1 Department of Internal Medicine, Far-Eastern Memorial Hospital, Pan-Chiao, 2 Department of Internal Medicine, 3 Center for Optoelectronic Biomedicine and 4 Department of Surgery, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
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Abstract |
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Methods. HPMC was cultured from human omentum by an enzyme digestion method. The expression of collagen 1(I) mRNA was determined by northern blotting, while the SMAD proteins and the MAPK kinase activity were determined by western blotting.
Results. TGF-ß-stimulated collagen 1(I) mRNA expression of HPMC was inhibited by PTX. The Smad2, ERK1/2 and p38HOG pathways were activated in response to TGF-ß. However, TGF-ß displayed no activation of the JNK pathway in HPMC. The addition of PD98059 and SB203580, which blocked the activation of ERK1/2 and p38HOG, respectively, suppressed the TGF-ß-induced collagen
1(I) mRNA expression. At a concentration (300 µg/ml) that inhibited the collagen gene expression, PTX suppressed the ERK1/2 and p38HOG activation by TGF-ß. In contrast, PTX had no effect on the TGF-ß-induced activation of Smad2, under the same concentration.
Conclusion. PTX inhibits the TGF-ß-induced collagen gene expression in HPMC through modulating the ERK1/2 and p38HOG pathways. Our study of PTX may provide the therapeutic basis for clinical applications in the prevention of EPS.
Keywords: encapsulating peritoneal sclerosis; mesothelial cell; pentoxifylline; signal transduction; TGF-ß
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Introduction |
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EPS is a serious complication that develops in long-term PD patients [2]. Fibrogenic matrix accumulation is important in the pathogenesis of EPS [3]. Transforming growth factor-ß (TGF-ß) has been regarded as the central mediator of the fibrosing process in clinical diseases. PD patients with persistent TGF-ß in their drained effluent were found to associate with an increased risk of EPS [4]. In addition, we had reported previously that TGF-ß stimulates the expression of types I and III collagen mRNA in cultured HPMC [5]. These observations imply that TGF-ß may mediate the development of EPS in PD patients. Pharmacological agents that attenuate TGF-ß-induced matrix synthesis of HPMC may have clinical implications in the prevention or retardation of EPS.
Despite the well-recognized association between TGF-ß and matrix accumulation, limited information is available regarding the mechanisms of TGF-ß to induce this process [6]. The SMAD family members have been identified as major intracellular mediators of TGF-ß signalling [7]. TGF-ß first binds to the type II receptor on the cell membrane, then recruits the type I receptor into a complex. The phosphorylated type I receptor activates Smad2 and allows it to form a heteromultimer with Smad4. This complex is then translocated to the nucleus to regulate the transcription of target genes. In addition to the SMAD proteins, the mitogen-activated protein kinase (MAPK) pathways have been recently proposed to transmit parts of the downstream signallings of TGF-ß [6,8]. The MAPK pathways contain three phosphorylation cascades: the extracellular signal-regulated protein kinase (ERK), the c-Jun N-terminal kinase (JNK), and p38HOG. In our previous report [9], we demonstrated the presence of SMAD proteins, which acted in response to TGF-ß in HPMC. We also showed that ERK1/2 was activated by TGF-ß, and the blockade of ERK1/2 activity resulted in the decrease of TGF-ß-induced 1(I) collagen gene expression. In other cell systems, the p38HOG [10] and/or the JNK [11] pathways have been demonstrated to be one of the downstream targets required for TGF-ß-mediated matrix expansion. In HPMC, however, the role of the JNK and the p38HOG pathways in response to TGF-ß has never been investigated.
Pentoxifylline (PTX) is a widely used anti-platelet agent. In addition to its anti-platelet effect, we demonstrated previously in human vascular smooth muscle cells [12] and in HPMC [5] that PTX may attenuate TGF-ß-induced collagen synthesis. Nevertheless, the molecular mechanism of this inhibitory effect of PTX on TGF-ß-induced collagen gene expression in HPMC remains undetermined.
In this work, we initially elucidated the intra-cellular signalling network (SMAD proteins, ERK1/2, JNK and the p38HOG pathways) downstream of TGF-ß in HPMC. Next, we further explored the inhibitory mechanism of PTX on TGF-ß-treated HPMC by modulating these signalling pathways. Our results may provide a pharmacological basis of PTX for the treatment of EPS.
