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Ubiquitin carboxyl-terminal hydrolase-L5 promotes TGF β -1 signaling by de-ubiquitinating and stabilizing Smad2/Smad3 in pulmonary fibrosis

View Article: PubMed Central - PubMed

ABSTRACT

Transforming growth factor β-1 (TGFβ-1)-induced phosphorylation of transcription factors Smad2 and Smad3 plays a crucial role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, the molecular regulation of Smad2/Smad3 proteins stability remains a mystery. Here, we show that ubiquitin carboxyl-terminal hydrolase-L5 (UCHL5 or UCH37) de-ubiquitinates both Smad2 and Smad3, up-regulates their stability, and promotes TGFβ-1-induced expression of profibrotic proteins, such as fibronectin (FN) and α-smooth muscle actin (α-SMA). Inhibition or down-regulation of UCHL5 reduced Smad2/Smad3 levels and TGFβ-1-induced the expression of FN and α-SMA in human lung fibroblast. We demonstrate that Smad2 and Smad3 ubiquitination was diminished by over-expression of UCHL5, while it was enhanced by inhibition or down-regulation of UCHL5. UCHL5 is highly expressed in IPF lungs. UCHL5, Smad2, and Smad3 levels were increased in bleomycin-injured lungs. Administration of UCHL5 inhibitor, b-AP15, reduced the expression of FN, type I collagen, Smad2/Smad3, and the deposition of collagen in lung tissues in a bleomycin-induced model of pulmonary fibrosis. Our studies provide a molecular mechanism by which UCHL5 mitigates TGFβ-1 signaling by stabilizing Smad2/Smad3. These data indicate that UCHL5 may contribute to the pathogenesis of IPF and may be a potential therapeutic target.

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Administration of b-AP15 reduces bleomycin-induced pulmonary fibrosis in mice.(A) C57BL/6 mice were intraperitoneally injected with b-AP15 (2.5 mg/kg or 5.0 mg/kg, 3 × 1 every other day) for 8 days, and then lung tissues were collected. Lysates from lung tissues were analyzed by immunoblotting with antibodies to Smad2, Smad3, p38, and β-actin. (B) Intensities of blots in the A were quantified by imageJ software. (C) C57BL/6 mice were intranasally challenged with bleomycin (0.045 U/mice). Starting from day 11, mice were intraperitoneally injected with DMSO (0.25%) or b-AP15 (5.0 mg/kg in DMSO) 4 times of every other day. At day 21, lung tissues were collected and lysates from lung tissues were analyzed by immunoblotting with FN, type I collagen, Smad2, Smad3, UCHL5, and β-actin antibodies. Western blot images were cropped to improve the conciseness of the data; samples derived from the same experiment and the blots were processed in parallel. (D) Intensities of blots in the (C) were quantified by imageJ software. (E) The right lungs were fixed with 3.7% formaldehyde. Masson’s trichrome staining was performed to detect the collagen in the marine lung slices. (F) The percentages of collagen deposition in the E were analyzed by NIS-Elements software.
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f7: Administration of b-AP15 reduces bleomycin-induced pulmonary fibrosis in mice.(A) C57BL/6 mice were intraperitoneally injected with b-AP15 (2.5 mg/kg or 5.0 mg/kg, 3 × 1 every other day) for 8 days, and then lung tissues were collected. Lysates from lung tissues were analyzed by immunoblotting with antibodies to Smad2, Smad3, p38, and β-actin. (B) Intensities of blots in the A were quantified by imageJ software. (C) C57BL/6 mice were intranasally challenged with bleomycin (0.045 U/mice). Starting from day 11, mice were intraperitoneally injected with DMSO (0.25%) or b-AP15 (5.0 mg/kg in DMSO) 4 times of every other day. At day 21, lung tissues were collected and lysates from lung tissues were analyzed by immunoblotting with FN, type I collagen, Smad2, Smad3, UCHL5, and β-actin antibodies. Western blot images were cropped to improve the conciseness of the data; samples derived from the same experiment and the blots were processed in parallel. (D) Intensities of blots in the (C) were quantified by imageJ software. (E) The right lungs were fixed with 3.7% formaldehyde. Masson’s trichrome staining was performed to detect the collagen in the marine lung slices. (F) The percentages of collagen deposition in the E were analyzed by NIS-Elements software.

