Published ahead of print on September 11, 2003, doi:10.1165/rcmb.2003-0013OC
© 2004 American Thoracic Society DOI: 10.1165/rcmb.2003-0013OC Hepatoma-Derived Growth Factor Is Involved in Lung Remodeling by Stimulating Epithelial GrowthDepartment of Molecular Medicine, and Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan Address correspondence to: Masahide Mori, M.D., Ph.D., Department of Internal Medicine, Osaka Chuo Hospital, Umeda, 3-3-30, Kita-ku, Osaka, 530-0001, Japan. E-mail: m-mori{at}osaka-centralhp.jp
Lung epithelial cells have an integral role in the maintenance of lung homeostasis; however, the regulatory mechanism thereof has not been fully clarified. Recently, hepatoma-derived growth factor (HDGF) was reported to be involved in organ development and remodeling through its mitogenic effect. We investigated the biological role of HDGF in lung remodeling. HDGF was more highly expressed in the lungs of idiopathic pulmonary fibrosis, chiefly in the epithelial cells, than in control nonfibrotic lungs. We also confirmed the expression of HDGF protein and mRNA in the lungs of bleomycin-instilled mice, mainly in the bronchial and alveolar epithelial cells, by immunohistochemical analysis and in situ hybridization. We found that recombinant HDGF promoted DNA synthesis in rat alveolar epithelial cells and A549 cells in vitro. Endogenous HDGF overexpressed by gene transfer was translocated into the nucleus and promoted the proliferation of A549 cells. In vivo intratracheal instillation of recombinant HDGF induced the proliferation of bronchial and alveolar epithelial cells without causing marked interstitial inflammation. These findings suggest that HDGF may be involved in lung remodeling after injury by promoting the proliferation of lung epithelial cells, probably in an autocrine manner.
Abbreviations: alveolar epithelial cell, AEC • bronchoalveolar lavage, BAL • bronchoalveolar lavage fluid, BALF • bronchial epithelial cell, BEC • 5-bromo-2'-deoxyuridine, BrdUrd • 3,3'-diaminobenzidine tetrahydrochloride, DAB • hepatoma-derived growth factor, HDGF • hematoxylin-eosin staining, H-E staining • hepatocyte growth factor, HGF • immunohistochemical, IHC • in situ hybridization, ISH • idiopathic pulmonary fibrosis, IPF • keratinocyte growth factor, KGF • 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT • short interfering RNA, siRNA • surfactant protein C, SP-C • transforming growth factor-ß, TGF-ß
Integral roles of the alveolar epithelium in the maintenance of lung homeostasis have been gradually revealed. The alveolar epithelium is composed of flat type I cells (type I pneumocytes) and cuboidal type II cells (type II pneumocytes). Type II pneumocytes cover < 5% of the alveolar surface. The primary functions of type II pneumocytes are the synthesis and secretion of surfactant proteins, and repair of the alveolar epithelium. In the case of lung damage due to various types of injury (e.g., oxygen, drugs, silica, chemical agents), type I penumocytes detach, and subsequently the type II pneumocytes proliferate, differentiate into type I pneumocytes, and then cover the defected surface (1, 2). Type I pneumocytes are incapable of mitosis; accordingly, type II pneumocytes have a critical role in the maintenance of lung homeostasis during the remodeling phase after lung injury in adults (3, 4). Denudation of the basement membrane without repair of the epithelium may induce interstitial inflammation and fibrosis of the alveoli wall. Idiopathic pulmonary fibrosis (IPF) is one of the representative diseases of lung injury and remodeling (5, 6). The hyperplastic lesions of type II pneumocytes, which are frequently observed in IPF lung tissues, reflect the process of repair from lung damage (7), and animal model investigations revealed that the DNA synthesis of type II pneumocytes is upregulated after lung injury (8). Some of the alveolar epithelial cells undergo apoptosis in IPF lungs (9) and in animal models such as bleomycin-induced lung (10). The putative role of alveolar epithelium in the induction of interstitial inflammation is also supported by Fas ligand–induced interstitial fibrosis (11). The proliferation of type II pneumocytes is thought to be an integral step in lung remodeling after injury (7). Hence, elucidation of the precise mechanism of regeneration and hyperplasia of the alveolar epithelium is