RESUMO
Osteoporosis (OP) is a bone disorder characterized by decreased bone mineral density (BMD). Bone Morphogenetic Protein-2 (BMP-2) injections are used to promote bone formation in OP patients. However, patients are unresponsive to BMP-2 while displaying an upregulation of BMP Receptor Type 1a (BMPRIa) and protein kinase CK2α (CK2α). A synthetically produced peptide named casein kinase 2.3 (CK2.3) utilizes the BMP-signaling pathway as it enhances osteogenesis of primary osteoblasts isolated from OP patients, whereas BMP-2 does not. Although shown in OP patients, there is currently no reliable mouse model to study BMP-2 and CK2.3 signaling. In this publication, we show that BMPRIa was required for CK2.3-mediated osteogenesis in C2C12 cells with a CRISPR-Cas9-mediated gene knockout for BMPRIa. We utilized the C57BL/6 (B6) mouse strain as an aging-model to study aberrant BMP-2 signaling, demonstrating that, like OP patients, in 15 and 20-month mice, BMP-2 did not increase bone growth and displayed upregulated BMPRIa and CK2α protein expression. Furthermore, CK2.3 enhanced osteogenesis and decreased osteoclastogenesis in all age groups, whereas BMP-2 only increased mineralization in 6-month mice while increasing osteoclast formation in all age groups. These data demonstrated that aging B6 mice were a reliable model and mimicked data obtained from OP patients.
Assuntos
Caseína Quinase II , Osteoporose , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Caseína Quinase II/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteogênese/fisiologia , Osteoporose/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Osteoporosis is a bone disease that has no definite cure. Current treatments for osteoporosis are divided into two categories: anti-resorptive and anabolic. However, these treatments are not perfect and have considerable risks. In addition, bone quality often declines over time with these treatments. We designed a peptide, CK2.3, that has both anabolic and anti-resorptive effects on bone. We reported that CK2.3 induced osteoblastic mineralization, promoted bone formation, and suppressed osteoclastogenesis in vivo. The effect of CK2.3 to rescue an osteoporosis phenotype model has never been shown. In this study, we demonstrated the effect of CK2.3 in ovariectomized rats, a standard model of osteoporosis. We systemically injected CK2.3 at 2.3 µg/kg each day for five consecutive days. Micro-computed tomography indicated that CK2.3 increased bone mineral density, (bone volume/tissue volume) BV/TV and (trabecular number) TbN, and decreased (trabecular space) TbSp in the femoral head. Similarly, single photon absorptiometry showed that treatment with CK2.3 increased bone mineral density in the lumbar spine and the pelvis. Additionally, we observed increased femoral shaft stiffness with ovariectomized rats treated with CK2.3. We also detected no significant changes in the weight of organs such as the heart, lung, liver, kidney, and spleen. An advantage of CK2.3 over current treatments was that it not only promoted bone formation but also improved fracture resistance. In conclusion, we demonstrated CK2.3 as a new anabolic treatment for osteoporosis.
Assuntos
Fêmur/efeitos dos fármacos , Vértebras Lombares/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Ovariectomia/métodos , Pelve/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
The most common bone disease in humans is osteoporosis (OP). Current therapeutics targeting OP have several negative side effects. Bone morphogenetic protein 2 (BMP2) is a potent growth factor that is known to activate both osteoblasts and osteoclasts. It completes these actions through both SMAD-dependent and SMAD-independent signaling. A novel interaction between the BMP type Ia receptor (BMPRIa) and casein kinase II (CK2) was discovered, and several CK2 phosphorylation sites were identified. A corresponding blocking peptide (named CK2.3) was designed to further elucidate the phosphorylation site's function. Previously, CK2.3 demonstrated an increased osteoblast activity and decreased osteoclast activity in a variety of animal models, cell lines, and isolated human osteoblasts. It is hypothesized that CK2.3 completes these actions through the BMP signaling pathway. Furthermore, it was recently discovered that BMP2 did not elicit an osteogenic response in osteoblasts from patients diagnosed with OP, while CK2.3 did. In this study, we explore where in the BMP pathway the signaling disparity or defect lies in those diagnosed with OP. We found that osteoblasts isolated from patients diagnosed with OP did not activate SMAD or ERK signaling after BMP2 stimulation. When OP osteoblasts were stimulated with BMP2, both BMPRIa and CK2 expression significantly decreased. This indicates a major disparity within the BMP signaling pathway in patients diagnosed with osteoporosis.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Sistema de Sinalização das MAP Quinases , Osteoblastos/metabolismo , Osteoporose/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/biossíntese , Caseína Quinase II/biossíntese , Feminino , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Osteoblastos/patologia , Osteoporose/diagnóstico , Osteoporose/patologiaRESUMO
