In Vivo Localization of Fas-Associated Death Domain Protein in the Nucleus and Cytoplasm of Normal Thyroid and Liver Cells
IIn vivo localization of FADD Tourneur et al. Letter
In vivo localization of Fas-associated death domain protein in the nucleus and cytoplasm of normal thyroid and liver cells
Tourneur, Léa , Alain Schmitt , and Gilles Chiocchia
1, 2, 3
From Inserm, U567, Institut Cochin, Paris, France . Université Paris Descartes, CNRS (UMR 8104), Paris, France Service de rhumatologie, Hôpital Ambroise Paré, Boulogne, France . Running title : In vivo localization of FADD Key words: Fas-associated death domain; Thyroid; Cellular localization; Nucleus; MBD4 Address all correspondence and requests for reprints to: Dr. Gilles Chiocchia, Institut Cochin, Département d'Immunologie, INSERM U 567, 27 rue du Faubourg Saint-Jacques, 75674 Paris Cedex 14, France. Phone: (33) 1 40 51 66 15. Fax: (33) 1 40 51 66 41. E-mail: [email protected]. n vivo localization of FADD Tourneur et al. Abstract
FADD (Fas-associated death domain) is the main death receptor adaptor molecule that transmits apoptotic signal. Recently, FADD protein was shown to be expressed both in the cytoplasm and nucleus of in vitro cell lines. In contrast to the cytoplasmic FADD, the nuclear FADD was shown to protect cells from apoptosis. However, in vivo subcellular localization of FADD was still unknown. Here, we demonstrated that FADD protein was expressed in both cytoplasmic and nuclear compartment in ex vivo thyroid cells demonstrating that nuclear sublocalization of FADD protein was a relevant phenomenon occurring in vivo. Moreover, we showed that in the nucleus of untransformed thyroid cells FADD localized mainly on euchromatin. We confirmed the nuclear localization of FADD in ex vivo liver and showed that in this organ FADD and MBD4 interact together. These results demonstrate that FADD is physiologically expressed in the nucleus of cells in at least two mouse organs. This particular localization opens new possible role of FADD in vivo either as an inhibitor of cell death, or as a transcription factor, or as a molecular link between apoptosis and genome surveillance. n vivo localization of FADD Tourneur et al. Introduction
Normal thyrocytes constitutively express the Fas death receptor but not the Fas ligand (FasL) [1-3]. However, FasL expression in thyroid follicular cells (TFC) can occur under numerous thyroid pathological conditions, including autoimmune diseases and cancer [4-6]. For instance, FasL expression has been reported in TFC from patients suffering of Graves' disease [7] and Hashimoto's thyroiditis [8], the two main thyroid autoimmune disorders in humans, and thyroid carcinomas [6]. In these pathologies, thyrocytes appear to be relatively insensitive to Fas-mediated cell death although expressed FasL is functional [9-12]. Reasons for such phenomenon are not clearly established, and probably involve several mechanisms such as expression of FLIP (FLICE-inhibitory protein) and Bcl-xL anti-apoptotic molecules [11], or loss of FADD (Fas-associated death domain) pro-apoptotic protein [13]. Interestingly, normal TFC are naturally resistant to Fas-induced apoptosis suggesting that the observed high insensitivity of pathological thyrocytes could result from an intrinsic property of normal thyroid cells. Since FADD physically interacts with death receptors located at the cell membrane, FADD protein was thought to be mainly cytoplasmic.
Recently, we reported by mean of an in vitro organ culture the existence of a new regulatory mechanism of FADD protein expression following adenosine receptor signaling [14].
Furthermore, the human FADD protein contains both a nuclear export and a nuclear localization sequence which account for FADD localization both in the cytoplasm and the nucleus, respectively [15]. Whereas cytoplasmic FADD possesses pro-apoptotic functions, it was recently reported that FADD expression in the nucleus protects cells from apoptosis [15]. The mechanism implicated in this survival function has not been investigated. As dogmas can be challenged, FADD nuclear sublocalization has been debated. It was found that FADD primarily resided in the nucleus of cells and thereafter shuttled from the nucleus to the cytoplasm [16]. In contrast, other report showed that FADD was exclusively n vivo localization of FADD Tourneur et al.
