Upregulation of UHRF1 in TGF-β1–stimulated fibroblasts and fibrotic lungs. First, we examined UHRF1 expression in fibroblasts induced with different concentrations of TGF-β1 (0, 1, 2, and 5 ng/mL) for 48 hours. As expected, both fibrotic markers (fibronectin, collagen I, and α-SMA) and UHRF1 were increased in a dose-response manner (Figure 1, A–C) in human embryonic lung fibroblasts (MRC-5 cells) and primary murine lung fibroblasts (PLFs). In addition, UHRF1 mRNA levels were markedly upregulated in fibrotic mouse lung tissues and TGF-β1–treated fibroblasts (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.162831DS1). Immunofluorescence assays revealed that UHRF1 was highly expressed following TGF-β1 treatment in fibroblasts and silica- and BLM-induced fibrotic lung tissues, respectively (Figure 1, D and E). Moreover, the coimmunostaining results further reinforced the conclusion that UHRF1 was highly expressed in α-SMA+ myofibroblasts in silica-induced mouse lung tissues (Supplemental Figure 1C). These data supported that UHRF1 was activated in fibroblasts. Additionally, H&E and IHC staining for UHRF1 reinforced that UHRF1 was highly expressed in patients with silicosis, mainly in the fibrotic area (Figure 1F). Furthermore, overexpression of UHRF1 was noted in the mouse tissues following silica and BLM induction, along with increased expression of fibrotic markers (Figure 1, G and H, and Supplemental Figure 1, D and E). Collectively, these findings suggested that UHRF1 may play an essential role in regulating the procession of pulmonary fibrosis.

UHRF1 is overexpressed in TGF-β1–stimulated fibroblasts and fibrotic lungs. (A–C) Western blot and corresponding densitometry analysis of fibronectin, collagen I, α-SMA, and UHRF1 in TGF-β1–treated (0, 1, 2, 5 ng/mL for 48 hours) MRC-5 cells and PLFs. Data are shown as the mean ± SEM (n = 3 in each group). (D) Immunofluorescence staining of UHRF1 in MRC-5 cells. Red represents UHRF1; blue represents nuclear DNA staining by DAPI. (E) Immunofluorescence staining of UHRF1 in mouse lung tissues. Red represents UHRF1; blue represents DAPI. (F) Representative results of H&E and UHRF1 IHC staining in lung sections from patients with silicosis and idiopathic pulmonary fibrosis (IPF) and normal participants. (G and H) Western blot and densitometric analysis of fibronectin, collagen I, α-SMA, and UHRF1 protein in saline- or silica-treated mouse lung tissues. Data are shown as the mean ± SEM (n = 3 in each group). Scale bar: 25 μm (D); 100 μm (E and F). P values were from (B and C) a 1-way ANOVA post hoc test with Tukey’s correction or (H) 2-tailed unpaired Student’s t test.

UHRF1 played an essential role in TGF-β1–induced lung fibroblast activation. Next, to investigate whether UHRF1 is required for fibroblast activation, we performed loss-of-function experiments using siRNAs. First, the mRNA and protein expression levels of UHRF1 were significantly downregulated in fibroblasts by UHRF1 siRNA (Figure 2A and Supplemental Figure 2A). Then, we observed decreases in fibronectin, collagen I, and α-SMA protein levels after UHRF1 knockdown (Figure 2, B and C, and Supplemental Figure 2B). As a selective UHRF1 inhibitor, compound NSC232003 exerted markedly antifibrotic effects by inhibiting the protein expression of fibrotic markers (Supplemental Figure 2C). Moreover, UHRF1 knockdown substantially reduced the expression of α-SMA via α-SMA staining in lung fibroblasts (Figure 2, D and E, and Supplemental Figure 2D). The EdU and CCK8 assays revealed that knockdown of UHRF1 inhibited lung fibroblast proliferation activity stimulated by TGF-β1 (Figure 2, F and G, and Supplemental Figure 2, E–G). The collagen gel contraction assay further confirmed that UHRF1 knockdown inhibited TGF-β1–induced fibroblast-mediated collagen contraction (Figure 2H). These results indicated that the low expression of UHRF1 inhibited fibroblast activation.