BACKGROUND: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. METHODS: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. RESULTS: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2ß from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. CONCLUSION: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osteogênese/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Animais , Células da Medula Óssea/metabolismo , Linhagem Celular , Feminino , Sistema de Sinalização das MAP Quinases , Camundongos , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/fisiopatologia , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais , Proteínas Smad Reguladas por Receptor/metabolismoRESUMO
Bone is one of the most important organs in the human body. It provides structure, function, and protection for other vital organs; therefore, bone maintenance and homeostasis are critical processes. As humans age, their bone mineral density decreases, which leads to diseases like osteoporosis. This disease affects one in two women and one in five men aged 50 and over. As the aging population increases, the interest and significance of studying this debilitating bone disease becomes more relevant. Current therapeutic products for osteoporosis have many side effects and can be taken for a limited number of years. Most therapeutic products only focus on decreasing bone resorption, not increasing bone formation. Bone morphogenetic protein 2 is an essential growth factor that drives osteoblast differentiation and activity and is essential for bone formation. However, usage in the clinic is unsuccessful due to several side effects. Recently, a signaling disparity in bone marrow stromal cells within the bone morphogenetic protein pathway that led to decreased bone morphogenetic protein 2 responsiveness was identified in patients diagnosed with osteoporosis. However, it is unclear how other cell populations, especially osteoblasts, which are key players in bone remodeling, are affected and whether the bone morphogenetic protein pathway is affected during osteoporosis. Our research group designed a novel peptide, casein kinase 2.3, that acts downstream of the bone morphogenetic receptor type Ia and increases bone mineralization in murine cells and primary bovine osteoblasts. The aim of the study presented here was to compare the responsiveness of osteoblasts to bone morphogenetic protein 2 and casein kinase 2.3, especially in patients diagnosed with osteoporosis. Mature osteoblasts were extracted from patients diagnosed with osteoporosis or osteoarthritis from Christiana Care Hospital in Newark, Delaware. They were stimulated with either bone morphogenetic protein 2 or casein kinase 2.3, and their effect on osteoblast activity was determined. The osteoporotic patients showed no mineralization response to bone morphogenetic protein 2 stimulation, while the osteoarthritis patients significantly responded to bone morphogenetic protein 2 stimulation. Furthermore, markers for osteoblast activity were increased by casein kinase 2.3, which was in sharp contrast to bone morphogenetic protein 2. This further supports a major bone morphogenetic protein signaling disparity in both the elderly and those suffering with osteoporosis. Both patient types did significantly respond to casein kinase 2.3. Further analysis of the bone morphogenetic protein pathway could lead to new therapeutic products for osteoporosis.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osteoblastos/metabolismo , Fragmentos de Peptídeos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/metabolismo , Células Cultivadas , Feminino , Humanos , Modelos Lineares , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteoporose/metabolismoRESUMO
The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.
Assuntos
Calcificação Fisiológica , Sinalização do Cálcio , Cavéolas/metabolismo , Caveolina 1/metabolismo , Osteoblastos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Células 3T3 , Animais , Calcificação Fisiológica/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cavéolas/efeitos dos fármacos , Caveolina 1/genética , Endocitose , Camundongos , Osteoblastos/efeitos dos fármacos , Transporte Proteico , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Interferência de RNA , Receptores Purinérgicos P2X7/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
Spatial and temporal changes of cytoplasmic calcium ions ([Ca(2+)]c), caused by external stimuli, are known as the Ca(2+) signature and presumably control cellular and developmental responses. Multiple types of ion channels, pumps, and transporters on plasma and organellar membranes modulate influx and efflux of Ca(2+) to and from the extracellular environment and internal Ca(2+) stores to form Ca(2+) signatures. Expression of a fluorescent protein-based Ca(2+) probe, Cameleon YC3.60, in Fusarium oxysporum enabled us to study how disruption of three Ca(2+) channel genes, including FoCCH1, FoMID1 and FoYVC1, affects Ca(2+) signature formation at polarized hyphal tips and whether specific changes in the Ca(2+) signature caused by these mutations are related to growth-related phenotypes. Resulting mutants displayed altered amplitude, interval, and duration of Ca(2+) pulses under various external Ca(2+) concentrations as well as changes in sporulation and growth. Loss of FoMID1 and FoCCH1, genes encoding putative plasma membrane channel proteins, had a major impact on Ca(2+) signatures and growth, while disruption of FoYVC1, which encodes a vacuolar channel, only subtly affected both traits. Results from our study provide new insights into the underpinning of Ca(2+) signaling in fungi and its role in controlling growth and also raise several new questions.
