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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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Related in: MedlinePlus

b-AP15 attenuates TGFβ-1 signaling in HLF cells.(A) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h, and then cells were treated with TGFβ-1 (2 ng/ml) for 24 h. Cell lysates were analyzed by immunoblotting with the antibodies to FN, α-SMA, and β-actin. (B) HLF cells were pre-treated with DMSO and b-AP15 (50 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 24 h, and then cells were fixed with 3.7% formaldehyde. Expressions of FN and α-SMA were detected by immunostaining with antibodies to α-SMA and FN. DAPI was used for nuclei staining (blue). Scale bar, 50 μm. (C) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 30 minutes. Cell lysates analyzed by immunoblotting with the antibodies to p-Smad2, p-Smad3, Smad2, Smad3, Smad7, ALK5, UCHL5, and β-actin. 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. Representative of experiments performed at least 3 independent times.
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f1: b-AP15 attenuates TGFβ-1 signaling in HLF cells.(A) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h, and then cells were treated with TGFβ-1 (2 ng/ml) for 24 h. Cell lysates were analyzed by immunoblotting with the antibodies to FN, α-SMA, and β-actin. (B) HLF cells were pre-treated with DMSO and b-AP15 (50 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 24 h, and then cells were fixed with 3.7% formaldehyde. Expressions of FN and α-SMA were detected by immunostaining with antibodies to α-SMA and FN. DAPI was used for nuclei staining (blue). Scale bar, 50 μm. (C) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 30 minutes. Cell lysates analyzed by immunoblotting with the antibodies to p-Smad2, p-Smad3, Smad2, Smad3, Smad7, ALK5, UCHL5, and β-actin. 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. Representative of experiments performed at least 3 independent times.

Mentions: b-AP15, a nitrophenylpiperidine small molecule, inhibits the deubiquitinating activity of UCHL5 and USP1423. Recently, b-AP15 has been shown to have activity against multiple myeloma in a human xenograft mouse model25. To examine the effect of b-AP15 on TGFβ-1-induced signaling, HLF cells were pretreated with increasing doses of b-AP15 (0, 1, 5, 10 μM) for 1 h, and then treated with TGFβ-1 (2 ng/ml) for additional 24 h. It is well known that FN and α-SMA are up-regulated by TGFβ-1 and contribute to the pathogenesis of pulmonary fibrosis. First, we investigated the effect of b-AP15 on TGFβ-1-induced FN and α-SMA expression in HLF. TGFβ-1 induced FN and α-SMA expression, which were attenuated by b-AP15 in a dose-dependent manner (Fig. 1A). The data was confirmed by immunostaining (Fig. 1B). Smad2 and Smad3 are downstream signaling of TGFβ-1/TβR. Next, we examined whether b-AP15 affects Smad2/Smad3 activity. As shown in Fig. 1C, b-AP15 attenuated phosphorylation of Smad2 and Smad3 induced by TGFβ-1 treatment in a dose-dependent manner. Interestingly, total Smad2 and Smad3 levels, not Smad7, ALK5, and TβRII, were decreased by b-AP15 in both TGFβ-1 treated and untreated cells (Fig. 1C), indicating that b-AP15 may regulate Smad2 and Smad3 levels.


Ubiquitin carboxyl-terminal hydrolase-L5 promotes TGF β -1 signaling by de-ubiquitinating and stabilizing Smad2/Smad3 in pulmonary fibrosis
b-AP15 attenuates TGFβ-1 signaling in HLF cells.(A) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h, and then cells were treated with TGFβ-1 (2 ng/ml) for 24 h. Cell lysates were analyzed by immunoblotting with the antibodies to FN, α-SMA, and β-actin. (B) HLF cells were pre-treated with DMSO and b-AP15 (50 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 24 h, and then cells were fixed with 3.7% formaldehyde. Expressions of FN and α-SMA were detected by immunostaining with antibodies to α-SMA and FN. DAPI was used for nuclei staining (blue). Scale bar, 50 μm. (C) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 30 minutes. Cell lysates analyzed by immunoblotting with the antibodies to p-Smad2, p-Smad3, Smad2, Smad3, Smad7, ALK5, UCHL5, and β-actin. 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. Representative of experiments performed at least 3 independent times.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5015047&req=5

f1: b-AP15 attenuates TGFβ-1 signaling in HLF cells.(A) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h, and then cells were treated with TGFβ-1 (2 ng/ml) for 24 h. Cell lysates were analyzed by immunoblotting with the antibodies to FN, α-SMA, and β-actin. (B) HLF cells were pre-treated with DMSO and b-AP15 (50 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 24 h, and then cells were fixed with 3.7% formaldehyde. Expressions of FN and α-SMA were detected by immunostaining with antibodies to α-SMA and FN. DAPI was used for nuclei staining (blue). Scale bar, 50 μm. (C) HLF cells were pre-treated with increasing doses of b-AP15 (0, 1, 5, and 10 μM) for 1 h followed by TGFβ-1 (2 ng/ml) for 30 minutes. Cell lysates analyzed by immunoblotting with the antibodies to p-Smad2, p-Smad3, Smad2, Smad3, Smad7, ALK5, UCHL5, and β-actin. 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. Representative of experiments performed at least 3 independent times.
Mentions: b-AP15, a nitrophenylpiperidine small molecule, inhibits the deubiquitinating activity of UCHL5 and USP1423. Recently, b-AP15 has been shown to have activity against multiple myeloma in a human xenograft mouse model25. To examine the effect of b-AP15 on TGFβ-1-induced signaling, HLF cells were pretreated with increasing doses of b-AP15 (0, 1, 5, 10 μM) for 1 h, and then treated with TGFβ-1 (2 ng/ml) for additional 24 h. It is well known that FN and α-SMA are up-regulated by TGFβ-1 and contribute to the pathogenesis of pulmonary fibrosis. First, we investigated the effect of b-AP15 on TGFβ-1-induced FN and α-SMA expression in HLF. TGFβ-1 induced FN and α-SMA expression, which were attenuated by b-AP15 in a dose-dependent manner (Fig. 1A). The data was confirmed by immunostaining (Fig. 1B). Smad2 and Smad3 are downstream signaling of TGFβ-1/TβR. Next, we examined whether b-AP15 affects Smad2/Smad3 activity. As shown in Fig. 1C, b-AP15 attenuated phosphorylation of Smad2 and Smad3 induced by TGFβ-1 treatment in a dose-dependent manner. Interestingly, total Smad2 and Smad3 levels, not Smad7, ALK5, and TβRII, were decreased by b-AP15 in both TGFβ-1 treated and untreated cells (Fig. 1C), indicating that b-AP15 may regulate Smad2 and Smad3 levels.

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