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Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, ChinaDepartment of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Reprint requests: Jungang Xie, Jianping Zhao, Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China.
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Reprint requests: Jungang Xie, Jianping Zhao, Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China.
Department of Respiratory and Critical Care Medicine, National Clinical Research Center of Respiratory Disease, Key Laboratory of Pulmonary Diseases of Health Ministry, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Dysregulation of type II alveolar epithelial cells (AECII) plays a vital role in the initiation and development of pulmonary fibrosis (PF). Dachshund homolog 1 (Dach1), frequently expressed in epithelial cells with stem cell potential, controls cell proliferation, apoptosis, and cell cycle in tissue development and disease process. In this study, we demonstrated that the lungs collected from PF patients and mice of Bleomycin (BLM)-treated were characterized by low expression of Dachshund homolog 1 (Dach1), especially in AECII. Dach1 deficiency in the alveolar epithelium exacerbated PF in BLM-treated mice, as evidenced by reduced pulmonary function and increased expression of fibrosis markers. Rather, treatment with lung-specific overexpression of Dach1 alleviated histopathological damage, lung compliance, and fibrosis in BLM-treated mice. Moreover, overexpression of Dach1 could inhibit epithelial apoptosis in vitro. Conversely, primary AECII with Dach1 depletion were more susceptible to apoptosis in vivo. Mechanically, Dach1 combined with C-Jun protooncogene selectively bound to the promoter of B-cell lymphoma 2 interacting mediators of cell death (Bim), by which it repressed Bim expression and alleviated epithelial apoptosis. Taken together, our data support that Dach1 in AECII contributes to the progression of PF and may be a viable target for the prevention and treatment of PF.
Dachshund homolog 1 (Dach1), frequently expressed in epithelial cells with stem cell potential, controls cell proliferation, apoptosis and cell cycle in tissue development and disease process. The role of Dach1 in type 2 alveolar epithelium (AEC2) remains unknown during pulmonary fibrotic processes.
Translational Significance
In this study, we revealed that Dach1 deficiency in AECII exacerbated pulmonary fibrosis (PF) in bleomycin (BLM)-treated mice. In contrast, overexpression of Dach1 in the lung protected mice against PF. Further mechanism study revealed that Dach1 inhibited epithelial apoptosis by blocking the binding of Jun proto-oncogene (C-Jun) to B-cell lymphoma 2 interacting mediator of cell death (Bim). Collectively, the present study suggests that Dach1 may be a viable target for the prevention of PF.
INTRODUCTION
Idiopathic pulmonary fibrosis (IPF) is a chronic, age-related, progressive, lethal interstitial lung disease with unknown etiology.
Chronic injury and dysregulation of type II alveolar epithelial cells (AECII) during repair processes play vital roles in the initiation and development of IPF.
However, the regulatory mechanism by which AECII guides successful or aberrant repair processes remains largely elusive.
Dachshund homolog 1 (Dach1), an essential component of the Retinal Determination Gene Network (RDGN), is commonly expressed in epithelial cells with stem cell properties.
Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.
Previous data have demonstrated that Dach1 participates in tumorigenesis and metastasis by regulating the proliferation, apoptosis, and cell cycle progression of tumor cells.
Functional studies have identified Dach1 as a regulator of the TGF-β signaling, which is vital to the progression of IPF in different types of cancers.
However, the biological role of Dach1 in the AECII in IPF has not been explored.
In this study, we established an AECII-specific Dach1 knockout mouse model and found that Dach1 deficiency in AECII exacerbated pulmonary fibrosis (PF) in bleomycin (BLM)-treated mice. In contrast, overexpression of Dach1 in the lung protected mice against PF. Further mechanism study revealed that Dach1 inhibited epithelial apoptosis by blocking the binding of Jun proto-oncogene (C-Jun) to B-cell lymphoma 2 interacting mediator of cell death (Bim). Collectively, the present study suggests that Dach1 may be a viable target for the prevention of PF.
RESULTS
Dach1 was downregulated in PF
The pathogenic mechanisms of IPF are poorly understood.