Assuntos
Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Fusarium/fisiologia , Meios de Cultura , Expressão Gênica , Hifas , Mutação , Imagem com Lapso de TempoRESUMO
The contribution of caveolae in Bone Morphogenetic Protein 2 (BMP2) activated Smad signaling was quantified using a system biology approach. BMP2 plays crucial roles during processes such as hematopoiesis, embryogenesis, and skeletal development. BMP2 signaling is tightly regulated on the plasma membrane by its receptors. The localization of BMP receptors in caveolae and endocytosis through clathrin-coated pits are thought to regulate the signaling; however the conclusions in the current literature are inconsistent. Therefore published literature was used to establish a mathematical model that was validated using confocal AFM (atomic force microscopy), confocal microscopy, and sucrose density centrifugation followed by Western blots, and reporter gene assays. The model and experiments confirmed that both caveolae and CCPs regulate the Smad-dependent signaling pathway, however caveolae are centers at the plasma membrane where receptor-ligand interaction is crucial, Smad phosphorylation occurs, and a high degree of Smad signaling is regulated. This demonstrates a role for caveolae that needs to be considered and further studied.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Cavéolas/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Proteínas Smad , Animais , Proteína Morfogenética Óssea 2/genética , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/genética , Endocitose , Regulação da Expressão Gênica , Camundongos , Microscopia de Força Atômica , Fosforilação , Ligação Proteica , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Biologia de Sistemas/métodosRESUMO
Experimental evidence is presented on the translocation of vitamin D metabolite, 1,25-(OH)2D3, from the membrane to the nucleus in osteoblast progenitor cells. A mathematical model permitting traversal of the cytoplasm at either a fixed velocity or by diffusion is formulated in order to determine whether transport along the cytoskeletal tracks is more consistent with the observed spatial-temporal distribution than diffusion, and it is so found. The model includes reactions in the nucleus involving D3 to form other compounds, such as protegerin, and thus also makes predictions of the concentrations of these compounds in various regions of the cell.
Assuntos
Colecalciferol/metabolismo , Modelos Biológicos , Osteoblastos/metabolismo , Animais , Transporte Biológico Ativo , Compartimento Celular , Linhagem Celular , Forma Celular , Biologia Computacional , Cinética , Conceitos Matemáticos , Camundongos , Osteoblastos/citologia , Pontos QuânticosRESUMO
Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for cell fate determination. It directs the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes, and myocytes. Initiation of BMP2 signaling pathways occurs at the cell surface through type I and type II serine/threonine kinases housed in specific membrane domains such as caveolae enriched in the caveolin-1 beta isoform (CAV1ß, caveolae) and clathrin-coated pits (CCPs). In order for BMP2 to initiate Smad signaling it must bind to its receptors on the plasma membrane resulting in the phosphorylation of the BMP type Ia receptor (BMPRIa) followed by activation of Smad signaling. The current model suggests that the canonical BMP signaling pathway, Smad, occurs in CCPs. However, several recent studies suggested Smad signaling may occur outside of CCPs. Here, we determined; (i) The location of BMP2 binding to receptors localized in caveolae, CCPs, or outside of these domains using AFM and confocal microscopy. (ii) The location of phosphorylation of BMPRIa on the plasma membrane using membrane fractionation, and (iii) the effect of down regulation of caveolae on Smad signaling. Our data indicate that BMP2 binds with highest force to BMP receptors (BMPRs) localized in caveolae. BMPRIa is phosphorylated in caveolae and the disruption of caveolae-inhibited Smad signaling in the presence of BMP2. This suggests caveolae are necessary for the initiation of Smad signaling. We propose an extension of the current model of BMP2 signaling, in which the initiation of Smad signaling is mediated by BMPRs in caveolae.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Cavéolas/metabolismo , Membrana Celular/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Células Cultivadas , Regulação para Baixo , Camundongos , Mioblastos/metabolismo , Fosforilação , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transdução de Sinais , Proteínas Smad/metabolismoRESUMO
