PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53
© You et al; licensee BioMed Central Ltd. 2006
Received: 05 July 2006
Accepted: 02 October 2006
Published: 02 October 2006
PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and invasion into cartilage and bone. Inadequate apoptosis of fibroblast-like synoviocytes (FLS) could contribute to this process by increasing the accumulation of cells in the intimal lining . As a result of the aggressive nature of rheumatoid synovium and the relatively low level of apoptosis, interventions designed to increase programmed cell death of synoviocytes have been considered in treating RA. Several genes have been evaluated as potential gene therapy targets, including Fas , TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) , p53 , and PUMA (p53 up-regulated modulator of apoptosis) . The latter is an especially interesting target because it rapidly induces apoptosis in cultured synoviocytes . PUMA is a Bcl-2 homology 3 (BH3)-only pro-apoptotic Bcl-2 family member recently identified as a principal mediator of p53-dependent apoptosis . The in vivo effects on apoptosis observed in PUMA-/- mice are similar to those in p53-/- animals, suggesting that PUMA can serve as an effector of p53 function [7, 8]. However, our previous studies showed that p53 is only a weak inducer of PUMA in FLS, which could account for the variable pro-apoptotic effect of p53 in this cell lineage, with no significant apoptosis induced by p53 overexpression in some studies [9, 10].
The mechanism of PUMA-mediated apoptosis has been extensively evaluated. PUMA expression leads to apoptosis by displacing p53 from Bcl-XL and allowing p53 to increase mitochondrial permeability . The need for functional p53 raises significant concerns about the utility of PUMA as a therapeutic target in RA because deficient p53 expression or function in the rheumatoid synovial intimal lining has been described [11–14]. To address this issue, we determined whether PUMA requires functional p53 in cultured FLS. These studies show that PUMA-induced apoptosis can occur despite defects in the p53 pathway.
Materials and methods
Human and murine cultured fibroblast-like synoviocytes
Synovial tissues were obtained from patients with rheumatoid arthritis and osteoarthritis at joint replacement surgery. The diagnosis of RA conformed to the American College of Rheumatology 1987 revised criteria . The protocol was approved by the University of California at San Diego Human Subjects Research Protection Program. FLS were isolated from individual tissues with 1 mg/ml collagenase and cultured in DMEM supplemented with 10% fetal calf serum, penicillin, streptomycin, and L-glutamine as described previously. Cell lines were used from the third to ninth passage, when they are a homogeneous population of fibroblast-like cells . Although the origin of these cells cannot be certain, they probably derive from the intimal lining, on the basis of vascular cell adhesion molecule (VCAM)-1 and CD55 expression. In addition to RA FLS, we also examined FLS derived from osteoarthritis FLS in most experiments. No differences were observed between RA and osteoarthritis FLS in these assays. p53+/+ and p53-/- murine synoviocytes were obtained as described previously from DBA/1J wild-type mice (Jackson Laboratory, Bar Harbor, ME, USA) and DBA/1J p53-/- mice .
Affinity-purified rabbit polyclonal anti-p53 (for immunohistochemistry), mouse monoclonal anti-p53 (for Western blotting), and rabbit polyclonal antibodies against p21 and hemagglutinin (HA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse and anti-rabbit IgG secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Rabbit anti-PUMA polyclonal antibody was purchased from ProSci, Inc. (Poway, CA, USA).
Scrambled RNA and p53 siRNA were purchased from Dharmacon Research, Inc. (Lafayette, CO, USA). Plasmids encoding HA-tagged full-length PUMA (HA-PUMA) and PUMA with a deletion of the BH3 domain (HA-PUMA-dBH3) were kindly provided by Dr B Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA) . R213* encoding mutant p53 was isolated from a patient with RA and has previously been characterized as dominant-negative . Bax-luc (BF72-2 PGL3) is a reporter construct containing the p53-responsive promoter for bax with the luciferase cDNA . The control construct contains the β-Gal cDNA and the cytomegalovirus (CMV) promoter in pCI. Cells were transfected with the use of the Amaxa Human Dermal Fibroblast Nucleofactor kit (NHDF-adult) with program U-23 for human FLS. Murine FLS were transfected with the use of the Mouse Embryonic Fibroblasts kit (MEF1) with program T-20. Cells (2 × 105 to 106) were transfected with siRNAs, cDNAs, or control plasmids in each reaction.
