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Aberrant activation of stress-response pathways leads to TNF-α oversecretion in Fanconi anemia

Delphine Briot, Gaëtane Macé-Aimé, Frédéric Subra and Filippo Rosselli

Article Figures & Data

Figures

  • Figure 1

    Overproduction of biologically active TNF-α by FA-C cells. (A) TNF-α accumulation in the supernatant of normal cells (HSC93), and FA-C (HSC536) and its derived cell lines, HSC536Neo and HSC536Corr. HSC536 cells were transfected with an empty vector (HSC536Neo) or with a vector bearing the wild-type FANCC cDNA (HSC536Corr). Cells were collected by centrifugation, washed, and resuspended in fresh medium (3 × 105 cells/mL/well in 3 mL medium in 6-well tissue culture plates). Supernatants were collected 24 hours and 48 hours after subculturing. The level of TNF-α in supernatants was determined by ELISA. Data presented are the mean (± SD) of at least 5 independent experiments. *P < .01. (B) Luciferase activity in 293T/NF-κB-Luc cells cultured in conditioned medium from lymphoblasts. 293T/NF-κB-Luc cells were plated (106 cells/well in 6-well tissue culture plates) 48 hours before treatment with supernatant collected from 48-hours-old lymphoblasts cultures. One volume of supernatant, equivalent to 106 lymphoblasts, was added per well of 293T/NF-κB-Luc cells. Cells were harvested 24 hours after treatment for luciferase measurement. Luciferase activity of 293T/NF-κB-Luc cells cultured in supernatant from HSC536Corr cells was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05.

  • Figure 2

    Analysis of TNF-α mRNA expression and level and protein expression and secretion. (A) TNF-α promoter activity in FA and corrected lymphoblasts. Cells were cotransfected with the reporter pGL3-hTNF-α-LucF and phRL-TK, used as internal control. Luciferase activity in HSC536Corr cells was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .01. (B) Steady-state level of TNF-α mRNA in FA and corrected lymphoblasts assessed by semiquantitative RT-PCR. The histogram represents the relative level of TNF-α mRNA in FA-C–deficient compared with FANCC-corrected cells. The level in HSC536Corr was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05. (C) Immunoblot showing the level of TNF-α in total cell extracts prepared from HSC536 cell line either untreated (Unt) or treated for 18 hours with brefeldin A (Bref A) or dexamethasone (Dexa). A cross-reactive band (marked with asterisk) was used as a loading control. (D) Intracellular TNF-α accumulation in FA and corrected lymphoblasts. Cells were treated with dexamethasone (10 μg/mL) for 3 hours. After this incubation, cells were collected by centrifugation, washed, and resuspended in fresh medium in presence of brefeldin A (1 μg/mL) for 24 hours. Proteins were extracted at the indicated time points. TNF-α content was measured by ELISA. Each point represents the mean (± SD) of at least 3 independent experiments. (E) Kinetics of TNF-α accumulation in HSC93, HSC536Corr, HSC536, and HSC536Neo cell lines. Supernatants were collected 3, 6, and 9 hours after cell subculturing in fresh medium and cytokine release was determined by ELISA. Each point represents the mean (± SD) of at least 3 independent experiments. *P < .01.

  • Figure 3

    Overexpression of MMP-7 in FA-C cells and its involvement in the aberrant secretion of the TNF-α. (A) Immunoblot showing the level of TACE in total cell extracts. Immature and mature forms of TACE are indicated by i and m, respectively. The doublets observed for each form on Western blot reflect protein glycosylation. Equal loading of the membrane was verified by ponceau red staining and/or evaluated by the intensity of the indicated (*) aspecific band. (B) Immunoblot showing the level of MMP-7 in total cell extracts (top panel) and in supernatant (bottom panel). Equal loading of the membrane was verified by ponceau red staining and/or evaluated by the intensity of the indicated (*) aspecific band. Vertical lines have been inserted to indicate a repositioned gel lane. (C) MMP-7 promoter activity in normal, corrected, and FA cells. Cells were cotransfected with the reporter pGL3-hMMP-7 and phRL-TK, as internal control. Luciferase activity in AHH1 cells was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05. (D) MMP-7 mRNA steady-state levels as determined by quantitative RT-PCR on mRNA extracted from exponentially growing lymphoblasts and normalized to 18S rRNA content. To normalize the values among the different experiments, each time the ratio MMP-7/18S was considered equal to 1 in AHH1 cells. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05. (E) TNF-α release in MMP-7 inhibited FA-C cells. HSC536 and HSC536Neo cells were treated with MMP inhibitor (50 μM) or its solvent (DMSO) or left untreated. Supernatants were collected 24 hours after subculturing and cytokine release was determined by ELISA. Data presented are the mean (± SD) of at least 3 independent experiments. *P < .01. (F) TNF-α release in FA-C cells after the down-regulation of MMP-7 expression. Immunoblot shows the level of MMP-7 in supernatant of FANCC siRNA–MMP-7 down-regulated cells. A cross-reactive band (*) was used as a loading control. TNF-α accumulation in the supernatant of infected HSC536Neo cells was evaluated by ELISA. Histogram represents the mean reduction (in percentage) plus or minus SD of TNF-α accumulation observed in at least 3 independent experiments. *P < .01.