Bone morphogenetic protein 2 (BMP2) is a growth factor that initiates osteoblast differentiation. Recent studies show that BMP2 signaling regulates bone mineral density (BMD). BMP2 interacts with BMP receptor type Ia (BMPRIa) and type II receptor leading to the activation of the Smad signaling pathway. BMPRIa must shuttle between distinct plasma membrane domains, enriched of Caveolin-1 alpha and Caveolin-1 beta isoforms, and receptor activation occurs in these domains. Yet it remains unknown whether the molecular mechanism that regulates BMP2 signaling is driving mineralization and BMD. Therefore, the B6.C3H-1-12 congenic mouse model with increased BMD and osteoblast mineralization was utilized in this study. Using the family image correlation spectroscopy, we determined if BMP2 led to a significant re-localization of BMPRIa to caveolae of the alpha/beta isoforms in bone marrow stromal cells (BMSCs) isolated from B6.C3H-1-12 mice compared to the C57BL/6J mice, which served as controls. The control, C57BL/6J mice, was selected due to only 4 Mb of chromosome 1 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Using reporter gene assays, the B6.C3H-1-12 BMSCs responded to BMP2 with increased Smad activation. Furthermore, disrupting caveolae reduced the BMP2-induced Smad signaling in BMSCs isolated from B6.C3H-1-12 and C57BL/6J. This study suggests for the first time a regulatory mechanism of BMPRIa signaling at the plasma membrane of BMSCs that (i) associated with genetic differences in the distal Chromosome 1 segment carried by the B6.C3H-1-12 congenic and (ii) contributes to increase BMD of the B6.C3H-1-12 compared to the C57BL/6J control mice.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osso e Ossos/metabolismo , Animais , Densidade Óssea , Medula Óssea/metabolismo , Calcificação Fisiológica/fisiologia , Cavéolas/metabolismo , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Feminino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Osteogênese/fisiologia , Fenótipo , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Células Estromais/metabolismoRESUMO
Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca(2+)-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca(2+) (i.e., "Ca(2+) signature"), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca(2+) biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca(2+) ([Ca(2+)](c)) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca(2+) signature. Furthermore, occurrence of pulsatile Ca(2+) signatures was age and development dependent, and major [Ca(2+)](c) transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell-cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca(2+)-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca(2+) signaling across eukaryotic kingdoms.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Fungos/metabolismo , Proteínas Luminescentes/metabolismo , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Calmodulina/metabolismo , Adesão Celular , Divisão Celular , Fungos/citologia , Fungos/genética , Fungos/crescimento & desenvolvimento , Expressão Gênica , Hifas/citologia , Hifas/metabolismo , Proteínas Luminescentes/genética , Raízes de Plantas/microbiologia , Ligação Proteica , Transporte ProteicoRESUMO
1,25 dihydroxyvitamin D3 (Calcitriol), one of the active forms of Vitamin D, plays a vital role not only in calcium absorption but also during neuromuscular function and regulation of inflammation. Epidemiological studies suggest a preventive effect of Calcitriol in breast, colon and prostate cancer, however high concentrations of Calcitriol are necessary. Therefore targeted biologically active probes must be designed to determine Calcitriol distribution and dynamics in vitro and in vivo. Our Calcitriol probe remained stable over 2 days at 37 degrees C. When added to live C2C12 cells, the Calcitriol probe can be seen entering the nucleus within 2 hours and the probe activated the expression of the Vitamin D Response Element (VDRE), one of the major transcription elements. The Calcitriol probe provides a novel imaging tool that can be used to view Calcitriol localization and dynamics.