indispensable for devising treatment strategies for lung injury, especially for IPF. A number of investigations have revealed that humoral factors such as keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and transforming growth factor (TGF)-ß are critically relevant to the regulation of the proliferation of type II pneumocytes. KGF and HGF exert mitogenic effects on type II pneumocytes in vitro and in vivo (8, 12–14). In contrast, TGF-ß is known to induce apoptosis of alveolar epithelial cells (15), and we have confirmed that gene transfer of the TGF-ß gene into the lungs of rats induced interstitial fibrosis without the proliferation of alveolar epithelial cells (16). In a previous investigation, we demonstrated that mutation of the type II receptor for TGF-ß is relevant to the regeneration of type II pneumocytes in IPF (17). Nevertheless, the control mechanism of physiologic and pathologic turnover of alveolar epithelial cells has not been fully clarified. Recently, a new growth factor, hepatoma-derived growth factor (HDGF), has been reported to exert mitogenic effects on several types of cells. HDGF is a heparin-binding protein that was purified from the conditioned medium of HuH-7 hepatoma cells, and its cDNA was cloned from HuH-7 cells (18, 19). HDGF stimulates the proliferation of fibroblasts, endothelial cells, vascular smooth muscle cells, and some hepatoma cells, including HuH-7 (18–21). Its primary sequence contains no previously identified domain motif but does contain putative nuclear localization signals, and its translocation to the nucleus is essential for its mitogenic activity, indicating that HDGF is a unique factor belonging to a nuclear/growth factor group (22, 23). Recent studies have identified several proteins that share N-terminal amino acid sequences highly homologous to that of mouse HDGF, and therefore, HDGF and its related proteins (HDGF-related proteins) form a new gene family (22, 24, 25). Although HDGF was initially identified in human hepatoma-derived cells, HDGF mRNA is expressed in various normal adult tissues of mice and humans, including lung, suggesting that HDGF has some physiologic functions in these normal organs. In addition, HDGF is involved in vascular smooth muscle cell proliferation during neointimal formation in response to injury, namely vascular remodeling (21). Thus, regarding the maintenance of the alveolar epithelium, it is possible that HDGF also plays important roles in lung remodeling by regulating the proliferation of type II pneumocytes. In the present study, we first confirmed that HDGF is expressed in the proliferating epithelial cells in the lung tissues of patients with IPF. We then attempted to clarify the biological roles of HDGF in lung epithelial cells and to elucidate the pathophysiology of lung epithelium repair and remodeling.
Preparation of IPF Tissue Sections Patients were diagnosed with IPF based on clinical findings, diagnostic imaging, and histologic examinations. Lung specimens were obtained by video-assisted thoracoscopic lung biopsy or open lung biopsy and diagnosed histologically as usual interstitial pneumonitis (UIP) (5, 6). For controls, surgically resected lung specimens from patients with lung cancer were also examined. All specimens were kindly provided by Dr. S. Yamamoto (National Kinki-chuo Hospital for Chest Diseases, Sakai, Osaka, Japan) and Dr. H. Hirano (National Toneyama Hospital, Toyonaka, Osaka, Japan). Three-micrometer-thick tissue sections were prepared for histologic examinations from paraffin-embedded tissues.
Preparation of Animals
Recombinant Protein and Antibody against Human HDGF
HDGF Expression Vector
Short Interfering RNA Expression Vector
Lung Injury Model We performed Western blot analysis of whole lungs of bleo-instilled mice that were killed on Days 3 and 7, bronchoalveolar lavage (BAL) and in situ hybridization (ISH) on Day 7, and immunohistochemical (IHC) analysis on Days 7 and 20. The lungs were intratracheally treated with 20% neutralized formalin (Wako, Osaka, Japan) for IHC analysis or 4% paraformaldehyde/PBS (pH7.4) for ISH, and subsequently removed from the thoracic cavity. For Western blot analysis of whole lungs, the lungs were resected after removal of the blood from the right ventricle. For BAL, 750 µl of saline was instilled twice and withdrawn from the lungs via an intratracheal cannula.
