PDGF-AA promotes cell-to-cell communication in osteocytes through PI3K/Akt signaling pathway

Osteocytes are the main sensitive cells in bone remodeling due to their potent functional cell processes from the mineralized bone matrix to the bone surface and the bone marrow. Neighboring osteocytes communicate with each other by these cell processes to achieve molecular exchange through gap junction channels. Platelet-derived growth factor-AA (PDGF-AA) has been reported to enhance bone tissue remodeling by promoting cell proliferation, migration, and autocrine secretion in osteoid cell linage. However, the effect of PDGF-AA on intercellular communication between osteocytes is still unclear. In the present study, we elucidated that PDGF-AA could enhance the formation of dendritic processes of osteocytes and the gap junctional intercellular communication by promoting the expression of connexin43 (Cx43). This modulation process was mainly dependent on the activation of phosphorylation of Akt protein by phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) signaling. Inhibition of PI3K/Akt signaling decreased the Cx43 expression induced by PDGF-AA. These results establish a bridge between PDGF-AA and cell–cell communication in osteocytes, which could help us understand the molecular exchange between bone cells and fracture healing.

Determination of the Earliest time Point for Heat-shock Inducing Osteoporosis-like Phenotype in the rankl:HSE:CFP Medaka Fish Model for Osteoporosis

The transgenic medaka rankl:HSE:CFP expressing Rankl, a stimulator for osteoclastogenesis - the formation and activation of osteoclasts, bone “eating” cells, under control of a heat inducible promotor has been established as a model for osteoporosis to evaluate antiosteoporosis effects of substances. Transgenic larvae were usually heat-shocked for 90 minutes at 39oC when they were at 9 days post fertilization (dpf) and osteoporosis-like phenotype was analysed when they were at 11 dpf. In this study we investigated whether osteoporosis-like phenotype could be induced in the transgenic larvae when heat-shock was applied at earlier time points, when larvae were at 1, 2, 3, 4, 5, or 6 dpf. Results showed that heat-shocks from the time point of 3 dpf onwards resulted in osteoporosis-like phenotype while heat-shock at 1 or 2 dpf did not affect mineralized bone matrix in 11 dpf larvae. These provide important evidence for study on onset of Rankl induced osteoclasts in fish and help improving experimental procedures using this fish model for osteoporosis.

Ankylosis homologue mediates cellular efflux of ATP, not pyrophosphate

The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints, but we recently showed that ANKH releases large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. This ATP is converted extracellularly into PPi and AMP by the ectoenzyme Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANK. We now addressed this question by determining the effect of a complete absence of ENPP1 on ANKH-dependent extracellular PPi concentrations. Introduction of ANKH in ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1-proficient cells.Ank-activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained < 2.5% of the PPi found in bones of wild type mice, showing that Enpp1-activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60-70 % of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself.

The Osteocyte as a Signaling Cell

Osteocytes, former osteoblasts encapsulated by mineralized bone matrix, are far from being passive and metabolically inactive bone cells. Instead, osteocytes are multifunctional and dynamic cells capable of integrating hormonal and mechanical signals and transmitting them to effector cells in bone as well as in distant tissues. Osteocytes are a significant source of molecules that regulate bone homeostasis by integrating mechanical cues and hormonal signals that coordinate the differentiation and function of osteoclasts and osteoblasts. Osteocyte function is altered in rare and common bone diseases, suggesting that osteocyte dysfunction is directly involved in the pathophysiology of disorders affecting the skeleton. Advances in osteocyte biology initiated the development of novel therapeutics interfering with osteocyte secreted molecules. Moreover, osteocytes are targets and key distributors of biological signals mediating the beneficial effects of various bone therapeutics used in the clinic. Herein, we review the most recent discoveries in osteocyte biology demonstrating that osteocytes regulate bone homeostasis and bone marrow fat via paracrine signaling, influence body composition and energy metabolism via endocrine signaling, and contribute to the damaging effects of diabetes mellitus and hematological and metastatic cancers in the skeleton.

Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin

Osteocytes use an elaborate network of dendritic connections to control bone remodeling. Some osteoblasts embed within mineralized bone matrix, change shape, and become osteocytes. The molecular circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we show that deletion of Sp7, a gene linked to rare and common skeletal disease, in mature osteoblasts and osteocytes causes severe defects in osteocyte dendrites. Unbiased profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues phenotypic and molecular defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates overt defects in osteocyte maturation in vivo in the absence of Sp7. Sp7-dependent gene networks enriched in developing osteocytes are associated with rare and common human skeletal traits. Moreover, humans homozygous for the osteogenesis imperfecta-causing SP7R316C mutation show dramatic defects in osteocyte morphology. Genes that mark osteocytes in vivo and that are regulated by Sp7 in vitro are highly enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. Taken together, these findings reveal a crucial role for Sp7 and its target gene Osteocrin in osteocytogenesis, demonstrating that pathways that control osteocyte development influence human bone diseases.

