|Molecular Vision 2007;
Received 10 July 2006 | Accepted 18 September 2007 | Published 18 September 2007
Inhibition of p38MAP kinase suppresses fibrogenic reaction in conjunctiva in mice
Anil K. Kamaraju,3
1Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan; 2Department of Pharmacology and Molecular Therapeutics, Kumamoto University Graduate School of Medical Science, Kumamoto, Japan; 3The Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD; 4Department of Anatomy, Graduate School of Medicine, Osaka City University, Osaka, Japan
Correspondence to: Osamu Yamanaka, M.D., Ph.D., Department of Ophthalmology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan; Phone: 81-73-441-0649; FAX: 1-73-448-1991; email: firstname.lastname@example.org
Purpose: To examine the effects of blocking p38 mitogen-activated protein kinase (MAPK) on post-injury conjunctival scarring in mice. Its effects on the behaviors of cultured subconjunctival fibroblasts were also investigated.
Methods: An in vivo study was conducted using an adenoviral vector carrying a dominant-negative (DN)-p38MAPK gene. A circumferential incision was made in the equatorial conjunctiva by scissors in the right eye of generally anesthetized adult C57BL/6 mice. DN-p38MAPK-expressing adenoviral vector was topically applied. The left control eye received non-functioning adenoviral vector. At 2, 5, and 7 days (each, n=22) the eyes were processed for histological or immunohistochemical examination to evaluate the tissue scarring. The expressions of type-I collagen and growth factors were evaluated by real time-reverse transcriptase-polymerase chain reaction. The effects of p38MAPK inhibitor on the proliferation, migration, and fibrogenic gene/protein expression of cultured human fibroblasts were also studied.
Results: The in vivo DN-p38MAPK gene introduction blocked the phospho-p38 expression with reduction of myofibroblast generation and suppression of mRNA expression of connective tissue growth factor (CTGF) and monocyte/macrophage chemoattractant protein-1 (MCP-1) in the mouse-injured conjunctiva. Blocking p38MAPK signal in the fibroblasts by a chemical inhibitor counteracted TGFβ1's enhancement of expressions of type-I collagen, fibronectin, and CTGF. It also retarded cell migration, but cell proliferation was unchanged.
Conclusions: Inhibiting p38MAPK signal impairs the fibrogenic reaction induced by the subconjunctival fibroblasts in vivo and in vitro, suggesting its potential effectiveness in preventing excessive scarring following glaucoma filtering surgery.
Scarring of the conjunctiva potentially reduces the filtration efficacy following glaucoma filtering surgery . Various growth factors, expressed by local cells and inflammatory cells, are considered to orchestrate the healing process in the conjunctiva .
Emerging evidences indicate that the transforming growth factor beta (TGF β) plays a critical role in the fibrogenic reaction in the injured conjunctiva. TGFβ is involved in fibroblasts-myofibroblasts conversion [3-5]. We previously reported that the fibrotic human conjunctival bleb tissue contains TGFβ1 and TGFβ2 [6,7]. The aqueous humor also contains abundant TGFβ2. This notion was further confirmed by the findings that signaling through Smad3, a key mediator of signals from the TGFβ and activin receptors, is required for the development of fibrotic lesions in the cornea and other ocular tissues on using Smad3-null mice or gene transfer of Smad7, an inhibitory Smad [8,9]. However, TGFβ can also activate other, non-Smad signaling cascades, including especially the mitogen-activated protein kinase (MAPK) pathways leading to activation of MAPK-extracellular signal-regulated kinase (Erk), Jun NH2-terminal kinase (JNK), and p38MAPK [10-13]. These pathways are also involved in the pro-fibrogenic reaction in vivo and in vitro dependently on or independently from Smad [14-20].
Recently, it has been reported that inhibition of p38MAPK inhibitor attenuates the fibrogenic reaction induced by subconjunctival fibroblasts . We also previously reported that gene introduction of dominant-negative (DN)-p38MAPK suppresses the fibrogenic reaction induced by retinal pigment epithelial cells in the mouse eye post-retinal detachment model .
Based on these findings, we hypothesized that blocking p38MAPK signal may suppress the injury-induced conjunctival scarring and provide a potential therapy to inhibit excessive bleb scarring in the conjunctiva following glaucoma surgery. In the present study, we first evaluated the anti-scarring effects of adenoviral DN-p38MAPK gene transfer using the mouse model of injury-induced conjunctival scarring and cultured human subconjunctival fibroblasts. Because a single administration of the chemical p38MAPK inhibitor in vivo might lose its inhibitory effect due to its short half-life in the tissue, we tried to inhibit p38MAPK by viral gene expression of DN isoform. To examine the role of TGFβ1-p38MAPK signaling in the regulation of the behavior of the human subconjunctival fibroblasts, we then used a specific inhibitor of p38MAPK and analyzed the role of this cascade in the pathogenesis of conjunctival fibrosis.
