Fas Associated Death Domain

Published Date: 02 Nov 2017

Rydant Michiel I, Prof. Geert van LooII

I Master student, Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical Research, Flanders Interuniversity, Institute for Biotechnology, University of Ghent, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium

II Senior Staff Scientist,VIB Department for Moleculair Biomediical Reasearch , UGent,Technologiepark 927,9052 Ghent,Belgium

Abstract: Apoptosis is the process of programmed cell death that occurs in the whole body. Understanding of how apoptosis arise, are the first steps to cure cancers. FADD has been suggested to been playing a crucial role in apoptosis. To study the mechanism behind apoptosis modified mice where used. Mice with a knockout for the FADD allel in the liver were tested if there were susceptible for liver apoptosis. Survival genes were inhibited so the only possible reaction way was the induction of apoptosis. Mice without the FADD allele did not shown apoptosis. This confirm the suggestion that FADD plays a crucial role in apoptosis.

Introduction

An accurate balance between cell proliferation and cell death by apoptosis is essential in living cells. The unbalance can lead to liver damage (by cell death) or cancer (by cell proliferation)(1). It has been shown that tumor necrosis factor (TNF) plays a crucial role in the balance between those two processes(2).

The process of apoptosis can happen both intrinsic and extrinsic. The intrinsic pathway is triggered by internal damage or deficiency of growth factors whereby the mitochondria plays a crucial role. The extrinsic apoptotic pathway occurs at the plasma membrane and contains Death Receptors (DR) and adapter molecules such as Fas Associated Death Domain (FADD)(3). Both playing an important role in receiving and passing on incentives. In this paper we examined whether FADD is indeed an essential protein in the pathway of apoptosis (fig.1). Both the in- and extrinsic pathways require caspases to guide the signalization pathway to a good end. Caspases 3 and 6 are typical effector/execution caspases. Effector caspases are activated by the initiator caspases. Execution caspases cut up numerous protein substrate(3).

In this paper we will examine whether caspases are present under different conditions. Thus allows us to determine whether or not the apoptosis pathway is activated.

Figure 1 - Signalization pathway Of Tumor necrosis factor (TNF) Activation of the TNF receptor (TNFR) by the TNF ligand leads to the recruitment of various proteins with a multiprotein-pathway as a result. Depending on the cellular context, two different signaling pathways can be monitored which lead to apoptosis or survival and inflammation. TNF binding to TNFR1/2 induces a range of inflammatory mediators and growth factors through activation of the AP1 transcription factor or IκB kinase (IKK) that, in turn, activate nuclear factor-κB (NF-κB) resulting in the standard pathway, that is, induction of the genes that promote inflammation and survival. However if NF-κB activation is inadequate, apoptosis is mediated through FAS-associated death domain (FADD) and caspase 8 and also the accumulation of intracellular reactive oxygen, Junamino-terminal kinase (JNK). Other compounts: TRADD,TNFR-associated death domain; TRAF2,TNF receptor-associated factor 2; RIP, receptor (TNFRSF)-interacting; MEKK; Mitogen-activated Protein/ERK Kinase Kinases; MKK, MAPK kinase (Based on Balkwill, Frances R., 1992)

TNF - In the liver and Kupffer cells

At the same time TNF plays an essential role in the liver(2). In nearly all liver diseases, an imbalance occur between cell damage and cell death. When inflammation occurs in the liver, this is accompanied by an increase in cytokines, such as TNF, IL1 and IFN(4).

Hepatocytes contains various receptors such as Toll-like receptor 4 (TLR4). This receptor is capable to recognize endotoxin lipopolysaccharide (LPS). Binding of LPS has the same effects as binding TNF. For economic reasons, we will work with LPS. In healthy mice Galactosamine (GalN) will be added,

GalN is a hepatoxine that inhibits transcription in hepatocyte(5). Consequently, the survival way trough nuclear factor kappa B (NfκB) is blocked and only apoptosis will occur (fig.1). LPS/GalN caused inflammatory hepatitis which will result in massive cell death(2). Conditioned mice who lacks FADD protein could survive when LPS/GalN treated. This will be investigated and so we can determine whether FADD plays a crucial role(3).

Material and methods

Testing animals

Conditioned KO mice were created in the "Department for Molecular Biomedical Research, VIB/Ghent University '. Induced mice were injected intraperitoneally with LPS (100 ng/mouse) and Galactosamine (GalN) (20 mg/mouse). Because the addition of TNF both induce apoptosis and the survival genes we added GalN so. This hepatoxine blocks the transcription in hepatocytes so only the apoptosis pathway will be examined(6).

Mouse Tail fragments and liver extracts were kindly provided by Professor Geert van LooII.

PCR genotyping

To make sure the provided mice lack the FADD allel in the liver. PCR genotyping was done.The four different mice tail fragments (9701, 9699, 9703, 9704) were separately lysed with 500 µl DNA lysis buffer and 20 µl proteinase K. DNA lysis buffer consists of: 100mM Tris-HCl (pH 8.5), 5 mM EDTA, 200 mM NaCl and 0.2% SDS. Proteinase K, a broad-range serine protease dissolve residual mouse hair (keratin). The mice tails were incubated at 56 ° C until they were completely lysed. After this, the supernatant was removed and transferred to new eppendorfs. Subsequently, to each eppendorfs 500 µl of isopropanol was added and it was spun for 5 minutes at 20800x g , so that the DNA precipitated. The supernatant was removed and the pellet was washed with 70% ethanol to then be evaporated to dryness. afterwards Resuspension of the pellet in 100 ul bidi happened to be followed by a PCR applied on the four samples for the detection of the absence of the FADD allel. The PCR mix consist of: 2 µl DNA extract, 3 µl 10x DNA Running Buffer, 0.3 µl of 20 mM dNTPs, 0.1 µl Left Primer, 0.1 ul Right Primer, 0, 1 µl Mid Primer , 0.9 µl of 25 mM MgCl2, 0.3 µl 5 U/µl TAQ polymerase, and 23.4 µl Bidi. The total volume was 30 µl. Consequently from each sample 20 µl was loaded on a 2% agarose gel. To each gel 5 µl CyberSafe(Invitrogen) was added. This product intercalates with DNA and will be visible under the UV- light. The gel was run for 30 minutes at 100 volts.

