Understanding and Exploiting Wnt5a and GSK3 Signaling in Inflammatory Disease

Atherosclerosis, a progressive inflammatory disease, is a leading cause of cardiovascular related death worldwide. Uptake of oxidized-low density lipoprotein (ox-LDL) by monocytes/macrophages plays a critical role in the pathogenesis of atherosclerosis. The importance of macrophages in the development of an atherosclerotic plaque has been well documented. Wnt5a, a secreted glycoprotein, is highly present in atherosclerotic lesions, and is known to be upregulated in other inflammatory diseases like rheumatoid arthritis and sepsis. Interleukin-6 (IL-6), an inflammatory cytokine secreted by activated macrophages, is also known to be expressed in these lesions. However, to date, the exact source of Wnt5a in these atherosclerotic lesions has not been established. The above observations motivated us to determine whether ox-LDL stimulated macrophages are an important source of Wnt5a, and can its increased expression be used as a marker for diagnostic identification of atherosclerosis. Using quantitative real time-polymerase chain reaction (RT-PCR), we demonstrated that lipopolysaccharide (LPS) and ox-LDL, but not native-LDL, induces Wnt5a and IL-6 mRNA expression in human monocyte-derived macrophages. Furthermore, we found increased expression of Wnt5a protein in human atherosclerotic serum samples as compared to control serum samples using an enzyme-linked immunosorbent assay (ELISA), suggesting a possible diagnostic/prognostic approach for the detection of atherosclerosis. Combined, the results of this study indicate that Wnt5a plays an active role in the pathophysiology of atherosclerosis.Glycogen synthase kinase (GSK3), a ubiquitously expressed multifunctional serine/threonine kinase, participates in a multitude of cellular and physiological processes, ranging from cell cycle, both cell death and survival, transcription, translation, microtubule stability and beta catenin Wnt signaling. Given its constitutive expression and diversity of putative substrates, GSK3 is regulated at various levels. Increased GSK3 activity is linked to neurological pathologies (e.g., Alzheimer’s disease, bipolar disorder), inflammatory diseases (e.g., sepsis, cirrhosis), and cancer. Due to this association, GSK3 is emerging as a potential therapeutic target for the treatment of these diseases. We worked with two potential GSK3 inhibitors. At the cellular level, we found via western blotting that these compounds dose dependently increase beta catenin expression in mouse and human macrophages, indicative of GSK3 inhibition. Additionally, given the link between GSK3, nuclear factor kappa B, and inflammation, in vitro models that mimic the inflammatory scenario of various pathological disorders (i.e. Alzheimer’s disease, LPS-induced inflammatory disorders) along with quantitative RT-PCR and ELISA were used to characterize the effect of these inhibitors. The results of this study established that amyloid beta and LPS induced human macrophages induce the production of IL-6, tumor necrosis factor-alpha, IL-1alpha, IL-1beta, and interferon-gamma. Treatment of amyloid beta and LPS induced human macrophages with the compounds resulted in differential inhibition of these inflammatory cytokines. Collectively, this part of the dissertation identified potential GSK3 inhibitors that could be used to differentially suppress induced inflammatory cytokine production.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Molecular and Cellular Biology (Arts and Sciences)

Grantor dc:publisher

Ohio University

Year dc:date

2013

Author dc:creator

• Bhatt, Pooja

Contributors dc:contributor

• Goetz, Douglas

• Biomedical Research
• Cellular Biology
• Molecular Biology
• Atherosclerosis
• Wnt5a
• Alzheimer's disease
• inflammation
• GSK3
• cytokines
• beta catenin
• inflammatory diseases

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1365012252

OAI identifier oai:identifier

oai:etd.ohiolink.edu:ohiou1365012252