All the experimental procedures were approved by the DNA Recombinant Experiment Committee and Animal Care and Use Committee of Wakayama Medical University, Wakayama, Japan, and conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Mouse model of conjunctival scarring and adenoviral gene introduction of DN-p38MAPK
A circumferential incision was made in conjunctiva at the equator by scissors in the right eye of generally anesthetized adult C57BL/6 mice (n=66). A mixture of Cre-Ad and DN-p38MAPK-Ad was topically administered (3 μl) one time after incision (DN-p38MAPK-Ad group) . Cre-Ad was used as non-functioning adenoviral vector in our current study. In the present project, we employed our established method of gene transfer as described previously using co-infection of Cre-Ad and green fluorescent protein (GFP)-Ad. On days 2 (n=22), 5 (n=22), or 7 (n=22) post-treatment, the eyes were enucleated, fixed in 4.0% paraformaldehyde and embedded in paraffin in each group at each time point.
Histology and immunohistochemistry
Deparaffinized sections (5 μm thick) were processed for indirect immunofluorescence microscopy as previously reported . The primary antibodies were mouse monoclonal anti-phosphorylated p38MAPK antibody (1:50 dilution in phosphate-buffered saline (PBS), Cell Signaling Technology, Danvers, MA), goat polyclonal anti-connective tissue growth factor (CTGF) antibody (1: 100 dilution in PBS, Santa Cruz Biotechnology Inc., Santa Cruz, CA), and mouse monoclonal anti-αsmooth muscle actin (SMA) antibody (1:100 dilution in PBS, Neomarker, Fremont, CA). They were used as previously reported. Reaction with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100 in PBS, Cappel, Aurora, OH) and nuclear staining with 4'-6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc., Burlingame, CA) were performed as described above. The histological features were observed by staining the tissues with hematoxylin and eosin (HE).
Expression of mRNA of type-I collagen α2 chain (col Ia2), CTGF, and monocyte/macrophage-chemoattractant protein-1 (MCP-1)
For RNA extraction and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), the eyes were mechanically injured in 12 mice from each treatment group. The animals were killed on days 2, 5, 7 and 14 using both CO 2 asphyxia and cervical dislocation, and the treated eyes were enucleated. The lens, uveal tissue, and retina were removed from the enucleated eye and processed for total RNA extraction. The untreated eyes of 5 C57BL/6 mice were also included to determine the baseline mRNA expression. Total RNA extraction and real-time RT-PCR for mRNAs of the mouse col1a2, ctgf, and mcp-1 were performed as previously reported . One eye served one RNA sample. Primers and oligonucleotide probes were designed according to the cDNA sequences in the GeneBank database, using the Primers Express software (P-E Applied Biosystems, CA) and listed in Table 1.
Primary cultures of the human subconjunctival fibroblasts were conducted as previously reported . In brief, redundant subconjunctival connective tissue was obtained from patients aged 4 to 9 years during strabismus surgery after obtaining informed consent from the parents of each patient. The tissue was explanted for cell outgrowth in a 25 ml culture bottle (Falcon; Becton Dickinson, Lincoln Park, NJ) and incubated until confluence in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum, antibiotics, and an antimycotic (MEM-10). After 2 or 3 passages, the cells were trypsinized for seeding for the experiments mentioned below.
TGFβ1 activation of p38MAPK; immunocytochemical analysis and western blotting
The cells were seeded in the wells of chamber slides (Nunc, Naperville, IL) and cultured in MEM-10. The subconfluent cells were then treated with TGFβ1 (5 ng/ml; R & D system, Minneapolis, MN) in the presence and absence of SB202190 (10 μM; Calbiochem, EMD Biosciences Inc., San Diego, CA) for specific intervals, and fixed in 4% paraformaldehyde. All the cultures contained the same concentration of dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO). The specimens were then processed for immunofluorescence staining with a monoclonal anti-phosphorylated p38MAPK antibody (1:50 dilution in PBS; Cell Signaling Technology) as previously reported . Negative control staining was performed using non-specific mouse IgG. The cells were also treated with SB202190 (10 μM) 1 h before addition of TGFβ1 (5 ng/ml). At different time points, cells were lysed in lysis buffer (25 mM HEPES, 150 mM NaCl, 10% Glycerol, 5 mM EDTA, 1% Triton X-100, 5 mM sodium orthovanadate, 50 mM NaF, 0.5 mg/ml AEBSF, 1 mg/ml pepstatin) and processed for western blotting as previously reported .