Western blot analysis
Cultured FLS were washed with phosphate-buffered saline, and protein was extracted with lysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1.5 mM EDTA, 20 mM β-glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotonin, 1 μM pepstatin A, 1 mM phenylmethylsulphonyl fluoride). The protein concentrations were determined with the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Whole cell lysates containing 50 μg of protein were fractionated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline plus 0.1% Tween 20 (TBST) containing 5% non-fat milk for 1 hour at room temperature followed by incubation overnight with the appropriate antibody at 4°C. The membrane was washed three times and incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. Immunoreactive protein was detected by chemiluminescence with Kodak X-AR film (Eastman Kodak, Rochester, NY, USA).
siRNA-transfected cells for immunostaining were cultured in four-well chamber slides at 4.0 × 104 cells per well. They were then fixed with methanol, permeabilized with 0.05% Triton X-100 and blocked with 10% human serum. The fixed cells were incubated overnight with anti-p53 antibody or matched control antibody at 4°C. Endogenous peroxidase was then depleted with 0.1% H2O2 and 0.1% NaN3. The cells were then washed and stained with biotinylated secondary antibody anti-mouse or anti-rabbit IgG and Vectastain ABC and developed with diaminobenzidine (Vector, Burlingame, CA, USA).
Cell viability and apoptosis assays
FLS were harvested and suspended in 0.2% trypan blue and counted with a hemocytometer. Cells that excluded dye were considered viable. Apoptosis was determined with a Cell Death Detection ELISAPLUS kit (Roche Applied Science, Mannheim, Germany). FLS (4 × 103) were seeded into each well of a 96-well plate after transfection. Nine hours later, samples were collected and ELISA was performed in accordance with the manufacturer's instructions. Results are presented as the fold induction compared with control. To confirm the role of apoptosis, caspase-3 activation was also determined in transfected cells with the use of the human active caspase-3 ELISA (R&D Systems, Minneapolis, MN, USA). PUMA or PUMA-dBH3 plasmids were transduced into p53 siRNA-transfected or scrambled siRNA-transfected cells. The cells were then cultured at 4.5 × 105 cells per well in six-well plates. Eight hours later, the cells were lysed and assayed as described by the manufacturer.
Cell proliferation assay
Alamar Blue assays incorporate a fluorimetric/colorimetric growth indicator based on the detection of metabolic activity. FLS (3 × 103) were plated into 96-well plate after siRNA transfection. At various time points, medium was replaced by DMEM without phenol red supplemented with 10% Alamar Blue. After incubation for 4 hours at 37°C, fluorescence was measured with a microplate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The number of cells is expressed as relative fluorescence units.
Data are expressed as means ± SEM. Statistics were performed with Student's t test, one-way analysis of variance and repeated-measures analysis of variance. A comparison was considered significant at p < 0.05.
Use of siRNA to knockdown p53 protein in fibroblast-like synoviocytes
Functional effects of p53 deficiency
p53-independent apoptosis induced by PUMA
p53-independent apoptosis induced by PUMA in murine fibroblast-like synoviocytes
PUMA-mediated apoptosis in the presence of mutant p53
Several therapeutic approaches to RA have focused on inducing apoptosis in the synovium, especially the intimal lining [2, 4]. This region is populated by macrophage-like and fibroblast-like synoviocytes and is a primary source of cytokines and enzymes that degrade the extracellular matrix. The accumulation of cells in the lining can be due to ingress of cells from the blood, local proliferation, or insufficient deletion through apoptosis. The latter is especially intriguing in view of the observation that many pro-apoptotic genes are either defective or minimally expressed in RA, including p53, sentrin , and PTEN (phosphatase and tensin homologue deleted on chromosome 10 .
