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Keywords:

  • Angiotensin;
  • Hepatocyte growth factor;
  • Mesothelial cell;
  • Peritoneal fibrosis;
  • Peroxisome;
  • Proliferator-activated receptor-γ

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an endogenous peroxisome proliferator-activated receptor γ (PPARγ) agonist that suppresses progressive matrix deposition; however, little is known about the effects of 15d-PGJ2 on human peritoneal mesothelial cells (HPMCs). We investigated the following: (i) the expression of PPARγ; (ii) the effect of 15d-PGJ2 on angiotensin II (Ang II)-induced fibronectin (FN) expression and secretion; (iii) the effect of 15d-PGJ2 (with or without Ang II and with or without the specific PPARγ antagonist GW9662) and pioglitazone, a synthetic PPARγ agonist, on hepatocyte growth factor (HGF) expression and secretion; (iv) the effect of HGF on Ang II-induced FN expression and secretion; (v) the expression of c-Met (a specific HGF receptor) and its phospho-signal; and (vi) the involvement of HGF in the effect produced by 15d-PGJ2 using selective c-Met inhibitor PHA-665752. The presence of PPARγ was detected by western blot analysis. 15d-PGJ2 inhibited Ang II-induced FN expression and increased HGF expression, even in the presence of Ang II. This effect of HGF expression was completely prevented by co-treatment with GW9662. Additionally, upregulation of HGF secretion induced by 15d-PGJ2 and HGF production induced by pioglitazone was revealed. We demonstrated the presence of c-Met, and presented evidence that HGF inhibits Ang II-induced FN expression and activates phosphorylation of c-Met, which is blocked by PHA-665752; 15d-PGJ2 also activated c-Met phosphorylation. Furthermore, PHA-665752 attenuates the inhibitory effects of 15d-PGJ2 on FN secretion. These findings suggest that 15d-PGJ2 has a novel and potent antifibrotic effect in HPMC and this action is likely mediated by HGF.

Peritoneal dialysis (PD) is a widely used renal replacement therapy. Peritoneal fibrosis (PF), characterized by submesothelial fibrosis and a thickening of the basement membrane, is an inevitable occurrence in PD patients over time (1). In turn, PF can lead to the development of encapsulating peritoneal sclerosis (EPS), a potentially fatal complication of long-term PD treatment. EPS can be avoided with the prevention of PF; however, the biological mechanisms underlying PF remain unclear and an effective treatment has not been identified.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor belonging to the nuclear hormone receptor superfamily (2). The most potent natural agonist for PPARγ is 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous prostaglandin D2 metabolite (3). A variety of synthetic thiazolidinediones, such as pioglitazone, rosiglitazone, and troglitazone, are also classified as PPARγ agonists and are used as insulin-sensitizing agents in patients with type 2 diabetes. Recent findings indicate that these agents play a role in the suppression of progressive fibrosis (4–6); however, there is scant data regarding the role of PPARγ agonists in human peritoneal mesothelial cells (HPMCs).

Accumulating evidence indicates that HPMCs may produce various cytokines (7) and extracellular matrix (ECM) proteins (8). Treatment of HPMCs with angiotensin II (Ang II) induces the expression of fibronectin (FN) (9), a key component of the ECM that serves as a scaffold for the deposition of other proteins. Therefore, this study focused on the effects of 15d-PGJ2 on FN expression in HPMCs.

Hepatocyte growth factor (HGF) was first purified as a potent mitogen for mature hepatocytes (10). Studies from numerous laboratories have shown that HGF is an endogenous antifibrotic factor that is capable of ameliorating fibrotic lesions and preserving organ function. Although two recent reports have suggested a beneficial effect of HGF upon HPMCs (11,12), the role of HGF in PF has not been thoroughly investigated. While the induction of HGF expression by PPARγ agonists has been reported (4,13,14), there are few reports on the production of HGF by HPMCs, and the association between PPARγ agonists and HGF is unclear.