Instillation of rhHDGF into Mouse Lungs
Histopathologic Evaluation
Immunohistochemical Staining To detect the uptake of BrdUrd, IHC analysis was performed using mouse anti-BrdUrd antibody (Dako Corp., Carpinteria, CA) at a dilution of 1:20 in PBS for primary antibody and biotinylated horse anti-mouse IgG (H+L) antibody (Vector Laboratories) at a dilution of 1:66 in PBS for secondary antibody. For double IHC staining, we used goat anti-SP-C antibody (sc-7705, Santa Cruz) at a dilution of 1:20–100 in PBS or goat anti-CC-10 antibody (sc-9772, Santa Cruz) at a dilution of 1:100 in PBS for primary antibody, and biotinylated rabbit anti-goat IgG (H+L) antibody (Santa Cruz) at a dilution of 1:200 in PBS for secondary antibody. The double-staining was visualized with DAB and vector SG kit (Vector Laboratories) as the substrate of the peroxidase.
In Situ Hybridization Mouse HDGF (mHDGF) cDNA was amplified by PCR using the full-length mHDGF cDNA (22) (GenBank No.D63707) as a template. The primers used for PCR were 5'-GGCTACCAGTCCTCCCAGAAAAAG-3' and 5'-GGTCTTTTCCTTTATGTCTGGGTG-3', which were designed not to contain the hath region, which is conserved among HDGF family members (22). The cDNA was cloned into pCRII vector using a TA cloning kit (Invitrogen, Carlsbad, CA). The orientation of the insert was determined by sequencing. The probes were synthesized by in vitro transcription using SP6 RNA polymerase after Xho I digestion for antisense and T7 RNA polymerase after BamH1 digestion for sense RNA production. Digoxigenin-labeled probes were prepared according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN). The procedures for hybridization and the staining reaction were previously described (28).
Cell Culture
Cell Proliferation (BrdUrd Incorporation) Assay and Cell Growth and Viability Assay For the cell growth and viability assay (MTT colorimetric assay), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well at a final concentration of 0.2 mg/ml. After 5 h of incubation at 37°C, the culture medium was replaced by 150 µl of dimethylsulfoxide for solubilization of the purple formazan crystals, and then the absorbance of the samples was measured at 540 nm using a microtiter plate reader.
The Mitogenic Effect of Exogenous HDGF In Vitro A549 cells were seeded in DMEM containing 10% FBS at a density of 5 x 103 cells/well for the cell proliferation assay and at a density of 2 x 103 cells/well for the MTT colorimetric assay in 96-well microtiter plates. For assessing the cell count, A549 cells were also seeded at a density of 1 x 104 cells/well in 24-well plates. Two days later, the medium was replaced with serum-free DMEM and the cells were maintained for 48 h. Thereafter, both AECs and A549 cells were incubated in serum-free DMEM containing 1–1,000 ng/ml of rhHDGF. The cell proliferation assay on AECs and A549 cells was performed after 24 h of incubation with rhHDGF. For the MTT and cell number assays, the medium was replaced by fresh medium every 48 h, and A549 cells were assessed after 96 h of incubation with HDGF. The effect was expressed as the percent absorbance of the treated cells relative to that of the cells incubated without serum and rhHDGF in the cell proliferation assay and MTT colorimetric assay.
The Mitogenic Effect of Endogenous HDGF In Vitro The effect was expressed as the percent absorbance of the cells transfected with the GFP-HDGF gene relative to that of the cells transfected with the GFP gene in the cell proliferation assay and MTT assay.
The Blockage of Endogenous HDGF by siRNA In Vitro Two days later, the cells were transfected with pSUPER or pSUPER-HDGF-KD vector using a lipofection kit (LIPOFECTAMIN 2000 Reagent; GibcoBRL). The medium was replaced by DMEM without serum after 2 h of transfection. The cell lysates were prepared from the transfected cells after an additional 48-h incubation. The medium of the other dishes was replaced by fresh medium every 48 h. Ninety-six hours later, the expression of HDGF was confirmed by immunocytochemical staining, and the cell proliferation assay (BrdUrd) in the transfected A549 cells was performed using the Cell Proliferation ELISA System, ver 2. Six days later, cell growth and viability was assessed by the MTT colorimetric assay and the cell number was counted. For immunocytochemical staining with anti-HDGF antibody, the cells were fixed with 3% paraformaldehyde and then permeabilized with 0.5% Triton X-100. The subsequent methods were described above. The stained cells were observed under a light microscope, and more than 400 cells were counted. The effect was expressed as the percent of the cells transfected with the pSUPER-HDGF-KD vector relative to that of the cells with the control pSUPER vector.
