Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin–α2-antiplasmin (α2AP) complex, and we recently implicated α2AP in the development of fibrosis through transforming growth factor β (TGFβ) production. This study was undertaken to clarify how α2AP induces TGFβ production and the development of fibrosis.
To clarify the detailed mechanism by which α2AP induces TGFβ production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A2 (iPLA2) and examined whether ATGL/ iPLA2 is associated with α2AP-induced TGFβ production. The mouse model of bleomycin-induced SSc was used to evaluate the role of α2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSc-like fibroblasts from the bleomycin-treated mouse skin and examined the production of TGFβ and prostaglandin F2α (PGF2α).
We found that α2AP binding to ATGL promoted PGF2α synthesis through iPLA2 in fibroblasts, and the PGF2α synthesis that was promoted by α2AP induced TGFβ production in fibroblasts. In addition, the neutralization of α2AP attenuated the production of TGFβ and PGF2α in SSc-like fibroblasts from mice. The α2AP deficiency attenuated bleomycin-induced fibrosis and PGF2α synthesis, while the administration of PGF2α to α2AP-deficient mice facilitated α2AP deficiency–attenuated fibrosis.
These findings suggest that α2AP regulates the development of fibrosis by PGF2α synthesis through ATGL/iPLA2. The inhibition of α2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.
Systemic sclerosis (SSc) affects the skin and the internal organs, resulting in tissue fibrosis. Although the disease process involves immunologic mechanisms, vascular damage, and activation of fibroblasts, the pathogenesis of SSc remains to be further elucidated. Fibrotic diseases are characterized by excessive scarring due to excessive production, deposition, and contraction of the extracellular matrix (ECM). This process usually occurs over many months and years, and can lead to organ dysfunction or death. Although it is known that the multiple biologic actions of transforming growth factor β (TGFβ) contribute to the central role that it plays in many fibrotic diseases (1), the regulation and mechanism responsible for TGFβ production in fibrotic diseases remain poorly understood.
The α2-antiplasmins (α2AP) are serpins with a molecular weight of 65–70 kd (2) that rapidly inactivate plasmin, resulting in the formation of a stable inactive complex, plasmin–α2AP (3). Many studies have shown that the levels of the plasmin–α2AP complex in plasma are elevated in fibrotic diseases, including SSc, diabetic nephropathy, liver cirrhosis, and rheumatoid arthritis (4–7). In addition, we have shown that α2AP is associated with the development of fibrosis through TGFβ production (8, 9). However, the detailed mechanism by which α2AP induces TGFβ production and the development of fibrosis is unclear.
Phylogenetically, α2AP is most closely related to the noninhibitory serpin pigment epithelium–derived factor (PEDF) (10). The structures of α2AP (11) and PEDF (12) are very similar, and they both have 3 β-sheets and 9 α-helices. It has recently been reported that adipose triglyceride lipase (ATGL), which has been described as a member of the calcium-independent phospholipase A2 (iPLA2)/nutrin/patatin-like phospholipase domain–containing protein 2 family, is a receptor for PEDF, and that PEDF binding stimulates the enzymatic PLA2 of ATGL (13).
Release of arachidonic acid (AA) from membrane glycerophospholipids is catalyzed by PLA2 enzymes, and AA is sequentially metabolized to prostaglandin G2 (PGG2) and then to PGH2 by cyclooxygenase 1 (COX-1) and/or COX-2. PGH2 is then converted to various bioactive PGs (thromboxane A2, PGD2, PGE2, PGF2α and PGI2) (14). The loss of PLA2 suppressed bleomycin-induced fibrosis (15). In addition, PGF2α, which is one of the PGs that stimulates TGFβ production (16), is associated with the development of fibrosis (17). PLA2/PGF2α plays an important role in the pathogenesis of fibrotic diseases.
In this study, we focused on the PEDF receptor, ATGL, and found that α2AP promoted PGF2α synthesis through ATGL/iPLA2. In addition, we demonstrated that the α2AP-promoted PGF2α synthesis regulates TGFβ production and the development of fibrosis.