  • Figure 4

    Aberrant activation of stress-response pathways in FA-C cells. (A) NF-κB transcriptional activity in lymphoblasts. NF-κB transcriptional activity was evaluated as increased luciferase activity in cell extracts isolated 48 hours after transfection with the reporter pNF-κB-Luc and phRL-TK. Luciferase activity in AHH1 cells was fixed to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. (B) Immunoblot showing the level of phosphorylation of key players of the MAPK pathways. Proteins were isolated from exponentially growing lymphoblasts. (C) TNF-α level in FA-C cells as function of MAPK activity. HSC536 and HSC536Neo cells were treated with SB203580 (p38 inhibitor, 10 μM), PD98059 (ERK inhibitor, 30 μM), and SP600125 (JNK inhibitor, 25 μM). Supernatants were collected 24 hours after subculturing and cytokine release was determined by ELISA. Data presented are the mean (± SD) of at least 3 independent experiments. *P < .01.

  • Figure 5

    FANCC down-regulation leads to aberrant activation of stress-response pathways and MMP-7 overexpression in HeLa cells. (A) Immunoblot showing monoubiquitination of FANCD2 in FANCC siRNA–transfected cells. Forty-eight hours after siRNA transfection, cells were treated with mitomycin C (MMC) (500 ng/mL) or 8-methoxypsoralen (10−5M) + UVA (10 kJ/m2) (8-MOP) and lysed 24 hours later. FANCC down-regulation impacts on DNA damage–induced FANCD2 monoubiquitination. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Western blot analysis of IκBα in HeLa cells depleted of FANCC for 3 days. Actin was used as a loading control. (C) Detection of p50 and p65 subcellular localization by confocal immunofluorescence microscopy. HeLa cells treated with FANCC siRNA were subjected to immunostaining of p50 (green fluorescence) and p65 (red fluorescence) 72 hours after transfection. (D) NF-κB transcriptional activity in HeLa cells depleted for FANCC. Twenty-four hours after siRNA treatment, cells were cotransfected with the reporter pNF-κB-Luc and phRL-TK for 48 hours before the analysis of the luciferase activity. Luciferase activity in GFP-siRNA–transfected cells was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .05. (E) Immunoblot showing the level of phosphorylation of MAPK players in cells with a reduced FANCC expression compared with mock-transfected and unperturbated cells. Cells were collected 72 hours after transfection and actin was used as a loading control. Vertical lines have been inserted to indicate a repositioned gel lane. (F) MMP-7 promoter activity in mock- and siRNA FANCC–transfected HeLa cells. Twenty-four hours after siRNA treatment, cells were cotransfected with the reporter pGL3-hMMP-7 and phRL-TK, and induced luciferase activity was measured 24 hours later. Luciferase activity in GFP-siRNA–transfected cells was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .01. (G) MMP-7 mRNA steady-state level in FANCC-deprived or mock-transfected HeLa cells was determined by quantitative RT-PCR. The relative mRNA level (normalized as function of 18S rRNA content) in GFP-siRNA–transfected cells was fixed to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .01. (H) MMP-7 promoter activity in 50 μM curcumin-treated FANCC-deprived cells assessed by luciferase activity as in panel F. *P < .01. (I) MMP-7 mRNA steady-state level in FANCC-deprived HeLa cells treated with curcumin (50 μM) as assessed by quantitative RT-PCR as in panel G. *P < .01. (J) MMP-7 promoter activity in siRNA FANCC–transfected HeLa cells treated with SB203580 (p38 inhibitor, 10 μM) and PD98059 (ERK inhibitor, 30 μM) for 18 hours. *P < .05 compared with DMSO treatment.

  • Figure 6

    FA gene mutation leads to MMP-7 overexpression and MAPK pathway activation. (A) TNF-α accumulation in the supernatant of AHH1 (normal), PD4L (FA-C), PD4L/FANCC (FA-C corrected), HSC72 (FA-A), and HSC72/FANCA (FA-A corrected) lymphoblasts. Supernatants were collected as described in Figure 1A. Data presented are the mean (± SD) of at least 3 independent experiments. *P < .01 compared with gene-corrected cells. (B) TNF-α accumulation in the supernatant of AHH1 (normal), HSC99 (FA-A), GM16757 (FA-F), and EUFA143 (FA-G) cell lines. Supernatants were collected as described in Figure 1A. Data presented are the mean (± SD) of at least 3 independent experiments. *P < .01. (C) HSC72, HSC99, PD4L, GM16757, and EUFA143 cells were treated with MMP inhibitor as described in Figure 3F and TNF-α content in supernatants was measured by ELISA. Data presented are the mean (± SD) of at least 3 independent experiments. *P < .01. (D) MMP-7 promoter activity in siRNA FANC–transfected HeLa cells. Cells were cotransfected with the reporter pGL3-hMMP-7 and phRL-TK, and induced luciferase activity was measured 24 hours later. Luciferase activity in GFP-siRNA–transfected cells was considered equal to 1 in each experiment. Data presented are the mean (± SD) of at least 3 independent experiments done in triplicate. *P < .01. (E) Western blot analysis of MAPK phosphorylation in siRNA FANC–transfected HeLa cells. Cells were collected 72 hours after transfection, and actin was used as a loading control.

  • Figure 7

    A model for TNF-α oversecretion in FA. (A) The FANC–TNF-α pathway. (B) The FA network (see “Discussion”).