Assuntos
Calcitriol/química , Pontos Quânticos , Animais , Linhagem Celular , Genes Reporter , Camundongos , Microscopia de Força Atômica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Protein kinase CK2 (CK2) is a ubiquitous holoenzyme involved in a wide array of developmental processes. The involvement of CK2 in events such as neurogenesis, cardiogenesis, skeletogenesis, and spermatogenesis is essential for the viability of almost all organisms, and its role has been conserved throughout evolution. Further into adulthood, CK2 continues to function as a key regulator of pathways affecting crucial processes such as osteogenesis, adipogenesis, chondrogenesis, neuron differentiation, and the immune response. Due to its vast role in a multitude of pathways, aberrant functioning of this kinase leads to embryonic lethality and numerous diseases and disorders, including cancer and neurological disorders. As a result, CK2 is a popular target for interventions aiming to treat the aforementioned diseases. Specifically, two CK2 inhibitors, namely CX-4945 and CIBG-300, are in the early stages of clinical testing and exhibit promise for treating cancer and other disorders. Further, other researchers around the world are focusing on CK2 to treat bone disorders. This review summarizes the current understanding of CK2 in development, the structure of CK2, the targets and signaling pathways of CK2, the implication of CK2 in disease progression, and the recent therapeutics developed to inhibit the dysregulation of CK2 function in various diseases.
RESUMO
Proper formation of the skeleton during development is crucial for the mobility of humans and the maintenance of essential organs. The production of bone is regulated by osteoblasts and osteoclasts. An imbalance of these cells can lead to a decrease in bone mineral density, which leads to fractures. While many studies are emerging to understand the role of osteoblasts, less studies are present about the role of osteoclasts. This present study utilized bone marrow cells isolated directly from the bone marrow of femoral heads obtained from osteoarthritic (OA) patients after undergoing hip replacement surgery. Here, we used tartrate resistant acid phosphatase (TRAP) staining, Cathepsin K, and nuclei to identity osteoclasts and their functionality after stimulation with macrophage-colony stimulation factor (M-CSF) and receptor activator of nuclear factor kappa-ß ligand (RANKL). Our data demonstrated that isolated cells can be differentiated into functional osteoclasts, as indicated by the 92% and 83% of cells that stained positive for TRAP and Cathepsin K, respectively. Furthermore, isolated cells remain viable and terminally differentiate into osteoclasts when stimulated with RANKL. These data demonstrate that cells isolated from human femoral heads can be differentiated into osteoclasts to study bone disorders during development and adulthood.
RESUMO
Bone morphogenetic proteins (BMPs) are pleiotrophic growth factors that influence diverse processes such as skeletal development, hematopoiesis, and neurogenesis. They play crucial roles in diseases such as pulmonary arterial hypertension (PAH). In PAH, mutants of the BMP type II receptors (BMPR2) were detected, and their functions were impaired during BMP signaling. It is thought that expression levels of these receptors determine the fate of BMP signaling, with low levels of expression leading to decreased Smad activation in PAH. However, our studies demonstrate, for the first time, that the localization of receptors on the plasma membrane, in this case BMPR2, was misdirected. Three BMPR2 mutants, D485G, N519K, and R899X, which are known to be involved in PAH, were chosen as our model system. Our results show that all three BMPR2 mutants decreased BMP-dependent Smad phosphorylation and Smad signaling. Although the three mutants reached the cell membrane and their expression was lower than that of BMPR2, they formed smaller clusters and associated differently with membrane domains, such as caveolae and clathrin-coated pits. The disruption of these domains restored the Smad signaling of D485G and N519K to the level of wild-type BMPR2, showing that these mutants were trapped in the domains, rather than just expressed at a lower level on the surface. Therefore, new treatment options for PAH should also target receptor localization, rather than just expression level.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Membrana Celular/metabolismo , Hipertensão Pulmonar/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cavéolas/metabolismo , Linhagem Celular , Invaginações Revestidas da Membrana Celular/metabolismo , Hipertensão Pulmonar Primária Familiar , Humanos , Mutação , Fosforilação , Transdução de Sinais , Proteínas Smad/metabolismo , Distribuição Tecidual , Regulação para CimaRESUMO
The osteogenic effects of Bone Morphogenetic Proteins (BMPs) were delineated in 1965 when Urist et al. showed that BMPs could induce ectopic bone formation. In subsequent decades, the effects of BMPs on bone formation and maintenance were established. BMPs induce proliferation in osteoprogenitor cells and increase mineralization activity in osteoblasts. The role of BMPs in bone homeostasis and repair led to the approval of BMP2 by the Federal Drug Administration (FDA) for anterior lumbar interbody fusion (ALIF) to increase the bone formation in the treated area. However, the use of BMP2 for treatment of degenerative bone diseases such as osteoporosis is still uncertain as patients treated with BMP2 results in the stimulation of not only osteoblast mineralization, but also osteoclast absorption, leading to early bone graft subsidence. The increase in absorption activity is the result of direct stimulation of osteoclasts by BMP2 working synergistically with the RANK signaling pathway. The dual effect of BMPs on bone resorption and mineralization highlights the essential role of BMP-signaling in bone homeostasis, making it a putative therapeutic target for diseases like osteoporosis. Before the BMP pathway can be utilized in the treatment of osteoporosis a better understanding of how BMP-signaling regulates osteoclasts must be established.