MATERIALS AND METHODS
Deficient mice were generated by homologous recombination using embryonic stem cells, as previously described (18, 19). All experiments were performed in accordance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
The α2AP was purchased from Calbiochem. Other chemical substances were obtained from Sigma.
Induction of dermal fibrosis.
We induced dermal fibrosis in mice as previously described (8). Briefly, dermal fibrosis was induced in 8-week-old male C57BL/6J mice by administration of phosphate buffered saline (PBS) alone, bleomycin alone, or bleomycin and the iPLA2-specific inhibitor bromoenol lactone (BEL; Cayman Chemical) (n = 7 mice per group). Bleomycin was dissolved in PBS at 1 mg/ml. A total of 100 μl of PBS, 100 μl of bleomycin, or 100 μl of bleomycin and 5 mg/kg of BEL was administered subcutaneously into the shaved backs of the mice. In an additional experiment, dermal fibrosis was induced in 8-week-old male α2AP-deficient (α2AP−/−) mice and 8-week-old wild-type mice (α2AP+/+) by administration of PBS alone, bleomycin alone, or bleomycin and PGF2α (n = 4 α2AP−/− mice and n = 4 wild-type mice per treatment group). A total of 100 μl of PBS, 100 μl of bleomycin alone, or 100 μl of bleomycin and 400 μg/kg of PGF2α was administered subcutaneously into the shaved backs of the mice. In both experiments, administration in the same site was carried out daily for up to 3 weeks. At the end of different observation periods, the mice were killed at the indicated times using an overdose of pentobarbital, and a skin sample was carefully collected from each mouse. These samples were used for the protein preparations. For extraction of the protein, skin samples, including the scab and the complete epithelial margins, were trimmed to specimens measuring 7–8 mm in diameter, placed immediately in liquid nitrogen, and stored at −80°C until used.
NIH3T3 fibroblasts (1 × 105) were seeded into dishes measuring 35 mm in diameter and maintained in 2 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere with 5% CO2/95% air. After 6 days, the medium was replaced with serum-free DMEM. The cells were then used for experiments.
Primary cultured cells.
Normal fibroblasts and SSc-like fibroblasts were obtained from the skin of wild-type mice as previously described (20). SSc-like fibroblasts were also obtained from the bleomycin-treated skin of wild-type mice. Bleomycin administration in the same site was carried out daily for up to 3 weeks.
Binding assay for α2AP and ATGL.
Immunoprecipitation was performed after incubating 800 ng of α2AP or bovine serum albumin (BSA; negative control) with 200 μg of fibroblast lysates for 30 minutes. Immunoprecipitation was performed using an Immunoprecipitation Kit (Invitrogen).
ATGL small interfering RNA (siRNA) experiments.
NIH3T3 cells were transfected with ATGL siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen) according to the recommendations of the manufacturer. A nonspecific siRNA was used as the control. Three days after transfection, the cells were stimulated with α2AP and then evaluated by Western blot analysis.
Enzyme-linked immunosorbent assay (ELISA).
PGF2α levels in the medium or mouse skin samples were measured using a PGF2α enzyme immunoassay kit (Enzo Life Sciences). The absorbance of the ELISA samples was measured at 405 nm using an EL340 Biokinetic Reader (BioTek Instruments).
Western blot analysis.
Western blot analysis was undertaken as previously described (21). We detected TGFβ, ATGL, phospho-serine, phospho–ERK-1/2, phospho-JNK, ERK-1/2, and JNK by incubation with anti-TGFβ antibody (R&D Systems), anti-ATGL antibody (Santa Cruz Biotechnology), anti–phospho-serine antibody (Invitrogen), anti–phospho–ERK-1/2 antibody (Cell Signaling Technology), anti–phospho-JNK antibody (Cell Signaling Technology), anti–ERK-1/2 antibody (Cell Signaling Technology), and anti-JNK antibody (Cell Signaling Technology), respectively, followed by incubation with horseradish peroxidase–conjugated antibody to rabbit IgG (Amersham Pharmacia Biotech).