Western Blot Analysis Samples containing 20 µg of protein from lung homogenates, 20 µl of BALF, or 10 µg of protein from cultured cell lysates were mixed with an equivalent volume of sample buffer (50 mM Tris-HCl, pH 6.5, 10% glycerol, 2% SDS, 10% 2-mercaptoethanol and 0.1% bromophenol blue), incubated at 98°C for 5 min, electrophoresed on 11% SDS-polyacrylamide gels under reducing conditions, and transferred to Immobilon-P membranes (Millipore, Bedford, MA). One hundred or 10 ng of recombinant hHDGF was used as a positive control. After washing, nonspecific binding sites of the membranes were blocked by incubation in PBS containing 50 mg/ml nonfat skim milk for more than 30 min. The membranes were then probed with anti-hHDGF antibody at a dilution of 1:1,000 for 1 h at room temperature, and then incubated with peroxidase-conjugated goat anti-rabbit IgG (H+L) (BIO-RAD) at a dilution of 1:30,000 for 1 h. Immunoreactive bands were visualized with Renaissance Chemiluminescent Reagent (NEN Life Science Products, Boston, MA), and the density of the bands was quantitated.
Statistical Analysis
HDGF Expression in the Bronchial and Alveolar Epithelial Cells of IPF First, to clarify the role of HDGF in the pathogenesis of IPF, we examined HDGF expression in lung tissues of normal subjects and patients with IPF by IHC analysis using anti-human HDGF (hHDGF) antibody. In the lungs of normal control individuals, the expression of HDGF was detected in some cells, exclusively in the nucleus. About half of the bronchial epithelial cells were positive for staining (53.7 ± 3.1%, > 200 cells were counted each; n = 4 individuals), but the staining intensity was not strong (Figure 1A). The staining intensity of the alveolar epithelial cells was much weaker than that of the bronchial epithelial cells (Figure 1B). A few endothelial cells and smooth muscle cells in the vessels were positively stained (Figure 1C).
In contrast, in the lungs of patients with IPF, HDGF was highly expressed as compared with the level in normal lung tissue. Numerous bronchial epithelial cells were positively stained (81.0 ± 3.2%, > 200 cells; n = 4; Figure 1E), and the positive rate was significantly larger than nonfibrotic control lungs (P < 0.001). The expression of HDGF was also increased in the epithelial cells of destroyed alveolar spaces. Many alveolar epithelial cells, which were identified with anti–SP-C antibody, were also positively stained with anti-HDGF antibody in the relatively early stage of fibrosis with septal thickening (Figures 1F and 1G). A lot of the alveolar lining epithelial cells were positive in the hyperplastic lesions (84.2 ± 2.3%, > 200 cells; n = 4), one of the characteristic lesions in IPF (5) (Figures 1H and 1I). Strong staining was also observed in most of the epithelial cells of the bronchiolization at the dilated air space as well as in the bronchial epithelial cells (79.7 ± 5.3%, > 200 cells; n = 4) (Figure 1J). In the interstitium, focally infiltrating mononuclear cells were almost all negative for staining. Some of the smooth muscle cells in the vessels of the IPF lungs were faintly, but not significantly, stained (Figure 1K). About half of the fibroblasts in the interstitium weakly expressed HDGF (Figure 1L).
Upregulation of HDGF Expression in the Bleomycin-Instilled Lungs
HDGF Expression in the Lung Epithelial Cells of Bleo-Instilled Mice Next, IHC analysis of the lungs from bleo-instilled mice was performed with anti-hHDGF antibody to analyze the localization of HDGF expression. In the saline-instilled control mice, HDGF was expressed locally in the nucleus in some bronchial epithelial cells, and the proportion of positive alveolar epithelial cells was less than that of bronchial cells, a finding similar to that in the normal human lungs (Figures 3A–3C).
In the lungs 7 d after bleo instillation, most of the bronchial epithelial cells strongly expressed HDGF as shown by IHC analysis (Figures 3E and 3F). Also in the alveoli, several types of cells expressed HDGF, including alveolar epithelial cells, which were confirmed by double IHC staining with anti–SP-C antibody (Figures 3G and 3H). On Day 20 after bleo instillation, most of the bronchial and alveolar epithelial cells were intensely stained (Figure 3H). The epithelial cells covered with the dilated air space were also positively stained (Figure 3J). In contrast, some of the fibroblasts were weakly stained. The pattern of staining for HDGF was similar to that in the lungs of patients with IPF.