UHRF1 regulates TGF-β1–induced lung fibroblast proliferation. (A) qRT-PCR analysis of UHRF1 expression in MRC-5 cells and PLFs transfected with UHRF1 siRNA or its negative control (NC) siRNA, Data are shown as the mean ± SEM (n = 3 in each group). (B and C) Western blot and corresponding densitometry analysis of fibronectin, collagen I, and α-SMA in MRC-5 cells and PLFs transfected with UHRF1 siRNA and its negative control siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. Data are shown as the mean ± SEM (n = 3 in each group). (D) The expression of α-SMA was detected by immunofluorescence staining in MRC-5 cells transfected with UHRF1 siRNA and its negative control siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. (E) Mean fluorescence intensity of α-SMA in MRC-5 cells from the different groups. Data are shown as the mean ± SEM (n = 3 in each group). (F and G) Proliferation of MRC-5 cells transfected with UHRF1 siRNA and its negative control siRNA, as assessed by EdU assays. Data are shown as the mean ± SEM (n = 3 in each group). (H) Effect of UHRF1 siRNA and its negative control siRNA on the contractility of TGF-β1–induced fibroblasts. Scale bar: 50 μm (D); 100 μm (F). (A, C, E, and G) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.

The YAP/TEAD pathway contributed to the elevated expression of UHRF1 in activated fibroblasts. We have confirmed the antifibrotic role of UHRF1 on TGF-β1–induced fibroblasts, but the mechanism behind this regulatory effect remains unclear. By using the JASPAR database (https://jaspar.genereg.net/), we predicted that TEAD1 and TEAD4, the members of the TEAD protein family, could directly bind to the UHRF1 promoter region (Figure 3A). Previous studies have indicated that the YAP/TEAD pathway involved fibroblast activation and promoted pulmonary fibrosis (14). Therefore, we hypothesized that the YAP/TEAD pathway might contribute to the upregulation of UHRF1 in the process of fibroblast activation. First, RNA interference was used to knockdown the mRNA levels of TEAD1, TEAD4, and YAP in lung fibroblasts (Figure 3B and Supplemental Figure 3A). Interference with TEAD1, TEAD4, and YAP expression resulted in marked downregulation of UHRF1 mRNA and protein levels (Figure 3, C–G, and Supplemental Figure 3, B–F). Furthermore, ChIP experiments indicated that UHRF1 is the key downstream target of the YAP/TEAD pathway (Figure 3H). Altogether, these data suggested that UHRF1 mediated the activation of fibroblasts through the YAP/TEAD pathway.

The YAP/TEAD pathway contributes to the expression of UHRF1 in fibroblast activation. (A) The potential binding sites (including TEAD1 and TEAD4) at the UHRF1 promoter region by using the JASPAR database. (B) Target siRNA transfection significantly decreased the expression of TEAD1, TEAD4, and YAP in MRC-5 cells. Data are shown as the mean ± SEM (n = 3 in each group). (C) qRT-PCR detection of UHRF1 expression in MRC-5 cells after transfection with TEAD1, TEAD4, and YAP siRNA. Data are shown as the mean ± SEM (n = 3 in each group). (D–G) Western blot and corresponding densitometry analysis of TEAD1, TEAD4, YAP, and UHRF1 in MRC-5 cells and PLFs transfected with TEAD1, TEAD4, and YAP siRNA and their negative control (NC) siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. Data are shown as the mean ± SEM (n = 3 in each group). (H) Chromatin was harvested for immunoprecipitation with IgG, an anti-TEAD1 antibody, an anti-TEAD4 antibody, an anti-YAP antibody, and an anti–histone H3 antibody. The expression of UHRF1 was detected by qRT-PCR analysis. Data are shown as the mean ± SEM (n = 3 in each group). P values were from (B) a 2-tailed unpaired Student’s t test and (C and E–H) a 1-way ANOVA post hoc test with Tukey’s correction.