p53 is an interesting potential therapeutic gene because it can induce apoptosis in many cell types. Although controversial, defects in p53 structure and function in RA have been described, suggesting that forced expression of the tumor suppressor protein could be beneficial [11, 12, 21, 22]. However, enhancing p53 gene expression in synovium with an adenoviral construct had only modest efficacy in a rabbit model of arthritis , and a similar approach was not effective in the rat adjuvant arthritis model (P.P. Tak, D.L. Boyle, G.S. Firestein, unpublished data). One potential explanation for the limited effect is that p53 does not readily induce apoptosis in synoviocytes, probably because PUMA expression is not increased . In contrast, directly transducing cells with PUMA leads to rapid synoviocyte death in vitro.
One issue that could potentially interfere with the efficacy of PUMA gene therapy in RA is that this protein usually requires the p53 to induce apoptosis . Elegant studies have demonstrated that the mechanism of PUMA action is through the release of p53 from inhibitory interactions with Bcl-XL in the cytoplasm . Unbound p53 protein can then directly activate Bax. If p53 is defective or deficient, the benefit of forced PUMA expression would potentially be lost.
PUMA accounts for many of the apoptotic activities attributed to p53 [9, 10], although it can serve as a mediator of some apoptotic pathways that do are not initiated by p53 induction, including glucocorticoids and serum deprivation [7, 23]. PUMA-mediated apoptosis can also bypass p53 in unusual situations, especially in tumor cells. For instance, p53 expression does not require PUMA in melanoma and glioma cell lines [24, 25] or human leukemia cells . Hence, the utility of PUMA as an apoptosis-inducing protein and its relationship to p53 depends on the cell lineage, the status of p53 (deficiency versus mutation), and the type of stimulus. Therefore p53 has a dual role related to PUMA gene expression and function. In most cell types, p53 expression leads to increased PUMA gene expression and subsequent PUMA-mediated apoptosis requires functional p53. It is of interest that neither of these relationships is effective in cultured FLS. This cell lineage can also be distinguished from other cells in that p53 is expressed constitutively  even though the short half-life of wild-type p53 protein generally limits detection in non-cycling cells.
These highly variable data imply that tissue-specific cells should be studied to determine the potential applicability of PUMA gene therapy to RA. Our experiments using siRNA to decrease p53 expression show that FLS are very sensitive to PUMA-induced death and that p53 expression has no influence on this effect. Because siRNA does not completely deplete p53 levels, we confirmed these results in p53-/- murine FLS. Finally, we showed that PUMA could function even in cells transfected with a known dominant-negative p53 mutant. These data demonstrate that PUMA-induced apoptosis in synoviocytes does not require p53 and that PUMA gene transfer could be effective regardless of the p53 status of the synovium.
These data support the potential use of PUMA as a local gene therapy approach to RA. By circumventing possible abnormalities in p53 and inducing extensive apoptosis of synoviocytes, intra-articular gene transfer could decrease the hyperplasia of the synovial intimal lining. Although not feasible for systemic administration, local therapy could debulk the synovium in RA and serve as an alternative to synovectomy or intra-articular corticosteroids.
PUMA efficiently induced apoptosis in control and p53 deficient human FLS after PUMA overexpression. Similar results were observed in p53-/- murine FLS and in human FLS transfected with the R123* mutant p53 gene. PUMA-induced apoptosis is therefore independent of p53 in FLS. These data suggest that PUMA gene therapy could be effective in RA regardless of the p53 status of the synovium.
