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First published online 22 January 2008
doi: 10.1242/jcs.017202

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ER stress is associated with dedifferentiation and an epithelial-to-mesenchymal transition-like phenotype in PC Cl3 thyroid cells

¹ Istituto di Endocrinologia ed Oncologia Sperimentale "G. Salvatore", Via S. Pansini 5, 80131 Napoli, Italy
² Dipartimento di Biologia e Patologia Cellulare e Molecolare `L. Califano', Via S. Pansini 5, 80131 Napoli, Italy
³ Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Facoltà di Scienze Matematiche Fisiche e Naturali, Università degli Studi di Lecce, Strada Provinciale Lecce-Monteroni, 73100 Lecce, Italy

View larger version (51K): [in this window] [in a new window]   Fig. 1. Thapsigargin (TH) or tunicamycin (TN) induce ER stress in PC Cl3 cells. (A) TH and TN induce upregulation of mRNA encoding BiP. Northern blot analysis of total RNA extracted from PC Cl3 cells vehicle treated (0) or treated with increasing concentrations of TH or TN for 30 minutes, followed by 12 and 24 hours in medium without TH or TN. GAPDH was analyzed on the same filters. (B) RT-PCR analysis for XBP-1 and β-actin of the same total RNAs used in (A).

View larger version (80K): [in this window] [in a new window]   Fig. 2. TH and TN induce inhibition of thyroid differentiation markers in PC Cl3 cells. (A) Northern blot analysis of total RNA extracted from PC Cl3 cells vehicle treated (0) or treated with increasing concentrations of TH and TN for 30 minutes, followed by 12 and 24 hours in medium without TH or TN. (B) Western blots of total protein extracts from PC Cl3 cells vehicle treated or treated with 0.5 or 5 µM TH or 0.5 or 10 µg/ml TN for 30 minutes, followed by 12 and 24 hours in medium without TH or TN. Filters were probed with antibodies against TTF-1, TTF-2, Pax8 and β-actin.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Time-course of total protein and thyroglobulin (TG) synthesis following ER stress. (A) PC Cl3 cells were treated with 0.5 µM TH for the times indicated and labeled for 5 minutes with [35S]-met/cys mix. Cell lysates were subjected to SDS-PAGE. (B) Values shown represent the mean (±s.d.) of three independent experiments. (C) PC Cl3 cells were treated and labeled as in (A), TG was immunoprecipitated and subjected to SDS-PAGE. (D) Values shown represent the mean (±s.d.) of three independent experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 4. ER stress induced by TH or TN inhibits thyroid-specific gene expression, at least in part, at the transcriptional level. (A) Relative luciferase activity of extracts of PC Cl3 cells transfected in triplicate with 2.5 µg NISLUC2 luciferase reporter plasmid. 24 hours after transfection, cells were vehicle treated (`C') or treated with 0.5 µM TH or 0.5 µg/ml TN for 30 minutes and harvested after 24 hours in medium without TH or TN. Measurements were normalized for β-galactosidase activity driven by a co-transfected plasmid encoding RSV-βgal. Values shown (in arbitrary units) represent the mean (±s.d.) of at least three independent experiments. (B) Run-on assay performed on nuclei prepared from PC Cl3 cells vehicle treated (`C') or treated with 0.5 µg/ml TN for 30 minutes, followed by 24 hours in medium without TN. Pax8 and GAPDH probes were immobilized on filters.

View larger version (78K): [in this window] [in a new window]   Fig. 5. ER stress induces downregulation of E-cadherin and formation of stress fibers in PC Cl3 cells. (A) PC Cl3 cells were grown on glass coverslips for 48 hours, then were vehicle treated (i) or treated with 0.5 µg/ml TN for 30 minutes, followed by 24 hours in medium without TN (ii). Cells were stained with antibodies against E-cadherin. Following TN treatment, the signal for E-cadherin decreased. Arrows in (ii) indicate residual E-cadherin localized at the remaining cell-cell contacts. Bars, 15 µm. (B) PC Cl3 cells were grown and treated as above. Cells were double stained with antibodies against TG and rhodamine-conjugated phalloidin. Bars, 30 µm (i, ii, iii), 15 µm (iv, v, vi). In control cells, rhodaminated phalloidin staining is mainly at the level of cortical actin. Following TN treatment, the signal for TG decreased and stress fibers were formed. Arrows indicate: the few cells expressing various amounts of residual TG (iv), the correlation between residual TG expression and partially formed, not fully formed, stress fibers (v) and, consequently, the lack of overlap between TG and actin signals (vi). A greater magnification for TN-treated cells was intentionally used to show better the coordinate variations of TG, cortical actin and stress fibers.

