Elucidating the mechanism of CK2.3 mediated osteogenesis
Date
2019
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Osteoporosis is a skeletal disorder that is associated with loss of bone structure, integrity, and bone mass occurring over a period of years; thereby, increasing the incidence of fractures either at proximal femur, distal radius, thoracic, and lumbar vertebrae. It affects one in two women and one in four men, age 50 and older. The underlying cellular pathology causing osteoporosis are a) excessive bone resorption by osteoclasts and/or b) failure of osteoblasts to produce and replace the resorbed bone leading to weak and porous bone. There is a need for a novel drug that can increase bone mineral density (BMD) in osteoporotic patients. ☐ Dr. Nohe has developed a bone morphogenetic protein receptor type IA (BMPRIA) mimetic peptide called CK2.3, it functions by mimicking the casein kinase 2 (CK2) phosphorylation site and prevents the interaction between CK2 and BMPRIA. In vitro experiments conducted in C2C12 cells, primary calvarial osteoblasts, and bone marrow stromal cells (BMSCs) revealed that 100 nM of CK2.3 induced significant mineralization. Furthermore, subcutaneous injection into the right side of the calvaria of 4-week-old female C57BL/6J mice and systemic injection of CK2.3 into 8-week-old and 6-month-old female C57BL/6J mice increased total bone area, areal bone mineral density (aBMD), and mineral apposition rate (MAR) compared to control and bone morphogenetic protein 2 (BMP2) injected mice. It also elevated the levels of osteoblast markers and reduced the number of osteoclasts. Thus far, Nohe lab has established that CK2.3 has osteogenic potency, but the mechanism of CK2.3 driving the differentiation of C2C12 cells and primary BMSCs towards osteoblastic lineage remains elusive. As part of Dr. Nohe’s research group, we had not yet delineated if CK2.3 functions intracellularly or extracellularly, nor had we identified the signaling pathway(s) involved in CK2.3 mediated calcification that were observed in in vitro and in vivo studies. More importantly, for CK2.3 to be considered as a potential candidate for the treatment of osteoporosis, detailed molecular events implicated in the differentiation of MSCs into osteoblasts by CK2.3 must be delineated, along with determining the appropriate dosage that would yield a favorable outcome without inducing a toxic reaction or any other side effects. ☐ The goal of my doctoral research was to determine the internalization and signaling mechanisms activated by CK2.3 in vitro. To this end, a functional fluorescent CK2.3 analog (CK2.3-Qdot®s) was synthesized (Chapter 3). Then using CK2.3-Qdot®s as a fluorescent bio-conjugate, I determined the internalization mechanism of CK2.3 into C2C12 cells and its interaction with intracellular CK2 protein. I identified CK2.3 mediated activation of BMPRIA downstream signaling pathways and osteoblast specific genes. These results were further verified in primary BMSCs isolated from 4-month-old female C57BL/6J mice and female osteoarthritic patients (Chapter 4). I also investigated the effect of CK2.3 on the expression and activity of endogenous CK2 protein and its substrate (Chapter 5). ☐ In Chapter 3, to demonstrate CK2.3’s internalization within the cells, I conjugated CK2.3 to quantum dot®s (Qdot®s), which are fluorescent semiconductor nanoparticles. Using Qdot®s as a bio-imaging probe, enabled me to indirectly track CK2.3 within the cells and visualize its interaction with endogenous proteins. First, I conjugated CK2.3 to Qdot®s using N,N’-Dicyclohexylcarbodiimide (DCC) as the linker agent, then I separated CK2.3-Qdot®s from non-specific reactants based on size exclusion chromatography (SEC), and verified the conjugation and stability using UV/VIS spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and confocal laser scanning microscopy. My results showed that CK2.3 was conjugated to the Qdot®s and the conjugate was stable in water for at least 4 days at 37°C and 12 hours within C2C12 cells. Furthermore, CK2.3-Qdot®s exerted mineralization activity similar to CK2.3. ☐ In Chapter 4, I investigated the mechanism of CK2.3 mediated induction of osteogenesis. Data from this study revealed that addition of CK2.3-Qdot®s to C2C12 cells resulted in its internalization starting at 6 hours post stimulation. Furthermore, using pharmacological inhibitors against endocytic pathways and using caveolin-1 (cav-1) siRNA, I demonstrated that CK2.3-Qdot®s were internalized by caveolae mediated endocytosis. Furthermore, using confocal microscopy, I visualized a time dependent co-localization of CK2.3 with CK2α within C2C12 cells. Next, using the Western blot technique, I determined CK2.3 mediated activation of BMPRIA downstream signaling pathways and utilizing quantitative reverse transcription polymerase chain reaction (RT-qPCR), I examined the expression of osteoblast specific genes such as RUNX2, OSX, ALP, and OCN following stimulation with CK2.3. Furthermore, by using specific inhibitors/siRNA against BMP signaling pathways, I have shown that ERK1/2 and Smad1/5/8 signaling pathways are crucial for CK2.3 mediated osteogenesis in C2C12 cells. This was further verified by the downregulation of ALP protein expression. I further bolstered my results by showcasing that CK2.3 induced mineralization also occurs via ERK1/2 and Smad1/5/8 signaling pathways in primary BMSCs isolated from the femur and tibia of 4-month-old female C57BL/6J mice and femoral heads of female osteoarthritic patients. ☐ Using CK2.3-Qdot®s as a bio-fluorescent probe, I have shown the interaction between CK2.3 and endogenous CK2 protein. Moreover, a Korean group have published research articles where they have reported that inhibition of CK2 using either CX-4945 or dovitinib, enhanced BMP2 mediated osteogenesis in C2C12 cells via activation of Smad1/5/8 and ERK1/2 signaling pathways. Therefore, in Chapter 5, I investigated the effect of CK2.3 on the activity and expression of CK2. In my study, CK2.3 appeared to upregulate the expression of CK2α but had no effect on the expression of CK2β. Furthermore, CK2.3 significantly inhibited the activity of purified CK2α protein and the IC50 was determined to be 65.15 μM. Furthermore, CK2.3 may have an influence on the expression of p-RelA/ NF-κB p65; one of the substrates of CK2. ☐ In conclusion, the majority of the prescribed drugs against osteoporosis such as parathyroid hormone (PTH), bisphosphonates, selective estrogen receptor modulators (SERMS) have failed to find a durable treatment for this disease, and in many cases, have led to adverse side effects. I have reported that CK2.3 is a novel and potential bone-formation inducing drug. However, its mechanism of action has not been studied. For my doctoral research, I have established the molecular sequence of events that are triggered from the stimulation of cells with CK2.3 till the induction of mineralization. Now that I have been able to characterize the activity of CK2.3, it can be considered as a potential therapeutic candidate for osteoporosis, subject to further research in advanced animal models. At the very least, the information gained from this research can be implemented in the development of future treatments of osteoporosis and it can be used to alleviate some of the physical pain and financial burdens endured by the osteoporotic patients.
Description
Keywords
Bone Morphogenetic Protein 2, CK2.3, Mesenchymal Stem Cells, Osteoblasts, Osteoporosis, Protein kinase 2