In this study, we first performed a bioinformatics analysis of a human IPF dataset (GSE47460) to screen DEGs between patients with IPF and HC (Supplemental Table I, Additional file 1). A total of 364 upregulated and 326 downregulated genes were identified (Supplemental Fig 1A, Additional file 2-4). Dach1 was significantly downregulated in patients with IPF compared with HC (Fig. 1, A, Supplemental Fig 1C). Correlation analysis revealed that pulmonary expression of DACH1 was negatively correlated with the levels of fibrosis-related genes in the lung, including MMP9, COL1A2, CDH2, TIMP1, FN, ACAT2, and COL3A1 (Supplemental Fig 1B), and positively correlated with pulmonary function parameter DLCO% (Fig. 1, B). In the validated datasets GSE32537 and GSE110147, the expression of Dach1 was decreased in IPF populations compared to heathy controls, though not significantly decreased in non-IPF ILD patients (Supplemental Fig 1D and 1E, Additional file 5 and 6).
Fig 1Decreased expression of Dach1 in pulmonary fibrosis. (A) Relative log2 expression of DACH1 between patients with IPF (n = 112) and HC (n = 108). (B) Correlation of log2 expression of Dach1 in lung tissues with DLCO% predicted. Pearson's correlation, *P = 0.0003. (C) Western blot analysis of Dach1 in BLM-induced lung tissues (n = 3) and control lungs (n = 3). (E–F) Representative fluorescence microscopy images of Dach1 (Cy3, red), pro-Spc (FITC, green), and DAPI (blue) in control (n = 4) and BLM-induced PF (n = 6), the scale bar 10um. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney's unpaired non-parametric test (A), Student's unpaired t-test (D and F) (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Consistently, expression of Dach1 was significantly downregulated in BLM-treated mice compared with the control group, along with markedly increased expression of Fibronectin, a fibrosis marker (Fig. 1, C and D). Dach1 was widely expressed in control mice, especially in pro-Spc+ AECII (Fig 1, E and F). After BLM treatment, the expression of Dach1 in AECII was significantly decreased. The expression of Dach1 was also decreased in Epcam+ lung epithelial (Bronchial epithelial cells) and Fsp1+ fibroblast (Supplemental Fig 2A–D), but not CD68+ macrophage (Supplemental Fig 1E and F). In line with the above results, TGF-β1 stimulation markedly decreased the level of Dach1 in MLE-12 cells in a time- and dose-dependent manner (Supplemental Fig 2G and H). Taken together, our data indicate that PF is characterized by low DACH1 expression in AECII.
Dach1 deficiency in AECII exacerbated PF in BLM-treated mice
To further investigate the role of Dach1 in PF, we generated Dach1 CKO mice with AECII-specific deletion of Dach1 (Fig. 2, A, Supplemental Fig 3A and B). Their littermates (Dach1 C mice) were used as the control group. Western blot analysis of primary AECII also indicated a significant decrease of Dach1 expression, while its levels in the fibroblast were unaltered (Fig. 2, B). Consistently, co-immunostaining of lung tissue sections validated Dach1 deficiency in the pro-Spc+ AECII of Dach1 CKO mice (Fig. 2, C).
Fig 2Dach1 deficiency in alveolar epithelium exacerbated pulmonary fibrosis in BLM-treated mice. Mice with AECII-specific knockout of Dach1 (SftpcCreERT+/−Dach1flox/flox) were developed. (A) Schematic diagram of transgenic mice used to generate Dach1 C and Dach1 CKO mice. (B) Western blot analysis of Dach1 expression in primary AECII and fibroblast to determine the specific deletion of Dach1 in AECII. (C) Co-immunostaining of Dach1 (Cy3, red), pro-Spc (FITC, green), and DAPI (blue) in lung tissue sections from Dach1 C and Dach1 CKO mice, the scale bar 20um. (D-E) Dach1 CKO and Dach1 C mice were exposed to BLM to induce PF. On day 21, the lungs of all mice were harvested and stained with Masson's trichrome (n = 6 per group). (F) The content of hydroxyproline in the right middle lung was measured (n = 6 per group). (G) The BALF total cells were counted (n = 6 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA (E–G) (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
At 21 days following the BLM challenge, Dach1 CKO mice showed significantly aggravated lung injury and fibrosis, as shown in Masson's trichrome staining (Fig. 2, D). Phenotypically, the Ashcroft score was much higher in Dach1 CKO mice compared with the Dach1 C group (Fig. 2, E). Consistently, Dach1 C mice showed lower hydroxyproline content in comparison with Dach1 CKO mice (Fig. 2, F). The number of inflammatory cells in the BALF of BLM-treated Dach1 CKO mice was much higher than that in Dach1 C mice (Fig. 2, G). Moreover, the parameters of lung compliance, lung elastance, and inspiratory capacity were decreased in BLM-treated mice. In line with these observations, Dach1 deficiency further exaggerated BLM-induced lung function loss in mice (Fig. 3, A–D). Consistently, western blot (Fig. 3, E–G) and RT-PCR (Fig. 3, H–K) analysis showed upregulated Fibronectin and Collagen I in the lung tissues of Dach1 CKO mice. At 14 days after BLM treated, the fibrosis degree of Dach1 CKO mice was much higher than that of Dach1 C mice, while the tendency was not significantly at 7 days (Supplemental 4). Considering that inflammation also played important role in PF, we analyzed the expression of classic inflammatory cytokines (IL-1β and IL-6). Just as RT-PCR analysis showed, the expression of IL-1β and IL-6 were significantly increased in BLM-treated mice, but there was no significant increase in the Dach1 CKO group. Collectively, these data support that specific deletion of Dach1 in AECII exacerbated lung injury and fibrosis.
