Notch Signaling Activation Suppresses v-Src-Induced Transformation of Neural Cells by Restoring TGF-β-Mediated Differentiation

BACKGROUND: We have been investigating how interruption of differentiation contributes to the oncogenic process and the possibility to reverse the transformed phenotype by restoring differentiation. In a previous report, we correlated the capacity of intracellular Notch (ICN) to suppress v-Src-mediated transformation of quail neuroretina (QNR/v-src(ts)) cells with the acquisition by these undifferentiated cells of glial differentiation markers. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we have identified autocrine TGF-β3 signaling activation as a major effector of Notch-induced phenotypic changes, sufficient to induce transition in differentiation markers expression, suppress morphological transformation and significantly inhibit anchorage-independent growth. We also show that this signaling is constitutive of and contributes to ex-vivo autonomous QNR cell differentiation and that its down-regulation is essential to achieve v-Src-induced transformation. CONCLUSIONS/SIGNIFICANCE: These results support the possibility that Notch signaling induces differentiation and suppresses transformation by a novel mechanism, involving secreted proteins. They also underline the importance of extracellular signals in controlling the balance between normal and transformed phenotypes

L' activation constitutive de la voie Notch supprime la transformation induite par v-Src (implication de la signalisation TGF-b)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Inhibition of QNR cell differentiation correlates with TGF-β3 down-regulation by v-Src.

<p>(A) QPCR analysis of TGF-β3 mRNA levels in 7-day-old (E7) embryonic QNR seeded for 1 (E7+1), 8 (E7+8) or 18 (E7+18) days. Results were normalized based on HPRT and TBP transcript levels and presented in relative arbitrary units, using values obtained for E7+1 QNR cells as reference. (B) Detection of Pax6 expression by immunofluorescence in QNR (E7+18) (a) and QNR(E7+18)/v-src^ts cells, maintained at 37°C (b-e) or transferred to 41°C for 7 (c–f) and 15 days (d–g), in presence of DMSO (control) or 10 µM SB431542. At 41°C (c–d), nuclear Pax6 expression progressively decreased in control cells, but we reproducibly observed an unusual Pax6 peri-nuclear labeling. Insets represent DAPI staining. Magnification x40 (C) Expression of Glutamine synthetase (GS) in QNR (E7+18) and QNR (E7+18)/v-src^ts cells at 37°C or following their transfer to 41°C for 7 and 15 days, in presence of DMSO (c) or 10 µM of SB431542 (SB) Erk was used to normalize protein loading. (D) TGF-β3 mRNA levels were analyzed by QPCR in uninfected (N.I) QNR (E7+18) cells or in QNR (E7+18)/v-src^ts cells at 37°C. (E) TGF-β3 mRNA levels in QNR(E7+18)/v-src^ts cells at 37°C or transferred to 41°C for 7 and 15 days. Values are represented in relative arbitrary units after HPRT and TBP normalization.</p

QNR/v-src^ts/ICN cells secrete and respond to mature TGF-β3.

<p>(A) Media were collected after a 30-hour incubation of QNR/v-src^ts (v-src^ts medium) and QNR/v-src^ts/ICN (ICN medium) cells at 37°C or 41°C. QNR/v-src^ts cells were incubated at 37°C in normal medium (BME) or conditioned media for 1 hour, before lysis. Phosphorylated Smad2 was detected by western-blot. β–actin was used for protein level normalization. (B) QNR/v-src^ts cells were treated at 37°C with increasing concentrations of SB431542 inhibitor for 2 hrs and then incubated in ICN medium during 1 hour in presence of inhibitor. Effects of this treatment on Smad2 phosphorylation were analyzed by western-blot. (C) Comparison of mature TGF-β3 levels in v-Src and ICN media. Cells were plated at 2.10⁶ cells per 100 mm dish at 37°C or 41°C. One day after plating, growth media were replaced with serum-free BME. After a 30-hour incubation, media were collected and concentrated 50 times using Vivaspin6 columns. 20 µg of proteins were migrated under non-denaturing conditions. Mature TGF-β3 migrates as 25 kDa band. (D) QNR/v-src^ts cells were treated, one hour before lysis, with ICN medium preincubated at 37°C for one hour with increasing amounts of neutralizing antibodies directed against TGF-β3. Levels of Smad2 phosphorylation were analyzed by western-blot. Erk was used to normalize protein loading. (E) Levels of phosphorylated Smad2 in QNR/v-src^ts/ICN cells treated at 37°C or 41°C with DMSO or 10 µM of SB431542 inhibitor for 24 hrs.</p