The HGF protein binds to its high-affinity receptor c- Met tyrosine kinase, leading to phosphorylation of multiple serine and tyrosine residue sites. Studies have been advanced, particularly in oncogenes (15), that suggest that c-Met would be an important target for cancer therapy; however, the evidence is limited for the c-Met/HGF pathway in HPMCs. Therefore, the goal of this study was to evaluate the antifibrotic effect of 15d-PGJ2, an endogenous PPARγ agonist, in HPMCs through the c-Met/HGF pathway.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

Reagents

Ang II was purchased from Sigma (St Louis, MO, USA). Both 15d-PGJ2 and GW9662 were obtained from Calbiochem (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO) (Sigma). PHA-665752 was obtained from Tocris Bioscience (Ellisville, MO, USA) and dissolved in DMSO. Recombinant human HGF (rhHGF) was obtained from R&D Systems (Minneapolis, MN, USA) and dissolved in phosphate-buffered saline including 0.1% bovine serum albumin (Sigma). Pioglitazone, kindly provided by Takeda Chemical Industries (Osaka, Japan), was dissolved in DMSO.

Cell culture

HPMCs were isolated from pieces of human omentum and maintained as previously described (9). All data presented are from experiments performed with confluent HPMCs from the second to third passage. All cells were washed and growth was arrested for 48 h in control standard M199 medium (Invitrogen Corporation, Carlsbad, CA, USA) containing 0.1% (vol/vol) fetal calf serum (Sigma) prior to each stimulation. Harvesting of the omentum was permitted by the Medical Ethics Committee of Hiroshima Graduate School of Biomedical Sciences and informed consent was obtained from all patients.

RNA extraction and quantitative real-time RT-PCR

Total RNA was extracted as previously described (9) and used to synthesize cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed on a 7500 Fast Real-Time PCR system using TaqMan Universal PCR Master Mix and universal cycling conditions (Applied Biosystems). Gene-specific oligonucleotide primers and probes for FN (assay ID: Hs00365058_m1), HGF (assay ID: Hs00300159_m1), and 18S rRNA (the endogenous control) were obtained as TaqMan Gene Expression Assays (Applied Biosystems). Data analysis was performed using 7500 Fast System Sequence Detection Software version 1.4 (Applied Biosystems).

Western blot analysis

Sample collection and immunoblotting were performed as previously described (9). Total protein content of the HPMC supernatants and lysates was determined using the BCA protein assay reagent (Pierce, Rockford, IL, USA). For detection of PPARγ, lysates were immunoblotted using anti-PPARγ antibody (Cell Signaling Technology, Beverly, MA, USA) at 1:1000 dilution overnight at 4°C. For detection of HGF, lysates and supernatants were immunoblotted using anti-human HGF-α antibody (Immuno-Biological Laboratories, Gunma, Japan) and monoclonal anti-human HGF antibody (R&D Systems) at 2 µg/mL overnight at 4°C. For detection of c-Met and its phosphorylation, lysates were immunoblotted using c-Met polyclonal antibody (C-12; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 : 750 dilution overnight at 4°C and phosphospecific c-Met antibodies against the phosphoepitopes [pY1230/1234/1235] and [pY1003] (BioSource International, Camarillo, CA, USA) at 1 : 1000 dilution for 2 h at room temperature. The detection of supernatant FN was performed as previously described (9). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (Dako, Carpinteria, CA, USA) or goat anti-mouse IgG antibody (Dako) were used as secondary antibodies. Monoclonal anti-actin antibody (Sigma) was used as the internal control for analysis of HGF production. The intensity of each band was estimated using National Institutes of Health Image software (version 1.6; NIH, Bethesda, MD, USA).

Statistical analysis

The individual experiments were usually performed at least thrice, and the results were expressed as mean ± SD. Statistical analysis was performed by analysis of variance (anova) followed by Tukey's post hoc test, unless otherwise indicated. P-values of <0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

Based on preliminary experiments to examine the effect of various concentrations of Ang II on FN expression, a concentration of 100 nmol/L Ang II was chosen for the following experiments.

Expression of PPARγ in HPMCs

Western blot analysis in HPMCs with anti-PPARγ antibody revealed the presence of two proteins (Fig. 1). The proteins were identified as PPARγ1 (53 kDa) and PPARγ2 (57 kDa).

image

Figure 1. Peroxisome proliferator-activated receptor γ (PPARγ) expression in human peritoneal mesothelial cells (HPMCs). Western blotting shows that PPARγ is expressed in HPMCs as 53 kDa (PPARγ1) and 57 kDa (PPARγ2) proteins.

