Date of Award

Spring 2025

Rights

Access is available to all users

Document Type

Thesis

Degree Name

Master of Science (MS) in Biology

Department

Biology

Abstract

Bone remodeling is a dynamic process that requires a balance between bone resorption by osteoclasts and bone formation by osteoblasts to maintain skeletal integrity. Disruptions to this equilibrium can lead to pathological conditions such as osteoporosis. Osteoclasts originate from macrophages, and their differentiation is regulated by several signaling pathways, notably the notch signaling pathway. Gamma-secretase, an enzyme crucial for initiating notch signaling, can be inhibited by DAPT. However, DAPT also targets non-Notch substrates, complicating the identification of genes specifically regulated by Notch. To address this limitation, this study employed IMR-1A, a selective Notch pathway inhibitor that disrupts the assembly of the Notch transcriptional activation complex by blocking the recruitment of Mastermind-like 1 (MAML1). By comparing the transcriptomic effects of DAPT and IMR-1A during osteoclast differentiation, we aimed to isolate notch-specific genes. Bone marrow-derived precursors from mice were differentiated into osteoclasts and treated with DMSO (control) or DAPT to generate differentially expressed genes (DEGs) via RNA sequencing. These DEGs were analyzed using g:Profiler2 for functional enrichment. Selected genes from enriched metabolic and immune pathways were then validated by RT-qPCR following treatment with DMSO, DAPT, or the more specific Notch inhibitor IMR-1A. Results revealed that Notch inhibition modulates metabolic, mitochondrial, and immune gene networks. Notably, NFATc1, CYC1, and ITGB3 emerged as strong responders to Notch inhibition, whereas Hes1, a classical notch target, showed stable expression, indicating possible compensatory regulation. Functional assays confirmed that DAPT strongly inhibited osteoclast formation and activity, while IMR-1A showed moderate effects, supporting its specificity. This study highlights the role of Notch signaling in osteoclast differentiation and identifies NFATc1 and ITGB3 as potential biomarkers of Notch-regulated osteoclastogenesis. By isolating Notch-specific DEGs, this work enhances our understanding of the molecular basis of osteoclast differentiation and reveals potential therapeutic targets for treating osteoporosis and related bone disorders.

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