Musculoskeletal disorders affect skeletal muscle, bone, cartilage, and connective tissue. Disorders such as osteoporosis (OP), osteoarthritis (OA), and rheumatoid arthritis (RA) can cause chronic pain, hamper mobility, and reduce quality of life. They are also among the costliest co-morbidities [1]. Traditionally, musculoskeletal disorders were identified as structural dysfunctions of respective physiologies. However, recently, metabolic dysregulation has come up as a significant risk factor in these diseases. Metabolic dysregulation is caused by the defective regulation of biochemical pathways required for homeostasis ^[2][3][2,3]. Emerging studies are investigating the underlying mechanism by which metabolic disorders increase the severity of musculoskeletal disorders. Rheumatoid arthritis (RA) is an autoimmune disorder affecting joints. Researchers are exploring the link between autoimmunity and altered metabolism ^[4][5][4,5]. Likewise, osteoarthritis (OA) is characterized by the progressive degeneration of joints. The pathogenesis of OA is affected by metabolic disorders ^[6][7][6,7]. Small molecular metabolites are assessed for OA-associated biomarker identification [8]. Altered lipid metabolism and immune response during obesity increase the severity of OA symptoms [9]. Osteoporosis (OP) is a disorder associated with low bone mineral density. Several studies investigate the complex relationship between bone homeostasis and metabolic disorders, especially in the case of OP ^{[10][11][12][13][14][15]}[10,11,12,13,14,15]. Patients diagnosed with OP are at a higher risk of fractures. An impaired immune system affects the natural process of fracture healing [16]. Defects in fracture healing in obese and insulin-resistant individuals are attributed to the dysregulated innate immunity during the healing-associated inflammatory response [17].

Protein kinase CK2 (formerly called “Casein Kinase 2”) is a ubiquitous serine/threonine kinase. The kinase has no extracellular activator [18]. It is not regulated hierarchically by other kinases of vertical signaling cascades. Thus, the regulatory role of CK2 is not limited to the activation of molecular signaling from the plasma membrane to the nucleus. Instead, it crosswise associates and integrates multiple pathways ^[19][20][19,20]. Hence, it is a ‘Lateral player’ in many biochemical pathways ^[21][22][21,22]. Expression and activity of CK2 are dysregulated in musculoskeletal disorders [23]. Deviation in metabolic processes affects tissue homeostasis and response to endocrine regulation [24]. The role of the inflammatory system and senescence in these diseases is also linked with metabolic dysregulation ^[25][26][25,26]. Moreover, the role of CK2 in modulating immune response, adipogenesis, and mitochondrial function has long been studied ^[27][28][29][27,28,29]. The kinase can identify and phosphorylate a vast range of substrates, estimated to be more than 400 today ^[30][31][30,31]. It is predicted to account for almost 20% of the phosphoproteome of cells [32]. Since CK2 is a lateral player, the effects of its dysregulation are manifold.

CK2 is ubiquitously present and expressed in almost all types of cells. The mechanism of pathway regulation by CK2 is different from that of other kinases. The following factors summarize the complexity of CK2’s function. First, while most kinases are activated in response to particular stimuli, CK2 is constitutively active [33] and requires no specific stimulus ^[18][34][35][18,34,35]. Second, the localization and form of CK2 change based on cell type and metabolic status [36]. CK2 has two catalytic subunits, CK2α and CK2α’, and a regulatory CK2β subunit. In vivo, it exists as a heterotetramer [37]. The tetramer form of CK2 is also termed CK2 holoenzyme. Holoenzyme comprises catalytic subunits α and α’ and two regulatory β subunits. The kinetics of holoenzyme formation and cellular distribution of these subunits change under different stimuli [38]. Third, the β subunit is not strictly a regulatory subunit. It is required to recognize substrates and structural stability of catalytic subunits. However, few substrates are phosphorylated without the β subunit, which makes its role elusive. Fourth, the catalytic subunits CK2α and CK2α’ are almost the same in structure, but their involvement in catalytic activity is different [39]. One is preferred over the other in some processes. Finally, the expression of subunits is regulated by various growth factors, and a single subunit’s expression level affects the expression and activity of other subunits.