Treatment liver extracts

Liver extract were taken from the mice after death. 1 ml of phosphate buffered saline (PBS) was added to the four different liver extracts (1, 2, 3, and 4) while working on ice. Working on ice is recommended during the entire experiment so the protease activity is minimised. All liver extracts were homogenised using a douncer ultil a cloudy substance was optained. The samples were then centrifuged for 5 minutes at 5000xg. The supernatant was removed and 500 µl of caspase lysis buffer was added. Caspase lysis buffer consisting of: 1% Nonidet P-40 (non-ionic detergent), 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0,3 mM aproprotin, 1 mM leupeptin. After a resting periode of 10 minutes, samples were centrifuged for 10 minutes at 20.000x g

The supernatans, containing the lysate was split into two eppendorfs, respectively, 200 µl and 270µ. The eppendorf with 270 µl was used for the DEVD cutting assay. The eppendorfs which contain 200 µl supernatant were treated with 50 µl 5x laemlli solution. Finally, those samples were boiled for 5 min at 95 ° C, in such a way the proteins are fully denatured. These samples were used for Western Blot analysis.

Sample 1 and 2 were not treated with LPS/GALN

Sample 3 and 4 were treated with LPS/GALN

Western blot analysis

Of each sample 50 µg proteins were used for the Western blot analysis. Each sample thus contained an equal concentration of proteins. This is essential for standardization.

27 µl DNA extract was loaded per sample Next, they were loaded onto a 15% SDS-PAGE stacking gel. The gel ran for 45min at 200 V .

Fig. 2: FADD Genotyping. Samples of 4 mice were loaded on a 2% agarosegel. Control contain a heterozygous mice(KO/WT). Kock-Out(KO) is 280bp, Wild Type(WT) 208bp long. If mice are homozygous only 1 band appears, if not 2. Subsequently, semi-dry blotting was performed, for 30 minutes at 120 mA. The blot was then immersed in Ponceau reagent. This allows us a rapid visualization of the proteins. The blot was washed with small amount of PBS and Tween (0.15%) until ponceau reagens was washed away. Next, the blot was treated with blocking buffer. Blocking buffer containing : 94.9%PBS, and 5% milk powder and 0.1% Tween).

Hereafter the primary antibody (anti-caspase-3) was added. After 2 days blot were washed 3 times 10 minutes with PBS. To continue, the secondary antibody (anti-rabbit-HRP) was added.

In the presence of luminol Horse Radish Peroxidase (HRP) transmits light that is detectable so we can easely find our protein of interest. Finally, the blots were incubated for about an hour. After this, the blots were washed again and developed using Enhanced Chemiluminescence (ECL) myECL Imager,thermo scientific.

Results

PCR genotypering

Using PCR we make sure that the mice were homozygous for the FADD Knock Out. This was the case for all samples (fig.2). Al samples contained a very clear single band of about 208 bp. We can conclude that all samples are Homozygous for the FADD Allel.

Western Blot analyse

Sample 1 and 2 were used to test. Random samples (A and B) of WT mice were use.

For positive control active caspase-3 was added. This gives a reference where we suppose to find active caspase-3 on the blot (Fig. 3). We expect that active caspase-3 is located at the 17 kDa. This is also confirmed by sample 2 where caspase-3 is clearly present. Sample 2 and B have not been treated with LPS, and show a similar pattern of bands (Fig.3). This refers to the fact that there isn't activity of apoptosis.

A clear difference between Samples 1 and A (treated with LPS) has been observed. The antibodies against active caspase-3 give only arise in A.This can be explained as followed, sample 1 is a liver extract of the conditioned KO mice. The absence of the FADD protein makes apoptosis impossible.Sample A is wild type, apoptosis occur. Hence we can conclude that the pressence of fadd plays a crucial role in the apoptose pathway !

Conclusion and discussion

Non-treated mice (KO and WT) show no significant differences in cell death. When we deal with the same mice, however, LPS-GalN added the WT mice exhibit very high levels of apoptosis, the KO mice remain spared. This indicates that FADD plays an essential role in the recruitment of caspases and finally cell death. The active caspase-3 concentrations are markedly higher in treated WT mice than in those from treated KO mice. The western blot allow us to conclude that when the FADD gene is abcense, the cells can no longer proceed induction of apoptosis stimulated by TNF.

We conclude that TNF plays a major role in the preservation of the homeostasis of the liver. It poorly understood how TNF can cause cancer and liver damage.(2)

Fig3. Westerblot Presence of active caspase-3 shows that the apoptose pathway is active. Sample 1 and A are threaded with Lipopolysachariden(lps). Sample 2 and B not. Visualisation of caspase-3 occurred with anti-caspase-3 Antibody.Pos. Control contains active Caspase, Neg control contain sample buffer.

Further studies on the use of TNF would be a step towards a cure for cancer. (1) Further studies on how tumor cells avoid apoptosis may bring more insight in this topic.