Treatment with anti-TGF-β antibody ameliorates chronic progressive nephritis by inhibiting Smad/TGF-β signaling

Fukasawa, Hirotaka; Yamamoto, Tatsuo; Suzuki, Hiroyuki; Togawa, Akashi; Ohashi, Naro; Fujigaki, Yoshihide; Uchida, Chiharu; Aoki, Michiko; Hosono, Mareto; Kitagawa, Masatoshi; Hishida, Akira

doi:10.1111/j.1523-1755.2004.00393.x

Cited by 98 publications

(78 citation statements)

References 34 publications

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“…Kidney tissues were dissolved in Triton X-100 lysis buffer (13)(14)(15). Equal amounts of proteins (40 g) were loaded for SDS͞PAGE, as described (13)(14)(15). The primary antibodies were rabbit anti-human Smad7 (Santa Cruz Biotechnology), goat anti-human Smurf1 (Santa Cruz Biotechnology), goat anti-human Smurf2 (Santa Cruz Biotechnology), and mouse anti-␤-actin mAb (Sigma).We used ␤-actin as an internal control.…”

Section: Methodsmentioning

confidence: 99%

“…Tissue sections (3 m thick) were stained with Masson's trichrome for histopathological analysis. Immunoreactivity for phosphorylated Smad2, Smad3, Smad7 proteins was determined by using a standard biotin-streptavidin-peroxidase method, as described (13,14). The primary antibodies were rabbit anti-human phosphorylated Smad2 and Smad3 (Santa Cruz Biotechnology) and rabbit anti-human Smad7 (Santa Cruz Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

“…Kidney tissues were dissolved in Triton X-100 lysis buffer (13)(14)(15). Equal amounts of proteins (40 g) were loaded for SDS͞PAGE, as described (13)(14)(15).…”

Section: Methodsmentioning

confidence: 99%

“…As a control, pcDNA3 lacking Flag-tagged mouse Smad7 was used. The cells were lysed with Triton X-100 lysis buffer (13,15) after incubation for 48 h. The cell lysates (1,000 g͞100 l) were immunoprecipitated with 2 g of anti-Flag M2 mAb (Sigma) and 30 l of protein A-Sepharose beads (Bio-Rad) for 3 h at 4°C. The resulting immunoprecipitates were washed thoroughly four times with cold lysis buffer and then used for in vitro ubiquitination assays.…”

Section: Rna Isolation and Quantitative Analysis Of Mrna By Real-timementioning

confidence: 99%

“…Total RNA isolation, reverse transcription of the RNA, and all PCR experiments were performed as described (13). The PCR primer sequences were as follows: mouse TGF-␤1, 5Ј-CCTGAGTGGCTGTCTTTTGACG-3Ј (sense) and 5Ј-AGT-GAGCGCTGAATCGAAAGC-3Ј (antisense); mouse Smad7, 5Ј-TCGGACAGCTCAATTCGGAC-3Ј (sense) and 5Ј-GGTA-ACTGCTGCGGTTGTAA-3Ј (antisense); and mouse GAPDH, 5Ј-TGCACCACCAACTGCTTAG-3Ј (sense) and 5Ј-GAG-GCAGGGATGATGTTC-3Ј (antisense).…”

Section: Rna Isolation and Quantitative Analysis Of Mrna By Real-timementioning

confidence: 99%

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Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice

Fukasawa

Yamamoto

Togawa

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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197

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Overexpression of transforming growth factor ␤ (TGF-␤) has been shown to play pathogenic roles in progression of renal fibrosis, and the severity of tubulointerstitial fibrosis correlates better with renal function than the severity of glomerulosclerosis. Smad proteins are signaling transducers downstream from TGF-␤ receptors. Three families of Smad proteins have been identified: receptorregulated Smad2 and Smad3, common partner Smad4, and inhibitory Smad7 (part of a negative-feedback loop). We investigated Smad-mediated TGF-␤ signaling pathway and regulatory mechanisms of inhibitory Smad7 in unilateral ureteral obstruction (UUO) kidneys in mice, a model of progressive tubulointerstitial fibrosis. Compared with sham-operated kidneys, the level of Smad7 protein, but not mRNA, decreased progressively in UUO kidneys, whereas immunoreactivity for nuclear phosphorylated Smad2 and Smad3 and renal fibrosis were inversely increased. Furthermore, we demonstrated that both the degradation and ubiquitination activity of Smad7 protein were increased markedly in UUO kidneys compared with sham-operated ones. We also found that both Smurf1 and Smurf2 (Smad ubiquitination regulatory factors), which are E3 ubiquitin ligases for Smad7, were increased and that they interacted with Smad7 in UUO kidneys. Our results suggest that the reduction of Smad7 protein resulting from enhanced ubiquitin-dependent degradation plays a pathogenic role in progression of tubulointerstitial fibrosis.transforming growth factor ␤ ͉ Smad proteins ͉ tubulointerstitial fibrosis