Materials and methods
Mice Different strains of mice (CBA/J, DBA/1, DBA/2, C57BL/6 or BALB/c mice) (Iffa Credo, L’Arbresle, France, and Harlan Olac, Bicester, GB) were used at 7-15 weeks of age. All mice were maintained in standard environmental conditions, and allowed to adapt to their environment at least for one week before the experiments. The studies were approved by the Cochin institute committee on animal care. Agreement number to perform experiments on living animals: n° 75-777, and animal facility agreement number n° 3991. n vivo localization of FADD Tourneur et al. Immunofluorescence
Animals were sacrificed and the two thyroid lobes removed. Collected lobes were immediately covered in optimal temperature medium (Tissue-Tek; Bayer, Elkhart, IN), slowly frozen by floating in isopentane on liquid nitrogen, and stored at -80°C until use. Sections of 5–6 µ m were cut on a cryostat at -18°C and collected onto SuperFrostplus slides (Roth Sochiel, Lauterbourg, France). Sections were dried overnight and stored at -80°C until use. Before staining, sections were fixed for 15 min in PBS with 2% paraformaldehyde (PFA) at 4°C, and incubated for 30 min in PBS with 2% bovine serum albumin. Then, sections were stained (60 min) with primary goat polyclonal IgG anti-mouse FADD antibody (10 µ g/ml, clone M19, Santa Cruz Biotechnology, TebuBio, Le Perray en Yvelines, France) or with isotype-matched control antibody at the same concentration (Vector Laboratories, AbCys, Paris, France). The secondary biotin-conjugated anti-goat IgG antibody (Vector Laboratories) was used at 1 µ g/ml (30 min). Alexa Fluor
488 conjugate streptavidin (10 µ g/ml, Molecular Probes Inc., Interchim, Montluçon, France) was used to visualize specific staining (30 min incubation at room temperature and protected from light). After washings in PBS, sections were mounted in VECTASHIELD Mounting Medium with DAPI (4’,6-diamidino-2-phenylindole) (Vector Laboratories) to counter-stain DNA. Cells were analyzed by confocal fluorescence microscopy (Bio-Rad
MRC1024, Bio-Rad Laboratories) equipped with a digital Diaphot 300 system. Digital pictures were analyzed using
LaserSharp software and processed using Adobe Photoshop . Immunogold electron microscopy
Thyroid lobes were fixed in 1% glutaraldehyde in 0.1 M phosphate buffer pH 7.4, then embedded in sucrose and frozen in liquid nitrogen. Cryosections were made using an ultracryomicrotome (Reichert Ultracut S.) and ultrathin sections mounted on Formvar-coated n vivo localization of FADD Tourneur et al. µ g/ml in PBS 15% glycine, 0.1% BSA, 4% normal donkey serum. After extensive rinsing in PBS 15% glycine, 0.1% BSA, sections were incubated for 1 h with gold-labeled secondary rabbit anti-goat antibody with a gold particle size of 10 nm (GAM 10, British Biocell, Cardiff, Wales). Sections were then washed for 30 min with PBS 15% glycine, stained with 2% uranyl acetate for 10 min and air dried. Examination was performed with a Philips CM 10 electron microscope. Preparation of protein extracts
Proteins of ex vivo mouse liver were extracted with the Nuclear Extract Kit (Active Motif, Europe, Rixensarf, Belgium) following manufacturer’s instructions. Sample concentration was determined using micro BCA protein assay reagent kit (Pierce , Rockford, IL, USA).
