Osteocytes and Cancer.

PURPOSE OF REVIEW: While the function of osteocytes under physiologic conditions is well defined, their role and involvement in cancer disease remains relatively unexplored, especially in a context of non-bone metastatic cancer. This review will focus on describing the more advanced knowledge regarding the interactions between osteocytes and cancer. RECENT FINDINGS: We will discuss the involvement of osteocytes in the onset and progression of osteosarcoma, with the common bone cancers, as well as the interaction that is established between osteocytes and multiple myeloma. Mechanisms responsible for cancer dissemination to bone, as frequently occur with advanced breast and prostate cancers, will be reviewed. While a role for osteocytes in the stimulation and proliferation of cancer cells has been reported, protective effects of osteocytes against bone colonization have been described as well, thus increasing ambiguity regarding the role of osteocytes in cancer progression and dissemination. Lastly, supporting the idea that skeletal defects can occur also in the absence of direct cancer dissemination or osteolytic lesions directly adjacent to the bone, our recent findings will be presented showing that in the absence of bone metastases, the bone microenvironment and, particularly, osteocytes, can manifest a clear and dramatic response to the distant, non-metastatic tumor. Our observations support new studies to clarify whether treatments designed to preserve the osteocytes can be combined with traditional anticancer therapies, even when bone is not directly affected by tumor growth.

E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models.

The transmembrane glycoprotein E11 is considered critical in early osteoblast-osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO-A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post-translational degradation. We found that exogenous treatment of MLO-A5 and osteocytic IDG-SW3 cells with 10 µM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome-selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin-A) produced similar dose-dependent increases in E11 protein levels in MLO-A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO-A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome-mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches.

Dmp1 Promoter-Driven Diphtheria Toxin Receptor Transgene Expression Directs Unforeseen Effects in Multiple Tissues.

Mice harbouring a dentin matrix protein 1 (Dmp1) promoter-driven human diphtheria toxin (DT) receptor (HDTR) transgene (Tg) have recently been used to attain targeted ablation of osteocytes by diphtheria toxin (DT) treatment in order to define osteocyte function. Use of these Tg mice has asserted mechano- and novel paracrine regulatory osteocyte functions. To explore osteocyte roles fully, we sought to confirm the selectivity of DT effects in these transgenic mice. However, our findings revealed incomplete DT-induced osteocyte ablation, prevalent HDTR misexpression, as well as more prominent histopathological DT-induced changes in multiple organs in Tg than in wild-type (WT) littermate mice. Mechanistic evidence for DT action, via prominent regulation of phosphorylation status of elongation factor-2 (EF-2), was also found in many non-skeletal tissues in Tg mice; indicative of direct "off-target" DT action. Finally, very rapid deterioration in health and welfare status in response to DT treatment was observed in these Tg when compared to WT control mice. Together, these data lead us to conclude that alternative models for osteocyte ablation should be sought and caution be exercised when drawing conclusions from experiments using these Tg mice alone.

Deletion of FNDC5/Irisin modifies murine osteocyte function in a sex-specific manner.

Irisin, released from exercised muscle, has been shown to have beneficial effects on numerous tissues but its effects on bone are unclear. We found significant sex and genotype differences in bone from wildtype (WT) mice compared to mice lacking Fndc5 (KO), with and without calcium deficiency. Despite their bone being indistinguishable from WT females, KO female mice were partially protected from osteocytic osteolysis and osteoclastic bone resorption when allowed to lactate or when placed on a low-calcium diet. Male KO mice have more but weaker bone compared to WT males, and when challenged with a low-calcium diet lost more bone than WT males. To begin to understand responsible molecular mechanisms, osteocyte transcriptomics was performed. Osteocytes from WT females had greater expression of genes associated with osteocytic osteolysis and osteoclastic bone resorption compared to WT males which had greater expression of genes associated with steroid and fatty acid metabolism. Few differences were observed between female KO and WT osteocytes, but with a low calcium diet, the KO females had lower expression of genes responsible for osteocytic osteolysis and osteoclastic resorption than the WT females. Male KO osteocytes had lower expression of genes associated with steroid and fatty acid metabolism, but higher expression of genes associated with bone resorption compared to male WT. In conclusion, irisin plays a critical role in the development of the male but not the female skeleton and protects male but not female bone from calcium deficiency. We propose irisin ensures the survival of offspring by targeting the osteocyte to provide calcium in lactating females, a novel function for this myokine.

L-BAIBA Synergizes with Sub-Optimal Mechanical Loading to Promote New Bone Formation.

The L-enantiomer of ß-aminoisobutyric acid (BAIBA) is secreted by contracted muscle in mice, and exercise increases serum levels in humans. In mice, L-BAIBA reduces bone loss with unloading, but whether it can have a positive effect with loading is unknown. Since synergism can be more easily observed with sub-optimal amounts of factors/stimulation, we sought to determine whether L-BAIBA could potentiate the effects of sub-optimal loading to enhance bone formation. L-BAIBA was provided in drinking water to C57Bl/6 male mice subjected to either 7 N or 8.25 N of sub-optimal unilateral tibial loading for 2 weeks. The combination of 8.25 N and L-BAIBA significantly increased the periosteal mineral apposition rate and bone formation rate compared to loading alone or BAIBA alone. Though L-BAIBA alone had no effect on bone formation, grip strength was increased, suggesting a positive effect on muscle function. Gene expression analysis of the osteocyte-enriched bone showed that the combination of L-BAIBA and 8.25 N induced the expression of loading-responsive genes such as Wnt1, Wnt10b, and the TGFb and BMP signaling pathways. One dramatic change was the downregulation of histone genes in response to sub-optimal loading and/or L-BAIBA. To determine early gene expression, the osteocyte fraction was harvested within 24 hours of loading. A dramatic effect was observed with L-BAIBA and 8.25 N loading as genes were enriched for pathways regulating the extracellular matrix (Chad, Acan, Col9a2), ion channel activity (Scn4b, Scn7a, Cacna1i), and lipid metabolism (Plin1, Plin4, Cidec). Few changes in gene expression were observed with sub-optimal loading or L-BAIBA alone after 24 hours. These results suggest that these signaling pathways are responsible for the synergistic effects between L-BAIBA and sub-optimal loading. Showing that a small muscle factor can enhance the effects of sub-optimal loading of bone may be of relevance for individuals unable to benefit from optimal exercise. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.