UHRF1 induced de novo promoter-specific methylation to suppress beclin 1 in lung fibroblasts. UHRF1 is acknowledged as a key regulator of DNA methylation combined with Dnmt1 (15), and a CpG island also exists on the beclin 1 promoter (Figure 4A). Thus, we asked whether UHRF1-mediated beclin 1 expression is associated with DNA methylation. Using 5-aza-dC, an inhibitor of methylation, we observed that beclin 1 expression was augmented compared with that in the TGF-β1 treatment group (Figure 4B). Next, we conducted a methylation-specific PCR assay and revealed that low expression of UHRF1 contributed to a remarkable decrease of beclin 1 promoter methylation (Figure 4C). Bisulfite-sequencing PCR was performed to detect beclin 1 CpG island methylation status, and the results illustrated that the knockdown of UHRF1 decreased the methylation of CpG nucleotides with elevated expression of beclin 1 (Figure 4D). Furthermore, ChIP-qPCR assays for beclin 1 were performed with chromatin from lung fibroblasts treated with anti-UHRF1 antibody in UHRF1-knockdown and TGF-β1–treated cells. As it turned out, UHRF1 could directly bind to the beclin 1 promoter in the TGF-β1–treated group compared with that in the UHRF1 siRNA–treated group (Figure 4E). Similarly, the interaction of beclin 1 and Dnmt1 also was identified by ChIP assay (Figure 4F). Thus, we concluded that UHRF1 regulated beclin 1 expression by recruiting Dnmt1 to its promoter to mediate beclin 1 methylation. Besides, UHRF1 knockdown significantly reversed TGF-β1–induced beclin 1 downregulation both at mRNA and protein levels (Figure 4, G–I, and Supplemental Figure 4A). compound NSC232003 could also increase the beclin 1 protein level (Supplemental Figure 4, B–D). Meanwhile, immunofluorescence staining for beclin 1 showed that UHRF1 knockdown reversed the expression of beclin 1 in lung fibroblasts (Supplemental Figure 4, E and F). These results prove that UHRF1 raised beclin 1 expression by epigenetically mediating its methylation.

UHRF1 epigenetically mediates beclin 1 methylation in lung fibroblasts. (A) Prediction of beclin 1 methylation CpG sites by using http://www.urogene.org/methprimer/ (B) Beclin 1 expression in MRC-5 cells and PLFs before and after 5-aza-2′-deoxycytidine treatment. Data are shown as the mean ± SEM (n = 3 in each group). (C) CpG island methylation status of the beclin 1 gene analyzed by a methylation-specific PCR assay in UHRF1 siRNA–treated PLFs. (D) A bisulfite sequencing assay was performed to reveal the CpG methylation status in the beclin 1 promoter region in the PLFs. (E) Chromatin was harvested for immunoprecipitation with IgG, an anti-UHRF1 antibody, after being transfected with TGF-β1 or TGF-β1 plus UHRF1 siRNA. The expression of beclin 1 was detected by qRT-PCR analysis. Data are shown as the mean ± SEM (n = 3 in each group). (F) Chromatin was harvested for immunoprecipitation with IgG, an anti-Dnmt1 antibody, and an anti–histone H3 antibody after TGF-β1 treatment in the fibroblasts. The expression of beclin 1 was detected by qRT-PCR analysis. Data are shown as the mean ± SEM (n = 3 in each group). (G) qRT-PCR analysis of beclin 1 expression in MRC-5 cells and PLFs after transfection with UHRF1 siRNA or its negative control (NC) siRNA. Data are shown as the mean ± SEM (n = 3 in each group). (H and I) Western blot and corresponding densitometry analysis of UHRF1 and beclin 1 in MRC-5 cells and PLFs transfected with UHRF1 siRNA and its negative control (NC) siRNA and then treated with 5 ng/mL TGF-β1 for 48 hours. Data are shown as the mean ± SEM (n = 3 in each group). P values were from (E) a 2-tailed unpaired Student’s t test and (B, F, G, and I) a 1-way ANOVA post hoc test with Tukey’s correction.

