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

doi:10.1182/blood-2007-07-099218Prepublished online November 30, 2007;

Delphine Briot, Gaetane Mace-Aime, Frederic Subra and Filippo Rosselli oversecretion in Fanconi anemia

αAberrant activation of stress-response pathways leads to TNF-

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1

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

Delphine Briot 1, Gaëtane Macé-Aimé 1 Frédéric Subra 2 and Filippo Rosselli 1 1CNRS FRE2939 – Univ Paris-Sud – Institut Gustave Roussy, Villejuif, France. 2CNRS UMR8113 – LBPA – ENS Cachan, Cachan, France. Corresponding author: Filippo Rosselli,

FRE2939 du CNRS – Institut Gustave Roussy PR2 39 rue Camille Desmoulins 94805 Villejuif Cedex (FRANCE). Tel.: 33.1.42.11.51.16; Fax: 33.1.42.11.50.08; e-mail: [email protected]

Running Title: Origins of TNF-α overproduction in Fanconi anemia

Blood First Edition Paper, prepublished online November 30, 2007; DOI 10.1182/blood-2007-07-099218

Copyright © 2007 American Society of Hematology

For personal use only.on May 19, 2014. by guest bloodjournal.hematologylibrary.orgFrom



2

ABSTRACT

Fanconi anemia (FA), an inherited syndrome that associates bone marrow failure, cancer

predisposition and genetic instability, is characterized by an overproduction of the

myelosuppressive cytokine TNF-α through unknown mechanisms. We demonstrate here that FANC

pathway loss-of-function results in the aberrant activation of two major stress-signaling pathways:

NF-κB and MAPKs. These responses are independent on TNF-α expression. On the contrary,

inhibition of the MAPK pathways normalizes TNF-α oversecretion in FA. Moreover, our data show

that the overexpression of the matrix metalloproteinase MMP-7 is the key event directly responsible

for the high rate of TNF-α shedding and release from the cytoplasmic membrane in FA. TNF-α

overproduction is, indeed, normalized by MMP-7 inhibition. Finally, MAPKs inhibition impacts on

MMP-7 overexpression. Evidences are provided of the existence of a linear pathway in which

FANC mutations activate MAPKs signaling that induces MMP-7 overexpression leading, in fine, to

TNF-α oversecretion. TNF-α may, in turn, sustain or amplify both MAPKs and NF-κB activation.

Aberrant expression or activity of NF-κB and/or MAPKs have been already involved in bone

marrow failure and leukemia and their inhibition offered clinical benefit for patients. In conclusion,

our data provide a strong rationale for new clinical trials on FA patients.




3

INTRODUCTION

Fanconi anemia (FA) is a rare recessive syndrome featuring progressive bone marrow

(BM) failure, multiple developmental abnormalities and cancer predisposition 1-3. BM failure

and its related consequences, such as pancytopenia or acute myeloid leukemia (AML) are the

major cause of morbidity and mortality of FA patients 4. The cellular phenotype is

characterized by chromosomal instability and hypersensitivity to DNA interstrand cross-link

(ICL) inducing agents such as mitomycin C (MMC), diepoxybutane and cisplatin 5-8. At least

13 complementation groups have been identified (FA-A, B, C, D1, D2, E, F, G, I, J, L, M and

N) and the genes for all of these groups have been cloned 9. One of the major functions of

FANC proteins is to deal with DNA damage, thus participating in an as yet undefined manner

to the repair of DNA lesions induced by cross-linking agents 10-12. However, the spectrum of

clinical and cellular abnormalities of the syndrome suggests that FANC proteins could have

other functions or participate in pathways other than DNA repair 13,14. Whether the

hematological problems of the FA patients are a consequence of a defect in DNA repair or in

other potential functions of the FANC proteins remains to be determined.

Tumor Necrosis Factor-α (ΤΝF−α) is a major cytokine involved in hematopoiesis,

inflammation and apoptosis 15,16. TNF-α is synthesized as a membrane-bound precursor of

26kDa that can be processed to generate a secreted 17kDa mature TNF-α 17,18. Soluble mature

TNF-α is released from the cells by cleavage of the precursor at the Ala76-Val77 bond by the

TNF-α converting enzyme (TACE or ADAM17) 19,20 or, less efficiently, by the matrix

metalloproteinase 7 (MMP-7 or matrilysin) 21,22. TNF-α signals through two distinct cell

surface receptors, TNFR-1 and TNFR-2 16. The binding of TNF-α to its receptors results,

among other events, in the activation of both the mitogen-activated protein kinases (MAPKs)

stress signaling cascade 16 and the NF-κB transcription factor 23. Activation of the MAPKs

and NF-κB plays an important role in the induction of many cytokines including the TNF-α

itself 24,25. TNF-α negatively regulates the expansion and self-renewal of pluripotent

hematopoietic stem cells (HSC) 26,27 and has inhibitory effects on normal human

hematopoietic progenitor cells as well as leukemia progenitor cells 28-30. Consistently, TNF-α

overproduction has been associated to different hematopoietic disorders such as

myelodysplastic syndrome (MDS), AML and aplastic anemia 31-33. FA syndrome recapitulates

all these abnormalities, i.e. impaired HSC expansion and development of the myeloid

lineages, MDS, aplastic anemia and AML 2-4. FA is also characterized by TNF-α

overproduction, both in vivo and in vitro. Indeed, it has been reported that TNF-α is (a)




4

overexpressed in BM of FA patients 34,35, (b) increased in the serum of patients 35,36 and (c)

overproduced by EBV-transformed FA lymphoblasts 36. Moreover, hematopoietic progenitors

from Fancc knockout mice, that fail to spontaneously overproduce the cytokine, show

hypersensitivity to the TNF-α myelosuppressive action 37,38. In light of these observations,

abnormalities in TNF-α expression could be considered at the origin of the progressive BM

failure observed in FA patients. Understanding the origins of TNF-α overproduction could

open new therapeutic roads for the BM failure in FA.