Immunohistochemical staining for α2AP, TGFβ, α-smooth muscle actin (α-SMA), and phospho-Smad2/3.
Specimens from the central area of the mouse skin treated with PBS or bleomycin were excised by using a punch biopsy instrument (Techne). Paraffin-embedded sections were labeled with anti-α2AP, TGFβ α-SMA, or phospho-Smad2/3 primary antibody, then secondarily labeled with Cy3-conjugated anti-rabbit IgG (Molecular Probes). The signals were then detected using a laser scanning microscope. The stained images obtained from separate fields on the specimens (n = 4) were analyzed using ImageJ software.
Measurement of dermal thickness.
The dermal thickness (distance from the epidermal–dermal junction to the dermal–muscle junction) was measured in mouse skin sections (n = 6 mice).
Measurement of collagen content in mouse skin sections by Sircol biochemical assay.
The collagen content was measured as previously described (22). Briefly, collagen content was assessed using sirius red staining. This approach was chosen because it accurately reflects the collagen content assessed with a hydroxyproline assay and allows areas of localized collagen accumulation to be specifically evaluated. In these assays, sections of mouse skin were stained with sirius red as described by Junqueira et al (23). After deparaffinization, the skin sections were treated with 0.2% phosphomolybdic acid for 5 minutes. Next, the skin sections were stained with 0.1% sirius red for 90 minutes and 0.01N HCl for 2 minutes. The red staining was then detected using a laser scanning microscope. In each section, the sirius red–positive area was measured in 7 randomly chosen fields and was expressed as a percent of that observed in the PBS-treated mice.
Calcium-independent PLA2 activity.
The activity of iPLA2 in the mouse skin samples was determined as described by Smani et al (24). The activity of iPLA2 was measured using a modified kit that was originally designed for cytosolic PLA2 (cPLA2) using a cPLA2 assay kit (Cayman Chemical). To detect the activity of iPLA2, and not of cPLA2, phospholipase activity was assayed by incubating the samples with the substrate arachidonoyl Thio-PC for 1 hour at 20°C in a modified calcium-free buffer (300 mM NaCl, 0.5% Triton X-100, 60% glycerol, 4 mM EGTA, 10 mM HEPES [pH 7.4], and 2 mg/ ml BSA). The reaction was stopped by the addition of 5,5′-dithiobis(2-nitrobenzoic acid) for 5 minutes, and absorbance was determined at 405 nm using an EL340 Biokinetic Reader. The activity of iPLA2 was expressed in absorbance per milligrams of protein.
All data are expressed as the mean ± SEM. The statistical significance of the effect of each treatment was determined by analysis of variance followed by Student-Newman-Keuls test. P values less than 0.01 were considered significant.
Binding and activation of ATGL by α2AP induces TGFβ production.
In previous studies, we showed that α2AP induced TGFβ production (8, 9). To clarify the detailed mechanism underlying how α2AP induces TGFβ production, we focused on PEDF, which is closely related to α2AP, and the PEDF receptor, ATGL, and examined whether ATGL acts as a receptor for α2AP.
First, we confirmed that ATGL is present in the skin of PBS-treated or bleomycin-treated wild-type mice by a Western blot analysis. We observed that ATGL was present in the skin, and that the expression of ATGL in the skin samples from bleomycin-treated mice was higher than in those from PBS-treated mice (Figure 1A). Second, to determine the ability of α2AP to bind ATGL, we performed immunoprecipitation of ATGL from fibroblast lysates in the presence and absence of recombinant α2AP by using control IgG or an anti-ATGL antibody. We showed that α2AP interacted with ATGL (Figure 1B). Third, we investigated whether α2AP binding to ATGL triggered the activation of ATGL. Mass spectrometry analysis detected 2 phosphorylated serine residues in murine ATGL (25). We showed that α2AP induced the phosphorylation of serine in ATGL (Figure 1C).