Beclin 1 downregulation arrested fibroblast proliferation. Above, we have confirmed that beclin 1 might be a downstream gene of UHRF1; its expression accompanies cell autophagy and proliferation (16, 17). Additionally, we detected the downregulation of beclin 1 in TGF-β1–induced fibroblasts (Figure 5A). In addition, beclin 1 was decreased in silica- and BLM-treated lung tissues through immunofluorescence staining and qRT-PCR assays (Supplemental Figure 5, A and B). Next, we used siRNAs to knockdown beclin 1 expression in MRC-5 cells and PLFs and then detected the knockdown efficiency at mRNA and protein levels (Supplemental Figure 5C and Figure 5, B–D). Similarly, beclin 1 siRNA induced a robust increase of fibrotic marker protein levels and staining of α-SMA in lung fibroblasts (Figure 5, E and F, and Supplemental Figure 5D). Next, collagen gel contraction, EdU, and CCK8 assays showed that loss of beclin 1 promoted collagen contraction and increased the proliferation activity of cells (Figure 5, G–J). Besides, overexpression of P62 and downregulation of LC3B (the markers of autophagy) were also observed in beclin 1 siRNA–treated fibroblasts (Supplemental Figure 5, E–G). Similarly, our previous study demonstrated that beclin 1 facilitates PARK2 translocation to mitochondria, involving the mitophagy process in TGF-β1–stimulated fibroblasts (18).

Beclin 1 is a functional downstream gene of UHRF1 and negatively regulates cell proliferation. (A) qRT-PCR analysis of beclin 1 expression in MRC-5 cells and PLFs after treatment with TGF-β1. Data are shown as the mean ± SEM (n = 3 in each group). (B–D) Western blot and corresponding densitometry analysis of fibronectin, collagen I, α-SMA, and beclin 1 in beclin 1 siRNA–treated MRC-5 cells and PLFs or those treated with its negative control (NC) siRNA. Data are shown as the mean ± SEM (n = 3 in each group). (E) Immunohistochemical staining of α-SMA in MRC-5 cells after beclin 1 siRNA or its negative control siRNA treatment. Red represents α-SMA; blue represents DAPI. (F) Mean fluorescence intensity of α-SMA in MRC-5 cells from the different groups. Data are shown as the mean ± SEM (n = 3 in each group). (G) Collagen gel contraction assay was performed to detect the effect of beclin 1 siRNA and its negative control siRNA on the contractility of fibroblasts. (H and I) Proliferation of MRC-5 cells transfected with beclin 1 siRNA and its negative control siRNA, as assessed by EdU assays. Data are shown as the mean ± SEM (n = 3 in each group). (J) CCK8 assays were performed to evaluate cell proliferative ability in fibroblasts. Data are shown as the mean ± SEM (n = 3 in each group). Scale bar: 50 μm (E and H). P values were from (A) a 2-tailed unpaired Student’s t test and (C, D, F, I, and J) a 1-way ANOVA post hoc test with Tukey’s correction.

To determine whether beclin 1 acts as a functional gene downstream of UHRF1, we simultaneously knocked down UHRF1 and beclin 1 in cells. Notably, low expression of UHRF1 could inhibit fibroblast activation, α-SMA expression, cell proliferation, and cell viability, whereas the effects were reversed by simultaneous knockdown of UHRF1 and beclin 1 (Figure 6 and Supplemental Figure 6, A and B). Furthermore, beclin 1 plasmid significantly reduced the effect of UHRF1 in fibroblasts (Supplemental Figure 6, C and D). These results demonstrated that beclin 1 is a functional target gene of UHRF1 that negatively regulates cell activation.