View larger version (45K): [in this window] [in a new window]   Fig. 6. ER stress upregulates EMT markers in PC CL3 cells. (A) RT-PCR analysis of total RNA extracted from PC Cl3 cells vehicle treated or treated with 0.5 µM TH or 0.5 µg/ml TN for 30 minutes, followed by 24 and 48 hours in medium without TH or TN. (B) Western blots of total protein extracts from PC Cl3 cells vehicle treated or treated with 0.5 µM TH or 0.5 µg/ml TN for 30 minutes, followed by 24 and 48 hours in medium without TH or TN. Filters were probed with antibodies against E-cadherin and β-actin (control). (C) Real-time RT-PCR analysis of total RNA isolated from PC Cl3 cells vehicle treated or treated with 0.5 µM TH or 0.5 µg/ml TN for 30 minutes, followed by 24 hours in medium without TH or TN. Each bar represents the mean±s.d. of four independent experiments, each performed in triplicate. β-actin was used as an internal standard (β-actin values were not affected by TH or TN treatments) (REU, relative expression units). Asterisks indicate statistically significant differences (***, P<0.001). (D) RT-PCR analysis of total RNA extracted from PC Cl3 cells vehicle treated or treated with 0.5 µM TH and 0.5 µg/ml TN for 30 minutes, followed by 6, 24 and 48 hours in medium without TH or TN.

View larger version (69K): [in this window] [in a new window]   Fig. 7. ER stress induces an EMT-like phenotype in FRT cells. (A) Northern blot analysis of total RNA extracted from FRT cells vehicle treated or treated with increasing concentrations of TH and TN for 30 minutes, followed by 24 hours in medium without TH or TN. (B) FRT cells were grown on glass coverslips for 48 hours, then were vehicle treated or treated with 0.5 µg/ml TN for 30 minutes, followed by 24 hours in medium without TN. Cells were stained with antibodies against E-cadherin and rhodamine-conjugated phalloidin. Following TN treatment, the signal for E-cadherin decreased and stress fibers were formed. Bars, 20 µm. (C) RT-PCR analysis of total RNA extracted from FRT cells vehicle treated or treated with 0.5 µM TH or 0.5 µg/ml TN for 30 minutes, followed by 24 and 48 hours in medium without TH or TN.

View larger version (62K): [in this window] [in a new window]   Fig. 8. TN decreases transepithelial resistance in FRT cells. (A) 2x106 cells were plated on 24.5 mm diameter Transwell filters. At confluency, transepithelial resistance was measured every 12 hours, until a plateau was reached (3-4 days). Then, cells were vehicle treated (`C') or treated with 0.5 µg/ml TN in the inferior chamber (TN inf) or in inferior plus superior chambers (TN inf + sup), and transepithelial resistance measured every 12 hours over a 4 day period. (B) FRT cells, vehicle treated and treated as in A, were observed daily by light microscopy to verify the integrity of the monolayer. At day four (last transepithelial resistance measurement), the cells were photographed.

View larger version (43K): [in this window] [in a new window]   Fig. 9. c-Src is involved in ER stress-induced thyroid dedifferentiation. (A) Northern blot analysis of total RNA extracted from PC Cl3 cells pretreated or not for 30 minutes with 5, 10 or 20 µM PP2, then vehicle treated or treated for 30 minutes with 0.5 µM TH or 0.5 µg/ml TN in the presence or absence of PP2, followed by 24 hours in medium without TH or TN, but with PP2. The same filter was probed with Pax8 and BiP. (B) RT-PCR analysis of total RNA extracted from PC Cl3 cells treated as in A. (C) Western blots of total protein extracts from PC Cl3 cells starved for 48 hours in hormone- and serum-free medium, pretreated with 5 µM PP2, then vehicle treated or treated for 30 minutes with 0.5 or 1.0 µM TH or 0.5 or 1 µg/ml TN in the absence or presence of 5 µM PP2. Filters were probed with antibodies against phosphorylated Src (p-Src; Tyr416) or total Src.

View larger version (77K): [in this window] [in a new window]   Fig. 10. Stable expression of SrcDN prevents TH- or TN-induced downregulation of TG and E-cadherin. (A) Western blots of total protein extracts from PC pSG5 cells, clones (Cl.) 12, 20 and 15 starved for 48 hours in hormone- and serum-free medium, then vehicle treated or treated with 5 nM EGF for 5 minutes. Filters were probed with antibodies against phosphorylated Src (p-Src; Tyr416) or total Src (c-Src). (B) Western blots of total protein extracts from PC pSG5 cells and clones 12, 15, and 20 vehicle treated or treated with 0.5 µg/ml TN for 30 minutes, followed by 24 hours in medium without TN. Filters were probed with antibodies against TG, E-cadherin and β-actin.

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