Activation of Src Mediates PDGF-Induced Smad1 Phosphorylation and Contributes to the Progression of Glomerulosclerosis in Glomerulonephritis

Platelet-derived growth factor (PDGF) plays critical roles in mesangial cell (MC) proliferation in mesangial proliferative glomerulonephritis. We showed previously that Smad1 contributes to PDGF-dependent proliferation of MCs, but the mechanism by which Smad1 is activated by PDGF is not precisely known. Here we examined the role of c-Src tyrosine kinase in the proliferative change of MCs. Experimental mesangial proliferative glomerulonephritis (Thy1 GN) was induced by a single intravenous injection of anti-rat Thy-1.1 monoclonal antibody. In Thy1 GN, MC proliferation and type IV collagen (Col4) expression peaked on day 6. Immunohistochemical staining for the expression of phospho-Src (pSrc), phospho-Smad1 (pSmad1), Col4, and smooth muscle α-actin (SMA) revealed that the activation of c-Src and Smad1 signals in glomeruli peaked on day 6, consistent with the peak of mesangial proliferation. When treated with PP2, a Src inhibitor, both mesangial proliferation and sclerosis were significantly reduced. PP2 administration also significantly reduced pSmad1, Col4, and SMA expression. PDGF induced Col4 synthesis in association with increased expression of pSrc and pSmad1 in cultured MCs. In addition, PP2 reduced Col4 synthesis along with decreased pSrc and pSmad1 protein expression in vitro. Moreover, the addition of siRNA against c-Src significantly reduced the phosphorylation of Smad1 and the overproduction of Col4. These results provide new evidence that the activation of Src/Smad1 signaling pathway plays a key role in the development of glomerulosclerosis in experimental glomerulonephritis

Importance of autophagyssociated molecules in lung cancer cases

科学研究費補助金研究成果報告書研究種目: 基盤研究(C)研究期間: 2011～2013課題番号: 23590395研究代表者: 瀧北 幹子（滋賀医科大学・医学部・助教）研究分担者: 茶野 徳宏（滋賀医科大学・医学部・准教授

Association of p62/SQSTM1 Excess and Oral Carcinogenesis

<div><p>p62/SQSTM1 (sequestosome1) has never been evaluated in oral epithelium. In order to clarify the role of p62/SQSTM1 in carcinogenesis in oral epithelium, both p62/SQSTM1 and Nrf2 were immunohistochemically evaluated in 54 carcinomas and 14 low grade dysplasias. p62/SQSTM1 knockdowns were also designed in oral cancer cells, and we analyzed the Nrf2 pathway, GSH contents and ROS accumulation. The association between p62/SQSTM1 excess and prognosis was addressed in a clinical cohort of oral carcinoma cases. p62/SQSTM1 excess was more obvious in carcinomas, but Nrf2 was abundant in almost all samples of the oral epithelium. In oral carcinoma cells, p62/SQSTM1 knockdown did not affect the Nrf2-Keap1 pathway but did significantly reduce GSH content with subsequent ROS accumulation, and caused cell growth inhibition in the irradiated condition. Finally, p62/SQSTM1 excess was associated with poor prognosis in a clinical cohort. In oral epithelial carcinogenesis, p62/SQSTM1 excess played a role in GSH induction rather than Nrf2 accumulation, and may cause resistance to cytotoxic stresses such as radiation or chemotherapy. Immunohistochemical evaluation of p62/SQSTM1 may be a potential significant marker to identify early carcinogenesis, chemo-radiotherapeutic resistance or poor prognosis of oral squamous cell carcinomas.</p></div

p62/SQSTM1 excess predicts clinically worse prognoses in oral cancer cases.

<p>Forty-nine cases of oral cancer were immunohistochemically evaluated for p62/SQSTM1 and statistically analyzed relative to the clinical outcomes. Kaplan-Meier curve with log-rank tests was performed for disease-specific survival in relation to p62/SQSTM1 level. (Chi-Square value = 4.640, p = 0.0312).</p

Nrf2 was abundantly expressed in carcinomas, low grade dysplasias, and non-atypical epithelia of oral tissue.