Mentions: To further investigate the role of UCHL5 in TGFβ-1 signaling and pulmonary fibrosis, first, we examined the UCHL5 expression in lungs from IPF patients. As shown in Fig. 6A, UCHL5 is highly expressed in lungs from IPF patients, compared with its expression in lungs from non-IPF subjects. Next, we examined levels of UCHL5, Smad2 and Smad3 in murine lungs from a bleomycin-induced pulmonary fibrosis model. The UCHL5, Smad2 and Smad3 levels, not USP14, were increased in lung tissue lysates from the mice challenged by bleomycin for three weeks (Fig. 6B,C). Consistent with in vitro studies, b-AP15 intraperitoneal treatment (3 × 1 every other day) at doses of 2.5 mg/kg and 5.0 mg/kg could lower the levels of Smad2 and Smad3, without altering p38 MAPK in murine lungs (Fig. 7A,B). To investigate whether b-AP15 may have therapeutic potential in pulmonary fibrosis, b-AP15 (2.5 mg/kg, 4 × 1 every other day) was intraperitoneally injected beginning 11 days after bleomycin challenge. b-AP15 dramatically attenuated the levels of FN and type I collagen, and also decreased the levels of Smad2 and Smad3, not UCHL5, at 21 days after bleomycin challenge (Fig. 7C,D). Trichrome staining shows that b-AP15 decreased the deposition of collagen compared to DMSO control treatment in bleomycin-induced pulmonary fibrosis (Fig. 7E). b-AP15 may be effective therapy for pulmonary fibrosis.


Ubiquitin carboxyl-terminal hydrolase-L5 promotes TGF β -1 signaling by de-ubiquitinating and stabilizing Smad2/Smad3 in pulmonary fibrosis
Administration of b-AP15 reduces bleomycin-induced pulmonary fibrosis in mice.(A) C57BL/6 mice were intraperitoneally injected with b-AP15 (2.5 mg/kg or 5.0 mg/kg, 3 × 1 every other day) for 8 days, and then lung tissues were collected. Lysates from lung tissues were analyzed by immunoblotting with antibodies to Smad2, Smad3, p38, and β-actin. (B) Intensities of blots in the A were quantified by imageJ software. (C) C57BL/6 mice were intranasally challenged with bleomycin (0.045 U/mice). Starting from day 11, mice were intraperitoneally injected with DMSO (0.25%) or b-AP15 (5.0 mg/kg in DMSO) 4 times of every other day. At day 21, lung tissues were collected and lysates from lung tissues were analyzed by immunoblotting with FN, type I collagen, Smad2, Smad3, UCHL5, and β-actin antibodies. Western blot images were cropped to improve the conciseness of the data; samples derived from the same experiment and the blots were processed in parallel. (D) Intensities of blots in the (C) were quantified by imageJ software. (E) The right lungs were fixed with 3.7% formaldehyde. Masson’s trichrome staining was performed to detect the collagen in the marine lung slices. (F) The percentages of collagen deposition in the E were analyzed by NIS-Elements software.
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Related In: Results  -  Collection