Effect of the p38MAPK inhibitor on Smad signal -dependent gene expression
To assess effects of the p38MAPK inhibitor on transcriptional activity of TGFβ1/Smad-dependent gene expression, we performed a reported assay as previously reported . The cells seeded in 6 well plates were transiently transfected with 4 mg/well CAGA12-Luc, a reporter gene containing 12 repeats of the Smad binding element, CAGAC together with pRL-TK plasmid DNA using FuGene (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. pcDNA3 was used either as a control or as filler. Twenty-four h after transfection, the medium was replaced with medium containing 0.2% serum and cells were either treated with 2 ng/ml of TGFβ1 or left untreated for 16-18 h and then were lysed in passive lysis buffer (Promega, Madison, WI). Luciferase and renila activities were determined by using VICTOR (Perkin-Elmer Life Sciences, Boston, MA).
Production of extracellular matrix component
mRNA expression of collagen Ia2 chain (col1a2) and ctgf and production of type-I collagen and fibronectin, the major matrix components of scarring bleb tissue, were assayed. The cells were grown to confluence in 60 mm culture dish for real time RT-PCR or a 6 well culture plate for enzyme-linked immunosorbent assay (ELISA) in MEM-10 supplemented with 10% fetal calf serum and then further incubated in 500 ml serum-free medium supplemented with SB202190 (10 μM), TGFβ1 (5 ng/ml), and TGFβ 1 (5 ng/ml) plus SB202190 (10 μM) for 48 h. All the cultures contained the same concentration of DMSO. The total RNA from the cultured cells (each, n=4) was extracted as previously reported  and processed for real-time RT-PCR for mRNA of human col1a2 and ctgf.
Nine wells were prepared for each culture condition for ELISA. At the end of the culture interval, the medium was harvested and the cells were sonicated in 500 μl of PBS as previously reported . The medium and cell lysate were processed for ELISA kits for collagen type-I COOH-terminal peptide and fibronectin (Takara, Tokyo, Japan) according to the protocol provided by the manufacturer. The color reaction was assessed at 450 nm.
Cell migration assay
Cell migration was evaluated by assaying the closure of a liner defect produced in a cell monolayer culture as previously reported . A defect was made in a confluent culture of fibroblasts by scraping with a silicone rubber needle (Terry's needle; Alcon Surgical, Fort Worth, TX). The culture was incubated in serum-free medium in the presence of SB202910 (10 μM). All cultures were normalized to the same concentration of DMSO. After specific intervals of culture at 6, 12, 18, 24, and 48 h post-wounding, the cells were photographed and the defect closure was evaluated.
Cell proliferation assay
The cells were seeded into the wells of a 96 well cell culture microplate (Becton Dickinson) at density of 5.0x104/ml. The culture was grown in the presence or absence of SB202190 (10 μM, n=8) without serum. After 2, 4, and 7 days, the effects of SB202190 on fibroblast proliferation were examined. The numbers of cells in each well were counted with a hemocytometer after trypsinization. Every 2 days, the medium was exchanged under similar conditions.
All data were statistically analyzed by analysis of variance using StatView statistics software (Abacus Concepts Inc., Berkley, CA).
Histological and immunohistochemical observation of a mouse conjunctival wound healing model treated with adenoviral gene transfer of DN-p38MAPK
The expression of the introduced DN-p38MAPK gene was examined by immuno-detection with DAB color reaction for the HA-Tag. The expression of HA-Tag was readily detected up to day 7 (data not shown), indicating successful introduction of DN-p38MAPK via adenoviral gene transfer.
HE staining showed that DN-p38MAPK introduction also apparently suppressed the conjunctival edema and inflammatory cell infiltration as compared to the Cre-Ad group (Figure 1A-L). HE staining also showed that epithelial closure of the mechanical incision in the mouse conjunctiva seemed to be facilitated with topical application of adenoviral vector of DN- p38MAPK at day 5 (Figure 1E-H). On day 7 in both the control and treatment groups, the conjunctival epithelium was resurfaced (Figure 1I-L). Although these histology findings suggested the effectiveness of DN-p38MAPK-Ad in suppression of conjunctival scarring, immunohistochemical examination was required to further evaluate its effectiveness as follows.