In the present work, we investigated the molecular origins of TNF-α overproduction in

FA. Our data indicate that the loss-of-function of FANC proteins leads to overactivation of

two major stress-response pathways: the MAPK and NF-κB networks. As a consequence of

MAPKs activation, MMP-7 is expressed over the level normally observed in FANC-proficient

cells and it sheds TNF-α from cytoplasmic membrane leading to the extracellular

accumulation of the cytokine. Importantly, we show that the inhibition of MAPKs or MMP-7

activity in FA cells leads to a significant decrease in TNF-α overproduction.




5

MATERIALS AND METHODS

Cell lines and reagents

EBV-transformed lymphoblasts and their growth conditions were previously described 36,39.

GM16757 and 293T/NF-κB-Luc cells were purchased from Coriell Cell Repositories (Camden, NJ,

USA) and Panomics (Freemont, CA, USA), respectively. HeLa and 293T/NF-κB-Luc cells were

grown in DMEM (Life Technologies, Invitrogen, Carlsbad, CA, USA) supplemented with 10%

FCS (Dutcher, Brumath, France) and antibiotics (Gibco-BRL, Gaithersburg, MD, USA).

Hygromycin (100µg/ml, Sigma, St. Louis, MO, USA) was added to 293T/NF-κB-Luc cultures.

Dexamethasone, brefeldin A and curcumin (Sigma, St. Louis, MO, USA) were dissolved in

ethanol and MMP inhibitor II, SB203580, PD98059 and SP600125 (Calbiochem, San Diego, CA,

USA) in DMSO.

Lentiviral shRNA vector construction, production and transduction

Lentivirus vectors were obtained from http://tronolab.epfl.ch/. MMP-7 and untargeted

shRNA oligonucleotide sequences can be provided on request. Vectors were constructed by cloning

the sequences into the Mlu1-Cla1 site of the pLV-TH plasmid and then sequenced. Lentiviruses

were produced by transient transfection of 293T cells according to standard protocols 40. For

transduction, lentiviruses were added to cells for 4h. Cells were sorted 96h later for green

fluorescence using a FACSDiva flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

RNA extraction and semiquantitative RT-PCR analysis

Total RNA was extracted using the RNeasy kit (Qiagen, Valencie, CA, USA), following

manufacturer’s instructions including the optional DNase treatment. PCR reactions were performed

as previously described 39. Primers sequences were: TNF-α forward 5’-

CAGAGGGCCTGTACCTCATC-3’, reverse 5’-GGTTGAGGGTGTCTGAAGGA-3’ and actin

forward 5’-AGAGCTACGAGCTGCCTGAC-3’, reverse 5’-AGTACTTGCGCTCAGGAGGA-3’.

RT-PCR quantification was done with Fluor-S-Multilmager (Biorad, Hercules, CA, USA).

Real-time PCR amplification using taqman assay

Primers were purchased from Applied Biosystems (Foster city, CA, USA). Real-time PCR

were performed on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster

city, CA, USA) following manufacturer's instructions.

Protein extraction and Western blot analysis

Whole-cell extracts were prepared in lysis buffer (50mM Tris-HCl pH 8.0, 150mM NaCl,

1% NP-40) supplemented with protease inhibitors (Roche Diagnostic, Meylan, France), 10mM NaF

and 1mM Na3VO4 (Sigma, Sigma, St. Louis, MO, USA). Supernatants from lymphoblasts cultured




6

in serum-free RPMI for 24h were collected by centrifugation, placed in Vivaspin 6 (Sartorius,

Goettingen, Germany) and centrifuged to remove proteins with molecular weights of less than 5

kDa. Blots were incubated with primary antibodies: TNF-α (R&D Systems, Minneapolis, MN,

USA), TACE, MMP-7 (Oncogene research, Merck, Darmstadt, Germany), phospho-p38, p38,

phospho-ERK, ERK, phospho-JNK, JNK (Cell Signaling, Danvers, MA, USA), FANCA, IκBα,

actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FANCD2 (Abcam, Cambridge, UK),

FANCC (FA Research Fund, Eugene, OR, USA) followed by species-specific secondary antibodies

coupled to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Signals were

detected by chemiluminescence using the ECL kit (Pierce, Rockford, IL, USA).

TNF-α titration

Cell suspensions were collected by centrifugation and supernatants were filtered (0,45µm,

Millipore Filter, Millipore, Paris, France) before storage at –20°C. TNF-α content in supernatants

or protein extracts was evaluated by Quantikine Elisa assay as described by the supplier (R&D

Systems, Minneapolis, MN, USA).

siRNA

siRNA duplexes (FANCA 41, GFP 42) were synthesized by Eurogentec (Angers, France) and human

FANCC smart pool siRNA was purchased from Dharmacon (Lafayette, CO, USA). Transfections

were performed with 100nM siRNA using Oligofectamine (Invitrogen, Carlsbad, CA, USA)

according to the instructions of the manufacturer.