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f7: Administration of b-AP15 reduces bleomycin-induced pulmonary fibrosis in mice.(A) C57BL/6 mice were intraperitoneally injected with b-AP15 (2.5 mg/kg or 5.0 mg/kg, 3 × 1 every other day) for 8 days, and then lung tissues were collected. Lysates from lung tissues were analyzed by immunoblotting with antibodies to Smad2, Smad3, p38, and β-actin. (B) Intensities of blots in the A were quantified by imageJ software. (C) C57BL/6 mice were intranasally challenged with bleomycin (0.045 U/mice). Starting from day 11, mice were intraperitoneally injected with DMSO (0.25%) or b-AP15 (5.0 mg/kg in DMSO) 4 times of every other day. At day 21, lung tissues were collected and lysates from lung tissues were analyzed by immunoblotting with FN, type I collagen, Smad2, Smad3, UCHL5, and β-actin antibodies. Western blot images were cropped to improve the conciseness of the data; samples derived from the same experiment and the blots were processed in parallel. (D) Intensities of blots in the (C) were quantified by imageJ software. (E) The right lungs were fixed with 3.7% formaldehyde. Masson’s trichrome staining was performed to detect the collagen in the marine lung slices. (F) The percentages of collagen deposition in the E were analyzed by NIS-Elements software.
Mentions: To further investigate the role of UCHL5 in TGFβ-1 signaling and pulmonary fibrosis, first, we examined the UCHL5 expression in lungs from IPF patients. As shown in Fig. 6A, UCHL5 is highly expressed in lungs from IPF patients, compared with its expression in lungs from non-IPF subjects. Next, we examined levels of UCHL5, Smad2 and Smad3 in murine lungs from a bleomycin-induced pulmonary fibrosis model. The UCHL5, Smad2 and Smad3 levels, not USP14, were increased in lung tissue lysates from the mice challenged by bleomycin for three weeks (Fig. 6B,C). Consistent with in vitro studies, b-AP15 intraperitoneal treatment (3 × 1 every other day) at doses of 2.5 mg/kg and 5.0 mg/kg could lower the levels of Smad2 and Smad3, without altering p38 MAPK in murine lungs (Fig. 7A,B). To investigate whether b-AP15 may have therapeutic potential in pulmonary fibrosis, b-AP15 (2.5 mg/kg, 4 × 1 every other day) was intraperitoneally injected beginning 11 days after bleomycin challenge. b-AP15 dramatically attenuated the levels of FN and type I collagen, and also decreased the levels of Smad2 and Smad3, not UCHL5, at 21 days after bleomycin challenge (Fig. 7C,D). Trichrome staining shows that b-AP15 decreased the deposition of collagen compared to DMSO control treatment in bleomycin-induced pulmonary fibrosis (Fig. 7E). b-AP15 may be effective therapy for pulmonary fibrosis.

View Article: PubMed Central - PubMed

ABSTRACT

Transforming growth factor β-1 (TGFβ-1)-induced phosphorylation of transcription factors Smad2 and Smad3 plays a crucial role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, the molecular regulation of Smad2/Smad3 proteins stability remains a mystery. Here, we show that ubiquitin carboxyl-terminal hydrolase-L5 (UCHL5 or UCH37) de-ubiquitinates both Smad2 and Smad3, up-regulates their stability, and promotes TGFβ-1-induced expression of profibrotic proteins, such as fibronectin (FN) and α-smooth muscle actin (α-SMA). Inhibition or down-regulation of UCHL5 reduced Smad2/Smad3 levels and TGFβ-1-induced the expression of FN and α-SMA in human lung fibroblast. We demonstrate that Smad2 and Smad3 ubiquitination was diminished by over-expression of UCHL5, while it was enhanced by inhibition or down-regulation of UCHL5. UCHL5 is highly expressed in IPF lungs. UCHL5, Smad2, and Smad3 levels were increased in bleomycin-injured lungs. Administration of UCHL5 inhibitor, b-AP15, reduced the expression of FN, type I collagen, Smad2/Smad3, and the deposition of collagen in lung tissues in a bleomycin-induced model of pulmonary fibrosis. Our studies provide a molecular mechanism by which UCHL5 mitigates TGFβ-1 signaling by stabilizing Smad2/Smad3. These data indicate that UCHL5 may contribute to the pathogenesis of IPF and may be a potential therapeutic target.

No MeSH data available.


Related in: MedlinePlus