ISH Analysis of HDGF mRNA Expression in the Lung Epithelial Cells of Bleo-Instilled Mice
Exogenous HDGF Promotes DNA Synthesis and Cell Proliferation of Lung Epithelial Cells In Vitro We examined the mitogenic effect of exogenous rhHDGF on lung epithelial cells in vitro using AECs and the A549 cell line. Exogeneously supplied HDGF upregulated the DNA synthesis which monitored as BrdUrd uptake in AECs by 19–27% (P < 0.01; n = 6 wells each, Figure 5A). HDGF also promoted the DNA synthesis in A549 cells in a dose-dependent manner at up to 100 ng/ml HDGF, with maximal upregulation of 58% (P < 0.05; n = 6 wells each, Figure 5B).
The effect of HDGF on the proliferation of A549 cells was assessed by the MTT colorimetric assay. The proliferation of A549 cells was increased by 14–25% compared with the control by incubation with 1–100 ng/ml rhHDGF (P < 0.05; n = 6, Figure 5C); however, at an overdose (1,000 ng/ml) of HDGF, no significant mitogenic effect was seen. The effect was also confirmed by monitoring the cell count, which was increased by 80% with 10 ng/ml rhHDGF (P < 0.05; n = 6, Figure 5D).
Endogenous HDGF Is Translocated to the Nucleus and Promotes DNA Synthesis and Cell Proliferation of A549 Cells In Vitro GFP protein was distributed throughout the cells transfected with pCAGGS-GFP, whereas GFP-hHDGF fusion protein was translocated into the nucleus of the cells transfected with pCAGGS-GFP-hHDGF (Figure 6A). Western blot analysis of the gene-transfected cells showed out that the expression of endogenous hHDGF was detected in A549 cells transfected with pCAGGS-GFP (Figure 6B, lane 1); however, the level of expression of GFP-hHDGF fusion protein was much higher than that of the endogenous hHDGF in the cells transfected with the GFP-hHDGF gene (Figure 6B, lane 2).
The transfer of the GFP-hHDGF gene enhanced the DNA synthesis of A549 cells by 26% (P < 0.01; n = 6 wells each, Figure 6C), and also promoted the proliferation of A549 cells by 16% in the MTT assay (P < 0.001; n = 6, Figure 6D) and by 60% as indicated by the cell count (P < 0.05; n = 6, Figure 6E), compared with the GFP-control vector.
Blockage of Endogenous HDGF by siRNA Suppressed the Cell Proliferation Immunocytochemical analysis revealed that 77% of A549 cells transfected with pSUPER control vector on Day 4 were positively stained with anti-HDGF antibody, whereas the positive rate was significantly decreased to 61% in those transfected with pSUPER-HDGF-KD (knock-down) vector (77.6 ± 1.4% versus 61.4 ± 1.1%, P < 0.001; n = 4 wells each). The staining intensity was relatively weak in the A549 cells transfected with knock-down vector (representatively presented in Figure 7A).
The suppressive effect of pSUPER-HDGF-KD vector was comfirmed also by Western blot analysis. The expression of HDGF protein was 28% of reduction in the A549 cells transfected with pSUPER-HDGF-KD vector compared with control (100 ± 11.6% versus 72.0 ± 3.1%, P < 0.02; cell lysates from 4 wells each; Figure 7B).
After confirmation of suppressive effect by siRNA on the expression of endogeneous HDGF, we next examined whether siRNA could suppress the mitogenic effect of endogeneous HDGF. In the A549 cells transfected with pSUPER-HDGF-KD vector, there was a slight suppression of DNA synthesis compared with that in the control cells, as indicated by the uptake of BrdUrd on Day 4 (the percent absorbance of the cells transfected with control vector, 100 ± 3.0% versus 85.2 ± 5.8%, P = 0.056); however, the difference was not significant. Nonetheless, the MTT colorimetric assay revealed that the blockage of endogenous HDGF by siRNA inhibited cell growth and viability by 12% on Day 6 (P < 0.0001, Figure 7C). The inhibitory effect was confirmed also by monitoring the cell count. The number of the cells transfected with pSUPER-HDGF-KD was reduced 50% compared with the control on Day 6 (P < 0.01, Figure 7D). Moreover, addition of exogenous rhHDGF (10 or 100 ng/ml) resulted in
Instillation of rhHDGF into the Lung Induces the Proliferation of the Epithelial Cells In Vivo In the murine lung 3 d after the instillation of recombinant hHDGF, the bronchial epithelial cells were widely hyperplastic and tufting (Figures 8A–8C). In the alveolar fields, a fraction of the alveolar epithelial cells were proliferating (Figures 8D and 8E). However, there were no marked inflammatory changes such as infiltration of inflammatory cells, proliferation of fibroblasts, or thickening of the alveolar septae. The histologic changes were maintained at least until Day 7.