Cell transfection and Luciferase assay

Lymphoblasts were transfected using the Amaxa nucleofection technology (Amaxa,

Cologne, Germany). Briefly, 2x106 cells were resuspended in 100µl Amaxa solution kit V, mixed

with 2µg of plasmid (pGL3-hTNF-α 43, pGL3-hMMP-7 44, pNF-κB-Luc (Clontech, Mountain

View, CA, USA) and 10ng of phRL-TK (Promega, Madison, WI, USA). Cells were nucleofected

using the program T20, harvested 48h after transfection and 20µl of cell lysate was assayed for

luciferase activity using the dual luciferase assay system (Promega, Madison, WI, USA). Firefly

luciferase activity was corrected for transfection efficiency using the Renilla luciferase activity.

After 24h of siRNA transfection, HeLa cells were transfected with 0,8µg of plasmid (pNF-

kB-Luc or pGL3-hMMP-7) and 5ng of phRL-TK using FUGENE 6 (Roche Diagnostic, Meylan,

France) according to the manufacturer’s protocol. Cells were always harvested 72h after siRNA

transfection as above.




7

Immunofluorescence

Cells were fixed with 4% paraformaldehyde (10min) and permeabilized with 0,1% SDS

(12min) at room temperature. Coverslips were blocked with 2% BSA in PBS and then incubated

with primary antibody (p50 and p65, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1h.

Cells were washed and incubated with secondary antibody (Alexa Fluor, Molecular Probes,

Invitrogen, Carlsbad, CA, USA) for 1h before mounting with a solution containing DAPI

(Vectashield, Vector Laboratories, Peterborough, UK). Analysis was performed using a laser

confocal microscope (LSM 510, Zeiss, Oberkochen, Germany).

Statistical Analysis

Results are the mean of at least three independent experiments with error bars showing the

standard error. The t-Student test was used and p<0,05 was considered statistically significant.




8

RESULTS

FANCC gene product modulates the spontaneous production of biologically active TNF-α.

Overproduction of TNF-α in FA syndrome has been previously reported both in vivo and in

vitro 35,36. However, previous experimental approaches did not establish whether TNF-α

overproduction was directly linked to FA gene mutation. In order to address this point, we

compared TNF-α level and activity in the supernatant of exponentially growing normal (HSC93),

FANCC-corrected (HSC536Corr) and FA-C lymphoblasts (HSC536 and HSC536Neo) (Figure 1A

and 1B). FA-C cells accumulated, in a time-dependent manner, 5 to 8 times more TNF-α in their

growth medium than lymphoblasts from a normal donor (Figure 1A). The ectopic expression of

FANCC in FA-C cells reduced TNF-α production to the level observed in normal cells (Figure 1A).

TNF-α is a potent inducer of NF-κB activity 23. We evaluated the biological activity of the

secreted TNF-α by measuring NF-κB transcriptional activity using a 293T cell line expressing a

luciferase reporter construct under the control of NF-κB target sequence. 293T/NF-κB-Luc cells

were cultured for 24h in 48h-old supernatants collected from FA-C and corrected cells. As depicted

in Figure 1B, luciferase activity was 6 to 8 times more important when 293T/NF-κB-Luc cells were

cultured in supernatant from FA-C cells than in supernatant from FA-C corrected cells.

All together, these results demonstrate that TNF-α is overproduced in FA-C cells, is

biologically active and that its aberrant expression is directly linked to FANCC loss-of-function.

FA-C cells show a higher TNF-α shedding from cytoplasmic membrane.

TNF-α production is controlled at multiple levels from transcription to cytoplasmic

membrane shedding. To determine the molecular origin of TNF-α overproduction, we first

analyzed, using two different approaches, TNF-α mRNA intracellular production and accumulation

(Figure 2A and 2B). First, FA-C cells and their ectopically corrected counterpart were transfected

with a reporter plasmid expressing Firefly luciferase under the control of human TNF-α promoter

(pGL3-hTNF-α) 43. Interestingly, relative TNF-α promoter activity in FA-C cells was 2 to 2,5 times

higher compared to corrected FA-C cells (Figure 2A). Then, we performed semi-quantitative RT-

PCR analysis to assess the steady-state level of TNF-α mRNA in lymphoblasts. Actin mRNA was

amplified in the same PCR reactions and used as an internal control. Quantification of RT-PCR

products revealed a slight (1,5 times higher) but constant and significant difference in TNF-α

mRNA content in FA-C cells compared to corrected cells (Figure 2B).




9

Having observed a slight increase in both TNF-α mRNA production and accumulation, we

then questioned whether this increase in mRNA could be responsible for the accumulation of TNF-

α in the supernatant of FA-C cells. In order to evaluate TNF-α protein synthesis, we first treated the

cells with dexamethasone which inhibits TNF-α mRNA translation 45 to reduce the steady-state

intracellular content of TNF-α protein (Figure 2C and 2D). After 3h exposure to dexamethasone

(10µg/ml), intracellular TNF-α content was reduced to about half of the starting quantity (Figure

2D). Dexamethasone was then removed and cells were treated with brefeldin A (1µg/ml). This

agent inhibits TNF-α protein transport to the membrane impeding its secretion 46, thereby leading to

its intracellular accumulation (Figure 2C). Proteins were extracted 3h, 6h, 9h and 24h following

brefeldin A addition, and the TNF-α protein level was assessed by ELISA. As reported in Figure

2D, the kinetics of intracellular accumulation of TNF-α was similar in both FA-C and corrected

cells. Importantly, the content of TNF-α in the supernatant of brefeldin A-treated cells was below

the detection threshold, showing the ability of the drug to efficiently block TNF-α transport to the

cytoplasmic membrane and its consequent release (data not shown). We thus conclude that TNF-α

protein accumulated equally in cells independently of the FA gene status.