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15d-PGJ2 inhibits Ang II-induced upregulation of FN expression

The addition of 100 nmol/L Ang II induced a significant increase in FN mRNA expression at 6 h (1.86-fold; P < 0.01 vs control) (Fig. 2A) and FN secretion at 24 h (1.98-fold; P < 0.01 vs control) (Fig. 2B). Treatment with 15d-PGJ2 at a concentration of 5 µmol/L completely suppressed upregulation of FN at the mRNA and protein levels (P < 0.01 vs Ang II alone) (Fig. 2).

image

Figure 2. Effects of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on angiotensin II (Ang II)-induced fibronectin (FN) mRNA and protein expression levels in human peritoneal mesothelial cells. Cells were preincubated with 15d-PGJ2 (2.5 and 5 µmol/L) or vehicle control for 24 h before stimulation with Ang II (100 nmol/L). (A) Quantitative real-time PCR was performed to assess the expression of FN mRNA at 6 h after incubation. The graph shows the relative FN mRNA levels normalized to 18S rRNA. (B) After incubation for 24 h, the culture supernatants were blotted with antibody to FN (15 µg protein loaded per lane). The graph shows the densitometric analysis of the FN band. Values are the mean ± SD of three individual experiments. *P < 0.05, **P < 0.01.

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Upregulation of HGF expression by PPARγ agonists

To assess the association between 15d-PGJ2 and HGF expression, we evaluated the effects of 15d-PGJ2 on HGF mRNA expression and HGF production. 15d-PGJ2 was used as a concentration of 5 µmol/L, which completely suppressed the Ang II-induced upregulation of FN expression to basal levels. After exposure of HPMCs to 15d-PGJ2, the expression of HGF mRNA gradually increased and peaked at 3 h (2.04-fold; P < 0.01 vs 0, 1, and 6 h) with expression falling at 6 h (Fig. 3A). Treatment with 15d-PGJ2 resulted in a significant increase in the expression of HGF protein that reached a plateau at 16 h (2.71-fold; P < 0.01 vs 0 and 8 h) (Fig. 3B). The effects of pioglitazone on HGF production were also analyzed. Based on preliminary experiments, pioglitazone was used at a concentration of 10 µmol/L, which suppressed the Ang II-induced upregulation of FN secretion to basal levels. The production of HGF gradually increased and reached a plateau at 12 h (1.99-fold; P < 0.05 vs 0 h) (Fig. 3C). We then examined the secretion of HGF induced by 15d-PGJ2. HGF secretion gradually increased for 24 h (Fig. 3D), with significant secretion at 24 h (2.29-fold; P < 0.01 vs control) (Fig. 3E). 15d-PGJ2 also induced HGF mRNA and protein expression even in the presence of Ang II (Fig. 4).

image

Figure 3. Effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists on hepatocyte growth factor (HGF) mRNA and protein expression levels in human peritoneal mesothelial cells. Cells were incubated with 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) (5 µmol/L) or pioglitazone (10 µmol/L) at the indicated times. (A) Quantitative real-time PCR was performed to assess the expression of HGF mRNA. The graph shows the relative HGF mRNA levels normalized to 18S rRNA. (B,C) Cell lysates were blotted with HGF and actin antibodies. The authenticity of HGF was confirmed by loading purified human HGF protein on an adjacent lane (data not shown). The graph shows the densitometric analysis of the HGF band standardized to actin. (D) The culture supernatants were blotted with antibody to HGF (25 µg protein loaded per lane). (E) The culture supernatants were blotted after incubation with or without 15d-PGJ2 for 24 h. The graph shows the densitometric analysis of the HGF band. Values are the mean ± SD of at least three individual experiments. *P < 0.05, **P < 0.01. NS, not significant.

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image

Figure 4. Effect of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on hepatocyte growth factor (HGF) mRNA and protein expression levels stimulated with or without angiotensin II (Ang II) in human peritoneal mesothelial cells. Cells were incubated with 15d-PGJ2 (5 µmol/L) or vehicle control in the presence or absence of Ang II (100 nmol/L). (A) Quantitative real-time PCR was performed to assess the expression of HGF mRNA at 3 h after incubation. The graph shows the relative HGF mRNA levels normalized to 18S rRNA. Values are the mean ± SD of four individual experiments. (B) After incubation for 24 h, the cell lysates were blotted with HGF and actin antibodies. The graph shows densitometric analysis of the HGF band standardized to actin. Values are the mean ± SD of three individual experiments. **P < 0.01. NS, not significant.