The only clinically approved small molecular inhibitor of CK2, Silmitasertib (CX-4945), is a first-in-class small molecule of its kind [40]. It is an ATP-competitive inhibitor with the highest level of selectivity for CK2. Its use in cancer therapies is under investigation. However, like most currently available CK2 inhibitors, it inhibits CK2 catalytic activity completely. Controlling the inhibition of CK2 activity during specific cellular processes while sparing other activity is not possible. Furthermore, complete inhibition of the catalytic activity of CK2 causes cytotoxicity. This is due to the cells’ dependence on the basal phosphorylation level of CK2 substrates for survival [35]. Cytotoxicity is not always desirable, especially in treating musculoskeletal disorders. The technology of modulating the phosphorylation of CK2 substrates taking part in disease pathogenesis rather than inhibiting overall kinase activity is emerging. Focusing on specific CK2–substrate interaction will help bring a pathology-tailored approach for musculoskeletal disorders [41].

One such peptide inhibitor of CK2 named CIGB300 inhibits phosphorylation of specific CK2 substrates. This synthetic peptide binds to the phosphoacceptor domain of CK2 [42]. It was first discovered as an effective peptide in treating cervical cancer. It is also effective in treating Large Cell Lung Carcinoma (LCLC), Non-Small Cell Lung Cancer (NSCLC), advanced cervical cancer, and acute myeloid leukemia ^{[43][44][45][46]}[43,44,45,46]. Due to its substrate-specific inhibition, it is highly effective and has mild side effects. This drug is under investigation in Phase 2 clinical trials, and studies about its effectiveness will soon be available [29]. The substrate-specific rather than global inhibitory effect of CIGB300 is thus emerging in cancer treatment.

Disease-modifying drugs help reverse the symptoms of disorders; they do so because they are designed to target molecular interactions critical for pathogenesis [47]. This approach re-directs metabolic pathways towards homeostasis. Few disease-modifying drugs, like Sprifermin (recombinant human FGF18, rhFGF18) for OA and methotrexate for RA, are available to treat musculoskeletal diseases ^[48][49][48,49]. Interventions that can restore dysregulated metabolism in these diseases are necessary [47]. CK2 is a lateral player, restoring its activity will effectively re-direct the metabolism to a healthy state. However, Targeting CK2 is challenging due to the complexity of its function ^[21][22][50][21,22,50]. Efforts to reveal the complexities of CK2 function in musculoskeletal disorders will help design disease-modifying therapeutics.

For the development of new drugs, the success rate of candidate molecules to move from bench to bedside is only 10%, despite the robustness of preclinical studies and perfect administration of clinical trials. Moreover, an excellent pre-clinical performer may be withdrawn due to the long processing time between drug discovery and commercial production. Substantial costs are involved with this process, and more importantly, the high failure rate delays the advances in better treatment for affected individuals. Lack of biomarker identification, off-target activity, and excess toxicity are a few of the challenges identified that can be addressed while designing new candidates. This process can utilize advanced knowledge of the molecular interactions between the drug and target [51]. In the case of CK2, many small molecular inhibitors have been discovered, most of which are investigated for the treatment of cancers. Yet, very few are approved for clinical use, partly due to their high cytotoxicity. Re-purposing the knowledge about these CK2 inhibitory molecules for treating musculoskeletal disorders is possible. Understanding the critical interactions of CK2 with its substrates in musculoskeletal diseases becomes essential.

2. Implication of CK2 in Musculoskeletal Disorders

2.1. Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disorder affecting the joints. The exact mechanisms through which RA develops have yet to be fully understood ^[52][53][52,53]. One leading hypothesis is that the interactions between epigenetic modifications and environmental influences facilitate self-antigens production and induce autoimmunity. Self-antigens are then modified by citrullination [5]. As the body cannot recognize citrullinated antigens, the modified self-antigens are then recognized by antigen-presenting cells, which transport them to the lymph nodes. In the lymph nodes, helper T cells and B cells are activated through co-stimulation. The B cells differentiate into plasma cells that produce antibodies against the self-antigens [54]. In this manner, the body produces antibodies against self-antigens and induces tissue inflammation [55]. Synovial hyperplasia and similar disorders may also release cytokines contributing to joint inflammation and autoimmunity. Specifically, the synovial fluid and joint capsule become inflamed during RA, contributing to bone and cartilage erosion [56]. During RA, the balance between the Type I T Helper (Th1) and Type 2 T Helper (Th2) cells is disrupted in favor of the Th1 cells, causing inflammation. Type 17 T Helper (Th17) cells induce the proliferation of fibroblast-like synoviocytes (FLS) [57]. FLSs contribute to the progression of RA. FLSs are found in the synovial intimal lining of healthy tissue; they secrete products that contribute to the synovial fluid and articular cartilage. In RA, they secrete cytokines that promote inflammation and proteases that degrade cartilage [58]. Protein kinase CK2 plays a role in RA through CK2α’s control of the Interleukin-12 (IL-12) receptor, which regulates the IL-STAT4 signaling pathway through which Th1 cell differentiation is promoted. The IL-STAT4 pathway is regulated primarily by the expression of IL-12R in CD4+ T cells, though it is also regulated, to a lesser extent, by interferon-α (IFN-α) and interleukin 23 (IL-23). The phosphorylation of the STAT4 pathway induces downstream signaling that ultimately facilitates Th1 cell differentiation [59]. CK2α, CK2α’, and CK2β promote Th17 cell differentiation. CK2α also regulates Th1 cell differentiation and suppresses the activity of Th2 cells ^[59][60][59,60].