The Osteocyte: From “Prisoner” to “Orchestrator”

Osteocytes are the most abundant bone cells, entrapped inside the mineralized bone matrix. They derive from osteoblasts through a complex series of morpho-functional modifications; such modifications not only concern the cell shape (from prismatic to dendritic) and location (along the vascular bone surfaces or enclosed inside the lacuno-canalicular cavities, respectively) but also their role in bone processes (secretion/mineralization of preosseous matrix and/or regulation of bone remodeling). Osteocytes are connected with each other by means of different types of junctions, among which the gap junctions enable osteocytes inside the matrix to act in a neuronal-like manner, as a functional syncytium together with the cells placed on the vascular bone surfaces (osteoblasts or bone lining cells), the stromal cells and the endothelial cells, i.e., the bone basic cellular system (BBCS). Within the BBCS, osteocytes can communicate in two ways: by means of volume transmission and wiring transmission, depending on the type of signals (metabolic or mechanical, respectively) received and/or to be forwarded. The capability of osteocytes in maintaining skeletal and mineral homeostasis is due to the fact that it acts as a mechano-sensor, able to transduce mechanical strains into biological signals and to trigger/modulate the bone remodeling, also because of the relevant role of sclerostin secreted by osteocytes, thus regulating different bone cell signaling pathways. The authors want to emphasize that the present review is centered on the morphological aspects of the osteocytes that clearly explain their functional implications and their role as bone orchestrators.

Promotion of Osteogenesis by Sweroside via BMP-2 Related Signaling in Postmenopausal Osteoporosis

Phlomis umbrosa has been traditionally used for bone diseases such as bone fracture and rheumatism in traditional Korea Medicine. Sweroside (SOS), which is one of the active compounds of P. umbrosa, has been known to promote osteoblast differentiation. In this study, ameliorative effects of SOS on osteoporosis and potential target pathway were investigated. Ovariectomized mice were administered 3 doses of SOS for 4 weeks after inducing osteoporosis. Bone mineral content (BMC) and bone mineral density (BMD) were analyzed by dual energy X-ray absorptiometry. SaOS-2 osteoblasts were differentiated to clarify the promoting effects of SOS on osteoblast differentiation and bone formation. Osteoblastic bone-forming markers were evaluated by RT-PCR in lumbar vertebrae (LV) and mineralized SaOS-2 cells. Treatment of SOS increased BMC and BMD levels. SOS markedly attenuated the bone marrow adipocytes in the central bone cavity of the femoral shaft. SOS increased the formation of bone matrix in SaOS-2 cells. BMP-2 and RUNX2 in LV and SaOS-2 cells were up-regulated by treating with SOS. BMP-2/RUNX2-activated ALP, OPN and BSP-1 expressions were increased by SOS. In conclusion, SOS induced the formation of mineralized bone matrix by regulating BMP-2/RUNX2-mediated osteoblastic molecules. Therefore, SOS could be a therapeutic active compound of treatment for osteoporosis by producing the new bone matrix.

3D Printing of Anisotropic Bone-Mimetic Structure with Controlled Fluid Flow Stimuli for Osteocytes: Flow Orientation Determines the Elongation of Dendrites

Although three-dimensional (3D) bioprinting techniques enable the construction of various living tissues and organs, the generation of bone-like oriented microstructures with anisotropic texture remains a challenge. Inside the mineralized bone matrix, osteocytes play mechanosensing roles in an ordered manner with a well-developed lacunar-canaliculi system. Therefore, control of cellular arrangement and dendritic processes is indispensable for construction of artificially controlled 3D bone-mimetic architecture. Herein, we propose an innovative methodology to induce controlled arrangement of osteocyte dendritic processes using the laminated layer method of oriented collagen sheets, combined with a custom-made fluid flow stimuli system. Osteocyte dendritic processes showed elongation depending on the competitive directional relationship between flow and substrate. To the best of our knowledge, this study is the first to report the successful construction of the anisotropic bone-mimetic microstructure and further demonstrate that the dendritic process formation in osteocytes can be controlled with selective fluid flow stimuli, specifically by regulating focal adhesion. Our results demonstrate how osteocytes adapt to mechanical stimuli by optimizing the anisotropic maturation of dendritic cell processes.

An improved method to isolate primary human osteocytes from bone

Osteocytes are of high importance in bone metabolism as they orchestrate bone remodeling, react to mechanosensory stimuli and have endocrine functions. In vitro investigations with osteocytes are therefore of high relevance for biomaterial and drug testing. The application of primary human cells instead of rodent osteocyte cell lines like MLOY4 and IDG SW3 is desirable but provides the challenge of isolating these cells, which are deeply embedded into the mineralized bone matrix. The present study describes an improved protocol for the isolation of human primary osteocytes. In contrast to an already established protocol, resting steps between the demineralization /digestion steps of the bone particles considerably improved the yield of osteocytes. Real-time polymerase chain reaction (PCR) analysis revealed the expression of typical osteocyte markers like osteocalcin, E11/podoplanin and dentin matrix protein 1 (DMP-1).