Loss and gain of function of beclin 1 reversed the effect of UHRF1 in fibroblasts. (A–C) Western blot and corresponding densitometry analysis of UHRF1, fibronectin, collagen I, α-SMA, and beclin 1 in the MRC-5 cells and PLFs for the indicated groups. Data are shown as the mean ± SEM (n = 3 in each group). (D) Immunohistochemical staining of α-SMA in MRC-5 cells for the indicated groups. α-SMA stained red; DAPI stained blue. (E) Mean fluorescence intensity of α-SMA in MRC-5 cells from the different groups. Data are shown as the mean ± SEM (n = 3 in each group). (F and G) Proliferation of MRC-5 cells transfected with different treatment, as assessed by EdU assays. Data are shown as the mean ± SEM (n = 3 in each group). (H) CCK8 assays were performed to evaluate ability of MRC-5 cells and PLFs to proliferate. Data are shown as the mean ± SEM (n = 3 in each group). Scale bar: 50 μm (D and F). (B, C, E, G, and H) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.

In vivo functional validation of UHRF1 siRNA–loaded liposomes. Next, we sought to prepare for the delivery of UHRF1 siRNA to pulmonary fibrotic mice. As showed in Figure 7A, the prepared liposomes possessed a uniform size distribution of around 157 nm (PDI = 0.19) and displayed 87% entrapment efficiency for loading siRNA, with a ζ potential of 29.36 mv (Figure 7A). Next, we assessed the distribution of liposome size, fluorescence intensity, and the stability of loaded liposomes, as shown in Figure 7, B–D. Figure 7E shows a representative image, taken by transmission electron microscopy. The tail vein mainly accumulated fluorescence signal in the lung (Figure 7F). In addition, UHRF1 siRNA–loaded liposomes could be taken up by fibroblasts in a few hours and without cytotoxicity (Supplemental Figure 7, A and B). To further confirm this phenomenon, we collected organs, including the heart, liver, spleen, kidney, and lung, from mice 48 hours after tail vein injection of liposomes. As expected, ex vivo imaging only detected fluorescence in the lung and not in other organs (Figure 7G). To further investigate the liposome cellular localization in the fibrotic lung, we performed immunofluorescence staining assays by using mouse pulmonary tissue slides. Interestingly, liposomes were predominantly located in the fibrotic area of lung tissues and mainly overlapped with α-SMA+ cells, which indicated that the fibroblasts could efficiently absorb liposomes. However, liposomes could also target pulmonary epithelial cells, as shown by the costaining of E-cadherin and liposomes (Supplemental Figure 7C).

Characterization of UHRF1 siRNA–loaded liposomes. (A) The hydrodynamic diameter, PDI, ζ potential of the liposomes, and siRNA entrapment efficiency of UHRF1 siRNA–loaded liposomes. (B–D) Distribution of liposome size (B), fluorescence intensity (C), and stability of liposomes (D) loaded in UHRF1 siRNA–loaded liposomes. (E) Representative transmission electron microscopy image of UHRF1 siRNA–loaded liposomes. Scale bar: 200 nm. (F) Representative IVIS images of a mouse at different time points (0 hours, 24 hours and 48 hours) after the administration of DiR-labeled liposomes. (G) Ex vivo fluorescence images of major organs from mice.