= Bcl-2 homology 3
= Dulbecco's modified Eagle's medium
= enzyme-linked immunosorbent assay
= fibroblast-like synoviocytes
= p53-upregulated modulator of apoptosis
= rheumatoid arthritis
= small interfering RNA.
This work was supported by grant R01 AR45347 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases.
- Firestein GS: Invasive fibroblast-like synoviocytes in rheumatoid arthritis. Passive responders or transformed aggressors?. Arthritis Rheum. 1996, 39: 1781-1790.View ArticlePubMedGoogle Scholar
- Okamoto K, Asahara H, Kobayashi T, Matsuno H, Hasunuma T, Kobata T, Sumida T, Nishioka K: Induction of apoptosis in the rheumatoid synovium by Fas ligand gene transfer. Gene Ther. 1998, 5: 331-338. 10.1038/sj.gt.3300597.View ArticlePubMedGoogle Scholar
- Yao Q, Seol DW, Mi Z, Robbins PD: Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis. Arthritis Res Ther. 2005, 8: R16-10.1186/ar1867.PubMed CentralView ArticleGoogle Scholar
- Yao Q, Wang S, Glorioso JC, Evans CH, Robbins PD, Ghivizzani SC, Oligino TJ: Gene transfer of p53 to arthritic joints stimulates synovial apoptosis and inhibits inflammation. Mol Ther. 2001, 3: 901-910. 10.1006/mthe.2001.0343.View ArticlePubMedGoogle Scholar
- Cha HS, Rosengren S, Boyle DL, Firestein GS: PUMA regulation and proapoptotic effects in fibroblast-like synoviocytes. Arthritis Rheum. 2006, 54: 587-592. 10.1002/art.21631.View ArticlePubMedGoogle Scholar
- Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer DD, Green DR: PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Science. 2005, 309: 1732-1735. 10.1126/science.1114297.View ArticlePubMedGoogle Scholar
- Jeffers JR, Parganas E, Lee Y, Yang C, Wang J, Brennan J, MacLean KH, Han J, Chittenden T, Ihle JN, et al: Puma is an essential mediator of p53-dependent and -independent apoptotic pathways. Cancer Cell. 2003, 4: 321-328. 10.1016/S1535-6108(03)00244-7.View ArticlePubMedGoogle Scholar
- Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, Ausserlechner MJ, Adams JM, Strasser A: p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science. 2003, 302: 1036-1038. 10.1126/science.1090072.View ArticlePubMedGoogle Scholar
- Nakano K, Vousden KH: PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell. 2001, 7: 683-694. 10.1016/S1097-2765(01)00214-3.View ArticlePubMedGoogle Scholar
- Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B: PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell. 2001, 7: 673-682. 10.1016/S1097-2765(01)00213-1.View ArticlePubMedGoogle Scholar
- Yamanishi Y, Boyle DL, Rosengren S, Green DR, Zvaifler NJ, Firestein GS: Regional analysis of p53 mutations in rheumatoid arthritis synovium. Proc Natl Acad Sci USA. 2002, 99: 10025-10030. 10.1073/pnas.152333199.PubMed CentralView ArticlePubMedGoogle Scholar
- Firestein GS, Echeverri F, Yeo M, Zvaifler NJ, Green DR: Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium. Proc Natl Acad Sci USA. 1997, 94: 10895-10900. 10.1073/pnas.94.20.10895.PubMed CentralView ArticlePubMedGoogle Scholar
- Inazuka M, Tahira T, Horiuchi T, Harashima S, Sawabe T, Kondo M, Miyahara H, Hayashi K: Analysis of p53 tumour suppressor gene somatic mutations in rheumatoid arthritis synovium. Rheumatology (Oxford). 2000, 39: 262-266. 10.1093/rheumatology/39.3.262.View ArticleGoogle Scholar