In view of these results, a difference in the TNF-α shedding may be responsible for the

observed overproduction of TNF-α by FA-C cells. To address this point, we determined the kinetics

of TNF-α secretion after plating the cells in fresh, TNF-α free, medium (Figure 2E). As early as 3h

after cell plating, TNF-α accumulation in the supernatant of FA-C cells was significantly higher

(2,5 times more) than proficient FANCC cells. Moreover, TNF-α accumulation in the supernatant

of HSC93 and HSC536Corr cells reached a plateau after six hours whereas in FA-C cells this

accumulation was constantly increasing even after nine hours.

All together, these results indicate that an increased release of the cytokine from the

cytoplasmic membrane is responsible for TNF-α overproduction in FA-C cells.

MMP-7 overexpression links FANCC to TNF-α oversecretion.

To date, only two proteases have been described as able to produce a correctly processed

and bioactive TNF-α: TACE 19,20 and MMP-7 21. Western blot analysis showed that TACE

expression was independent of the FANCC status (Figure 3A). On the contrary, MMP-7 was clearly

overexpressed in FA-C cell lines as assessed by Western blot analysis on either whole cell lysates or

supernatant (Figure 3B, upper and lower panel, respectively). Then, the intracellular production and

accumulation of MMP-7 mRNA was analyzed (Figure 3C and 3D). First, lymphoblasts were




10

transfected with a reporter plasmid expressing Firefly luciferase under the control of human MMP-7

promoter (pGL3-hMMP-7) 44. As reported in Figure 3C, relative MMP-7 promoter activity in FA-C

lymphoblasts was higher than in corrected and normal cells. The steady-state level of MMP-7

mRNA in lymphoblasts was analyzed by quantitative RT-PCR. 18S rRNA was used as control gene

for normalization. As shown in Figure 3D and in agreement with our previous data 39, FA-C cells

showed a significant accumulation of MMP-7 mRNA compared to FANCC proficient cells.

To validate the role of MMP-7 in TNF-α secretion in FA, we analyzed TNF-α accumulation

in supernatant of FA-C cells in which MMP-7 activity has been downregulated either by

pharmacological inhibition (Figure 3E) or by knocking-down MMP-7 expression by shRNA

(Figure 3F). As reported, inhibition of MMP-7 activity or protein downregulation by shRNA

significantly reduced by 2 to 3 fold the level of TNF-α in the supernatant of FA-C cells. All

together, these data suggest that MMP-7 overexpression is involved in TNF-α oversecretion in FA-

C cells.

Stress-response pathways are aberrantly activated in FA-C cells.

We next wanted to determine what could be the link between loss of FANCC function and

MMP-7 overexpression / TNF-α oversecretion. Previous studies have implicated two masterpieces

of the stress responses, the NF-κB and MAPK pathways, in MMP-7 transcriptional induction 47-50.

TNF-α is able to activate NF-κB and MAPKs signaling, which in turn could regulate transcription

of genes including TNF-α itself 16,23-25. Consequently, we questioned about the relationship between

the stress-response pathways activity and the TNF-α overproduction in FA.

A constitutive NF-κB activity was already found in SV40-transformed FA fibroblasts 51. To

determine if this is also the case in EBV-transformed lymphoblasts, NF-κB transcriptional activity

was analyzed by transfecting FA-C cells with a luciferase reporter construct containing six NF-κB

consensus sequences (pNF-κB-Luc). As reported in Figure 4A, FA-C cells exhibited a higher level

of basal NF-κB transcriptional activity compared to FANCC proficient cells. Then, we attempted to

evaluate the consequences of the inhibition of NF-κB activity on MMP-7 promoter activity.

Unfortunately, since NF-κB activity is critical for the growth and survival of EBV-transformed B

lymphocytes 52, NF-κB inhibition was lethal impeding the evaluation of the implication of NF-κB

in MMP-7 overexpression in FA-C lymphoblasts.

Successively, we evaluated the basal phosphorylation status of p38, ERK and JNK, three

major players of the MAPKs pathway, in FANCC proficient and deficient lymphoblasts. As

reported in Figure 4B, all the three MAPK pathways appeared to be activated, i.e. phosphorylated in




11

absence of exogeneous stimuli, in FA-C cells compared to healthy donor-derived or corrected cells.

Having determined the spontaneous overactivation of MAPKs in FA-C cells, we sought to

determine if their activation was upstream or a consequence of the TNF-α oversecretion. For this

purpose, we inhibited MAPKs activity using SB203580, PD98059, and SP600125 that are,

respectively, p38, ERK and JNK specific inhibitors. As reported in Figure 4C, each of the MAPKs

inhibitors reduced TNF-α oversecretion in FA-C cells, suggesting that overactivation of MAPK

pathways leads to TNF-α oversecretion in FA-C cells.

FANCC downregulation leads to aberrant activation of stress-response pathways and

MMP-7 overexpression.