Moreover, to clarify whether α2AP induces TGFβ production through ATGL, we investigated the effects of α2AP-induced TGFβ production on the reduction of ATGL expression by siRNA. We confirmed that ATGL expression was attenuated by siRNA (Figure 1D). In addition, the siRNA-induced reduction in ATGL expression attenuated α2AP-induced TGFβ production (Figure 1E). These data suggest that α2AP interacts with ATGL, and that this interaction induces TGFβ production.
Activation of iPLA2 by α2AP induces TGFβ production.
ATGL has enzymatic iPLA2 activity (13). Therefore, we examined whether iPLA2 activation is associated with the α2AP-induced TGFβ production in fibroblasts by using an iPLA2-specific inhibitor, BEL. BEL attenuated the α2AP-induced TGFβ production in fibroblasts (Figure 2A). It has been demonstrated that PEDF binding stimulates the iPLA2 activity of ATGL (13). We also showed that PEDF induced TGFβ production in fibroblasts, and that the PEDF-induced TGFβ production was attenuated by BEL (Figure 2B). These data suggest that activation of iPLA2 induced TGFβ production.
Prevention of bleomycin-induced fibrosis through inhibition of iPLA2.
To verify the potential of iPLA2 as a novel target for antifibrotic therapies in vivo, we examined the effects of the iPLA2 inhibitor BEL on bleomycin-induced fibrosis. The administration of BEL attenuated bleomycin-induced fibrotic changes, such as increased dermal thickness and collagen production, in wild-type mice (Figures 3A–C). In addition, BEL attenuated bleomycin-induced α-SMA (a hallmark of the myofibroblast phenotype), TGFβ, and Smad2/3 phosphorylation (Figures 3D and E). However, BEL did not attenuate bleomycin-induced α2AP expression (Figures 3D and E).
Activation of iPLA2 by α2AP induces PGF2α synthesis.
PG synthesis is catalyzed by PLA2 activation (26). PGF2α stimulates TGFβ production (16) and is associated with the development of fibrosis (17). Therefore, we examined whether α2AP promoted PGF2α synthesis in fibroblasts. We observed that α2AP promoted PGF2α synthesis, and that the α2AP-promoted PGF2α synthesis was attenuated by BEL (Figure 4A). These data suggest that α2AP promotes PGF2α synthesis through iPLA2. Two isoforms of COX, COX-1 and COX-2, are involved in the synthesis of PGs. We examined whether each of these isoforms was associated with the α2AP-promoted PGF2α synthesis by using selective inhibitors of COX-1 (SC560) and COX-2 (NS398). Both SC560 and NS398 reduced the α2AP-promoted PGF2α synthesis, although SC560 had a stronger effect (Figure 4B).
PGF2α synthesis by α2AP induces TGFβ production.
We confirmed that PGF2α induces TGFβ production in fibroblasts (Figure 5A). We also examined the PGF2α-stimulated phosphorylation of ERK-1/2 and JNK and found that PGF2α activated both the ERK-1/2 and JNK pathways in fibroblasts (Figure 5B). In addition, we examined whether the ERK-1/2 and JNK pathways were associated with PGF2α-induced TGFβ production in fibroblasts by using a specific inhibitor of MEK (PD98059) and a specific inhibitor of JNK (SP600125). PD98059 and SP600125 attenuated the PGF2α-induced TGFβ production in fibroblasts (Figure 5C). Moreover, we examined whether α2AP-promoted PGF2α synthesis is associated with TGFβ production by using a selective FP receptor antagonist, AL8810 (27). AL8810 attenuated the α2AP-induced TGFβ production in fibroblasts (Figure 5D). These data suggest that α2AP induced TGFβ production through PGF2α synthesis. We also examined the effect of the selective inhibitors of COX-1 (SC560) and COX-2 (NS398) on α2AP-induced TGFβ production in fibroblasts. Both SC560 and NS398 reduced α2AP-induced TGFβ production, although SC560 had a stronger effect (Figure 5E).