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Upregulation of HGF expression is PPARγ-dependent

We then tested whether the effect of 15d-PGJ2 on HGF expression is mediated by PPARγ using GW9662, a PPARγ-specific antagonist. Preliminary experiments led to the use of GW9662 at a concentration of 1 µmol/L. As shown in Figure 5, GW9662 abolished the 15d-PGJ2-induced increase in HGF mRNA expression at 3 h and HGF production at 24 h, with HGF expression falling to basal levels.

image

Figure 5. Effect of GW9662 on 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2)-induced hepatocyte growth factor (HGF) mRNA and protein expression levels in human peritoneal mesothelial cells. Cells were preincubated for 30 min in the presence or absence of GW9662 (1 µmol/L) before the addition of 15d-PGJ2 (5 µmol/L). (A) Quantitative real-time PCR was performed to assess the expression of HGF mRNA at 3 h after incubation. The graph shows the relative HGF mRNA levels normalized to 18S rRNA. (B) After incubation for 24 h, the cell lysates were blotted with antibodies to HGF and actin. The graph shows the densitometric analysis of the HGF band standardized to actin. Values are the mean ± SD of five individual experiments. **P < 0.01.

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HGF inhibits Ang II-induced upregulation of FN expression

Since 15d-PGJ2 increased HGF expression, we next examined whether HGF treatment could inhibit Ang II-induced FN expression. These studies were performed using rhHGF at a concentration of 30 ng/mL as previously described (11). rhHGF treatment did not result in cell detachment or death (data not shown). As shown in Figure 6, incubation of HPMCs with rhHGF abolished the upregulation of FN mRNA expression such that FN secretion fell to basal levels. In addition, rhHGF had little effect on basal expression levels of FN.

image

Figure 6. Effect of recombinant human hepatocyte growth factor (rhHGF) on angiotensin II (Ang II)-induced fibronectin (FN) mRNA and protein expression levels in human peritoneal mesothelial cells. Cells were preincubated for 30 min in the presence or absence of rhHGF (30 ng/mL) before stimulation with Ang II (100 nmol/L). (A) Quantitative real-time PCR was performed to assess the expression of FN mRNA at 6 h after incubation. The graph shows the relative FN mRNA levels normalized to 18S rRNA. (B) After incubation for 24 h, the culture supernatants were blotted with FN antibody (15 µg protein loaded per lane). The graph shows the densitometric analysis of the FN band. Values are the mean ± SD of five individual experiments. **P < 0.01.

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Activation of c-Met tyrosine phosphorylation by HGF is blocked by PHA-665752

After revealing the antifibrotic effect of HGF, we next investigated the expression of c-Met and its activation resulting from phosphorylation. In addition to the detection of c-Met, significant induction of tyrosine phosphorylation of its major autophosphorylation site pY1230/1234/1235, as well as the juxtamembrane domain pY1003, was evident 5 min after HGF stimulation (Fig. 7A). A slight decrease in total c-Met levels was seen at 60 and 120 min after HGF induction, which may be a result of ligand-induced ubiquitination of c-Met mediated by c-Cbl binding to the pY1003 domain (16). Likewise, 15d-PGJ2 was able to induce c-Met receptor activation, as illustrated by its peak phosphorylation at 12 h (Fig. 7B). PHA-665752, a selective c-Met kinase inhibitor, abrogated the phosphorylation of pY1230/1234/1235 and pY1003 at a concentration of 20 nmol/L and 50 nmol/L, respectively (Fig. 7C).

image

Figure 7. Activation of tyrosine phosphorylation of c-Met and the effect of PHA-665752 on activated phosphorylation in human peritoneal mesothelial cells. (A) Cells were incubated with recombinant human hepatocyte growth factor (rhHGF) (30 ng/mL) at the indicated times and the cell lysates were blotted with two phospho-specific c-Met antibodies. Subsequently, the same lysates were blotted with antibodies to total c-Met and actin. (B) Cells were incubated with 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) (5 µmol/L) at the indicated times before the cell lysates were blotted. (C) Cells were preincubated with PHA-665752 (0, 10, 20 and 50 nmol/L) for 3 h and treated with rhHGF (30 ng/mL) for 5 min before blotting.