We then examined the expression of phospho-p38MAPK in the tissues to determine whether DN-p38MAPK suppressed the p38MAPK activity in the subconjunctival cell population. The epithelium and mesenchymal cells in the injured conjunctiva were labeled with anti-phospho-p38MAPK antibody markedly on day 5 and faintly on day 7 (Figure 2A,B), whereas those in the DN-p38MAPK-Ad group specimens were not, or just faintly, stained (Figure 2C,D). At day 2 DN-p38MAPK-Ad did not seem to affect the degree of inflammatory cell infiltration and of edema in the subepithelial tissue as examined by HE histology.
alpha SMA is the hallmark of myofibroblast generation and development of fibrotic tissue. Immunohistochemical analysis of alpha SMA showed that many fibroblasts in the subconjunctiva were labeled with anti- alpha SMA antibody in the control Cre-Ad specimens, but a few fibroblasts in DN-p38MAPK-Ad specimens were labeled on day 5 (Figure 2E,G). On day 7, a few cells were still positive in the control, but not in the DN-p38MAPK-Ad group (Figure 2F,H). The expression pattern of CTGF was similar to that of αSMA (Figure 2I-L).
Fibrogenic gene expression in healing tissue
Real-time RT-PCR was carried out to further evaluate the fibrogenic reaction in the conjunctiva. Overall, col Ia2 mRNA expression was lower in the DN-38MAPK-Ad group until day 7, but there was not apparent difference on day 14 (Figure 3A). CTGF mRNA was also suppressed by application of DN-p38MAPK-Ad on days 5 and 7 (Figure 3B). mRNA of MCP-1 was rapidly induced on day 2 and then declined in the control group. This up-regulation of MCP-1 mRNA was dramatically suppressed by DN-p38MAPK-Ad on day 2 (Figure 3C).
In vitro experiments; Activation of p38MAPK
To elucidate the mechanism of inhibitory effects of overexpression of suppression of p38MAPK on conjunctival scarring, we further conducted a cell culture experiment using subconjunctival fibroblasts and a specific p38MAPK inhibitor, SB2020190. Under quiescent conditions in the absence of exogenous TGFβ1 (Figure 4A,F), as well as at 0.5 h after TGFβ1 addition (Figure 4B,G), a low level of phosphorylated p38MAPK was detected in the cytoplasm, but not in the nuclei of the cells. At 1 h after addition of TGFβ1 (Figure 4C,H), phospho-p38MAPK was markedly upregulated in the nuclei and cytoplasm and lasted until 6 h (Figure 4D,I). At 12 h, the expression of phospho-p38MAPK returned to the basal level (Figure 4E,J). This activation of p38MAPK and its nuclear translocation were abolished by adding a chemical inhibitor of p38MAPK, SB202190 (Figure 4K,O, Figure 4L,P, Figure 4M,Q, and Figure 4N,R). Western blotting further showed that SB202190 suppressed phosphorylation of p38MAPK 1 and 2 h post-TGFβ1 addition (Figure 5).
Effect of the p38MAPK inhibitor on Smad signal-dependent gene expression
As shown in Figure 6, the p38MAPK inhibitor did not affect the Smad-dependent promoter activity.
Gene expression of col Ia2 and CTGF
The effect of adding SB202190 on the expression of col Ia2 and CTGF mRNAs was evaluated. SB202190 reduced the expression level of col Ia2 both in the presence and absence of exogenous TGFβ1, but the reduction was more marked in the TGFβ1-plus culture (Figure 7A). CTGF mRNA was faintly detected in the TGFβ1-minus culture, but adding TGFβ1 remarkably upregulated it. This upregulation of CTGF mRNA was counteracted by adding SB202190 (Figure 7B).
Production of extracellular matrix components
The effect of adding SB202190 on the expression of the extracellular matrix components, i.e., type-I collagen and fibronectin, was evaluated. SB202190 did not have any effect on the production of type-I collagen (Figure 7C,D) and fibronectin (Figure 7E,F), fibroblast cultures in the absence of exogenous TGFβ1 in the culture medium and cell lysate. Adding TGFβ1 increased both components in the medium and lysate, and this increment was reversed by further addition of SB202190.
Cell migration and proliferation
Cell migration was evaluated by a scratch assay. The closure of the liner defect produced in the fibroblast monolayer was delayed with SB202190. The control defect was completely healed within 48 h, but still not in the test culture (Figure 8A,B). Cell proliferation was not significantly affected by adding SB202190 up to day 7 (Figure 8C).