Role of α2AP in the production of TGFβ and PGF2α in SSc.
In a previous study, we showed that the expression of α2AP in the skin of mice with bleomycininduced SSc was significantly higher than that in the skin of control mice (9). To clarify the role of α2AP in the production of TGFβ and PGF2α in SSc, we cultured SSc-like fibroblasts from the skin of mice with bleomycin-induced SSc and examined the production of TGFβ and PGF2α. Although α2AP did not induce the production of TGFβ and PGF2α in the SSc-like fibroblasts, the production of TGFβ and PGF2α in the SSc-like fibroblasts was increased dramatically compared to that in normal fibroblasts (results are available from the corresponding author upon request). In addition, the expression of α2AP in the SSc-like fibroblasts was increased dramatically compared to that in normal fibroblasts (results are available from the corresponding author upon request), and the neutralization of α2AP attenuated the production of TGFβ and PGF2α in the SSc-like fibroblasts (results are available from the corresponding author upon request). These data suggest that α2AP regulates the production of TGFβ and PGF2α in SSc.
Effects of PGF2α on the attenuation of bleomycin-induced fibrosis by α2AP deficiency.
In a previous study, we showed that α2AP deficiency protected mice against bleomycin-induced fibrosis (8, 9). To clarify whether the protection against bleomycin-induced fibrosis in α2AP−/− mice is associated with iPLA2 activation and PGF2α synthesis, we measured iPLA2 activity and PGF2α levels in the skin of bleomycin-treated α2AP+/+ and α2AP−/− mice. The iPLA2 activity and PGF2α levels were lower in the skin of α2AP−/− mice than α2AP+/+ mice (Figures 6A and B). Next, we confirmed the effects of PGF2α on the bleomycin-induced fibrotic changes in α2AP−/− mice by administering PGF2α. Histologic examination of the skin of α2AP−/− mice treated with bleomycin and those treated with PGF2α demonstrated a considerable increase in dermal thickness in the mice treated with PGF2α (Figure 6C). Quantitative analysis showed that the dermal thickness and collagen content of the skin of α2AP−/− mice treated with bleomycin and PGF2α was increased compared with that of α2AP−/− mice treated with bleomycin alone (Figures 6C–E). In addition, the skin of α2AP−/− mice treated with both bleomycin and PGF2α showed increased expression of TGFβ, α-SMA, and phospho-Smad2/3 compared to the skin of α2AP−/− mice treated with bleomycin alone (Figures 6F and G). These data suggest that PGF2α is associated with the protection of mice against bleomycin-induced fibrosis that results from α2AP deficiency.
TGFβ has long been known to promote fibrosis by the induction of ECM synthesis and contraction in fibroblasts. In previous studies, we showed that α2AP is associated with the development of fibrosis through TGFβ production (8, 9). However, the mechanisms by which α2AP regulated TGFβ production and the development of fibrosis were not precisely understood. The structures of α2AP (11) and PEDF (12) are very similar. In addition, some studies have demonstrated that PEDF inhibits vascular endothelial growth factor (VEGF) expression and angiogenesis (28–30), and we previously showed that α2AP deficiency induced VEGF expression and angiogenesis (20). These findings suggest that α2AP and PEDF might have the same function. Moreover, PEDF is involved in fibrosis (31). Therefore, in the present study, we focused on PEDF and the PEDF receptor, ATGL, and found that α2AP could bind and activate ATGL. In addition, the reduction of ATGL by siRNA attenuated α2AP-induced TGFβ production. These data suggest that α2AP induces TGFβ production through ATGL.