2.2. Osteoarthritis

Osteoarthritis (OA) is a degenerative disease of the articular cartilage, which is increasingly common with advanced age, especially in women. OA is associated with loss in the number and activity of chondrocytes through increased apoptosis. Reduced activity of chondrocytes reduces the repair of the collagen matrix and diminishes the structural integrity of the cartilage. Oxidative stress is another hallmark of OA. Metabolic disorders affect the severity of OA. Increased serum glucose levels due to type 2 diabetes and metabolic syndrome increase advanced glycation products (AGESs) levels. The AGES are associated with the collagen matrix of cartilage and cause structural damage. They also interact with receptors of AGES called RAGE and cause oxidative stress in chondrocytes [61]. Excess reactive oxygen species (ROS) production leads to oxidative stress response, increasing chondrocyte senescence [62]. CK2 is an inhibitor of apoptosis, and its expression is reduced in chondrocytes of patients diagnosed with OA. Inhibition of CK2, sensitized chondrocytes to Tumor Necrosis Factor-alpha (TNFα)-mediated cell death [63]. Parathyroid hormone-related protein (PTHrP) is a parathyroid hormone homolog. It protects chondrocytes from undergoing excessive apoptosis [64]. PTHrP exerts its protective activity against mitochondria-dependent apoptotic pathway via upregulation of CK2 activity ^[65][66][65,66]. PTHrP is localized to nuclei due to its mid-region bipartite nuclear targeting sequence (NTS) [67]. In one study, treatment with synthetic peptide PTHrP-NTS (the NTS region of PTHrP) protected HEK293 cells from TNFα-activated apoptosis. Exogenous NTS increased nuclear accumulation of CK2. Nuclear retention and activity of CK2 were enhanced. Similar results were obtained in cells treated with intact PTHrP [68]. CK2 is also involved in the oxidative stress response. CK2 signaling is essential for activating transcription factor NF-E2-related factor 2 (Nrf2). Nrf 2 signaling is crucial for redox balancing within cells [69]. It reduces cellular ROS levels and suppresses nuclear factor-κB (NF-κB) localization in the nucleus. Nrf2 upregulates expression of Heme oxygenase-1 (HO-1). Enzyme HO-1 is vital in heme degradation and countering oxidative stress response [70]. This enzyme becomes activated in chondrocytes in response to peroxynitrite-induced oxidative stress [71]. In another study, inhibition of the catalytic activity of CK2 with 4,5,6,7-terabromo-2-azabenzimidazole (TBB) and 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) induced senescence and apoptosis in chondrocytes. Overexpression of HO-1 reduced the TBB-induced senescence. Chondrocytes overexpressing HO-1 had reduced sensitivity towards TBB-induced senescence. The knockdown of CK2 reduced type II collagen and increased β catenin expression [72]. The small heat shock protein αB-crystallin is a mediator for the expression of chondrogenic Bone Morphogenetic Protein-2 and collagen type II. Its expression is downregulated in OA [73]. Chondrocytes treated with CK2 inhibitors and αB-crystallin siRNA were sensitized to apoptosis. However, αB-crystallin had a protective effect. Its expression was modulated, and cellular localization was changed after CK2 inhibition [74]. The role of αB-crystallin in aiding CK2 to prevent chondrocyte apoptosis should be explored further. Thus, evidence suggests that CK2 protects chondrocytes from oxidative stress and apoptosis.