Fig 3Lung function and fibrotic gene expression following prophylactic deletion of Dach1 in BLM-treated mice. (A–D) The pressure-volume-loops (PV-loops), inspiratory capacity, static compliance (Cst), and respiratory system compliance (Crs) of mice were measured (n = 6 per group). (E–G) Western blot analysis of Fibronectin and Collagen I (n = 4 per group). (H-K) RT-PCR analysis of Fibronectin (FN), Collagen I (Col1a2), α-SMA (Acta2), and Tgf-β1 (Tgfb1) (n = 5–9 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA (B–D, F–G, H–K) or two-way ANOVA (A).
Lung-specific transgenic expression of Dach1 protected mice against PF
Subsequently, we generated mice with lung-specific transgenic expression of Dach1 via intratracheal instillation of AAV6-Dach1. Western blot and immunostaining analysis showed that Dach1 was overexpressed in the lung, especially in the epithelium (Fig. 4, B and C). The expression of Dach1 was also increased in isolated AECII from Dach1-OE mice (Supplemental Fig 5A). Intuitively, bodyweight reduction was also significantly improved by the overexpression of Dach1(Fig. 4, D). Overexpression of Dach1 significantly alleviated BLM-induced pulmonary injury and fibrosis, as shown by histological analysis (Fig. 4, E) and the Ashcroft score (Fig. 4, F). Mice with lung-specific overexpression of Dach1 displayed much less content of hydroxyproline (Fig. 4, G) and TGF-β1 in BALF (Supplemental Fig 5B), improved lung function (Supplemental Fig 5C–F), and reduced inflammatory cell infiltration (Fig. 4, H). Additionally, upregulation of Dach1 also decreased the expression of fibrosis markers in BLM-treated mice (Supplemental Fig 5G–I). These findings suggest that Dach1 overexpression in the lung might be used as a treatment option for PF.