Immunoprecipitation
The nuclear extracts were precleared with proteins G sepharose (P-3296, Sigma-Aldrich chimie SARL, Saint Quentin Fallavier, France) on rocking for 1 h at 4°C. Then, 1 µ g of either rabbit polyclonal anti-FADD (AB3102, Chemicon International, Temecula, CA, USA), or rabbit polyclonal anti-MBD4 (ab3756, abcam,Cambridge, United Kingdom), or isotype-matched control antibody (normal rabbit IgG, Santa Cruz Biotechnology) was added at the precleared nuclear extracts, and the mix were incubated for 2 h on ice. Thereafter, 16 µ g of clear proteins G sepharose was added. After an additional 2 h on rotating wheel at 4ºC, precipitates were washed five times with 500 µ l of lysis buffer (10 mM Tris HCl pH 7.8, 150 n vivo localization of FADD Tourneur et al. Western Blot µ g of cytoplasmic or nuclear proteins, and the immunoprecipitates were diluted in reducing sample buffer, subjected to 15 % SDS-PAGE, transferred to PVDF membrane (NEN Life Sciences, Boston, Massachusetts), and probed with specific primary anti-FADD antibody (M-19, 0.2 µ g/ml in TTBS 0.1% containing 5% milk) (Santa Cruz Biotechnology) following by peroxidase-conjugated anti-goat IgG (0.66 µ g/ml) secondary antibody (Sigma-Aldrich). Proteins were visualized using the enhanced chemiluminescence technique (Amersham Pharmacia Biotech, Orsay, France). Bands obtained were quantified by densitometry using biocapt and bio-profil bio1d software. The same amount of nuclear proteins and cytoplasmic proteins were loaded on gel although the total amount of nuclear protein was five to ten times lesser than the amount of cytoplasmic proteins. Results and discussion
Animals were sacrificed and the thyroid removed. The two thyroid lobes were either immediately covered in optimal temperature embedding medium for immunofluorescence analysis (Fig. 1), or immediately fixed with glutaraldehyde for immunogold electron microscopy (Fig. 2). To avoid the problems associated with permeabilisation reagents such as saponin [18, 19], we performed thyroid cryosections that we immunolabeled with the previously characterized specific anti-mouse FADD antibody [13]. n vivo localization of FADD Tourneur et al.
8 Confirming our previous study [13], confocal immunofluorescence microscopy analysis showed that FADD protein was expressed in the cytoplasm of all TFC (Figure 1 A, E, G), and in all thyroid examined independently of the genetic background of the mice (data not shown). Moreover, we observed that FADD was additionally localized in the nucleus of some, but not all, TFC (Fig. 1 A, B, E-G). In contrast, omission of the primary antibody or staining with an isotype-matched control antibody showed no positive reactivity in the nucleus (Fig. 1 C, D, H-J, and data not shown). To confirm these results, and to localize more precisely the FADD protein in TFC, we used immunogold electron microscopy. This method allowed us to detect FADD protein both in the cytoplasm and in the nucleus of TFC (Fig. 2 A and B), confirming the results obtained by confocal microscopy. Furthermore, the electron microscopy technique showed that FADD was expressed in the nuclear compartment of all the TFC examined (data not shown). The different cell-types in tissue samples provided useful internal comparators, when assessing FADD immunolabelling. We found that FADD protein was not, or barely detected in the nucleus of endothelial cells from blood vessel which are localized between thyroid follicles (Fig. 2 E and F). Since FADD was present in the nucleus of ex vivo mouse thyroid cells, our data demonstrated that nuclear localization of FADD protein was not an in vitro culture cell line dependent event. However, the TFC restricted pattern of expression of nuclear FADD protein in thyroid gland suggested that nuclear sublocalization of this protein may not be a common feature and may depend on cell type. Finally, we confirmed that FADD could be expressed in the nucleus of cells in vivo using a third approach which is cellular fractionation. Because of the large amount of protein needed for such technic, we used proteins of ex vivo mouse liver instead of thyroid. We chose liver to compare with thyroid because both hepatocytes and thyrocytes are epithelial cells. Whatever the amounts of protein extracts loaded on SDS-PAGE, we detected FADD expression both in the cytoplasmic and n vivo localization of FADD Tourneur et al. et al. et al. et al. Acknowledgements
