Determining the mechanism of CK2.3 signaling in cells isolated from patients diagnosed with osteoporosis
Date
2020
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Publisher
University of Delaware
Abstract
Bone is an extremely important organ as it provides structure, protects other internal organs, anchors muscles to assist in locomotion, and supports mineral homeostasis. Two major cell types found within bone that are responsible for its maintenance are osteoblasts (bone building cells) and osteoclasts (bone resorbing cells). However, there are many different bone diseases in humans where bone becomes unhealthy or the critical balance between osteoblasts and osteoclasts may become disrupted. The most common bone disease in humans is osteoporosis (OP), which is characterized by low bone mineral density. Current therapeutics treating OP have a laundry list of negative side effects, and a majority can be taken only for a limited number of years, with drug holidays interspersed throughout their use. Therefore, there is a need to develop new therapeutics that target both osteoblasts and osteoclasts to treat this debilitating bone disease. ☐ Bone morphogenetic protein 2 (BMP2) is a potent growth factor that is known to activate both osteoblasts and osteoclasts. Recently, many studies have showed a lack of BMP2 response in OP patients. While BMP2 may not be an effective or ideal treatment for OP, its signaling pathway is still of interest since it controls both osteoblasts and osteoclasts. The Nohe lab has extensively studied this protein and its signaling pathway. They previously discovered a novel interaction between the BMP type Ia receptor (BMPRIa) and an interacting protein called casein kinase II (CK2). Several CK2 phosphorylation sites were discovered on BMPRIa and corresponding blocking peptides (named CK2.3, CK2.2, and CK2.1) were designed to further elucidate their function. The peptides are hypothesized to bind to CK2 and block the interaction with BMPRIa at that site. Previously, CK2.3 has been shown to increase osteoblast activity and decrease osteoclast activity in a variety of animal models and cell lines. CK2.3’s effect on primary human cells has not yet been investigated. ☐ In this study, I investigated BMP2 and CK2.3’s effect on isolated osteoblasts from human femoral heads obtained from patients undergoing hip arthroplasty surgery at Christiana Care Hospital in Newark, DE. The patients were diagnosed with either OP or osteoarthritis (OA), however prior to experimentation the clinical diagnosis was confirmed through single photon absorptiometry by X-raying the femoral heads. After confirmation, cells were extracted from the femoral heads and stained for osteoblast specific markers osteocalcin (OC) and alkaline phosphatase (ALP) to determine if the extracted cell population was mature osteoblasts. The cells also were assessed for mineralization potential through a von Kossa assay after stimulation with either BMP2, CK2.3 or left unstimulated (US). Cells extracted from OA patients had increased their mineralization significantly after both BMP2 and CK2.3 stimulation. Cells extracted from OP patients only significantly increased their mineralization after CK2.3 stimulation. Cells from OP patients did not respond to BMP2 stimulation. Next, the cells were stained again for osteoblast specific markers following BMP2 and CK2.3 stimulations. CK2.3 significantly increased fluorescent intensity of the osteoblast specific markers, while BMP2 did not. ☐ BMPRIa levels were investigated in a variety of models using both in vitro and in vivo approaches. Increase fluorescent expression was observed in MMA embedded bone slices of OP patients when compared with control patients. Explants from both OP or control patients were stimulated as mentioned previously. In control explants both BMP2 and CK2.3 stimulation significantly increased BMPRIa and CK2 expression when compared to US. In OP explants only CK2.3 significantly increased expression of these two proteins, while BMP2 significantly decreased BMPRIa and CK2 expression. Steady state mRNA levels also showed a decrease in BMPRIa after BMP2 stimulation. The consequences of this decrease in expression could be causing an overall decrease in BMD; therefore, downstream signaling proteins like pSMAD and pERK were investigated. Both BMP2 and CK2.3 did not change the immunofluorescent or protein expression of pSMAD. CK2.3 significantly increased expression of pERK in both the fluorescent and protein expression studies, indicating that CK2.3 still acts through ERK signaling in humans. BMP2 significantly decreased expression of pERK in both experiments, indicating that this lack in response may be due to aberrant BMP signaling. ☐ Previously, CK2 was discovered to bind and phosphorylate ERK in several cell lines. Since CK2.3 is known to act through this signaling pathway, and directly binds and mediates CK2 activity, this could be a possible explanation as to how CK2.3 induces a response in cells derived from OP patients, while BMP2 does not. Therefore, this was investigated through a fluorescent colocalization study. No significant changes in colocalized pixel distribution was discovered between pSMAD and CK2. CK2.3 significantly increased pixel colocalization between CK2 and pERK, when compared with BMP2 and US cells, indicating a potential mechanism of action. Given the research presented here, CK2.3 continues to be a unique potential therapeutic for the treatment of OP. It is critical to continue exploring this mis-regulation in BMP signaling, as well as to further delineate the exact mechanism of CK2.3.
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Keywords
BMP2, CK2.3, Osteoarthritis, Osteoporosis