Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha-interaction

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPalpha) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPalpha-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPalpha strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)+ osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47-/- mice were strongly reduced, and bones of CD47-/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPalpha interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47-/- mice.     

Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.     

Serglycin induces osteoclastogenesis and promotes tumor growth in giant cell tumor of bone

Giant cell tumor of bone (GCTB) is an aggressive osteolytic bone tumor characterized by the within-tumor presence of osteoclast-like multinucleated giant cells (MGCs), which are induced by the neoplastic stromal cells and lead to extensive bone destruction. However, the underlying mechanism of the pathological process of osteoclastogenesis in GCTB is poorly understood. Here we show that the proteoglycan Serglycin (SRGN) secreted by neoplastic stromal cells plays a crucial role in the formation of MGCs and tumorigenesis in GCTB. Upregulated SRGN expression and secretion are observed in GCTB tumor cells and patients. Stromal-derived SRGN promotes osteoclast differentiation from monocytes. SRGN knockdown in stromal cells inhibits tumor growth and bone destruction in a patient-derived orthotopic xenograft model of mice. Mechanistically SRGN interacts with CD44 on the cell surface of monocytes and thus activates focal adhesion kinase (FAK), leading to osteoclast differentiation. Importantly, blocking CD44 with a neutralizing antibody reduces the number of MGCs and suppresses tumorigenesis in vivo. Overall, our data reveal a mechanism of MGC induction in GCTB and support CD44-targeting approaches for GCTB treatment.Subject terms: Bone cancer, Cancer microenvironment, Mechanisms of disease     

CD109 Plays a Role in Osteoclastogenesis

Osteoclasts are large multinucleated cells that arise from the fusion of cells from the monocyte/macrophage lineage. Osteoclastogenesis is mediated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) and involves a complex multistep process that requires numerous other elements, many of which remain undefined. The primary aim of this project was to identify novel factors which regulate osteoclastogenesis. To carry out this investigation, microarray analysis was performed comparing two pre-osteoclast cell lines generated from RAW264.7 macrophages: one that has the capacity to fuse forming large multinucleated cells and one that does not fuse. It was found that CD109 was up-regulated by>17-fold in the osteoclast forming cell line when compared to the cell line that does not fuse, at day 2 of the differentiation process. Results obtained with microarray were confirmed by RT-qPCR and Western blot analyses in the two cell lines, in the parental RAW264.7 cell line, as well as primary murine monocytes from bone marrow. A significant increase of CD109 mRNA and protein expression during osteoclastogenesis occurred in all tested cell types. In order to characterize the role of CD109 in osteoclastogenesis, CD109 stable knockdown cell lines were established and fusion of osteoclast precursors into osteoclasts was assessed. It was found that CD109 knockdown cell lines were less capable of forming large multinucleated osteoclasts. It has been shown here that CD109 is expressed in monocytes undergoing RANKL-induced osteoclastogenesis. Moreover, when CD109 expression is suppressed in vitro, osteoclast formation decreases. This suggests that CD109 might be an important regulator of osteoclastogenesis. Further research is needed in order to characterize the role played by CD109 in regulation of osteoclast differentiation.     

Expression and possible role of PVR/CD155/Necl-5 in osteoclastogenesis

Osteoclasts, the bone-resorbing cells, are differentiated from hematopoietic precursors via two-step cell–cell interactions. One is the interaction between the osteoclast precursor and the stromal cell to initiate differentiation. The other is the interaction among osteoclast precursors to form multinucleated osteoclasts. Recently, the poliovirus receptor (PVR, CD155, Necl-5) was reported to play important roles in cell adhesion and migration. However, there are no reports of PVR in osteoclastogenesis. In this paper, we examined the expression of PVR and its ligand, DNAX accessory molecule-1 (DNAM-1, CD226), in osteoclast precursors, mature osteoclasts, and stromal cells. We found that the PVR was constitutively expressed in both osteoclast cells and stromal cells. The expression of PVR was not changed at various stages of osteoclast formation. In contrast, the expression of DNAM-1 was observed in mononuclear cells and was down-regulated during osteoclastogenesis. Moreover, multinucleated osteoclast formation was inhibited by treatment with the extracellular domain of DNAM-1 (ED-DNAM-1) as a soluble ligand for PVR, but mononuclear preosteoclast formation was not affected. Especially, during the 7-day cultivation, osteoclast formation was suppressed by the treatment with ED-DNAM-1 on days 6 and 7, when the mononuclear preosteoclasts fused into multinucleated osteoclasts. This suppression was abrogated partially by a small interfering RNA specific for PVR. These results suggest that, at least in part, the binding of PVR with DNAM-1 negatively regulates osteoclast formation. Furthermore, our results indicate that the cellular fusion process may be inhibited by the PVR-mediated signaling.     

Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion

Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR(-/-)) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR(-/-) osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR(-/-) preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V(0) subunit d2 and dendritic cell-specific transmembrane protein in LDLR(-/-) plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR(-/-) mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.     

Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.     

IL-23 is critical for induction of arthritis, osteoclast formation, and maintenance of bone mass

The role of IL-23 in the development of arthritis and bone metabolism was studied using systemic IL-23 exposure in adult mice via hydrodynamic delivery of IL-23 minicircle DNA in vivo and in mice genetically deficient in IL-23. Systemic IL-23 exposure induced chronic arthritis, severe bone loss, and myelopoiesis in the bone marrow and spleen, which resulted in increased osteoclast differentiation and systemic bone loss. The effect of IL-23 was partly dependent on CD4(+) T cells, IL-17A, and TNF, but could not be reproduced by overexpression of IL-17A in vivo. A key role in the IL-23-induced arthritis was made by the expansion and activity of myeloid cells. Bone marrow macrophages derived from IL-23p19(-/-) mice showed a slower maturation into osteoclasts with reduced tartrate-resistant acid phosphatase-positive cells and dentine resorption capacity in in vitro osteoclastogenesis assays. This correlated with fewer multinucleated osteoclast-like cells and more trabecular bone volume and number in 26-wk-old male IL-23p19(-/-) mice compared with control animals. Collectively, our data suggest that systemic IL-23 exposure induces the expansion of a myeloid lineage osteoclast precursor, and targeting IL-23 pathway may combat inflammation-driven bone destruction as observed in rheumatoid arthritis and other autoimmune arthritides.     

Cover Image,Volume 120, Number 11, November 2019

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 ^−/−) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 ^−/− mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 ^−/− mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 ^−/− mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 ^−/− mice. Finally, bone marrow cells from Bif-1 ^−/− mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 ^−/− mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).     

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.     

Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin

Increasing evidence suggests the existence of osteoclast diversity. Here we investigated whether precursors obtained from marrow of the mandibula or long bone could give rise to phenotypically different osteoclasts. Formation of multinucleated cells was assessed after culturing mouse marrow cells of the two bone types with macrophage colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL) for up to 10 days on plastic, bone or dentin. Two times more osteoclasts formed from long bone marrow cells on bone compared to dentin, whereas higher numbers of jaw osteoclasts formed on dentin. Resorption of dentin or bone was similar for osteoclasts formed from both types of precursors. In contrast to jaw marrow derived osteoclasts, long bone osteoclasts predominantly had a multi-compartmented shape, with at least two nuclei containing compartments per cell. Osteoclasts on bone contained two times more actin rings than osteoclasts on dentin, regardless of their precursor origin. However, the area per osteoclast covered by actin rings was similar (20%) for both substrates. This study suggests that marrow cells obtained from different bones give rise to different osteoclasts. The substrate on which the osteoclasts are generated plays a role in steering their formation rather than their resorption.     

Direct cell–cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis

The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell–cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell–cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co‐culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule‐1 (ICAM‐1) and of osteoclastogenesis‐related genes (RANKL, RANK, TNF‐α, and IL‐1β) was highly up‐regulated in the co‐cultures compared to mono‐cultures and the 5–10‐fold up‐regulation reflected a synergistic increase due to direct cell–cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)₂VitD₃ and dexamethasone. In case of indirect cell–cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast‐like cells that were formed in co‐culture with periodontal ligament fibroblasts was significantly augmented compared to mono‐cultures. Our data indicate that cell–cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts. J. Cell. Physiol. 222: 565–573, 2010. © 2009 Wiley‐Liss, Inc.     

Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.     

LPS administration increases CD11b<Superscript>+</Superscript> c-Fms<Superscript>+</Superscript> CD14<Superscript>+</Superscript> cell population that possesses osteoclast differentiation potential in mice

Osteoclasts are multinucleated giant cells that originate from a monocyte/macrophage lineage, and are involved in the inflammatory bone destruction accompanied by periodontitis. Recent studies have shown that osteoclast precursors reside not only in the bone marrow, but also in the peripheral blood and spleen, though the precise characteristics of each precursor have not been analyzed. We hypothesized that the number of osteoclast precursors in those tissues may increase under pathological conditions and contribute to osteoclast formation in vivo in a mouse model. To test this hypothesis, we attempted to identify cell populations that possess osteoclast differentiation potential in the bone marrow, spleen, and blood by analyzing macrophage/monocyte-related cell surface markers such as CD11b, CD14, and colony-stimulating factor-1 receptor (c-Fms). In the bone marrow, the CD11b^? cell population, but not the CD11b⁺ cell population, differentiated into osteoclasts in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. On the other hand, in the spleen and blood, CD11b⁺ cells differentiated into osteoclasts. Interestingly, lipopolysaccharide (LPS) administration to the mice dramatically increased the proportion of CD11b⁺ c-Fms⁺ CD14⁺ cells, which differentiated into osteoclasts, in the bone marrow and spleen. These results suggest that LPS administration increases the proportion of a distinct cell population expressing CD11b⁺, c-Fms⁺, and CD14⁺ in the bone marrow and spleen. Thus, these cell populations are considered to contribute to the increase in osteoclast number during inflammatory bone destruction such as periodontitis.     

Interleukin-10 selectively inhibits osteoclastogenesis by inhibiting differentiation of osteoclast progenitors into preosteoclast-like cells in rat bone marrow culture system

Recombinant human interleukin-10 (hIL-10) inhibited the formation of osteoclast-like multinucleated cells in rat whole bone marrow cultures. The effect of hIL-10 on the process of osteoclast formation was further examined, since the process of osteoclast formation includes the proliferation and the differentiation of osteoclast progenitors into mononuclear preosteoclasts and the fusion of preosteoclasts into multinucleated osteoclasts. In the nonadherent bone marrow cell culture system, which was free of stromal cells and formed preosteoclast-like cells, hIL-10 significantly inhibited the formation of preosteoclast-like cells even at a very low concentration (0.5 U/ml). The strong inhibition appeared even after treatment with hIL-10 for only the first 24 h of the culture. However, hIL-10 did not affect the fusion process of preosteoclast-like cells to form osteoclast-like multinucleated cells in the rat coculture system of preosteoclast-like cells with primary osteo-blasts. Furthermore, hIL-10 completely inhibited the colony formation induced by granulocyte macrophage colony-stimulating factor (GM-CSF). These findings suggest that the inhibition of osteoclastogenesis by hIL-10 started at the early stage of the differentiation of osteoclast progenitors to preosteoclasts. © 1995 Wiley-Liss Inc.     

DAP12 Overexpression Induces Osteopenia and Impaired Early Hematopoiesis

ITAM-bearing transmembrane signaling adaptors such as DAP12 and FcRγ are important players in bone homeostasis, but their precise role and functions are still unknown. It has been shown that osteoclast differentiation results from the integration of the RANK and of the DAP12 and FcRγ signaling pathways. DAP12-deficient mice suffer from a mild osteopetrosis and culture of their bone marrow cells in the presence of M-CSF and RANKL, fails to give rise to multinucleated osteoclasts. Here, we report that mice overexpressing human DAP12 have an osteopenic bone phenotype due to an increased number of osteoclasts on the surface of trabecular and cortical bone. This enhanced number of osteoclasts is associated with an increased number of proliferating myeloid progenitors in Tg-hDAP12 mice. It is concomitant with an arrest of B cell development at the Pre-Pro B/Pre B stage in the bone marrow of Tg-hDAP12 mice and important decrease of follicular and marginal B cells in the spleen of these animals. Our data show that the overexpression of DAP12 results in both increased osteoclastogenesis and impaired hematopoiesis underlining the relationship between bone homeostasis and hematopoiesis.