TGF-β3 mRNA is upregulated in QNR/v-src^ts/ICN cells.

<p>QNR/v-src^ts and QNR/v-src^ts/ICN cells were incubated 72 hrs at 37°C or 41°C before extraction of total RNA. After reverse transcription of 1 µg of RNA, TGF-β3 cDNA was amplified by QPCR using specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013572#pone-0013572-t001" target="_blank">Table 1</a>) Results were normalized based on HPRT and TBP transcript levels and presented in relative arbitrary units. Normalized TGF-β3 mRNA levels detected in QNR/v-src^ts cells at 37°C were used as reference equal to 1. Each bar represents the mean -/+ S.E. of experiments realized on 3 different cultures per cell type.</p

TGF-β3 expression is controlled by a positive feed-back loop.

<p>QPCR analysis of TGF-β-3 mRNA levels in (A) QNR/v-src^ts treated with 2 µg/ml of recombinant TGF-β3 or (B) QNR/v-Src^ts/ICN cells at 37°C incubated with increasing doses of SB431542 during 24 hrs. After reverse transcription of 1 µg of RNA, TGF-β3 cDNA were amplified by QPCR. Results were normalized based on HPRT and TBP transcript levels and presented in relative arbitrary units using values obtained for untreated cells as reference equal to 1. Each bar represents the mean −/+ S.E. of three experiments.</p

TGF-β signaling induces α2-actin expression and MLC2 phosphorylation.

<p>QPCR analysis of α2-actin mRNA levels in (A) QNR/v-src^ts and QNR/v-src^ts/ICN cells at 37°C and 41°C, (B) QNR/v-src^ts cells treated at 37°C for 24 hrs with increasing concentrations (0.5–2 ng/ml) of recombinant TGF-β3 protein, or (C) QNR/v-src^ts/ICN treated at 37°C for 24 hrs with increasing concentrations of SB431452 (1–50 µM) Results are represented in relative arbitrary units after normalization using HPRT and TBP transcript levels. For (A), data presented are representative of three distinct experiments. For (B and C), each bar represents the mean −/+ S.E. of three experiments. (D) QNR/v-src^ts cells were treated with increasing doses of recombinant TGF-β3 protein for 24 hrs. One hour before lysis, TGF-β3 treatment was renewed. Phosphorylated MLC2 was detected by western-blot and Erk levels were used for normalization.</p

Recombinant TGF-β3 suppresses morphological transformation and induces cytoskeleton reorganization.

<p>QNR/v-src^ts cells were incubated at 37°C in absence or presence of 1 or 10 ng/ml of recombinant TGF-β3 protein. 24 hrs later, cells were fixed and F-actin stress fibers labeled with FITC-phalloïdin. Magnification: x20 for phase-contrast morphology (a–c); x40 for F-actin staining (d–f).</p

TGF-β signaling controls commitment into glial differentiation.

<p>(A) QNR/v-src^ts cells were treated at 37°C with increasing concentrations (0.2–2 ng/ml) of recombinant TGF-β 3 protein during 7 days. Pax6 and Glutamine Synthetase (GS) were detected by western-blot. Protein loading was normalized using Erk antiserum. (B) Pax6 expression was analyzed by immunofluorescence after treatment of QNR/v-Src^ts/ICN cells at 37°C with DMSO (control) or 10 µM SB431542 during 7 days. The majority of control QNR/v-Src^ts/ICN cells did not express nuclear Pax6 but we observed a weak peri-nuclear labeling. Magnification x40.</p