Halloran, Daniel R.2023-08-212023-08-212023https://udspace.udel.edu/handle/19716/33125Bones are complex systems that are necessary for the protection of internal organs, structure of the human skeleton, and locomotion. There are different types of bones comprised of multiple layers. Typically, the structure of bone is well-maintained by bone remodeling, and approximately 10% of the human skeleton is replaced every year. Unfortunately, as humans age and their bones naturally become weaker, certain bone disorders can occur. One of these bone disorders is osteoporosis (OP), which is a devastating bone disorder that affects most of the world’s population over the age of 50. Not only does OP lead to an increased occurrence of microfractures and bone breakage, it also leads to a decreased quality of life for those diagnosed with the disorder, and it costly. OP is characterized by a drastic reduction in bone mineral density (BMD) likely caused from decreased activity of osteoblasts, increased activity of osteoclasts, or both. Osteoblasts are responsible for forming new bone and derive from mesenchymal stem cells (MSCs), whereas osteoclasts resorb old or damaged bone and differentiate from hematopoietic stem cells (HSCs). Currently, there is no cure for OP, and the treatment options are short-term and limited. These treatments are either anabolic, which focus on increasing the production of bone, or antiresorptive, which targets the activity of osteoclasts. However, each available treatment leads to several adverse side-effects, and only one current therapeutic can target osteoblasts and osteoclasts simultaneously. ☐ Bone Morphogenetic Protein-2 (BMP-2) is a member of the Transforming Growth Factor-Beta (TGF-B) superfamily and is crucial for many developmental and adulthood processes. In 1965, BMP-2 was first identified in the osteogenesis pathway; since then, it has been implicated in neurogenesis, chondrogenesis, cardiogenesis, and more. Further, BMP-2 is necessary to differentiate MSCs into active osteoblasts. Due to its multifunctionality and osteogenic capacity, BMP-2 was approved by the Food and Drug Administration (FDA) in 2002 to be injected during anterior lumbar interbody fusion (ALIF) surgery to promote osteogenesis. However, shortly after BMP-2 began its debut as a therapeutic, several side-effects were reported. Some of these side-effects include hematoma formation, increased osteolysis, and interestingly, primary osteoblasts isolated from OP patients do not respond to BMP-2 stimulation. While exogenous BMP-2 may be ineffective in treating OP, the BMP-signaling pathway can still be implicated in the development of a future therapeutic. Specifically, the Nohe lab identified an interacting protein of the BMP-signaling pathway known as protein kinase CK2 (CK2). CK2 binds to the BMP Receptor Type Ia (BMPRIa), which is a serine/threonine kinase receptor, preventing it from phosphorylating downstream targets. Further, the Nohe lab demonstrated that when BMP-2 preferentially binds to BMPRIa and BMP Receptor Type II (BMPRII), CK2 is released from BMPRIa, allowing this receptor to be phosphorylated by BMPRII. To mimic a similar response, three custom designed peptides (CK2.1, CK2.2, and CK2.3) were each created with the three identified sequences for phosphorylation on BMPRIa. CK2.3 is effectively able to block CK2’s interaction with BMPRIa, causing an activation of downstream signaling pathways without the presence of exogenous BMP-2. While CK2.3 upregulates osteogenic genes, increases osteoblast activity, and decreases osteoclastogenesis in several models, its precise mechanism in humans and in vivo is unclear. ☐ In this dissertation project, the roles of BMP-2 and CK2.3 in aged-C57BL/6 (B6) mice, primary osteoblasts and osteoclasts isolated from human femoral heads, and C2C12 cells was explored. Specifically, as primary osteoblasts obtained from OP patients are unresponsive to BMP-2, we wanted to further investigate its potential mechanism of aberrant signaling in mice and humans. First, to create a viable method that can trace the localization and function of BMP-2, I created an analog of BMP-2 conjugated to fluorescent Quantum Dots (QDot®s). Here, it was determined that the BMP-2- QDot®s analog is fluorescently active, photostable, and allows BMP-2 to retain its biological function. This analog will be helpful for determining the precise localization and function of BMP-2 within primary osteoblasts and BMSCs to elucidate improper signaling. ☐ Next, to understand the role of BMP-2 in human osteoclasts and in B6 mice, human femoral heads and aged-mice were utilized, respectively. We determined that after isolating pre-osteoclasts from the bone marrow of human femoral heads of patients diagnosed with OP or osteoarthritis (OA), we could differentiate these cells into functional osteoclasts. I demonstrated that these cells become multinucleated and express two major osteoclast markers, Tartrate Resistant Acid Phosphatase (TRAP) and Cathepsin K (CathK). Further, I displayed that BMP-2 enhanced osteoclastogenesis, while CK2.3 decreased the formation of osteoclasts in both OA and OP patients. Next, to translate these findings into an in vivo model, we utilized B6 mice aged 6-, 15-, or 20-months. Interestingly, we discovered that similar to osteoblasts isolated from OP patients, the BMSCs isolated from 15-month mice displayed a significant upregulation of BMPRIa compared to 6-month mice as depicted by immunofluorescent staining and western blotting. In addition, we displayed that all mice aged 6-months responded positively to BMP-2 stimulation via von Kossa assays and micro computed tomography (µCT), but this effect was not the same in 15- and 20-month mice. However, CK2.3 improved bone volume/tissue volume (BV/TV) and mineralization in all age groups. Next, I uncovered that after treating BMSCs isolated from 15-month B6 mice with methyl-β-cyclodextrin (MβCD) followed by BMP-2 stimulation, there was an increase in BMP-2-QDot®s-BMPRIa colocalization and downstream mineralization compared to the BMP-2 only and control groups. This suggests that BMPRIa is mis-localized in aged-mice and can be rescued with MβCD. Further, regarding osteoclasts, BMP-2 enhanced osteoclastogenesis at all ages, whereas CK2.3 inhibited the formation of active osteoclasts. Finally, to further elucidate the precise mechanism of CK2.3, a BMPRIa knockout (KO) model was produced in C2C12 cells. Within these cells, CK2.3 did not increase mineralization, demonstrating its reliance on the presence of this receptor. These results were confirmed when utilizing small interfering RNA (siRNA) targeting BMPRIa in C2C12 cells. Taken together, this dissertation proposes that in aged-mice, BMPRIa is mis-localized on the cell surface, but can be rescued. Further, despite this mis-localization, CK2.3 can bypass extracellular binding to BMPRIa, it is able to effectively activate BMP-signaling to induce bone growth and mineralization. These data propose a potential intervention strategy for treating OP.BMP-2C57BL/6 MiceCK2.3OsteoporosisThesis1399968334https://doi.org/10.58088/c369-n0702023-06-26en