Since the observed anomalies in stress-response pathways could be simply related to the

EBV immortalization of B-lymphocytes, we decided to validate our data working with another

cellular model. The transient inhibition of the expression of a target protein by siRNA approach is a

powerful methodology allowing characterization of molecular pathways in heterologous cell

models. Downregulation of FANCC protein in HeLa cells recapitulates the major cellular

characteristics of FA, i.e. absence of FANCD2 monoubiquitination (Figure 5A) and cellular

hypersensitivity to DNA cross-linking agents (data not shown). Knocking-down FANCC

expression in HeLa cells results in 1) a decrease of the NF-κB inhibitor IκB (Figure 5B), 2) the

nuclear translocation of the NF-κB subunits p50 and p65 (Figure 5C), 3) an increased NF-κB

transcriptional activity (Figure 5D) and 4) a spontaneous activation of MAPKs as shown by the

phosphorylation status of p38, ERK and JNK (Figure 5E). All these data, combined with the

previously reported observations, are consistent with a basal overactivation of NF-κB and MAPK

pathways as a consequence of FANCC loss-of-function. Furthermore, a higher production and

intracellular accumulation of MMP-7 mRNA were also observed as evaluated by its promoter

activity and the consequent mRNA accumulation (Figure 5F and G).

HeLa cells used in this study are able to activate NF-κB transcriptional activity in response

to exogenous TNF-α, but were unable to secrete the cytokine after exposure to classical inducers

like phorbol-ester (PMA) or lipopolysaccharide (LPS). This suggests that HeLa cells are

intrinsically unable to produce TNF-α and, consequently, the basal overactivation of NF-κB and

MAPK pathways observed following FANCC downregulation can not be the consequence of TNF-

α production.

In order to determine whether NF-κB and/or MAPK hyperactivity were potentially involved

in FANCC – MMP-7 – TNF-α pathway, we measured MMP-7 promoter activity and mRNA




12

cellular content after inhibition of both NF-κB and MAPK pathways in FANCC-depleted HeLa

cells. For this purpose, we exposed cells to curcumin, a chemical agent able to inhibit both

pathways 53. Firstly, we analyzed MMP-7 promoter activity. Twenty-four hours after FANCC-

siRNA transfection, HeLa cells were transfected with the reporter of MMP-7 promoter, cultured for

24h and then incubated with 50µM of curcumin for 24h before evaluation of the luciferase activity.

As reported in Figure 5H, curcumin reduced MMP-7 promoter activity to basal level. Then, 48h

after FANCC-siRNA transfection, HeLa cells were incubated for 24h with 50µM curcumin before

RNA extraction for quantitative RT-PCR analysis. As shown in Figure 5I, curcumin clearly reduced

the amount of MMP-7 mRNA in FANCC-depleted cells. These results suggest that NF-κB, MAPKs

or both may be implicated in MMP-7 overexpression in FANCC-depleted HeLa cells.

To decipher which target(s) of curcumin is involved, we transfected a dominant-negative

mutant inhibitor of NF-κB activation (pCMV-IκBM) in HeLa cells. Overexpression of IκBM fails

to modify MMP-7 promoter activity whereas NF-κB transcriptional activity was significantly

reduced (data not shown). This demonstrates that NF-κB is not responsible for MMP-7

overexpression in FANCC-depleted HeLa cells. Next, MMP-7 promoter activity was measured in

these cells exposed to MAPK specific inhibitors. Treatment with PD98059 (ERK inhibitor) but not

SB203580 (p38 inhibitor) decreases MMP-7 promoter activity (Figure 5J). Curiously, treatment

with SP600125 (JNK inhibitor) leads to a basal overactivation of MMP-7 promoter activity even in

the siGFP-transfected cells, impeding the analysis of JNK involvement in MMP-7 expression in

HeLa cells (data not shown). Taken together, these results suggest that MMP-7 overexpression in

FANCC-deficient cells is mainly induced via ERK activation.

FA gene mutations lead to MMP-7 overexpression.

In order to generalize our findings, we extended our study to other FA complementation

groups. As it was observed for HSC536 cells, ectopic expression of FANCC in PD4L cells (another

FA-C cell line) or FANCA in HSC72 cells (a FA-A cell line) significantly reduced TNF-α

accumulation in the supernatant (Figure 6A). FA-A, FA-B, FA-C and FA-D1 cells were already

showed as overproducers of TNF-α (36 and Figure 1A). We demonstrated here that FA-F and FA-G

lymphoblasts display a similar characteristic. Indeed, FA-A (HSC99), FA-F (GM16757) and FA-G

(EUFA143) cells accumulated 4 to 10 times more TNF-α in the growth medium than FANC-

proficient lymphoblasts (AHH1) (Figure 6B). In addition, MMP-7 inhibition significantly reduced

the amount of TNF-α in supernatant of FA-A (HSC72 and HSC99), FA-C (PD4L), FA-F

(GM16757) and FA-G (EUFA143) cells as it was observed for the FA-C cell line HSC536 (Figure




13

6C). So, all the FANCcore complex lymphoblasts examined show the same defect in TNF-α

overproduction and MMP-7 overactivation. The consequences of FANCcore complex proteins

depletion on MMP-7 promoter activity and MAPKs network activation were evaluated using HeLa

cells subjected to siRNA-mediated FANCA knock-down. To assess MMP-7 mRNA production,

FANC-siRNA-downregulated cells were transfected with the reporter of MMP-7 promoter and

cultured for 48h before the analysis. MMP-7 promoter activity was clearly upregulated in HeLa

cells depleted for FA proteins (Figure 6D). FANCA-depleted HeLa cells also presented a

spontaneous overactivation of MAPKs as shown by phosphorylation status of p38, ERK and JNK

(Figure 6E).