The ligand binding to ATGL has been reported to stimulate enzymatic iPLA2 activity (13). The α2AP-induced TGFβ production observed in this study was attenuated by the inhibition of iPLA2. We also showed that the inhibition of iPLA2 attenuated the bleomycin-induced production of TGFβ, and protected mice against fibrosis. However, inhibition of iPLA2 did not attenuate bleomycin-induced α2AP expression. These data suggest that α2AP is an upstream regulator of iPLA2, and that the α2AP-mediated activation of iPLA2 is associated with TGFβ production and the development of fibrosis.
It has been reported that PGF2α stimulates TGFβ production (16) and plays a pivotal role in the development of fibrosis (17). The synthesis of PGF2α is involved in PLA2 activation and 2 isoforms of COX: COX-1 and COX-2. We showed that α2AP-mediated activation of iPLA2 is associated with PGF2α synthesis by using a specific iPLA2 inhibitor. In addition, the selective inhibitors of COX-1 and COX-2 reduced α2AP- promoted PGF2α synthesis. Moreover, we showed that α2AP-induced TGFβ production was attenuated by a specific iPLA2 inhibitor, a selective inhibitor of COX, and a selective FP receptor antagonist. These data suggest that α2AP induces TGFβ production through iPLA2 activation, COX, and PGF2α synthesis.
The effect of a specific COX-1 inhibitor was significantly stronger than that of a specific COX-2 inhibitor. PGF2α has been reported to induce COX-2 expression (32, 33). PGF2α synthesis was promoted by α2AP through iPLA2/COX-1, and α2AP-promoted PGF2α induced TGFβ production. At the same time, the α2AP-promoted PGF2α might induce COX-2 expression, and thereby enhance PGF2α synthesis. The enhancement of PGF2α synthesis might then lead to the further induction of TGFβ production. We also showed that PGF2α induced TGFβ production through the ERK-1/2 and JNK pathways. Both the ERK-1/2 and JNK pathways are associated with α2AP-induced TGFβ production (9). These data suggest that α2AP-mediated PGF2α synthesis might induce TGFβ production through the ERK-1/2 and JNK pathways, and regulate the development of fibrosis.
Furthermore, we showed that the production of TGFβ PGF2α, and α2AP in bleomycin-treated SSc-like fibroblasts was significantly higher than that in normal fibroblasts, and the neutralization of α2AP attenuated SSc-enhanced TGFβ and PGF2α synthesis. On the other hand, in a mouse model of SSc, the levels of PGF2α in the α2AP−/− mice decreased compared with those in wild-type mice, and the administration of PGF2α to α2AP−/− mice facilitated TGFβ production and the development of fibrosis. These data suggest that the enhancement of α2AP in SSc promotes PGF2α synthesis, which then facilitates TGFβ production and the development of fibrosis.
Plasmin is known to activate matrix metalloproteinases (MMPs) and a number of growth factors, including TGFβ and hepatocyte growth factor (HGF). Plasmin itself and plasmin-activated MMPs can degrade most ECM proteins, including collagen, which is the major proteinaceous component of fibrotic tissue (34). In addition, plasmin-activated HGF has been shown to contribute to antifibrosis (35, 36). Moreover, the overexpression of urokinase plasminogen activator (37, 38), and the deletion of plasminogen activator inhibitor 1 (PAI-1) (39) attenuated the development of fibrosis. Conversely, the overexpression of PAI-1 led to the development of a more pronounced fibrotic response (39). Therefore, α2AP might facilitate the development of fibrosis not only by producing TGFβ, but also by inhibiting plasmin activity. These findings indicate that α2AP expression may represent a target that can be used to suppress the development of fibrosis.
In conclusion, in the present study, α2AP activated PLA2 through ATGL and then promoted PGF2α synthesis. The α2AP-promoted PGF2α synthesis regulated TGFβ production and the development of fibrosis. These findings may provide new insights into the development of clinical therapies for the prevention of fibrosis.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Kanno had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Kanno.
Acquisition of data. Kawashita, Kokado, Okada.
Analysis and interpretation of data. Ueshima, Matsuo, Matsuno.