The authors would like to thank S Mistou for his great assistance with cell culture and protein studies and M. Garfa for his excellent technical assistance with confocal fluorescence microscopy. They are also indebted to F. Lager for help with animal care. They are grateful to C. Fournier for critical reading of the manuscript. Léa Tourneur was a recipient of a “Société Française d’Hématologie” (SFH) post-doctoral training fellowship. This work was supported by the Institut National de la Santé Et de la Recherche Médicale (INSERM) and by Ligue Nationale Contre le Cancer-Comité de Paris. n vivo localization of FADD Tourneur et al. References [1] Arscott PL, Knapp J, Rymaszewski M, Bartron JL, Bretz JD, Thompson NW, et al. Fas (APO-1, CD95)-mediated apoptosis in thyroid cells is regulated by a labile protein inhibitor. Endocrinology1997;138: 5019-27. [2] Tourneur L, Malassagne B, Batteux F, Fabre M, Mistou S, Lallemand E, et al. 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Death-effector filaments: novel cytoplasmic structures that recruit caspases and trigger apoptosis. J Cell Biol1998;141: 1243-53. [24] Nagata S. Apoptosis by death factor. Cell1997;88: 355-65. [25] Yeh WC, Pompa JL, McCurrach ME, Shu HB, Elia AJ, Shahinian A, et al. FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science1998;279: 1954-8. [26] Hendrich B, Hardeland U, Ng HH, Jiricny J, Bird A. The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites. Nature1999;401: 301-4. n vivo localization of FADD Tourneur et al. Figures Legend
Fig. 1. Analysis of FADD subcellular localization by immunofluorescence confocal microscopy. (A, E) E x vivo thyroid sections were stained with anti-FADD antibody (green fluorescence). (C, H) Staining of thyroid sections with Alexa Fluor
488 anti-goat antibody alone showed no positive reactivity. The same result was obtained using isotype-matched control antibody (data not shown). (B, D, F, I) Nucleus of TFC were counter-stained with DAPI (blue fluorescence). (G, J) FADD immunolabelling and DAPI merge. Co: colloid; Cy: cytoplasm; N: nucleus. Bars indicate scale. Fig. 2. Analysis of FADD subcellular localization by immunogold electron microscopy. FADD (indicated by arrowheads) is found both in the cytoplasm and the nucleus of TFC (A-D) but not in the nucleus of endothelial cells from blood vessel (E, F). C, D, and F represent higher magnification of B and E, respectively (see framed). Staining with secondary anti-goat antibody alone or isotype-matched control antibody showed no immunogold deposition (data not shown). Co: colloid; Cy: cytoplasm; N: nucleus; RBC: red blood cell. Bars indicate scale. Fig. 3. Analysis of FADD subcellular localization by western blot after cellular fractionation. (A) Western blot was performed using different amount of protein extracts from ex vivo liver, as noted in the figure. (B) Histogram represents quantification of bands obtained in (A). Results are expressed in arbitrary units. Full and empty histograms represent cytoplasmic and nuclear fractions, respectively. Fig. 4. FADD interacts with MBD4 in vivo. (A) Western blot analysis of FADD in the cytoplasm (C) and nucleus (N) of ex vivo liver. Nucleus lysates was immunoprecipitated (IP) n vivo localization of FADD Tourneur et al. µ g. Results are expressed in arbitrary units. DAPIFADDa Control DAPId c d DAPIFADD Mergee f g
Control DAPI Mergeh i j
Figure 1
Figure 1 cCy
200 nm dN d
200 nm 200 nm e fNc b Cy fN RBCRBC fCy Nc d
Figure 2