Taken together, present data indicate that TNF-α overproduction, MMP-7 overexpression

and basal MAPKs overactivation are general features of FANCcore complex depleted cells.




14

DISCUSSION

The aim of this work was to comprehend the origin of the TNF-α overproduction in FA.

Combining several approaches, we demonstrated that FANC pathway loss-of-function induces

elevated NF-κB transcriptional activity, overactivation of all three MAPK pathways,

overexpression of MMP-7 and oversecretion of TNF-α (Figure 7A). Using both molecular and

pharmacological inhibitors, we provided evidence that the ERK pathway is the main player

responsible for the elevated expression of the MMP-7 mRNA in FA, which is the key event linking

FANC mutation to the high TNF-α shedding from the cytoplasmic membrane in FA. Beside this

pathway, TNF-α oversecretion is reduced by p38 and JNK specific inhibitors. This suggests that the

aberrant activation of stress-response pathways participates to the clinical and cellular FA

phenotype acting on multiple targets and pathways. A speculative working model that recapitulates

in a hierarchical order the events induced by FANC loss-of-function is summarized in Figure 7A

and 7B. It is clear that MAPKs and NF-κB stress pathways are involved in the expression of several

myelosuppressive cytokines including interferons, TGF-β, MIP-α and MCP-1 and in this regard the

model in figure 7A is extremely simplified. However, we and others failed to detect the presence of

other cytokine than TNF-α in primary as well as in immortalized FA samples (34,35 and data not

shown), indicating that overexpression of TNF-α is specifically linked to FANC loss-of-function

mediated stress pathways activation

We used two complementary cellular models to perform our analysis: patient-derived

lymphoblasts and HeLa cells in which FANC expression was downregulated using siRNA

technology. Comparing FA-C lymphoblasts to their isogenic ectopically corrected counterpart, we

showed that wild-type FANCC cDNA is able to normalize TNF-α overproduction in FA-C cells,

demonstrating that TNF-α overproduction is directly linked to FANCC mutation. Same results were

observed with a FA-A cell line pair suggesting that at least FANCcore complex loss-of-function

leads to TNF-α overproduction. Although a slight increase in TNF-α mRNA

transcription/stabilization in FA-C cells was observed, it seems devoid of major biological

significance since we failed to observe any differences in protein synthesis between FANC-mutated

and wild-type cells. Instead, we demonstrated that the extracellular accumulation of the cytokine in

FA cells is the results of a high rate of cleavage from the cell membrane.

TNF-α shedding is mainly related to TACE activity 19,20. Among other proteases able to

cleave membrane-anchored TNF-α 21, MMP-7 is the only one known to correctly cleave TNF-α

precursor 22. It has been proposed that TACE and MMP-7 are part of independent mechanisms by

which TNF-α could be shed from the cell surface. TACE is involved in inducible release such as in




15

response to PMA or LPS treatment, while MMP-7 is involved in the constitutive release of the

cytokine 54,55. In line with these data, MMP-7 is overexpressed at both mRNA and protein level in

FA-C cells and its overexpression was obtained in HeLa cells by siRNA-mediated depletion of

FANCC. Furthermore, the pharmacological inhibition of MMP-7 activity as well as the

downregulation of its expression by specific shRNA significantly reduced the cytokine release in

the supernatant of FA-C cells. The key role of MMP-7 in TNF-α overproduction is also observed in

FA-A, FA-F and FA-G cells, suggesting that the aberrant expression of MMP-7 is a general

characteristic of FA. These results provide the first evidence of the mechanism involved in the

overproduction of TNF-α in FA syndrome.

Exposure of cells to TNF-α activates several signaling pathways, including NF-κB and

MAPK networks. Inversely, activation of both NF-κB and MAPKs plays an important role in the

induction of many cytokines, including TNF-α itself 16,23-25. Overactivation of NF-κB activity was

previously reported in SV40-transformed FA fibroblasts 51. Here we demonstrated that EBV-

transformed FA lymphoblastoid cells are also characterized by an increased NF-κB and MAPKs

activity and these phenotypic modifications have been reproduced in FANCC and FANCA siRNA-

depleted HeLa cells. Since these cells are intrinsically unable to secrete TNF-α, NF-κB and

MAPKs activation is not due to TNF-α overproduction but rather linked to FANC depletion. In line

with this data, using specific inhibitors of MAPKs in lymphoblasts and in FANC-depleted HeLa

cells, evidences are provided that the FANC-dependent TNF-α overproduction and MMP-7

overexpression are dependent on MAPK activity. These data do not exclude that TNF-α may still

play a role in maintaining or in exacerbating NF-κB and/or MAPK activity in FA cells.

Observations described in this study corroborate data from others published works and bring

arguments allowing to propose a functional connection between the TNF-α and the FANC

pathways. On one hand, the myelosuppresive cytokine TNF-α is increased in BM and serum of FA

patients, as well as in culture medium from EBV-transformed FA lymphoblasts 34-36 as a

consequence of MMP-7 induction (this work). Inversely, TNF-α acts on the FANC pathway in

normal cells. Indeed, the expression of FANCG and FANCA and the FANCA/FANCG complex in

the nucleus increase after TNF-α treatment in an NF-κB dependent-manner 56. In contrast to

FANCG, the amount of FANCC associated with FANCA is reduced following treatment with TNF-

α 57. Interestingly, in Fancc-null mice, that do not show spontaneously hematological

abnormalities58-61, TNF-α oversecretion was not observed 38. Recently, Pang and collaborators,

using different approaches in Fancc-/- mice, including treatment with LPS or TNF-α, reported that




16

the cytokine, besides its negative action on mice BM development, is also implicated in

overactivation of p38 and JNK, phosphorylation of p53 and H2AX, induced DNA damage,

chromosome aberrations and apoptosis 62,63. All these characteristics are shared by human FA cells

(64, this paper and unpublished data). Moreover, in agreement with a role of TNF-α in FA genetic

instability, our previous data have demonstrated that TNF-α inhibition partially rescues FA

hypersensitivity to cross-linking agents treatment at both cellular and chromosomal level 36.