- Han Z, Boyle DL, Shi Y, Green DR, Firestein GS: Dominant-negative p53 mutations in rheumatoid arthritis. Arthritis Rheum. 1999, 42: 1088-1092. 10.1002/1529-0131(199906)42:6<1088::AID-ANR4>3.0.CO;2-E.View ArticlePubMedGoogle Scholar
- Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, et al: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988, 31: 315-324.View ArticlePubMedGoogle Scholar
- Alvaro-Gracia JM, Yu C, Zvaifler NJ, Firestein GS: Mutual antagonism between interferon-γ and tumor necrosis factor-α on fibroblast-like synoviocytes: paradoxical induction of IFN-γ and TNF-α receptor expression. J Clin Immunol. 1993, 13: 212-218. 10.1007/BF00919974.View ArticlePubMedGoogle Scholar
- Yamanishi Y, Boyle DL, Pinkoski MJ, Mahboubi A, Lin T, Han Z, Zvaifler NJ, Green DR, Firestein GS: Regulation of joint destruction and inflammation by p53 in collagen-induced arthritis. Am J Pathol. 2002, 160: 123-130.PubMed CentralView ArticlePubMedGoogle Scholar
- Pap T, Aupperle KR, Gay S, Firestein GS, Gay RE: Invasiveness of synovial fibroblasts is regulated by p53 in the SCID mouse in vivo model of cartilage invasion. Arthritis Rheum. 2001, 44: 676-681. 10.1002/1529-0131(200103)44:3<676::AID-ANR117>3.0.CO;2-6.View ArticlePubMedGoogle Scholar
- Franz JK, Pap T, Hummel KM, Nawrath M, Aicher WK, Shigeyama Y, Muller-Ladner U, Gay RE, Gay S: Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis. Arthritis Rheum. 2000, 43: 599-607. 10.1002/1529-0131(200003)43:3<599::AID-ANR17>3.0.CO;2-T.View ArticlePubMedGoogle Scholar
- Pap T, Franz JK, Hummel KM, Jeisy E, Gay R, Gay S: Activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor PTEN at sites of invasive growth and destruction. Arthritis Res. 2000, 2: 59-64. 10.1186/ar69.PubMed CentralView ArticlePubMedGoogle Scholar
- Reme T, Travaglio A, Gueydon E, Adla L, Jorgensen C, Sany J: Mutations of the p53 tumour suppressor gene in erosive rheumatoid synovial tissue. Clin Exp Immunol. 1998, 111: 353-358. 10.1046/j.1365-2249.1998.00508.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Kullmann F, Judex M, Neudecker I, Lechner S, Justen HP, Green DR, Wessinghage D, Firestein GS, Gay S, Scholmerich J, et al: Analysis of the p53 tumor suppressor gene in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 1999, 42: 1594-1600. 10.1002/1529-0131(199908)42:8<1594::AID-ANR5>3.0.CO;2-#.View ArticlePubMedGoogle Scholar
- Yu J, Wang Z, Kinzler KW, Vogelstein B, Zhang L: PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci USA. 2003, 100: 1931-1936. 10.1073/pnas.2627984100.PubMed CentralView ArticlePubMedGoogle Scholar
- Karst AM, Dai DL, Martinka M, Li G: PUMA expression is significantly reduced in human cutaneous melanomas. Oncogene. 2005, 24: 1111-1116. 10.1038/sj.onc.1208374.View ArticlePubMedGoogle Scholar
- Ito H, Kanzawa T, Miyoshi T, Hirohata S, Kyo S, Iwamaru A, Aoki H, Kondo Y, Kondo S: Therapeutic efficacy of PUMA for malignant glioma cells regardless of p53 status. Hum Gene Ther. 2005, 16: 685-698. 10.1089/hum.2005.16.685.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu FT, Newland AC, Jia L: Bax conformational change is a crucial step for PUMA-mediated apoptosis in human leukemia. Biochem Biophys Res Commun. 2003, 310: 956-962. 10.1016/j.bbrc.2003.09.109.View ArticlePubMedGoogle Scholar
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