Fig 4Lung-specific transgenic expression of Dach1 protected mice against lung fibrosis. Mice were administered with AAV6-Dach1 via intratracheal instillation to induce Dach1 overexpression (Dach1-OE). After 21 days, mice were treated with BLM. After additional 21 days, mouse lung tissues were harvested. (A) The timeline of the experiment. (B) Western blot analysis of Flag in lung tissue lysates (n = 3–6 per group). (C) Immunostaining of Dach1-Flag Dach1 (FITC, green), pro-Spc (Cy3, red), and DAPI (blue) in mouse lung tissues, the scale bar 20um. (D) The body weight change during BLM treatment was monitored (n = 6–12 per group). (E–F) The lungs from Control and Dach1-OE mice were stained with Masson's trichrome (n = 6 per group). (G) The content of hydroxyproline in the right middle lung was measured (n = 6 per group). (H) The BALF total cells were counted (n = 6 per group). Data are presented as mean±SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA (F–H) or two-way ANOVA (D) (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
We firstly confirmed that overexpression Dach1 in epithelium alleviated the process of epithelial-mesenchymal-transition (Supplemental Fig 6A–C), with fibronectin (mesenchymal marker) increased and Zo-1 (epithelial marker) decreased. Then, to explore the potential mechanism involved in the downregulation of Dach1 in PF, we analyzed Dach1-associated DEGs from Chip-seq datasets (GSE127361 and GSE91962) and microarray datasets (GSE685) (Additional file 7–10). The Gene Ontology analysis of DEGs indicated that Dach1 was closely associated with apoptosis (Fig. 5, A and B) and transcriptional regulation. We firstly confirmed that BLM induced apoptosis of MLE-12 cells with insufficient Dach1 expression in a time- and dose-dependent manner (Supplemental Fig 6D and E). To investigate the effects of Dach1 overexpression, MLE-12 cells were transfected with cDNA-Dach1 or negative control (NC) plasmid for 24 hours before BLM treatment. Flow cytometry revealed that Dach1 overexpression reduced the apoptotic rate of MLE-12 cells (Fig. 5, C and D). The expression of apoptosis markers were also significantly decreased in Dach1-OE cells compared with the NC group (Fig. 5E–G). The above data indicate that overexpression of Dach1 protected MLE-12 cells against BLM-induced apoptosis. On the contrary, the deletion of Dach1 makes primary alveolar type II epithelial cells more prone to apoptosis (Supplemental Fig 6F-6H)
Fig 5Dach1 regulated AECII apoptosis. (A–B) Dach1 was overexpressed in different cells. Chip-seq data (GSE127361 and GSE91962) and microarray data (GSE685) were analyzed to identify DEGs. The results of Gene Ontology analysis of DEGs. (C–D) The apoptosis of MLE-12 cells transfected with cDNA-Dach1 or negative plasmid and treated with or without BLM for 24 hour was analyzed by flow cytometry (n = 5 per group). (E–G) Western blot analysis of Dach1, caspase-3, cleaved caspase-3, Bax, and Bcl-2 in MLE-12 cells treated as mentioned above (n = 4 per group). Data are presented as mean±SEM. *P < 0.05, **P < 0.01 by one-way ANOVA (D, F–G).
Dach1 protects MLE-12 cells against apoptosis by binding to C-Jun and transcriptionally inhibits Bim expression
To elucidate the mechanisms by which Dach1 ameliorated epithelial apoptosis, we first measured the mRNA levels of Foxo1, Bim, Bcl-2, Bax, Dusp1, Fosb, Junb, and Pdk1, as these genes were the DEGs associated with apoptosis in PF. The expressions of Foxo1 and Bim, 2 key regulators of apoptosis,
were significantly altered in cells transfected with cDNA-Dach1 compared with the NC group (Fig. 6, A and B, Supplemental Fig 7A–F). As Dach1 is a transcription factor, we speculated that Dach1 may directly interact with the Foxo1 or Bim promoters. However, bioinformatics analysis showed that the promoter of Foxo1 or Bim had no binding site for Dach1 (http://cistrome.org/db/#/), which led us to hypothesize that Dach1 may regulate the expressions of Foxo1 and Bim by interacting with other transcription factors. Immunoprecipitation analysis was then performed to explore whether Dach1 could interact with another classic, apoptosis-related transcription factors, such as C-Jun and Trp53.
The results showed that Dach1 pulled down C-Jun but not Trp53 in MLE-12 cells (Fig. 6, C, Supplemental Fig 7G), which was consistent with immunofluorescence staining, showing the co-localization of Dach1 and C-Jun in the nuclei of MLE -12 cells (Fig. 6, D). Western blot analysis confirmed that the protein levels of Bim, Foxo1 and C-Jun were negatively correlated with the expression of Dach1 (Fig. 6, D–H). Only the mRNA expression of Bim was parallel to its protein expression. Hence, we speculated that Dach1 is bound to C-Jun to regulate the expression of Bim in the pulmonary epithelium.