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Materials and methods |
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Establishment of HPMC culture
Specimens of human omentum were obtained from omentectomy for elective gastric cancer resection, in which the omentum was grossly normal. HPMC culture was carried out as we previously reported [5]. Briefly, the surgically removed human omentum was washed thrice with PBS and then digested with trypsin EDTA (0.125%, Gibco) for 15 min. After centrifugation, the cell pellet was washed with culture medium and then seeded into a gelatin-coated (1 mg/ml) flask. Medium was changed on the third day. RPMI-1640 medium containing 20% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml) and insulin (30 µg /ml) was used. After 24 days, the cells became confluent and were subcultured with medium containing 10% FCS. HPMC was identified by the presence of vimentin and cytokeratin but without desmin and factor VIII-related antigen by the immunofluorescence method. All experiences were performed in passages 13 cells.
Northern blot analysis
To determine the effect of PTX on collagen gene expression, HPMC was grown in RPMI supplemented with 10% FCS until subconfluent. HPMC was arrested by 0.5% FCS for 24 h and then treated with TGF-ß (12.5 ng/ml) in the absence or presence of various concentrations of PTX. After 24 h, cells were harvested for isolation of total RNA, as described previously [5,9]. The concentration of each sample was determined using spectrophotometry with the absorbance at 260 nm (A260). The purity of each sample was determined based on the ratio of A260 to A280. Ten micrograms of RNA were electrophoresed on a 1% agarose gel containing 1 M formaldehyde in MOPS buffer (0.2 M morpholinopropanesulfonic acid, 0.05M Na acetate, 0.01 M EDTA). The equivalency of sample loading and the lack of degradation were verified by ethidium bromide staining of 28S and 18S rRNA bands. The RNA was then transferred to nylon membranes by overnight capillary action and followed by fixation in a UV cross-linker.
For northern blotting, a 1.5-kb EcoRI fragment of collagen 1(I) was subcloned into pBSII/SK (Stratagene, La Jolla, CA) and used as templates for in vitro transcription of antisense digoxigenin-conjugated riboprobes according to the manufacturer's instructions (Boehringer Mannheim). The blots were developed using CSPD (Boehringer Mannheim) as the substrate for alkaline phosphatase, as described by the supplier. The signal intensity recorded on X-ray film was then quantified with computerized densitometry and was normalized against the signal of glyceraldehyde-3-phosphate dehydrogenase messages.
Cell preparations and protein extraction
HPMC was grown in 10 cm dishes until subconfluent, growth-arrested (0.5% FCS) for 24 h and then was harvested after treatment at indicated periods of time with 200 µl ice-cold lysis buffer. The lysis buffer contains: 50 mM TrisHCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 40 mM ß-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 200 µM sodium orthovanadate, 10 µg leupeptin/ml, 200 U aprotinin/ml, 1 µM pepstatin A, 1 mM PMSF, 100 nM okadaic acid. The obtained cell lysate was centrifugated at 14 000 r.p.m. for 5 min, and the protein concentration of the supernatant measured by BCA protein assay (Pierce).
Western blot analysis
The western blotting analysis of the MAPK pathways and SMAD proteins was performed by using phospho-specific antibodies according to the manufacturer's protocol. Briefly, HPMC was low serum (0.5% FCS) starved for 24 h, then stimulated with TGF-ß (2.5 ng/ml) for different time intervals. For evaluating the inhibitory effect of PTX on TGF-ß-stimulated activation of MAPK (ERK1/2, JNK, p38HOG) and/or SMAD pathways, we incubated HPMC with 300 µg/ml PTX for 30 min before stimulation with TGF-ß. The cells were harvested at the indicated time period then for western blotting. In addition, lanes of cells were treated with 3 or 10 µM H89 for 30 min before PTX. Cell lysates (30 µg protein) were separated by SDSPAGE (12%), then transferred to PVDF membrane (Millipore, Bedford, MA). For immunodetection, the membranes were probed with primary antibody followed by incubation with peroxidase-conjugated secondary antibodies. Bands were visualized by the ECL system (Amersham).
Statistical analysis
The results are expressed as mean±SEM, unless otherwise stated. These statistical analyses were carried out using StatView IV on a personal computer. Statistical significance (P<0.05) was evaluated using the Student's t-test or one way analysis of variance with modified t-test performed using the Bonferroni correction.
