Va sposter ricky_v9_11

The central role of FasL-Fas system in the physiological regulation of apoptosis via

FADD/Caspase8 signaling cascade is well known (Figure 2).

However, it is also known to transduce proliferative and activating signals through

pathways such as, ERK signaling and NF-kB by mechanisms that are poorly defined

(Figure 2).

FasL-Fas system is particularly prominent in liver.

Thus, it is likely that different chemokines trigger different downstream FasL-Fas

signaling events such as caspase dependent apoptosis, NF-kB dependent inflammation

or proliferation.

NF-kB dependent Fas signaling in hepatocytesQuoc Tran1,3, Rohini Mehta3, Aybike Birerdinc2,3,Ancha Baranova2,3

1. Biology Department, College of Science, George Mason University

2. School of Systems Biology, College of Science, George Mason University

3. Betty and Guy Beatty Center for Integrated Research, Inova Health System, Falls Church, VA, USA.

Cohen, J.C., Horton, J.D., Hobbs, H.H., 2011. Human Fatty Liver Disease: Old Questions and New

Insights. Science 332, 1519 -1523.

Feldstein, A.E., Canbay, A., Guicciardi, M.E., Higuchi, H., Bronk, S.F., Gores, G.J., 2003. Diet

associated hepatic steatosis sensitizes to Fas mediated liver injury in mice. Journal of Hepatology 39, 978-

983.

Pahl, H.L., 1999. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18,

6853-6866.

Ponton, A., Clément, M.-V., Stamenkovic, I., 1996. The CD95 (APO-1/Fas) Receptor Activates NF-

kappaB Independently of Its Cytotoxic Function. J. Biol. Chem. 271, 8991-8995.

Sahin, H., Trautwein, C., Wasmuth, H.E., 2010. Functional role of chemokines in liver disease models.

Nat Rev Gastroenterol Hepatol 7, 682-690.

http://www.sabiosciences.com/elisaprotocol.php

HepG2 cell line (ATCC No. HB-8065 Hep G2 ;Hepatocellular carcinoma, human) will be

used as in vitro model for the current study.

The cells will be subcultured according to the recommended protocol (ATCC).

For understanding the mechanism of chemokine CCL4 and CCL21 mediated hepatocyte

inflammation, Fas signaling will be monitored.

Cells will be subjected to varying concentration of individual chemokines in a time course

experiment of 8, 12 and 24 hours.

After appropriate stimulation as described above, supernatants will be collected for sandwich

ELISA

AIM

Study in our laboratory has shown positive correlation of chemokine CCL21 and negative

correlation of chemokine MIP-1b with advanced hepatic inflammation in obese patients with

biopsy proven NAFLD.

Delineating the role of these chemokines in Fas mediated inflammatory pathways could help

elucidate whether these chemokines act upstream of inflammation or are the targets of

inflammatory pathways.

METHODS

EXPECTED RESULTS

Figure 2: Fas signaling and canonical NF-kB pathway

Supernatant will be subjected to ELISA using custom Multi-analyte ELISAarray kits from Qiagen

according to the manufacturer's suggestions (Figure 3).

The cytokines represented by these arrays would include: IL1β, IL6, MIP-1α, MIP-1β, RANTES,

TNFα (Table 1).

Experiments will be performed in triplicates.

Figure 4: Multianalyte ELISAarray Workflow

REFERENCES

METHODSINTRODUCTION

NAFLD is considered as hepatic manifestation of metabolic syndrome affecting

both adults and children.

Association of NAFLD with obesity, particularly visceral obesity has been long

recognized (Figure 1).

The high prevalence and substantial morbidity and mortality accompanying NAFLD

makes it imminent to understand the mechanistic basis of this disorder.

Little is known about the progression of steatosis to inflammatory NASH, fibrosis

and ultimately cirrhosis.

One of the critical questions in progression of NAFLD is - which factors could be

the driving forces toward a more progressive, inflammatory disease phenotype?

Hepatocyte apoptosis, is an important mechanism in the pathogenesis of NAFLD.

Not only is hepatocyte apoptosis recognized as an important mechanism of liver

injury, but it is believed to contribute to liver inflammation, fibrogenesis and the

development of cirrhosis.

Hepatocyte apoptosis is an important mechanism in the pathogenesis of NAFLD. The

current study aims to determine whether chemokines CCL21 and MIP-1b induce Fas

ligation and, if so, whether the Fas signaling activates downstream NF-kB mediated

inflammatory pathway or caspase-3 dependent apoptosis.

Figure 1: Obesity and NAFLD Spectrum

Figure 3: Sandwich ELISA

Sandwich ELISA measures the amount of antigen

between two layers of antibodies (i.e. capture and

detection antibody). The detection antibody is linked

to an enzyme, and in the final step a substrate is added

for colorimetric detection of antigen. The advantage

of Sandwich ELISA is that the sample does not have

to be purified before analysis, and the assay

can be very sensitive.