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HGF may contribute to the effect of 15d-PGJ2

If the inhibitory effect of 15d-PGJ2 on Ang II-induced upregulation of FN is mediated by HGF, we would expect that a selective c-Met inhibitor would negate this effect. These studies were performed using PHA-665752 at a concentration of 50 nmol/L, which abrogated tyrosine phosphorylation of c-Met at both pY1230/1234/1235 and pY1003. Ang II stimulated FN secretion regardless of the presence of PHA-665752. PHA-665752 could attenuate the inhibitory effects of 15d-PGJ2 on Ang II-induced upregulation of FN, but FN was not upregulated to a level of Ang II stimulation without 15d-PGJ2. In addition, both 15d-PGJ2 and PHA-665752 had little effect on basal expression levels of FN (Fig. 8).

image

Figure 8. Effect of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) with or without PHA-665752 on angiotensin II (Ang II)-induced fibronectin (FN) secretion in human peritoneal mesothelial cells. After preincubation with or without PHA-665752 (50 nmol/L) for 3 h and then with or without 15d-PGJ2 (5 µmol/L) for 24 h, cells were stimulated with Ang II (100 nmol/L) for 24 h. The culture supernatants were blotted with FN antibody (15 µg protein loaded per lane). The graph shows the densitometric analysis of the FN band. Values are the mean ± SD of four individual experiments. *P < 0.05, **P < 0.01, P < 0.01 vs Ang II alone. NS, not significant.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

The goal of this study was to evaluate the antifibrotic effects of 15d-PGJ2 in HPMCs through the c-Met/HGF pathway. We demonstrated that 15d-PGJ2 reduces the Ang II-induced expression of FN and stimulates HGF production and secretion, and that HGF inhibits Ang II-induced FN expression. We also detected c-Met in HPMC lysates and found that its phosphorylation is induced by HGF and 15d-PGJ2. Furthermore, we found that HGF might contribute to the effects of 15d-PGJ2 using PHA-665752.

Ang II is a potent vasoactive peptide that also plays a regulatory role in the fibrotic process (17). The fibrotic effects of Ang II are related to the increased synthesis of ECM proteins. Increased levels of Ang II levels have been found in the supernatants of glucose-supplemented cultures of HPMCs (18), and an elevation of Ang II is evident in the PD effluent of patients with infectious peritonitis (9).

PPARγ agonists have garnered attention for their potential antifibrotic activity. Guo et al. reported that 15d-PGJ2 and pioglitazone have an inhibitory effect on transforming growth factor-β1 (TGF-β1)-induced FN expression in human mesangial cells (5), and pioglitazone, rosiglitazone and troglitazone inhibit Ang II-induced hypertrophy of neonatal rat cardiac myocytes (6). In this study, the expression of PPARγ protein was confirmed in HPMCs. Next, the inhibitory effect of 15d-PGJ2 on Ang II-induced FN expression was verified. The preincubation time of 15d-PGJ2 was set at 24 h for the production and secretion of HGF. The mRNA expression and protein levels were analyzed at 6 h and 24 h, respectively, based on data from a previous study (9). Since it appears that the cumulative production of FN contributes to the initial histopathological response observed in the development of PF, our results suggest that 15d-PGJ2 may have therapeutic potential as an antifibrotic agent in patients with PD. A recent study showed that rosiglitazone improves the structural and functional alterations of the peritoneum in an experimental rat model of EPS (19).

On the other hand, it is noteworthy that the PPARγ agonist pioglitazone provides limited benefit in the treatment of established liver fibrosis in rats (20). Pioglitazone arrested the progression of hepatic fibrosis only if the drug was introduced early enough in the course of disease progression (20). These data suggest that if PPARγ agonists are to be of therapeutic benefit in patients with liver disease, then treatment should be undertaken as early as possible in the course of the disease. Early treatment is also likely to be optimal in patients undergoing PD whose peritoneum is exposed to long-term bioincompatible dialysis fluids, high glucose concentrations, or refractory or repeated episodes of infectious peritonitis.

It is thus of interest that PPARγ binds to the putative PPAR responsive element in the promoter region of the HGF gene, with PPARγ ligand stimulation leading to increased HGF gene transcription, mRNA expression, and protein secretion (4,13). In addition, previous studies have reported that there are not only PPARγ-dependent but also PPARγ-independent pathways involved in the mechanism of 15d-PGJ2 action (21). We report here for the first time that 15d-PGJ2 increases HGF mRNA expression and HGF production through PPARγ-dependent pathways in HPMCs. The rapid increase in HGF mRNA expression was consistent with a recent observation demonstrating the peak level of HGF expression by human mesangial cells at 3 h following 15d-PGJ2 stimulation (4). As HGF inhibits Ang II-induced FN, we first thought that Ang II might inhibit HGF. In other words, we thought that HGF and FN would show opposite actions. It is notable that Ang II had little influence on HGF production and that 15d-PGJ2 was able to induce HGF production in the presence of Ang II. It is also important that 15d-PGJ2 extracellularly secreted HGF. This results in activation of c-Met and plays a role in the antifibrotic action. Furthermore, the finding that pioglitazone upregulates HGF production will be clinically valuable, because this agent is already in use.

