This web page was produced as an assignment for Gen677 at UW-Madison Spring 2012.
Conclusions
The goal of my in-depth, genomic and bioinformatic investigations was to learn more about ITGAM and systemic lupus erythematosus (SLE) and to search for a molecular connection between ITGAM, estrogen, and coronary heart disease in humans. ITGAM small nuclear polymorphisms (SNPs) have been shown in some studies to confer a higher risk for SLE, but there is little known about how ITGAM is involved in the pathogenesis of SLE. It is thought to inhibit ITGAM’s normal functions but it is still not fully understood. I hypothesize that ITGAM may be connected to the estrogen pathway at the molecular level. Due to the high prevalence of lupus in women, there may be a link between estrogen and SLE pathogenesis. I also hypothesize that ITGAM and estrogen play a role in the high risk of coronary heart disease seen in women with SLE.
To learn more about ITGAM and its role in SLE pathogenesis, I first decided to focus on ITGAM isoform-2. ITGAM has two isoforms, and I chose to focus on isoform-2 because there was more information available at the time of my project. Next, I investigated ITGAM isoform-2 homology, phylogeny, domains, and interaction networks. I found ITGAM homologs in many model organisms and observed that the domains found in human ITGAM are highly conserved among vertebrate model organisms. I also found that the STRING 9.0 mouse ITGAM protein-protein interaction network was similar to the STRING 9.0 human ITGAM protein-protein interaction network which provides more evidence that the most common model organism for SLE research, the murine model, is a useful model for SLE research [1].
I then looked at the ontology for ITGAM to try to determine the role of ITGAM in SLE pathogenesis. First, I found that ITGAM forms a complex with integrin beta 2 called integrin alpha m beta 2 (also known as Mac-1 or CR3). The complex is expressed on the surface of monocytes and granulocytes and is involved in glycoprotein binding and receptor activity. ITGAM is involved in blood coagulation, cell adhesion, integrin-mediated signaling pathway, and leukocyte migration. This information lead me to start to hypothesize that ITGAM could play a role in plaque formation and, therefore, coronary heart disease.
To determine the molecular link between ITGAM, estrogen, and coronary heart disease, I compared the STRING 9.0 protein-protein interaction networks of ITGAM, estrogen receptor 2 (ESR2), and fibrinogen gamma chain (FGG). I chose estrogen receptor 2 (ESR2) because it is a receptor for estrogen and its interaction network is essentially the “estrogen pathway”. I chose fibrinogen gamma chain (FGG) because it is a cofactor for platelet aggregation, yields fibrin, and has been linked to heart disease [2]. In the STRING interaction networks, I found that two heart disease related proteins, coagulation factor II, thrombin (F2) and glycoprotein 1b (GP1BA) are in ITGAM’s protein interaction network. F2 was also found in the FGG protein interaction network. Signal transducer & activator transcription 3 (STAT3) was found in both the FGG protein interaction network and the ESR2 protein interaction network. The link between STAT3 and FGG in the FGG interaction network is only based on scientific literature and not direct evidence; however, it demonstrates a potential link between the ITGAM, FGG, and ESR2 pathways.
Next, I found microarray data from previous experiments in breast cancer cells (MFC-7) that investigated the link between estrogen and ITGAM expression. I also looked into the expression of some of proteins that link the ITGAM, estrogen, and fibrinogen pathways in the presence of estrogen. I primarily focused on STAT3 and FGG because there is the least known about their interactions, and they were the only proteins I could find microarray data for. I looked at microarray data from the GEO database. First, I found a study that showed that ITGAM expression is not affected by estrogen. Also, I found microarray data that showed an increase in FGG expression in the presence of estrogen and a decrease in STAT3 expression in the presence of estrogen. This information led me to develop a model for a pathway responsible for coronary heart disease in SLE patients.
My model for how ITGAM, estrogen, and coronary heart disease are linked at the molecular level based on my bioinformatic analysis is:
Estrogen binds to estrogen receptor (ESR2) which causes interaction with tyrosine-protein kinase Src.
This interaction leads to a decrease in expression of signal transducer & activator transcription 3 (STAT3).
STAT3 normally has an inhibitory effect on fibrinogen gamma chain (FGG) expression.
When there is less expression of STAT3, FGG is expressed at a higher level.
Increased levels of FGG lead to more platelet aggregation and fibrin.
ITGAM expression is unchanged in the presence or absence of estrogen.
ITGAM interacts with one or more of the other proteins to promote plaque formation in the presence of estrogen. One protein in particular, GP1BA (a surface membrane protein of platelets known to interact with ITGAM), could also be involved in the process.
In conclusion, ITGAM appears to be linked to both the estrogen and fibrinogen pathway which may be the reason why lupus primarily affects women and causes heart disease…but there is still a lot of research that needs to be done!
Future Directions
The connections between ITGAM, SLE, estrogen, and coronary heart disease is far from being fully understood. My model is only a starting point based on the limited genomic and bioinformatic data I could obtain.
For the next step, I propose doing a microarray experiment using arterial cells from the coronary arteries of mice. I would like to use the murine model because ITGAM and its interaction network are conserved in this organism, SLE symptoms can be replicated in mice (seen in previous studies), and most importantly mice have a similar circulatory system [1]. All the microarray experiments presented on this website used human breast cancer cells which may not be a great indicator for what is going on inside of the coronary arteries of SLE patients. I would like to obtain cells for the microarray experiments from the arteries of male and female mice that are wild-type or knockouts of ITGAM or FGG. I would test gene expression in the presence and absence of estrogen. I would expect FGG expression to be increased in the presence of estrogen, and there to be no change on ITGAM expression.
As for additional experiments, I would like to do more microarray experiments to compare the expression of the other proteins (STAT3, F2, GP1BA, and etc.) in the presence and absence of estrogen in mouse arterial cells. Also, it would be interesting to look into other sex hormones such as prolactin and progesterone to see if they are linked to ITGAM, SLE, and/or coronary heart disease.
References
[1] Morel, L. (2010). Genetics of SLE: evidence from mouse models. Nature Reviews Rheumatology 6, 348-357 doi:10.1038/nrrheum.2010.63
[2] Valvi, D et al. (2012) Fibrinogen, chronic obstructive pulmonary disease (COPD) and outcomes in two United States cohorts.Int J Chron Obstruct Pulmon Dis. 7:173-82.