Fig 6Dach1 bound to C-Jun and transcriptionally inhibited Bim expression. RT-PCR analysis of DEGs (ie, Foxo1 and Bim) in MLE-12 cells transfected with cDNA-Dach1 or negative plasmid for 24 hour (n = 6 per group). (C) Co-immunoprecipitation analysis of Dach1 and C-Jun. (D) Representative fluorescence microscopy images of Dach1 (FITC, green), C-Jun (Cy3, red), and DAPI (blue), the scale bar 20um. (E–H) Western blot analysis of Dach1, Bim, C-jun, and Foxo1 in MLE-12 cells treated as described above (n = 3 per group). Data are presented as mean±SEM. **P < 0.01, ***P < 0.001 by Student's paired t-test (A–B, F–H) (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
To test this hypothesis, we performed the Bim promoter reporter assay. As expected, disruption of the C-Jun-binding site significantly suppressed Dach1-induced luciferase reporter activity in MLE-12 cells (Fig. 7, A and B). Dach1 overexpression-induced increase in the percentage of apoptotic cells was inhibited by Navitoclax (a molecule that mimics Bim) pretreatment (Fig. 7, C and D), which was consistent with the upregulation of apoptosis markers in Navitoclax-treated cells (Fig. 7, E–G). These data suggest that Dach1 protects MLE-12 cells against apoptosis by binding to C-Jun and transcriptionally inhibiting the expression of Bim.
Fig 7Dach1 protected MLE-12 cells against apoptosis by inhibiting Bim expression. (A–B) Results of the luciferase reporter assay of the Bim promoter in MLE-12 cells (n = 3 per group). (C–D) MLE-12 cells were transfected with cDNA-Dach1 or negative plasmid for 24 hour, followed by treatment with or without Navitoclax (10 μM, Bim mimics) for 12 hour and incubation with BLM for 24 hour. The apoptosis of MLE-12 cells was assessed by flow cytometry (n = 5 per group). (E–G) Western blot analysis of Dach1, caspase-3, cleaved caspase-3, Bax, and Bcl-2 in MLE-12 cells treated as described above (n = 4 per group). Data are presented as mean±SEM. **P < 0.01, ***P < 0.001 by one-way ANOVA (B, D, F–G).
The above findings prompted us to confirm the regulatory role of Dach1 in apoptosis in vivo. We found that the level of cleaved caspase-3/caspase3 was markedly increased in the lungs of BLM-treated mice, and further elevated in Dach1 CKO mice (Fig. 8, A and B). The expression of Bax/Bcl-2 showed a similar tendency though not significant (Fig. 8, C). The expression level of Bim was decreased in BLM-treated mice, while the level of Bim was increased in Dach1 CKO mice (Fig. 8, D). There was no significant difference in the expression of C-Jun among groups (Fig. 8, E). Mice with lung-specific transgenic expression of Dach1 showed lower expression of apoptosis markers (Supplemental Fig 8). Overall, it could be concluded that Dach1 regulated apoptosis in vivo.
Fig 8Dach1 regulated apoptosis in vivo. (A–E) Western blot analysis of caspase-3, cleaved caspase-3, Bax, Bcl-2, Bim, and C-Jun in normal saline- or BLM-treated Dach1 C and Dach1 CKO mice (n = 5–8 per group). (F) Pulmonary fibrosis represses the expression of Dach1, which reduces the inhibition of C-Jun on the promoter of Bim. Subsequently, C-Jun enhances the transcription of Bim, leading to alveolar epithelial apoptosis. Increased apoptosis of the alveolar epithelium drives the development of pulmonary fibrosis. Data are presented as mean±SEM. *P < 0.05, **P < 0.01 by one-way ANOVA (B–E).
The study herein investigated the effects of Dach1, a transcription cofactor, on the pathogenesis of PF. The lung tissues of patients with IPF and BLM-treated mice were characterized by low Dach1 expression. Specifically, the expression of Dach1 was decreased in AECII after lung injury. Dach1 deficiency in AECII exacerbated PF in BLM-treated mice, as shown by compromised pulmonary function and upregulated fibrosis markers. Lung-specific transgenic expression of Dach1 protected mice against PF. The mechanism study revealed that Dach1 protected the epithelium from apoptosis by inhibiting C-Jun-induced transcription of Bim (Fig. 8, F). These findings provide novel insights into the etiology of PF and suggest that Dach1 could be a viable strategy for the prevention and treatment of PF.
Dach1 was originally identified in Drosophila embryos as a regulator of development.
Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.