In vivo studies have shown that HGF has the potentially therapeutic effect of suppressing ECM overproduction (22). In PD, Nakamura et al. demonstrated that HGF expression is substantially increased in the damaged peritoneal tissues of PD patients in areas without severe structural changes (23), while HGF appears to have a beneficial effect in HPMCs (11,12) and in a rat model treated by peritoneal dialysate (24). In this study, treatment with rhHGF completely suppressed Ang II-induced FN expression with no evidence of cell injury. These results support a model where HGF acts as an intermediate effector mediating the antifibrotic actions of 15d-PGJ2.

Since PPARγ agonists upregulated HGF expression and HGF inhibited FN expression, we investigated the role of c-Met activated by HGF. Numerous studies have demonstrated that activation of c-Met, which results in the binding and phosphorylation of adaptor proteins and the subsequent activation of signal transducers, is involved in cell growth, cell survival, angiogenesis, cell motility, and migration. In this study, HGF dramatically induced phosphorylation of c-MET at both pY1230/1234/1235 and pY1003. Consistent with a previous study (4), we also found that 15d-PGJ2 activates c-Met phosphorylation, indicating that HGF levels secreted by 15d-PGJ2 were high enough for activation. PHA-665752, a selective small molecule inhibitor of c-Met, produced a dose-dependent reduction of these tyrosine phosphorylations. This is one of the first studies to show that HGF activates phosphorylation of c-Met in HPMCs, and that this activation can be blocked by PHA-665752.

Finally, this study investigated whether the antifibrotic effect of 15d-PGJ2 is mediated by HGF. Our findings suggest the possibility that HGF contributes to the effect of 15d-PGJ2. Although the mechanism of this action must be considered, there are few studies on the signaling pathways between Ang II and FN. One possibility is that the antifibrotic effect may contribute to inhibiting TGF-β1. TGF-β1 is the major profibrotic factor and is thus likely to be involved in the accumulation of ECM, such as FN, laminin, and collagen. Indeed, the beneficial action of HGF against TGF-β1 was shown in a previous report (11). We examined whether 15d-PGJ2 reduces Ang II-induced TGF-β1 mRNA and protein expression, resulting in a significant decrease at 24 h (data not shown); however, we could not find a significant difference in less than 24 h, which seems to be a little too late to accumulate FN. Alternatively, TGF-β1-independent signal transduction pathways, such as protein kinase C, protein tyrosine kinases, mitogen-activating protein kinases (MAPK), extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38 MAPK, may be involved (17). We previously reported that Ang II-induced FN expression is mediated by the activation of ERK1/2 and p38 MAPK (5), but not JNK (9). Guo et al. reported that 15d-PGJ2 significantly inhibits TGF-β1-induced phosphorylation of ERK1/2 and p38 MAPK. Likewise, the antifibrotic effects of HGF via ERK1/2 have been noted (25), but there are no published accounts regarding p38 MAPK. The contribution of p38 MAPK to the action of HGF has apparently gone unnoticed. Altogether, it is assumed that 15d-PGJ2-induced HGF might not regulate the activation of p38 MAPK, which leads to our result that the inhibitory effect of PHA-665752 was insufficient.

CONCLUSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

This study provides evidence that 15d-PGJ2, an endogenous PPARγ agonist, can exert antifibrotic effects on HPMCs, suggesting that this action is likely mediated by HGF.

Acknowledgments

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES

Acknowledgments:  This work was partly supported by the Terumo Foundation for Peritoneal Function Research and also by the Ryokufukai Research Grant. The authors wish to thank Dr Kazuhiro Yoshida (Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, during the study period, currently at the Department of Oncologic Surgery, Gifu University Graduate School of Medicine) and Dr Jun Hihara (Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University) for their assistance in collecting samples from patients undergoing abdominal surgery.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgments
  8. REFERENCES
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