Altogether, these data strongly suggest that TNF-α could play a role in both the BM failure and the

cellular and chromosomal hypersensitivity to DNA damage observed in FA.

FANC proteins play a role in defining cellular tolerance to cross-linking agents via their

function in DNA repair/cell cycle checkpoint 65,66. However, they are also functionally or

biochemically associated with several other proteins not involved in the enzymology of DNA

repair, including several redox regulators: NADPH cytochrome-P450 reductase, glutathione S-

transferase P1-1 and the peroxiredoxin-3 67-70. The reported interactions with redox regulators are

involved in both the pro-oxidant state and the oxygen hypersensitivity that characterize the FA

cells14. Both oxidative stress and impaired DNA repair may contribute to the activation of the ATM

pathway recently reported in FA 64. Interestingly, it has been described that both ATM and reactive

oxygen species (ROS) may induce NF-κB and MAPKs 71,72. Consequently, we speculate (Figure

7B) that FA cells suffer of a permanent stress due to both intracellular ROS increase and

accumulation of endogenous DNA damage that leads to MAPK and NF-κB signaling activation.

MAPKs activation, in turn, contributes, through the alteration of the MMP-7 expression, to the

TNF-α overproduction. Since TNF-α is able to activate NF-κB and MAPKs, these factors form an

autocrine loop that results in the escalation of their own levels and, consequently, of the severity of

the pathogenesis. In accord with this, it has been shown that NF-κB and MAPKs overactivation is

causative of BM failure, MDS and leukemia 73-75 and that MMP-7 overexpression promotes

leukemia, facilitating migration and invasion of the leukemic cells 76. Importantly, from a clinical

point of view, NF-κB and MAPK inhibition was reported as beneficial in BM failure and

leukemia77-79.

Currently bone marrow transplantation is the best treatment available for the hematopoietic

manifestations of FA. Our data provide a strong rationale for clinical strategies based on the use of

pharmacological approaches to block stress-response pathways and/or TNF-α activity or shedding

in FA patients.




17

ACKNOWLEDGMENTS

We thank J. Grosjean, G. Cherubini and J.H. Guervilly for helpful discussion and J.A.

Dugas Du Villard, Y. Lecluse, S. Queille and J. Abdelali for excellent technical assistance. We also

thank the following colleagues and organization for kindly sharing material: Dr. G.C. Bagby, Dr.

J.P. Bidwell, Dr. C. Dufour, Dr. L. Matrisian, Dr. D. Schindler, Dr. J. Surrallés, Dr. D. Trono, Dr. I.

Udalova and FARF association. This work was supported by grant from La Ligue contre le Cancer

(Equipe labellisée 2006). D. Briot was a fellow of Ministere de l’Enseignement Supérieur et de la

Recherche (France) and Association pour la Recherche sur le Cancer, France.

Author contribution statement :

D. Briot performed research, analyzed data and wrote the manuscript.

G. Macé-Aimé performed research.

F. Subra made lentiviral construction, production and transduction.

F. Rosselli supervised the research and wrote the manuscript.

The authors declare no competing financial interests.




18

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22

LEGENDS Figure 1. Overproduction of biologically active TNF-α by FA-C cells.

(A) TNF-α accumulation in the supernatant of normal (HSC93), 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 (3x105 cells/ml/well in three

ml of medium in six-well tissue culture plates). Supernatants were collected 24h and 48h after sub-

culturing. The level of TNF-α in supernatants was determined by ELISA. Data presented are the

mean +/- SD of at least five independent experiments. ∗, p < 0,01

(B) Luciferase activity in 293T/NF-κB-Luc cells cultured in conditioned medium from

lymphoblasts. 293T/NF-κB-Luc cells were plated (1x106 cells/well in six-well tissue culture plates)

48h before treatment with supernatant collected from 48h-old lymphoblasts cultures. One volume of

supernatant, equivalent to 1x106 lymphoblasts, was added per well of 293T/NF-κB-Luc cells. Cells

were harvested 24h after treatment for luciferase measurement. Luciferase activity of 293T/NF-κB-

Luc cells cultures in supernatant from HSC536Corr cells was considered equal to 1 in each

experiment. Data presented are the mean +/- SD of at least three independent experiments done in

triplicate. ∗, p < 0,05

Figure 2. Analysis of TNF-α mRNA, protein expression and secretion.