Our study showed that Dach1 was downregulated in patients with IPF and associated with the expression of fibrosis markers and DLCO% predicted. We also observed that there exists an association between Dach1 expression and disease in IPF but not in other ILDs. Clinical data suggest that patients with IPF have more severe fibrosis. We speculated that Dach1 expression level was significantly positively correlated with fibrosis degree. We further found that Dach1 was downregulated in mice with BLM- induced PF, especially in AECII. These findings led us to speculate that alveolar epithelial expression of Dach1 might be involved in the pathogenesis of PF. To address these questions, we generated mice with AECII-specific deletion of Dach1 by Tamoxifen treatment. Dach1 CKO mice displayed significantly aggravated PF and worsen lung function. The loss of Dach1 in AECII contributed to the apoptosis of AECII, as manifested by an increased level of cleaved caspase-3/caspase-3. In contrast, lung-specific overexpression of Dach1 protected mice against PF, indicating that Dach1 could be a promising target for the prevention of PF in clinical settings.
Accumulating evidence has indicated that apoptosis of AECII was closely associated with the pathogenesis of IPF.
which was consistent with the results of our bioinformatics analysis (Fig. 5, A and B). The most exciting finding in this study was that Dach1 regulated the apoptosis of AECII, which may be a common factor responsible for the initiation of PF. Significantly increased apoptosis was observed in mice with specific depletion of Dach1. Moreover, overexpression of Dach1 in pulmonary epithelium inhibited apoptosis both in vitro and in mice. These data support that Dach1 participates in the development of PF via regulating the apoptosis of AECII.
To elucidate the mechanisms by which Dach1 regulated apoptosis following BLM exposure, we firstly explored the effects of Dach1 overexpression on the levels of key genes related to apoptosis. Upregulation of Dach1 negatively regulated the expression of Bim both in vitro and in vivo. Bim is a potent pro-apoptotic protein belonging to the Bcl-2 family.
Navitoclax, a Bim mimics, restored apoptosis under Dach1 overexpression in vitro. Despite the established role of Dach1 as a transcriptional modulator, Chip-seq analysis revealed that there was no direct association between Dach1 and the Bim promoter. Instead, Dach1 interacted with C-Jun and transcriptionally regulated the expression of Bim.
Dach1 negatively regulated the expression of C-Jun in vitro but did not affect the protein level of C-Jun in vivo. Future studies using primary mouse AECII may explain the discrepancy. Although the activation of the c-Jun N-terminal kinase (JNK)/C-Jun pathway has been reported in PF,
Adeno-associated virus, as a means of gene overexpression/knockdown, has an important application prospect in gene therapy. Up to now, 5 drugs based on AAV have been approved by FDA.
We also demonstrated that adeno-associated virus-based lung overexpression of Dach1 can significantly alleviate the degree of pulmonary fibrosis and improve lung function in mice. Adeno-associated virus-coated Dach1 overexpression plasmid can be used as an effective treatment for fibrosis.
Our study has some limitations. Due to the difficulty in obtaining clinical samples, our research only carried out biogenic and animal experiments. The immunostaining of lung tissue sections from mice with BLM-induced fibrosis also showed low Dach1 expression in certain cells other than pro-Spc+ AECII. As the present study focused on AECII, the effects of Dach1 expression on fibroblasts in PF would be tackled in future investigations.
In conclusion, we demonstrate that altered Dach1 expression in AECII is a critical manifestation relevant to epithelial apoptosis in PF. Furthermore, mice with AECII-specific Dach1 deficiency showed exacerbated PF, which was abolished by Dach1 overexpression. Mechanically, Dach1 regulated the apoptosis of AECII in PF via modulating the activity of C-Jun/Bim. These data suggest that Dach1 might be used as a target for the prevention of PF.
METHODS
Bioinformatics analysis
IPF-related gene expression microarray datasets (GSE47460, GSE32537, GSE110147) and Dach1 overexpression datasets (GSE127361, GSE91962, and GSE685) were acquired from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/gds). After excluding samples with other chronic lung diseases, we included 112 IPF samples and 108 healthy controls (HC) samples from GSE47460,
50 HC and 119 IPF samples from GSE32537, 11 HC and 22 IPF samples from GSE110147. GSE685 consisted of 3 Dach1-overexpressing MDA-MB-231 cell samples and 3 control samples. GSE127361 was composed of 2 samples of Dach1 chromatin immunoprecipitation-sequence (Chip-seq) of K562 cells, whereas GSE91962 included 2 samples of control K562 cells. Differently expressed genes (DEGs) were screened in datasets with a fold-change of >1.5 and a P-value of <0.05. Significant DEGs of Dach1 target promoter (-1000 bp to 100 bp distance from the transcription start site) from the GSE127361 and GSE91962 datasets were identified.