The control lungs with saline instillation showed no remarkable histopathologic changes (Figures 8F and 8G). In addition, DNA synthesis was examined as the uptake of BrdUrd, which was administered before killing on Day 1 by IHC analysis with anti-BrdUrd antibody. No marked histologic changes were observed in the murine lungs with HDGF instillation on Day 1; however, the uptake of BrdUrd was detected in some bronchial epithelial cells (BECs) (Figure 8I) and in some of alveolar epithelial cells (Figures 8J and 8K). In murine lungs with saline instillation, only faint staining with anti-BrdUrd antibody was observed in a much lower number of bronchial epithelial cells (Figure 8L) and little of alveolar epithelial cells (Figure 8M).
A number of investigations have revealed that the network among the epithelial cells, mesenchymal cells, and components of the ECM is important for lung epithelial morphogenesis (for reviews see Refs. 7, 30). In the course of lung remodeling after lung injury, several cytokines play a key role in regulating the proliferation or suppression of type II pneumocytes. We have shown here that HDGF is a new autocrine growth factor for lung epithelial cells. First we showed that HDGF was highly expressed locally in the nuclei of the hyperplastic and proliferating alveolar epithelial cells of IPF lungs. Moreover, in an experimental mouse model, HDGF expression was markedly increased throughout the lung after the lung injury induced by the bleomycin instillation. HDGF expression is already known to be significantly induced by tissue injury and remodeling in other organs. HDGF is expressed in endothelial cells undergoing repair during the vascular remodeling process in response to injury (21). The fact that HDGF expression was predominantly observed in the bottom of intestinal crypts suggests a possible role of HDGF in intestinal regeneration (31). A noticeable and common feature among the lung, the vessels and the intestine is that expression of HDGF is localized in the nuclei of the proliferating cells. HDGF is a unique growth factor which trafficks to the nucleus and whose nuclear translocation is essential for its mitogenic activity (23). We demonstrated that the expression of HDGF was significantly increased in the nuclei of lung epithelial cells in vivo. Hence, HDGF was predicted to affect lung epithelial cells. Regarding the effects of HDGF on epithelial cells, a few results have been reported about the proliferation of renal epithelial cells (293 cells) and fetal hepatocytes (20, 32). In the present report, we demonstrated that exogenous HDGF promotes in vitro DNA synthesis in lung epithelial cells, rat alveolar epithelial cells and A549 cells. The mitogenic effect of recombinant HDGF on proliferating epithelial cells in vitro was not so remarkable: 10–100 ng/ml HDGF induced 20–50% increases of DNA synthesis and cell number. In addition, the extent of the effect was not increased in proportion with the concentration of recombinant HDGF, which was similar to the effects seen in previous in vitro investigations, for example, on fetal hepatocyte and hepatoma cell lines (32, 33). Endogenous HDGF overexpressed via transient gene transfer was translocated into the nucleus and promoted the proliferation of A549 cells. The extent of the mitogenic effect was modest (about a 26% increase of DNA synthesis) but significant. A high concentration of exogenous HDGF or hyperexpression of HDGF by gene transfer did not necessarily produce parallel mitogenic effects in A549 cells. It is possible that endogenous HDGF itself might have a mitogenic effect to some extent, and therefore that the effect of excess HDGF was not so clear. Enomoto and colleagues have confirmed this assumption by showing that the mitogenic effect of recombinant HDGF on fetal hepatocytes was only marginally significant, but was enhanced after inhibition of endogenous HDGF by adenoviral-mediated introduction of HDGF antisense cDNA (32). We also confirmed, using the siRNA technique, that endogenously produced HDGF has a mitogenic effect on A459 cells. Nevertheless, the direct instillation of recombinant HDGF into murine lungs clearly caused proliferation of the bronchial and alveolar epithelial cells. Considering that the area of diffusion and permeation by transbronchially administered HDGF is mostly limited to epithelial cells, the results of the in vivo direct instillation are thought to reflect the effect of HDGF in lung remodeling after injury. These results demonstrated the function of HDGF as a mitogen for epithelial cells in vivo, which has already been reported for various cells in vitro including lung cells, but no previous reports have shown such an effect of HDGF in vivo. In addition, no marked inflammatory changes were observed. It is thus at least clear that HDGF would not have effects on