In the present study, we first showed that inhibition of p38MAPK signal by overexpression of DN-p38MAPK via adenoviral gene introduction suppressed the fibrogenic reaction, as evaluated by immunohistochemistry for αSMA and CTGF as well as examination of the mRNA expressions of type-I collagen, CTGF, and MCP-1. The present study was undertaken to evaluate the effect of inhibition of p38MAPK on the conjunctival fibrotic reaction, because a previous in vitro study showed that p38MAPK inhibitor inhibits the fibrogenic behaviors of cultured subconjunctival fibroblasts . However, the chemical specific inhibitor might not be suitable for in vivo application because there is a possibility that such drug might be easily washed out from the local tissue. We therefore employed adenoviral gene transfer of DN-p38MAPK to suppress this signaling cascade. Our in vitro experiments also showed that inhibition of p38MAPK by a specific inhibitor resulted in reduction of the myofibroblast generation and CTGF expression. It is well known that myofibroblast generation and collagen up-regulation are both TGFβ-dependent , and consequently inhibition of p38MAPK signal may affect the TGFβ signal. MCP-1 reduction might further reduce the TGFβ level in tissue because (1) MCP-1 is chemotactic to the macrophages  that are a major source of TGFβ and (2) MCP-1 itself reportedly induces the TGFβ1 expression .
Inhibition of p38MAPK by the specific inhibitor, SB202190, interferes with the stimulatory effects of the exogenous TGFβ1 on production of ECM components, such as type-I collagen and fibronectin, while having no effects on the basal levels of these components. These findings suggest that stimulation of ECM production by TGFβ1 likely results from cooperative effects of multiple signaling inputs including p38MAPK, and that these signals are probably different from the signaling pathways utilized in maintaining the basal activity of these endpoints, which appears to be independent from p38MAPK. Nevertheless, the exact mechanism of the mediate action of p38MAPK on the fibrogenic response has not been clarified yet. A previous study showed that Smad and p38MAPK independently regulate collagen Ia1 mRNA in the hepatic stellate cells . It was reported that p38MAPK is directly required for TGFβ-dependent stimulation of matrix production by the dermal fibroblasts, and thus inhibition of p38MAPK may potentially be effective in preventing dermal scarring [17,19,27]. On the other hand, the cooperative effects of signaling through p38MAPK and the Smad cascade by exogenous TGFβ are also well documented. It has been shown that p38MAPK-dependent activation of Smad3 promoted deposition of the extracellular matrix by myofibroblasts both in vitro and in vivo [17,18,27]. Induction of MMP-13 or aggrecan gene expression by TGFβ requires activation of both Smad and p38MAPK cascades as does apoptosis in the hepatocytes [28,29]. Indeed, it has recently been demonstrated that p38MAPK can result in activating phosphorylation of Smad3 in the middle linker region which enhanced Smad3/4 complex formation and nuclear translocation , consistent with our finding of diminished Smad3/4 reporter gene activity in the presence of the p38MAPK inhibitor. It has been recently reported that such phosphorylation by MAPKs in the Smad3 linker region is required for full activation of Smad signaling [12,13]. In the present study Smad-dependent promoter activity was not affected by a p38MAPK inhibitor, different from ARPE-19 retinal pigment epithelial cell line . This suggests that the way of cross-talk between Smad signal and p38MAPK signal depends on cell types.
TGFβ1 enhanced the expression of CTGF mRNA even in the presence of the p38MAPK inhibitor, indicating that TGFβ1-dependent enhancement of CTGF mRNA expression depends on both non-p38MAPK and p38MAPK signaling. In contrast, the effects of TGFβ1 on type-I collagen and fibronectin were almost totally abrogated by the p38MAPK inhibitor, suggesting that TGFβ1-dependent induction of type-I collagen and fibronectin depends more on p38MAPK. Our previous study revealed that type-I collagen expression was less dependent on p38MAPK as compared with fibronectin in the ARPE-19 retinal pigment epithelial cell line . Explanations for this discrepancy include the dependency of a cell linage difference; namely, mesenchymal versus epithelial.
Migration of the subconjunctival fibroblasts was also dependent on p38MAPK, as SB202190 reversed the enhanced cell migration. This result is consistent with previous studies demonstrating an involvement of p38MAPK in promotion of migration of the epidermal keratinocytes, ARPE-19 cell, mammary epithelial cell line or others [13-18]. Such retarded migration may contribute to the DN-p38MAPK's suppressive effect of excess fibrosis.
These reports together with our findings lead us to hypothesize that inhibition of p38MAPK may be beneficial in preventing/treating bleb scarring following glaucoma filtration surgery. In the bleb tissue, however, the cell components must be affected by factors derived from the aqueous humor. Further studies are needed to establish the clinical application of this therapeutic strategy.
The authors thank Dr. Kathy Flanders, the Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD, for her valuable suggestions.
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