Pearson's correlation was performed to determine the correlations of Dach1 expression with fibrosis-related genes and diffusion capacity for carbon monoxide (DLCO). The DEGs were uploaded to the online software Database for Annotation, Visualization, and Integrated Discovery (http://david.abcc.ncifcrf.gov/, version 6.8) for typical batch annotation and Gene Ontology enrichment analysis. The R studio (version 3.6.2) was used for bioinformatics analysis.
Mouse models
All mice were housed in a specific-pathogen-free animal facility at the Tongji Hospital (Wuhan, China). The sample size of each group is 5–12. Mice were randomly assigned to different groups. The accessing process was conducted by an assessor blind to treatment allocation. All mice were 8–10 weeks male C57BL/6.
To create a Dach1 conditional knockout mouse by CRISPR/Cas9-mediated genome engineering (GemPharmatech, Nanjing, China), gRNA direct Cas9 endonuclease cleavage in Dach1 gene and create a DSB (double-strand break). Such breaks will be repaired by donor-mediated homologous recombination, and result in loxp sites inserted upstream and downstream of exon2 respectively. Exon2 will be floxed by loxP sites.
SftpcCreERT+/+ transgenic mice were also generated by GemPharmatech (Nanjing, China).
SftpcCreERT+/−Dach1flox/flox (Dach1 CKO) mice were generated by crossing SftpcCreERT+/+ mice with Dach1flox/flox mice, and the littermates SftpcCreERT-/−Dach1flox/flox (Dach1 C) were used as controls. To induce AECII-specific deletion of Dach1, tamoxifen (75 mg/kg; MCE) was dissolved in absolute ethanol and corn oil (MCE), and injected intraperitoneally in Dach1 CKO mice for 7 consecutive days.
To generate mice with lung-specific transgenic expression of Dach1 (Dach1-OE), adeno-associated virus 6 (AAV6)-CMV-Dach1-3xFlag-P2A-mNeonGreen-tWPA (OBIO, China) was delivered to mice via intratracheal instillation. The control mice were subjected to AAV6-CMV-3xFlag-P2A-mNeonGreen-tWPA (negative control).
Mice were anesthetized by isoflurane inhalation and then treated with BLM (2 mg/kg; MCE, USA) or normal saline via intratracheal instillation. The bodyweight was recorded twice a day. At 21 days, mice were anesthetized with 1% pentobarbital sodium (100 mg/kg). The lung function test was then performed using the FlexiVent system (SCIREQ, Canada). The Broncho alveolar lavage fluid (BALF) was collected. The supernatant of BALF samples was stored at -80°C and the total cells were counted. The right lungs were frozen in liquid nitrogen immediately and then transferred to -80°C for subsequent measurements. The left lungs were soaked in 4% paraformaldehyde
Reagents
Antibodies against Fibronectin, E-cadherin, α-SMA, Dach1, Fsp-1, Zo-1, Epcam and β-actin were purchased from ProteinTech. Antibodies against B-cell lymphoma 2 protein (Bcl-2), Bax, cleaved caspase-3, caspase-3, Bim, Foxo1, and C-Jun were obtained from Cell Signaling Technology. Anti-Pro-Spc antibody was purchased from Millipore (Germany). Anti-Collagen I antibody was obtained from Servicebio (China). Anti-Dach1 antibody for immunoprecipitation and anti-CD68 antibody were purchased from Santa Cruz Biotechnology.
Histological and immunohistochemical analysis
The left lung was fixed and then subjected to Masson's trichrome staining. Two pathologists who were blinded to the study assessed the degree of interstitial fibrosis using the Ashcroft scoring system.
Paraffin-embedded lung sections were stained with primary antibodies, followed by incubation with anti-mouse/rabbit FITC or anti-mouse/rabbit Cy3 antibody (Boster).
Hydroxyproline analysis
The content of hydroxyproline in the lung was determined using a Hydroxyproline Colorimetric Assay Kit (Biovison, Milpitas) following the manufacturer's instructions. Briefly, the vacuum-dried lung tissue was cracked with concentrated hydrochloric acid at 120°C. After adding the detection reagents to the supernatant, the absorbance was detected using a microplate reader at a wavelength of 560 nm.