epithelial cells leading to the induction of interstitial inflammation as indicated by the infiltration of inflammatory cells, proliferation of fibroblasts, or collagen synthesis. Thus, we demonstrated direct mitogenic effects of HDGF on lung epithelial cells in vitro and in vivo, and our findings suggest that HDGF plays an important role through its mitogenic effects on epithelial cells in the phase of the lung repair and the remodeling process after injury. Several factors have been reported to control the regulation of proliferation of type II pneumocytes through their mitogenic and/or morphogenetic effects. Especially, KGF and HGF are certainly important factors concerned with mesenchymal–epithelial interactions. Ulich and coworkers reported that intratracheal instillation of recombinant KGF induced alveolar epithelial cells to proliferate and differentiate to type II pneumocytes (13). Moreover, in the bronchus, the tufting of epithelial cells was also observed. Similar changes were observed after transfer of the gene encoding KGF using adenovirus vector (34). Another factor, HGF, also promoted DNA synthesis in rat alveolar epithelial cells when the recombinant protein was directly instilled into lungs (8). Thus HGF profoundly affects lung repair through its mitogenic and morphogenetic effects. It has been clearly demonstrated that some mesenchymal cells produce both KGF and HGF, and after secretion, these factors bind their respective receptors expressed on epithelial cells in the lungs to induce epithelial proliferation (14, 35). For instance, in the liver HGF is produced by nonparenchymal cells, and has paracrine mitogenic and morphogenic effects on hepatocytes (36). Thus, the effects of KGF and HGF on their target cells are exerted exclusively in a paracrine manner. With regard to epithelial cell proliferation, the effect of HDGF was akin to that of KGF and HGF. However, the manner of the secretion and intracellular transport of HDGF is quite different from that of these growth factors. Very few factors have been reported to exert an autocrine mitogenic effect on epithelial cells. Both HDGF protein and its mRNA were expressed in the bronchial and alveolar epithelial cells of bleo-instilled mice. HDGF mRNA was detected at least in the alveolar corner cells and alveolar wall, in cells which were tentatively identified as type II and type I pneumocytes. HDGF protein was also localized in the nuclei of epithelial cells by IHC analysis. In addition, we confirmed the existence of HDGF in the BALF of normal and bleo-instilled mice by Western blot analysis, which implies that HDGF was released from alveolar epithelial cells into the conducting airway and the alveolar space. Hence, these findings suggest the possibility that HDGF is a factor that stimulates the proliferation of lung epithelial cells, at least in part in an autocrine manner. With regard to other organs, HDGF is produced by fetal hepatocytes and has a mitogenic effect on hepatocytes themselves (32). Furthermore, lens epithelium–derived growth factor (LEDGF), which is a member of the HDGF family, has been shown to be an autocrine and intracrine factor for retinal pigment epithelial cells (25, 37). The autocrine manner of action may be characteristic of this family of molecules. In the current study, we demonstrated that HDGF plays an important role in lung remodeling after injury by promoting the proliferation of lung epithelial cells, probably in part in an autocrine manner. Little has been reported about autocrine factors for lung epithelial cells, and HDGF is a candidate for such a new type of growth factor in lung remodeling. Although the effects of HDGF itself have not been fully clarified, analyses of the mechanism of HDGF expression and clarification of its relationship to the pathophysiology of lung epithelium repair and remodeling might offer new insights into interstitial lung diseases with epithelial injury such as IPF.
The authors thank Dr. Reuven Agami (The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital) for providing pSUPER, an siRNA expression vector. They thank Dr. M. Shiratori (Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan) for teaching the AEC isolation method. They are grateful to Mr. T. Hashimoto (National Toneyama Hospital, Toyonaka, Osaka, Japan) and Miss M. Kim (Osaka Chuo Hospital, Kita-ku, Osaka, Japan) for their technical supports. They also thank Miss Y. Habe for her secretarial work. This study was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare of Japan.
* These authors contributed equally to this work.  Received in original form January 14, 2003 Received in final form August 3, 2003
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Abstract
Introduction
4.5-fold in the lungs 7 d after bleo instillation compared with the nontreated control (P < 0.05; n = 3 each, 