Intravenous administration of UHRF1 siRNA–loaded liposomes alleviated pulmonary fibrosis in multiple experimental mouse models. The therapeutic effects of UHRF1 siRNA–loaded liposomes were assessed in mice following silica (50 mg/kg) treatment for 28 days. Mice were administered with scrambled or UHRF1 siRNA–loaded liposomes by tail vein (dosage of siRNA, 1 mg/kg) on day 28, day 35, and day 42, respectively (Figure 8A). Administration of UHRF1 siRNA–loaded liposomes significantly attenuated silica-induced lung injury, as evidenced by the H&E, Sirius red, Masson’s trichrome staining and the Ashcroft score (Figure 8, B and C). Consistently, administration of UHRF1 siRNA–loaded liposomes decreased collagen content, as evidenced by hydroxyproline assays (Figure 8D), coupled with a marked reduction in the expression of fibrotic markers (Figure 8E). The body weight of mice indicated that the DiR-labeled liposomes could relieve weight loss caused by silica or BLM installation (Supplemental Figure 8A and Supplemental Figure 9A). Besides, an immunostaining assay and qRT-PCR assay showed that UHRF1, α-SMA, and collagen I in lung tissues from the UHRF1 siRNA–loaded liposome group were lower than in the scramble group, in contrast to the expression of beclin 1 (Figure 8, F and G, and Supplemental Figure 8, B–G).

Administration of UHRF1 siRNA liposomes attenuates silica-induced pulmonary fibrosis in mice. (A) Strategy for UHRF1 siRNA–loaded liposome administration in the silica-induced pulmonary fibrosis mouse model. (B) H&E, Sirius red, and Masson’s trichrome staining assays were performed to measure the severity of the lung fibrosis. (C) Semiquantitative Ashcroft scores indicating the severity of fibrosis. Data are shown as the mean ± SEM (n = 6 in each group). (D) The hydroxyproline content in the lungs of mice for the different groups. Data are shown as the mean ± SEM (n = 6 in each group). (E) Western blot of UHRF1, fibronectin, collagen I, α-SMA, and beclin 1 in mouse lung tissues. (F and G) Immunohistochemical staining of collagen I and α-SMA in mouse lung tissues for the indicated groups. Collagen I stained blue; α-SMA stained red; DAPI stained blue. Scale bar: 100 μm (B, F, and G). (C and D) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.

We also made another pulmonary fibrosis model by administering BLM (6 mg/kg) (Figure 9A). Similarly, lung injury and Ashcroft scores as well as collagen content in lungs of the UHRF1 siRNA–loaded liposome group were decreased compared with those of the scrambled siRNA–loaded liposome group in BLM-treated mice, as shown by H&E, Sirius red, and Masson’s trichrome staining and hydroxyproline assays (Figure 9, B–D). Furthermore, the expression of fibrotic markers and UHRF1 were also decreased both at protein and mRNA levels after UHRF1 siRNA–loaded liposome treatment, in contrast to the expression of beclin 1 (Figure 9E and Supplemental Figure 9, B–E). In addition, immunostaining of α-SMA and collagen I, key fibrotic markers, as well as UHRF1 and beclin 1, was conducted in BLM-induced mouse lung sections. Following UHRF1 siRNA–loaded liposome administration, levels of α-SMA, collagen I, and UHRF1 were downregulated and levels of beclin 1 were upregulated (Figure 9, F and G, and Supplemental Figure 9, F and G). Together, these data suggested that the administration of UHRF1 siRNA liposomes attenuates pulmonary fibrosis in multiple experimental mouse models.

Administration of UHRF1 siRNA liposomes attenuates BLM-induced pulmonary fibrosis in mice. (A) Strategy for UHRF1 siRNA–loaded liposome administration in the BLM-induced pulmonary fibrosis mouse model. (B) H&E, Sirius red, and Masson’s trichrome staining assays were performed to measure the severity of fibrotic lesions. (C) The severity of fibrosis was evaluated by Ashcroft scores. Data are shown as the mean ± SEM (n = 6 in each group). (D) Lungs of mice following different treatments were analyzed for hydroxyproline content. Data are shown as the mean ± SEM (n = 6 in each group). (E) Western blot of UHRF1, fibrotic markers, and beclin 1 in mouse lung tissues in the different groups. (F and G) The expression of collagen I and α-SMA was detected by immunofluorescence staining in mouse lung tissues. Collagen I stained blue; α-SMA stained red; DAPI stained blue. Scale bar: 100 μm (B, F, and G). (C and D) P values were from a 1-way ANOVA post hoc test with Tukey’s correction.