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Results |
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PTX inhibits TGF-ß-induced 1(I) mRNA expression in HPMCs
The inhibitory effect of PTX on TGF-ß-induced expression of collagen 1(I) mRNA level was analysed in HPMC. Similar to what we reported previously [5], TGF-ß induced collagen
1(I) mRNA expression in HPMC (Figure 1
). Compared with untreated cells (control), TGF-ß enhanced the collagen
1(I) mRNA levels. The treatment with PTX (300 µg/ml) potently down-regulated the collagen
1(I) mRNA expression by TGF-ß in HPMC. PTX had been reported to exert its biological effect through the cAMPPKA pathway [5,12]. Accordingly, we also examined the effect of DBcAMP, a membrane-permeable analogue of cAMP, on TGF-ß-induced collagen
1(I) mRNA expression and tested whether the blockade of the cAMPPKA pathway can overcome the inhibitory effect of PTX in TGF-ß-treated HPMC (Figure 1
). As expected, the exposure of HPMC to DBcAMP (1 mM) inhibited the induction of collagen
1(I) mRNA expression by TGF-ß. On the other hand, the combination of PTX and H-89 (10 µM), a non-specific PKA inhibitor, prevented the suppressive effect of PTX on collagen
1(I) mRNA expression by TGF-ß.
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TGF-ß activates ERK1/2, Smad2 and the p38HOG pathways in HPMC
We [9] demonstrated previously in HPMC the presence of ERK1/2 and Smad2/4 proteins, which were both activated in response to TGF-ß. Briefly, after adding TGF-ß (2.5 ng/ml), Smad2 (58 kDa) was rapidly phosphorylated, which began within 15 min, peaked within 1530 min, and persisted for 8 h. In contrast, the protein levels of Smad4 (62 kDa) were not changed in response to TGF-ß (data not shown). In HPMC, TGF-ß also activated ERK1/2, which began within 15 min after TGF-ß, peak occurring in 30 min, and persisted for around 48 h (data not shown).
In this work, we further evaluated the effect of TGF-ß on the other two MAPK pathways, the JNK and the p38HOG pathways, in HPMC. As shown in Figure 2A, p38HOG was activated within 15 min after TGF-ß (2.5 ng/ml), peak occurring in 30 min, and returned to baseline by 8 h. In contrast, the JNK1/2 pathway displayed no activation in response to TGF-ß (2.5 ng/ml) (Figure 2B
). Similar results were observed at a higher concentration of TGF-ß (5 ng/ml, data not shown). Protein levels of JNK1/2 and p38HOG remained constant during treatment with TGF-ß. Conclusively, we found that ERK1/2 [9], Smad2 [9] and p38HOG (Figure 2
) were activated by TGF-ß, which may be responsible for the consequence of enhanced collagen
1(I) gene expression in HPMC.
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PTX suppressed downstream signalling of TGF-ß through modulations of ERK1/2 and p38HOG pathways
To explore the inhibitory mechanism of PTX on collagen 1(I) gene induction by TGF-ß, we examined whether PTX modulates these intracellular signalling pathways downstream to TGF-ß in HPMC. As shown in Figure 3
, the activation of Smad2 by TGF-ß was not significantly altered by PTX (300 µg/ml) in the presence or absence of H-90. In contrast, PTX (300 µg/ml) markedly suppressed ERK1/2 and p38HOG activation by TGF-ß (2.5 ng/ml), which were rescued in the presence of H-89. We also tested the inhibitory effect of DBcAMP on activation of ERK1/2 or and p38HOG by TGF-ß (Figure 4
). In TGF-ß-treated HPMC, DBcAMP (0.31 mM) suppressed the activation of ERK1/2 and p38HOG. Pre-treatment with H-89 (10 µM) before the addition of DBcAMP preserved the activation of ERK1/2 and P38HOG by TGF-ß. We next examined the effects of blockade of these transduction pathways (ERK1/2 and p38HOG) on collagen
1(I) gene induction by TGF-ß. Similar to what we observed in a previous report [9], PD98059 (25 µM) markedly inhibited the induction of collagen
1(I) gene expression by TGF-ß (Figure 5
). Similarly, the ability of TGF-ß to stimulate collagen
1(I) gene expression was also reduced by SB203580 (30 µM), a selective inhibitor of the p38HOG pathway. These observations indicated that ERK1/2 and p38HOG, but not the Smad2 pathway, were involved in the inhibitory effect of PTX on TGF-ß-induced collagen
1(I) gene expression in HPMC. The modulations of ERK1/2 and p38HOG by PTX may mainly operate through the cAMPPKA pathway.
