, LLL-CHO, etoposide, and C2-ceramide between rheumatoid synovial cells and synovial cells isolated from patients with osteoarthritis (data not shown).

Table 2

TGFβ1 mediated inhibition in synovial cell apoptosis induced by LLL-CHO, etoposide and C2-ceramide

DISCUSSION

Our series of studies have indicated that the growth promoting activity of TGFβ1 for synovial cells can, in part, be mediated by inhibition of synovial cell apoptosis.2,3 As reported in other cell types,14,15 activation of caspase-3/-8/-9 as well as ΔΨm was found in anti-Fas monoclonal antibody treated synovial cells, whereas these hallmarks for apoptosis were markedly suppressed in TGFβ1 treated synovial cells.

Mitochondrial dysfunction in synovial cells was also induced by LLL-CHO, etoposide, and C2-ceramide, leading to the activation of caspase-3/-9 and DNA fragmentation. TGFβ1 treatment significantly suppressed apoptogenic activity of LLL-CHO, etoposide, and C2-ceramide. As we previously reported,3 relative expression of Bcl-2 and Bcl-xL was increased by TGFβ1, which may prevent the release of cytochrome c and apoptotic cell death of synovial cells induced by the above chemicals. TGFβ1 mediated protection for the mitochondria may also be effective in Fas induced apoptosis in synovial cells.

Impaired apoptosis of synovial cells in rheumatoid synovial tissues is supposed to have an important role in the progression of synovial cell hyperplasia of patients with RA, but recent investigations have suggested its characteristic may not be an intrinsic property of rheumatoid synovial cells, but that the rheumatoid synovial microenviroment affects the sensitivity of synovial cell apoptosis.1–5,12 One factor which may inhibit apoptosis of synovial cells is TGFβ1, but rheumatoid synovial tissue contains a variety of cytokines and growth factors other than TGFβ1, which also inhibit apoptotic cell death of synovial cells, including basic fibroblast growth factor and interleukin 1β.1,4 The present data are obtained only under bovine serum albumin culture conditions, thus, the in vivo role of TGFβ1 in rheumatoid synovial tissues remains obscure. Further investigations, including interactions between TGFβ1 and other humoral factors, are necessary to clarify the in vivo role of TGFβ1 in the regulation of apoptotic cell death of synovial cells of patients with RA.

Acknowledgments

We thank Misses Yoko Uchiyama and Nobuko Fukuda for their technical assistance.

REFERENCES

View Abstract

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.