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Kidney tissues were dissolved in Triton X-100 lysis buffer (13)(14)(15). Equal amounts of proteins (40 g) were loaded for SDS͞PAGE, as described (13)(14)(15).…”

Section: Methodsmentioning

confidence: 99%

Section: Rna Isolation and Quantitative Analysis Of Mrna By Real-timementioning

confidence: 99%

Section: Rna Isolation and Quantitative Analysis Of Mrna By Real-timementioning

confidence: 99%

See 3 more Smart Citations

Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice

Fukasawa

Yamamoto

Togawa

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

197

206

View full text Add to dashboard Cite

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Differential expression of long non‐coding RNAs in hyperoxia‐induced bronchopulmonary dysplasia

Bao

Cheng

et al. 2016

Cell Biochemistry & Function

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Bronchopulmonary dysplasia (BPD) is a common complication of premature birth that seriously affects the survival rate and quality of life among preterm neonates. Long non-coding RNAs (lncRNAs) have been implicated in many human diseases. However, the role of lncRNAs in the pathogenesis of BPD remains poorly understood. Here, we exposed neonatal C57BL/6J mice to 95% concentrations of ambient oxygen and established a mouse lung injury model that mimicked human BPD. Next, we compared lncRNA and messenger RNA (mRNA) expression profiles between BPD and normal lung tissues using a high-throughput mouse lncRNA + mRNA array system. Compared with the control group, 882 lncRNAs were upregulated, and 887 lncRNAs were downregulated in BPD lung tissues. We validated some candidate BPD-associated lncRNAs by real-time quantitative reverse-transcription polymerase chain reaction analysis in eight pairs of BPD and normal lung tissues. Gene ontology, pathway and bioinformatics analyses revealed that a downregulated lncRNA, namely AK033210, associated with tenascin C may be involved in the pathogenesis of BPD. To the best of our knowledge, our study is the first to reveal differential lncRNA expression in BPD, which provides a foundation for further understanding of the molecular mechanism of BPD development. Copyright © 2016 John Wiley & Sons, Ltd.

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Reduction of transforming growth factor‐β type II receptor is caused by the enhanced ubiquitin‐dependent degradation in human renal cell carcinoma

Fukasawa

Yamamoto

Fujigaki

et al. 2010

Intl Journal of Cancer

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Although dysregulation of transforming growth factor-b (TGF-b) signaling is implicated in renal carcinogenesis, its precise mechanism is unknown in renal cell carcinoma (RCC). In our study, we investigated Smad-mediated TGF-b signaling pathway and its regulatory mechanisms in surgical samples from patients with RCC. We found that immunoreactivity for nuclear phosphorylated Smad2 was significantly decreased in RCC compared to normal renal tissues, thereby TGF-b signaling was suggested to be attenuated in RCC tissues. In accordance with the result, transcriptional downregulation of Smad4 and posttranscriptional downregulation of TGF-b type II receptor (TbR-II) were frequently found in RCC tissues compared to normal renal tissues. Next, to clarify the reason why the protein level of TbR-II was decreased in RCC, we investigated the activities of degradation and ubiquitination of TbR-II. We found that both proteasome-mediated degradation and ubiquitination of TbR-II were markedly enhanced in RCC tissues. Moreover, we found that the level of Smad-ubiquitination regulatory factor 2 (Smurf2), the E3 ligase for TbR-II, was increased in RCC tissues of the patients with higher clinical stages compared to the normal tissues and was inversely correlated with the level of TbR-II. Our results suggest that the low TbR-II protein level is due to augmented ubiquitin-dependent degradation via Smurf2 and might be involved in the attenuation of TGF-b signaling pathway in RCC.

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Treatment with anti-TGF-β antibody ameliorates chronic progressive nephritis by inhibiting Smad/TGF-β signaling

Abstract: In chronic progressive ATS nephritis, the TGF-beta/Smad signaling was up-regulated. TGF-beta blockade by alphaT suppressed the progression of renal scarring, at least in part, via inhibition of activated TGF-beta/Smad signaling.

Cited by 98 publications

References 34 publications

Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice

Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice

Differential expression of long non‐coding RNAs in hyperoxia‐induced bronchopulmonary dysplasia

Reduction of transforming growth factor‐β type II receptor is caused by the enhanced ubiquitin‐dependent degradation in human renal cell carcinoma

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