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Interaction of the ERK1/2 pathway and p38HOG in HPMC
Although the ERK1/2 and the p38HOG have been recognized as two independent transduction pathways for intracellular signalling, the nature of their possible interaction in HPMC is not clear. We performed experiments to evaluate the potential interaction between these two pathways. HPMC pre-treated with PD98059 did not influence the activation of P38HOG upon TGF-ß treatment (data not shown). Similarly, the activation of ERK1/2 by TGF-ß was not altered in the presence of SB203580. These observations suggest that in HPMC, TGF-ß induced collagen 1(I) gene expression through two independent pathways: the ERK1/2 and p38HOG, which were both inhibited by PTX.
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Discussion |
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As PTX inhibited collagen 1(I) gene induction by TGF-ß, we next examined the effect of PTX tomodulate these intracellular signalling pathways (ERK1/2, Smad2 and p38HOG) downstream to TGF-ß in HPMC. As shown in Figure 3
, PTX (300 µg/ml) did not alter the activation of Smad2 by TGF-ß. In contrast, PTX (300 µg/ml) markedly suppressed ERK1/2 and p38HOG activation by TGF-ß. Furthermore, the blockade of ERK1/2 and p38HOG by PD98059 (25 µM) and SB203580 (30 µM), respectively, abrogated the induction of collagen
1(I) gene expression by TGF-ß (Figure 5
). These observations indicated that the inhibitory mechanism of PTX on TGF-ß-induced collagen
1(I) gene expression in HPMC were operated mainly through the ERK1/2 and p38HOG pathways.
PTX had been reported to exert its biological effect through the cAMPPKA pathway [5,10]. Accordingly, the role of the cAMPPKA pathway mediating the effect of PTX in TGF-ß-treated HPMC was also determined. In this study, we found that the suppressive effect of PTX on ERK1/2 and p38HOG activation by TGF-ß was reversed in the presence of H-89 (Figure 3). We also tested the inhibitory effects of DBcAMP and PTX on the induction of collagen
1(I) mRNA expression by TGF-ß (Figure 1
). PTX (300 µg/ml) as well as DBcAMP (1 mM) inhibited the enhancement of collagen
1(I) mRNA expression by TGF-ß, which were reversed by pre-treatment of HPMC with H-89 (10 µM) before the addition of DBcAMP or PTX. The proposed possible mechanism of PTX on collagen
1(I) mRNA expression by TGF-ß was described in Figure 6
.
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Another important issue we investigated in this work is the roles of the p38HOG and JNK pathways in TGF-ß-treated HPMC. Our data demonstrated the involvement of p38HOG, but not the JNK pathway, in TGF-ß-induced collagen 1(I) mRNA expression of HPMC (Figures 3
and 5
). During the preparation of our work, a similar observation in cultured myoblast was newly reported by Rodriguez-Barbero et al. [16]. They noticed that TGF-ß-induced collagen
2(I) mRNA expression through the activated p38HOG and ERK1/2 pathways, but independent of JNK phosphorylation. PTX (Figure 3
) and DBcAMP (Figure 4B
) both reduced the TGF-ß-induced phosphorylation of p38HOG, which were blunted by H-89. We believe that PTX modulated the p38HOG pathway through the cAMPPKA pathway. Nevertheless, the debate on possible cross-talk between the cAMPPKA and the MAPK family continues. The elevation of intracellular cAMP in cardiomyocyte [17] or T-cell [18] increased p38HOG activity. In contrast, activated cAMPPKA activity that abolished p38HOG phosphorylation was also noticed in ET-1 treated osteoblast [19]. Different cells under various stimulatory conditions may exhibit diverse intracellular responses. The possible cross-talk between PKA and MAPK families in HPMC deserves further study.
In summary, we explored in HPMC the downstream signalling pathways of TGF-ß. PTX, through modulations of the ERK1/2 and p38HOG pathways, inhibited TGF-ß-induced collagen 1(I) gene expression. Our observations may provide a therapeutic basis for PTX in the prevention or retardation of EPS.
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Acknowledgments |
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Notes |
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References |
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