Western blot
Protein was extracted from mouse lung tissues or cell lysates, separated by electrophoresis, transferred onto PVDF membranes, incubated overnight with primary antibodies, and visualized using the Gel Doc XR+ System (Bio-Rad) as reported elsewhere.
Total RNA was extracted from mouse lung tissues or cell lysates using Trizol (Takara, Japan) and then reverse transcribed. Quantitative RT-PCR was performed using SYBR Premix Ex Taq (Takara, Japan). The primers of the target genes are shown in Supplemental Table II (Additional file 1). Relative mRNA expression was calculated using the 2−ΔΔCT method with β-actin as the endogenous control.
Cell culture
Murine lung epithelial-12 (MLE-12) cells were obtained from ATCC and cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco). Cells were stimulated with TGF-β1 (5 or 10 ng/mL, MCE) for 24 or 48 hours. Cells were treated with BLM (5 or 10 μg/mL) for 12 or 24 hours. Cells were transfected with plasmid overexpressing Dach1 (cDNA-Dach1, CMV-MCS-IRES-EGFP-SV40-Neomycin, Jikai Gene, China) or negative control plasmid (NC, Jikai Gene) using Lipofectamine 3000 (Invitrogen, CA) for 24 hours and treated with or without BLM for 24 hours. In the cell function recovery experiment, Navitoclax (10 μg/mL, MCE) was added to cells for 24 hours before transfection and BLM treatment.
Apoptosis assay
Synchronized MLE-12 cells were collected and incubated with propidium iodide (PI) and Annexin V-APC (Biolegend, USA) at 4°C for 10 minute in the dark. The apoptosis of cells was analyzed by a flow cytometer (BD Biosciences).
Luciferase activity assay
The mouse Bim promoter site (Mus, chromosome 2, 127966925–127968184) was cloned into the KpnI and XhoI sites of the pGL3 luciferase reporter plasmid (Promega). The predicted Bim promotor binding site of C-Jun was “TGACTCT” (Mus, chromosome 2, 127968142–127968147). The mutant sequence of Bim promotor was the deletion of the wide-type plasmid sequence (Mus, chromosome 2, 127966925–127967684). MLE-12 cells were co-transfected with vectors harboring wild-type promoter of Bim, mutant promotor of Bim, or control vectors and plasmid overexpressing Dach1 or negative control plasmid. The luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison).
Immunostaining with anti-SPC or anti-Fsp1antibody confirms that >90% of cells are AECII or fibroblast (Supplemental Fig 2C).
Statistical analysis
No specific statistical tests were used to predetermine the sample size. Comparisons among groups were performed using the GraphPad Prism 7 software (GraphPad, San Diego, California). Data were expressed as the mean ±SEM values. Student's t-test or ANOVA were used to compare normally distributed data. Mann-Whitney U-test was performed to compare the data that were not normally distributed. Pearson's correlation was used for correlation analysis. A two-sided P-value of < 0.05 was considered statistically significant.
Study approval
All animal experiments were approved by the Animal Care and Use Committee of Tongji Hospital.
ACKNOWLEDGMENTS
Conflict of Interest: The authors have declared that no conflict of interest exists.
This study was supported by the National Natural Science Foundation of China (No. 81973986, 82170049, 82070032, 81800041), the Health Research Fund of Wuhan (No. WX21Q07), and Leading talents of public health in Hubei Province (2022SCZ047).
Author contributions are as follows: Yanjiao Lu: Conceptualization, Investigation, Formal analysis, Writing - original draft. Kun Tang: Conceptualization, Investigation, Formal analysis, Writing - original draft. Shanshan Wang: Conceptualization, Investigation, Formal analysis, Writing - original draft. Zhen Tian: Formal analysis. Yan Fan: Formal analysis, Writing - original draft. Boyu Li: Investigation. Meijia Wang: Formal analysis. Jianping Zhao: Conceptualization, supervision. Jungang Xie: Conceptualization, Supervision
The authors thank all the IPF patients and healthy controls involved in our study. All authors have read the journal's authorship agreement and that the manuscript has been reviewed by and approved by all named authors.
Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.
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