<p>(<b>A</b>) Case-frequencies (%) of Nrf2 staining grades in oral squamous cell carcinomas (blue columns; 54 cases), low grade dysplasias (red columns; 14 cases) and non-atypical epithelia (green columns; 29 cases). (<b>B</b>) Means ± S.E. of PLA signals for Nrf2 are displayed as bar graphs. The values (RCPs/cell) are 2.32±0.20, 2.10±0.32 and 1.24±0.17 in carcinomas (54 cases), low grade dysplasias (14 cases) and non-atypical epithelia (29 cases), respectively. There was a significant difference between carcinomas and non-atypical epithelia (<i>p</i> = 0.0029), using one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test. (<b>C</b>) Representative findings of Nrf2 staining in carcinoma (left), in low grade dysplasia (middle), and in non-atypical epithelium (right). Corresponding PLA signals are displayed in the lower row. Scale bar; 100 µm. (<b>D</b>) There was a weakly positive correlation between p62/SQSTM1- and Nrf2- PLA signals (Pearson’s correlation coefficient; r = 0.245, n = 97, <i>p</i> = 0.0265). (<b>E</b>) There was a strongly positive correlation between p62/SQSTM1- and GSH-PLA signals (Pearson’s correlation coefficient; r = 0.588, n = 97, <i>p</i><0.0001).</p

p62/SQSTM1 was abundantly stained in oral squamous cell carcinomas.

<p>(<b>A</b>) Case-frequencies (%) of p62/SQSTM1 staining grades in oral squamous cell carcinomas (blue columns; 54 cases), low grade dysplasias (red columns; 14 cases) and non-atypical epithelia (green columns; 29 cases). (<b>B</b>) Means ± S.E. of PLA signals for p62/SQSTM1 are displayed as bar graphs. The values (RCPs/cell) are 9.95±0.89, 3.90±0.48, 2.05±0.69 and 1.95±0.30 in the highest expression grade (++) carcinomas (24 cases), other carcinomas (30 cases), low grade dysplasias (14 cases) and non-atypical epithelia (29 cases), respectively. There was a significant difference between the highest expression grade (++) carcinomas and the other categories (<i>p</i><0.0001), using one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test. (<b>C</b>) Representative findings of p62/SQSTM1 staining in the highest expression grade (++) carcinoma (left), in low grade dysplasia (middle), and in non-atypical epithelium (right). Corresponding PLA signals and BlobFinder images are displayed in the middle and lower rows, respectively. Scale bar; 100 µm.</p

p62/SQSTM1 knockdown had little effect on the Nrf2-NQO1 pathway in oral cancer cells.

<p>However, p62/SQSTM1 knockdown affected the growth of the cells. (A) p62/SQSTM1 was abundantly expressed in SAS and CAL27 oral cancer cells. HeLa, endocervical carcinoma cells; TIG-108 and 121, normal human fibroblasts. (B) p62/SQSTM1 knockdown was performed by two kinds of shRNAs (sh-p62(1) and sh-p62(2)). shRNA for luciferase was used as control knockdown (sh-control). p62/SQSTM1 expression was significantly decreased by sh-p62(1) and sh-p62(2). Under no irradiation or 10 Gy X-ray irradiation, expressions of Nrf2 (pS40), Keap1, NQO1 and HO-1 were subtly affected by p62/SQSTM1 knockdown. Similar amounts of each cell protein were loaded in each lane of the SDS-PAGE. α-tubulin was used a loading control. Left and right panels indicate the data on SAS and CAL27, respectively. Three independent experiments were repeated, and the blotting photographs are representative ones. (C) p62/SQSTM1 knockdowns and X-ray irradiation were similarly performed. No radiation (upper); The effects of shRNA (sh-p62(1) and sh-p62(2)) were partial under normal culture condition. X-ray irradiations of 5 Gy (middle) and 10 Gy (lower); The cancer cell growth was significantly inhibited by two kinds of shRNA for p62/SQSTM1. Left and middle panels indicate the data on SAS and CAL27, respectively. The data on TIG-121 are displayed as a reference at the right panels. In the WST-8 assays, mean absorbance values (OD450) ± SE are shown vertically, and the number of days after exposure to radiation is indicated horizontally. The values are derived from quadruplicate experiments (*, <i>p</i><0.05, one-way factorial ANOVA and multiple comparison tests accompanied by Scheffe's significance test).</p

Clinical parameters of the patients with low grade dysplasias.

<p>Clinical parameters of the patients with low grade dysplasias.</p