RESUMO
Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate processes as diverse as neurogenesis, skeletal formation, and hematopoesis. They signal through a hetero-oligomer complex of BMP receptors. Binding of the ligand to the receptors activates several pathways, including Smad and p38. BMP signaling is controlled in the extracellular space, the plasma membrane, and the intracellular space; however, the mechanism of receptor signaling at the plasma membrane and proteins that regulate this process still need to be identified. The experiments presented here identify the protein kinase casein kinase II (CK2) as a BMP receptor type Ia (BRIa) interacting protein. Fluorescence resonance energy transfer revealed that this interaction occurs at the plasma membrane. BMP2 stimulation of C2C12 cells leads to the release of CK2 from BRIa. Blocking this interaction with specific peptides that inhibit the binding sites for CK2 on BRIa demonstrated a redistribution of BRIa on the plasma membrane. Signaling was initiated once CK2 was released from BRIa, leading to the mineralization of C2C12 cells. These data suggest that CK2 is a negative regulator of BMP signaling and osteoblast differentiation.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Caseína Quinase II/metabolismo , Transdução de Sinais , Animais , Proteína Morfogenética Óssea 2/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Genes Dominantes/genética , Camundongos , Modelos Biológicos , Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismoRESUMO
Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the Transforming Growth Factor-Beta (TGF-ß) superfamily. These proteins are essential to many developmental processes, including cardiogenesis, neurogenesis, and osteogenesis. Specifically, within the BMP family, Bone Morphogenetic Protein-2 (BMP-2) was the first BMP to be characterized and has been well-studied. BMP-2 has important roles during embryonic development, as well as bone remodeling and homeostasis in adulthood. Some of its specific functions include digit formation and activating osteogenic genes, such as Runt-Related Transcription Factor 2 (RUNX2). Because of its diverse functions and osteogenic potential, the Food and Drug Administration (FDA) approved usage of recombinant human BMP-2 (rhBMP-2) during spinal fusion surgery, tibial shaft repair, and maxillary sinus reconstructive surgery. However, shortly after initial injections of rhBMP-2, several adverse complications were reported, and alternative therapeutics have been developed to limit these side-effects. As the clinical application of BMP-2 is largely implicated in bone, we focus primarily on its role in bone. However, we also describe briefly the role of BMP-2 in development. We then focus on the structure of BMP-2, its activation and regulation signaling pathways, BMP-2 clinical applications, and limitations of using BMP-2 as a therapeutic. Further, this review explores other potential treatments that may be useful in treating bone disorders.
RESUMO
Quantum Dot®s (QDot®s) are novel, semi-conductive nanostructures that emit a certain fluorescence when excited by specific wavelengths. QDot®s are more photostable, brighter, and photobleach less than other fluorescent dyes. These characteristics give them the potential to be used in many biological applications. The shells of QDot®s are coated with functional groups, such as carboxylate and organic groups, allowing them to couple to peptides/proteins and be used for real-time imaging and high-resolution microscopy. Here, we utilize Quantum Dot®s and Bone Morphogenetic Protein-2 (BMP-2) to create a BMP-2-QDot®s conjugate. BMP-2 is a growth factor that drives many processes such as cardiogenesis, neural growth, and osteogenesis. Despite its numerous roles, the trafficking and uptake of BMP-2 into cells is not well-established, especially during progression of diseases. The results presented here demonstrate for the first time a fluorescent BMP-2 analog that binds to the BMP-receptors (BMPRs), remains biologically active, and is stable for long time periods. Previous attempts to develop a biological BMP-2 analog with Fluorescein isothiocyanate (FITC) or nanodiamonds lacked data on the analog's stability. Furthermore, these analogs did not address whether they can signal within the cell by binding to the BMPRs or were mediated by non-stable conjugates.