(A) TNF-α promoter activity in FA and corrected lymphoblasts. Cells were co-transfected 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 three independent experiments done in triplicate. ∗, p < 0,01

(B) Steady-state level of TNF-α mRNA in FA and corrected lymphoblasts assessed by semi-

quantitative RT-PCR. The histogram represents the relative level of TNF-α mRNA in FA-C

deficient compared to FANCC-corrected cells. The level in HSC536Corr was considered equal to 1

in each experiment. Data presented are the mean +/- SD of at least three independent experiments

done in triplicate. ∗, p < 0,05

(C) Immunoblot showing the level of TNF-α in total cell extracts prepared from HSC536 cell line

either untreated (Unt.) or treated for 18h with brefeldin A (Bref. A) or dexamethasone (Dexa.). A

crossreactive 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 (10µg/ml) for 3h. After this incubation, cells were collected by centrifugation,




23

washed and resuspended in fresh medium in presence of brefeldin A (1µg/ml) during 24h. Proteins

were extracted at the indicated time points. TNF-α content was measured by ELISA. Each point

represents the mean +/- SD of at least three independent experiments.

(E) Kinetics of TNF-α accumulation in HSC93, HSC536Corr, HSC536 and HSC536Neo cell lines.

Supernatants were collected 3h, 6h and 9h after cell sub-culturing in fresh medium and cytokine

release was determined by ELISA. Each point represents the mean +/- SD of at least three

independent experiments. ∗, p < 0,01

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 (upper panel) and in supernatant

(lower 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 co-transfected 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 three

independent experiments done in triplicate. ∗, p < 0,05

(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 three independent experiments done

in triplicate. ∗, p < 0,05

(E) TNF-α release in MMP-7 inhibited FA-C cells. HSC536 and HSC536Neo cells were treated

with MMP inhibitor (50µM), its solvent (DMSO) or left untreated. Supernatants were collected 24h

after sub-culturing and cytokine release was determined by ELISA. Data presented are the mean +/-

SD of at least three independent experiments. ∗, p < 0,01

(F) TNF-α release in FA-C cells after the downregulation of MMP-7 expression. Immunoblot

shows the level of MMP-7 in supernatant of FANCC siRNA-MMP-7 downregulated cells. A cross-




24

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) +/- SD of TNF-α accumulation observed in at least three independent experiments. ∗, p

< 0,01

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 48h 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 three 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, 10µM), PD98059 (ERK inhibitor, 30µM) and SP600125

(JNK inhibitor, 25µM). Supernatants were collected 24h after sub-culturing and cytokine release

was determined by ELISA. Data presented are the mean +/- SD of at least three independent

experiments. ∗, p < 0,01.

Figure 5. FANCC downregulation 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. 48h

after siRNA transfection, cells were treated with mitomycin C (MMC) (500ng/ml) or 8-

methoxypsoralen (10-5M) +UVA (10kJ/m2) (8-MOP) and lysed 24h later. FANCC downregulation

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 for FANCC during 3 days. Actin was

used as a loading control.

(C) Detection of p50 and p65 sub-cellular localization by confocal immunofluorescence

microscopy. Hela cells treated with FANCC siRNA were subjected to immunostaining of p50

(green fluorescence) and p65 (red fluorescence) 72h after transfection.

(D) NF-κB transcriptional activity in HeLa cells depleted for FANCC. Twenty-four hours after

siRNA treatment, cells were co-transfected with the reporter pNF-κB-Luc and phRL-TK for 48h

before the analysis of the luciferase activity. Luciferase activity in GFP-siRNA transfected cells was




25

considered equal to 1 in each experiment. Data presented are the mean +/- SD of at least three

independent experiments done in triplicate. ∗, p < 0,05

(E) Immunoblot showing the level of phosphorylation of MAPK players in cells with a reduced

FANCC expression compared to mock transfected and unperturbated cells. Cells were collected 72h

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

fours after siRNA treatment, cells were co-transfected with the reporter pGL3-hMMP-7 and phRL-

TK and induced luciferase activity was measured 24h 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 three independent experiments done in triplicate. ∗, p < 0,01

(G) MMP-7 mRNA steady-state level in FANCC deprived or unperturbed HeLa cells were

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 three independent experiments done in triplicate. ∗, p < 0,01

(H) MMP-7 promoter activity in 50µM curcumin-treated FANCC-deprived cells assessed by

luciferase activity as in F. ∗, p < 0,01

(I) MMP-7 mRNA steaty-state level in FANCC-deprived HeLa cells treated with curcumin (50µM)

as assessed by quantitative RT-PCR as in G. ∗, p < 0,01

(J) MMP-7 promoter activity in siRNA FANCC-transfected HeLa cells treated with SB203580

(p38 inhibitor, 10µM) and PD98059 (ERK inhibitor, 30µM) for 18h. ∗, p < 0,05 compared with

DMSO treatment.

Figure 6. FA gene mutation leads to MMP-7 overexpression and MAPK pathways activation.

(A) TNF-α accumulation in the supernatant of AHH1 (normal), PD4L (FA-C), PD4L/FANCC (FA-

C corrected), HSC72 (FA-A), HSC72/FANCA (FA-A corrected) lymphoblasts. Supernatants were

collected as described in Figure 1A. Data presented are the mean +/- SD of at least three

independent experiments. ∗, p < 0,01 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 three independent experiments. ∗, p < 0,01




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(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 three independent experiments. ∗, p < 0,01

(D) MMP-7 promoter activity in siRNA FANC-transfected HeLa cells. Cells were co-transfected

with the reporter pGL3-hMMP-7 and phRL-TK and induced luciferase activity was measured 24h

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 three independent experiments done in

triplicate. ∗, p < 0,01

(E) Western blot analysis of MAPKs phosphorylation in siRNA FANC-transfected HeLa cells.

Cells were collected 72h 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 text).




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

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