2.3. Bone Fracture

Healthy bone is excellent at self-healing in the event of injury. The process occurs roughly through the following phases. The first is the inflammatory phase and hematoma formation. Physical rupture of the bone causes infiltration of immunoregulatory cells, such as Macrophages, at the site. These cells secrete inflammatory cytokines IL-1, IL-6, and TNFα during the early phases of healing [75]. These factors recruit mesenchymal stem cells (MSC) to the fracture site. MSCs undergo chondrogenic and osteogenic differentiation through secreted growth factors. Next, angiogenesis and soft callus formation begin, at which point the central region of the fracture undergoes endochondral ossification during this phase [76]. The MSCs undergo differentiation into osteo-chondroprogenitor cells [77]. Thus, formed chondrocytes form the cartilaginous matrix. Angiogenesis and calcification of the cartilaginous matrix by hypertrophic chondrocytes marks the start of the next phase of hard bone formation. In this phase, the cartilage is replaced by bone via hard calcification. Here, the activity of osteoblasts increases. Finally, in the last phase, bone remodeling occurs. Here, the coordinated function between bone matrix-secreting osteoblasts and bone-resorbing osteoclasts takes place [64]. Bone remodeling is essential in maintaining bone shape. During aging, macrophages, chondrocytes, and osteocytes secrete senescence-associated factors [78]. Their secretion prolongs the inflammatory phase and hampers the bone healing process ^[79][80][79,80]. The bone morphogenic protein (BMP) pathway is essential for fracture healing ^[81][82][81,82]. BMP ligands are critical growth factors for bone, cartilage, joint development, and homeostasis. They drive MSC differentiation. BMP-2 stimulation of senescent bone marrow mesenchymal stem cells (BMSCs) activated adipogenic and cell death pathways like NF-kB or p38-mitogen activated protein kinase (MAPK). In non-senescent bone marrow mesenchymal stem cells (BMSCs), BMP-2 activated bone-forming pathways such as SMAD, BMP, and TGFβ [83]. CK2 negatively regulates BMP-2-activated signaling [84]. Casein kinase 2 interacting protein-1 (CKIP-1) is a negative regulator of BMP signaling [85]. CKIP-1 interacts with the CK2α subunit and regulates signaling between CK2α and effector molecules of BMP signaling ^[86][87][86,87]. CKIP-1 is known to inhibit BMP signaling by promoting Smad1 ubiquitination. The suppression of BMP signaling leads to suppressed osteogenic differentiation [23]. The CKIP-1 knockout mice had abnormally high levels of bone mass. The effects of CKIP-1 on bone growth and repair were age-dependent, with CKIP-1 having a more substantial impact on the bone mass of 18-month-old mice and comparatively little effect on 2-month-old mice. CKIP-1 is a negative regulator of osteogenic differentiation, and age-related inflammation causes an upregulation of CKIP-1, although the exact mechanisms of this upregulation remain unclear ^[88][89][88,89]. Inhibition of interaction between CK2α and CKIP-1 is a possible therapeutic strategy for bone fractures.

2.4. Osteoporosis

Osteoporosis (OP) is a degenerative disease that, as of 2023, affects more than 200 million people. The incidence rate of OP is 1.7% of men and 26% of women over age 50 worldwide [65]. OP is associated with a loss of bone mass and mineral density. This weakens the bones and predisposes them to fractures. It is a musculoskeletal disorder that is very closely related to metabolic dysregulation. OP is characterized by a disruption in the balance between osteoblasts and osteoclasts, cells that generate bone and cells that degrade bone, respectively. One common type of OP is glucocorticoid-induced osteoporosis (GIO). GIO is diagnosed in patients undergoing treatment with glucocorticoids for inflammatory or auto-immune diseases. Glucocorticoids inhibit osteoblast function and decrease the vascularity of the bone [66]. In one study, primary cells from human patients and mice with GIO had elevated levels of CKIP-1 and reduced levels of Smad1/5 compared to controls. CKIP-1 is a non-enzymatic protein that regulates the CK2α subunit of protein kinase CK2. It can be utilized in the localization of protein kinase CK2 in cell [67]. In vitro, elevated expression and activity of CKIP-1 in osteoblasts inhibited Smad-dependent BMP signaling [23]. CKIP-1 regulates BMP signaling and can recruit CK2α to the plasma membrane. Hence, its overexpression can affect the activation of BMP signaling in osteoblasts and osteoclasts. CKIP-1’s suppression of BMP signaling inhibits and affects the differentiation of MSC into osteoblasts. These alterations contribute to the reduction in bone mineralization observed in GIO patients. Suppressing the interaction between CKIP-1 and interaction with CK2 could be a novel therapeutic strategy for treating GIO. Upregulation of BMP signaling can increase bone formation and repair [16]. Blocking of the interaction between CK2 and CKIP-1 may induce BMP signaling [68]. A challenge in creating osteoporosis therapeutics is maintaining the optimal balance between osteoblasts and osteoclasts, cells that generate and degrade bone, respectively. Ideally, a therapeutic for OP would increase osteoblast activity while decreasing osteoclast activity, inducing bone formation and repair.