Regulation of bone metabolism by megakaryocytes in a paracrine manner

Abstract

Megakaryocytes (MKs) play key roles in regulating bone metabolism.To test the roles of MK-secreted factors, we investigated whether MK and promegakaryocyte (pro-MK) conditioned media (CM) may affect bone formation and resorption. K562 cell lines were differentiated into mature MKs. Mouse bone marrow macrophages were differentiated into mature osteoclasts, and MC3T3-E1 cells were used for osteoblastic experiments. Bone formation was determined by a calvaria bone formation assay in vivo. Micro-CT analyses were performed in the femurs of ovariectomized female C57B/L6 and Balb/c nude mice after intravenous injections of MK or pro-MK CM. MK CM significantly reduced in vitro bone resorption, largely due to suppressed osteoclastic resorption activity. Compared with pro-MK CM, MK CM suppressed osteoblastic differentiation, but stimulated its proliferation, resulting in stimulation of calvaria bone formation. In ovariectomized mice, treatment with MK CM for 4 weeks significantly increased trabecular bone mass parameters, such as bone volume fraction and trabecular thickness, in nude mice, but not in C57B/L6 mice. In conclusion, MKs may secrete anti-resorptive and anabolic factors that affect bone tissue, providing a novel insight linking MKs and bone cells in a paracrine manner. New therapeutic agents against metabolic bone diseases may be developed from MK-secreted factors.Bone metabolism is regulated mainly by the action of bone-resorbing osteoclasts and bone-forming osteoblasts. Increased bone resorption and/or decreased bone formation can lead to reduced bone mass and quality, resulting in high fracture risk. Typical conditions that induce this imbalance include estrogen deficiency and immobilization 1,2 . On the contrary, decreased bone resorption and/or increased formation with pharmacological interventions can reverse these imbalances. Synchronously inhibiting bone resorption and stimulating bone formation are regarded as an ideal therapeutic strategy against metabolic bone diseases, such as osteoporosis.Bone marrow, where most osteoclasts and osteoblasts exist, also contains many types of hematopoietic cells. The coexistence of the cells in the bone marrow allows these cells to influence one another by cell-to-cell contact or in a paracrine manner. Specifically, megakaryocytes (MKs), as one of hematopoietic cell residing in bone marrow, have been intriguing in the field of bone research. MKs, which are polyploid cells derived from MK/erythroid progenitors, generate platelets, which contribute to hemostasis and produce a number of growth factors 3-5 . Interestingly, estrogen deficiency and immobilization reduced the number of MKs 6,7 , and estrogen treatment increased MK number in postmenopausal women 8 . In addition, MK-related disorders are associated with osteosclerosis 9-11 . Thus, MKs may have a critical role in bone metabolism.Actually, it has been reported that MKs may act on both bone resorption and bone formation. A mouse model with increased MKs showed decreased osteoclast number and bone resorption 12 . ...

Muscle-Derived Lumican Stimulates Bone Formation via Integrin α2β1 and the Downstream ERK Signal

“…Cell viability was measured using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 10 μL of WST-8 dye [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] was added to each well of a 96-well plate ( Lee et al, 2020 ). The suspension was incubated for 1 h, and the absorbance was then read at 450 nm with a reference wavelength of 650 nm using a microplate reader (SPECTRAmax 340PC; Molecular Devices, Palo Alto, CA, United States).…”

“…After deparaffinization, sections were rehydrated and then stained with either hematoxylin and eosin (H&E, Sigma-Aldrich) following the manufacturer's instructions. Using Image-Pro Plus software (Media Cybernetics, Rockville, MD, United States), bone widths were measured at 5 adjacent locations that were at the same distance from the midline between the sagittal suture and the site of muscle attachment, and their mean values were calculated (Lee et al, 2020). To count the number of osteoblasts, the rehydrated sections were stained with toluidine blue O solution (Sigma-Aldrich), and images of the stained osteoblasts were captured using cellSens Standard BX53 software (Olympus, Tokyo, Japan).…”

S100 Calcium-Binding Protein P Secreted from Megakaryocytes Promotes Osteoclast Maturation

“…K562 cells were differentiated into mature MKs in phorbol 12-myristate 13-acetate (PMA)-supplemented medium for 7 days [ 26 ]; differentiation was determined by morphological changes, such as an increased nuclear/cytoplasmic ratio, large and multilobulated nuclei, and strong adhesion. Based on several reports that MKs may indirectly contribute to skeletal homeostasis [ 12 , 27 ], we investigated the effects of MK CM on osteoclast differentiation. Using tartrate-resistant acid phosphatase (TRAP) staining, TRAP-positive osteoclasts can be distinguished from TRAP-negative MKs [ 28 ].…”

“…Increased MKs in aged mice activate RANKL expression and signaling, thereby promoting osteoclastogenesis and bone resorption activity [ 27 ]. Another study showed that MK CM regulates osteoblast and osteoclast differentiation and increases bone mass in ovariectomized mouse models, although the MK-secreted factors in MK CM have not been identified [ 12 ]. Moreover, the role of MKs in bone metastasis remains unclear.…”

“…A direct interaction has been detected between MKs and osteoblasts, with MKs increasing osteoblast proliferation and bone mass, suggesting an anabolic role for MKs in skeletal homeostasis [ 10 , 11 ]. A recent study showed that MK-cultured conditioned medium (MK CM) affected bone cell differentiation in vitro and bone mass in ovariectomized mice, suggesting that MK-secreted factors play a role in bone metabolism [ 12 ]. Although some studies have focused on the indirect effects of MKs on bone cells or MK-secreted factors involved in bone metabolism [ 12 , 13 , 14 , 15 , 16 ], these effects and factors still remain unclear.…”