1. REGULATION OF MONOAMINE OXIDASE-A TRANSCRIPTION
Dilip Sarkar,Prof. E.Ellen Billett and Dr.Christoph Ufer
Nottingham Trent University,UK
In acquired as well as in innate immune system monocyte and macrophages play a significant role
(Stoy, 2001). During antigen-antibody dependent reactions, actions of monocytes and macrophages are
also found to be influenced greatly by the cytokines (Clericit et al.1994). Therefore, they are also
found to be interacting with the acquired immune responses. Cytokines or anti-inflammatory
messengers are powerful mediators in inflammation and immune reactions. Interleukin (IL4) is one of
the type I cytokines found to be produced by T-cells, mast cells in response to immune recognition
receptor engagement (Pernis et al. 1995). Study conducted by using quantitative PCR shows more than
2000 fold up-regulation of MAO-A enzyme. The other isoform i.e., MAO-B was not found to be up-
regulated upon IL4 or IL3 stimulation in U937 cells (Chaitidis et al 2004). Our study is based on
expression of specific MAO-A isoform at transcriptional level upon IL4 stimulation. Monoamine
oxidase enzyme is a mitochondrial flavoenzyme and primarily responsible for the degradation of the
dietary amines including dopamine, epinephrine, nor epinephrine and serotonin into corresponding
aldehydes, ammonia (Chen et. al. 1991) and hydrogen peroxide (Shih et. al. 1999)
From previous investigations it has been found that IL4 strongly up-regulates MAO-A in U937 cells
and there may be strong co-relation between immune responses with up-regulation of MAO-A. Our
target is to detect the specific transcription factor involved in the up-regulation of MAO-A
transcription. The successful investigation may produce useful information's about regulation of
immune responses upon IL4 stimulation in monocyte cells. The specific research upon completion may
allow us to analyze involvement of differential proteins and DNA interactions in the MAO-A promoter
region after IL4 stimulation and the method has been chosen for that is Electrophoretic Mobility Shift
Assay (EMSA).
We have investigated the protein-DNA interactions with all six DNA probes (Probe A to F) and nuclear
extracts of various time intervals using DIG-gel shift Electrophoresis assay technique. From our
investigation it is evident that MAO-A enzyme gets up-regulated upon IL4 stimulation. As our work
was mainly based on transcription factor identification which may regulate MAO-A in U937 monocyte
cells, the result confirms that SP1 transcription factor binds at DNA probes selected from MAO-A
promoter region along with other unidentified but significant proteins. A maximum upward shift can be
observed with day3 IL4 stimulated cells.
We have observed quite interesting facts during this research project especially the position of Sp1
transcription factor binding sites in non-stimulated as well as IL4 stimulated U937 cells under our
laboratory conditions. This transcription factor and its position can be important aspects in regulating
specific MAO-A expression in U937 cells. Sp1 transcription factor regulates lots of genes in monocytic
cells by its consensus binding site. This transcription factor may be useful in regulation of monoamine
oxidase A upon regulation of IL4 stimulation. A supershift assay technique can be performed further to
identify possible protein binding to the Sp1 transcription factor. A combination of RT-PCR and ELISA
technique can also can be taken into consideration to detect the unidentified proteins.