A selective ER-phagy exerts procollagen quality control via a Calnexin-FAM134B complex.

Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.

RANKL (Receptor Activator of NFκB Ligand) Produced by Osteocytes Is Required for the Increase in B Cells and Bone Loss Caused by Estrogen Deficiency in Mice.

The cytokine receptor activator of NFκB ligand (RANKL) produced by osteocytes is essential for osteoclast formation in cancellous bone under physiological conditions, and RANKL production by B lymphocytes is required for the bone loss caused by estrogen deficiency. Here, we examined whether RANKL produced by osteocytes is also required for the bone loss caused by estrogen deficiency. Mice lacking RANKL in osteocytes were protected from the increase in osteoclast number and the bone loss caused by ovariectomy. Moreover, these mice did not exhibit the increase in bone marrow B lymphocytes caused by ovariectomy that occurred in control littermates. Deletion of estrogen receptor α from B cells did not alter B cell number or bone mass and did not alter the response to ovariectomy. In addition, lineage-tracing studies demonstrated that B cells do not act as osteoclast progenitors in estrogen-replete or estrogen-deficient mice. Taken together, these results demonstrate that RANKL expressed by osteocytes is required for the bone loss as well as the increase in B cell number caused by estrogen deficiency. Moreover, they suggest that estrogen control of B cell number is indirect via osteocytes and that the increase in bone marrow B cells may be a necessary component of the cascade of events that lead to cancellous bone loss during estrogen deficiency. However, the role of B cells is not to act as osteoclast progenitors but may be to act as osteoclast support cells.

Cortical bone loss caused by glucocorticoid excess requires RANKL production by osteocytes and is associated with reduced OPG expression in mice.

Glucocorticoid excess is a major cause of low bone mass and fractures. Glucocorticoid administration decreases cortical thickness and increases cortical porosity in mice, and these changes are associated with increased osteoclast number at the endocortical surface. Receptor activator of NF-κB ligand (RANKL) produced by osteocytes is required for osteoclast formation in cancellous bone as well as the increase in cortical bone resorption caused by mechanical unloading or dietary calcium deficiency. However, whether osteocyte-derived RANKL also participates in the increase in bone resorption caused by glucocorticoid excess is unknown. To address this question, we examined the effects of prednisolone on cortical bone of mice lacking RANKL production in osteocytes. Prednisolone administration increased osteoclast number at the endocortical surface, increased cortical porosity, and reduced cortical thickness in control mice, but none of these effects occurred in mice lacking RANKL in osteocytes. Prednisolone administration did not alter RANKL mRNA abundance but did reduce osteoprotegerin (OPG) mRNA abundance in osteocyte-enriched cortical bone. Similarly, dexamethasone suppressed OPG but did not increase RANKL production in cortical bone organ cultures and primary osteoblasts. These results demonstrate that RANKL produced by osteocytes is required for the cortical bone loss caused by glucocorticoid excess but suggest that the changes in endocortical resorption are driven by reduced OPG rather than elevated RANKL expression.

Suppression of autophagy in osteocytes mimics skeletal aging.

Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66(shc) phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging.

Biomimetic coating with phosphoserine-tethered poly(epsilon-lysine) dendrons on titanium surfaces enhances Wnt and osteoblastic differentiation.

OBJECTIVES: Phosphoserine-based functionalization has been proposed as a tool to improve integration of endosseous implants by promoting osteoblast adhesion and differentiation in vitro. The present work investigates whether phosphoserine-tethered poly(epsilon-lysine) dendrons, when applied as a film to titanium surfaces, enhance the differentiation of osteoblastic cells and the activation of Wnt/ß-catenin signaling. MATERIALS AND METHODS: These films were tested in a murine model of calvaria-derived MC3T3 osteoblastic cells, primary bone marrow cells and mesenchymal, undifferentiated C2C12 cells. Gene expression was assayed by Real Time PCR, and activation of Wnt signaling pathway was measured with a reporter assay. RESULTS: Dendrons increased expression of alkaline phosphatase and osteocalcin, two osteoblastic markers, in both murine osteoblastic MC3T3 cells and primary bone marrow cells. The expression of osteoprotegerin, a protein opposing osteoclastogenesis was also significantly higher in cells growing on dendron-coated substrates both at 3 and 6 days of culture. Similarly, the mRNA levels of Wisp-2 and of ß-catenin, two Wnt target genes, were also markedly increased in this group at day 6. The activation of this signaling pathway in cells growing on the dendron-coated surfaces was confirmed by use of a TCF/ß-catenin reporter system in the C2C12 cell line. CONCLUSIONS: The findings of the present study show that phosphoserine-tethered poly(epsilon-lysine) dendron films act as stimuli for the activation of specific signal cascades and promote the differentiation of adhering progenitor cells into an osteoblastic phenotype.

Effect of surface treatment on cell responses to grades 4 and 5 titanium for orthodontic mini-implants.

INTRODUCTION: Mini-implants are used to improve orthodontic anchorage, but optimal composition and surface characteristics have yet to be determined. We investigated the behavior of osteoblast-like cells on grade 4 commercially pure titanium and grade 5 titanium alloy with different surface treatments for mini-implants. METHODS: MC3T3 cells were plated on machined, acid-etched, or acid-etched grade 4 titanium enriched with calcium phosphate, or machined, anodized, or anodized and calcium phosphate-enriched grade 5 titanium disks. Surface and cell morphologies were assessed by scanning electron microscopy. Cell viability was measured by chemiluminescence, cytoskeletal organization was investigated by immunofluorescence, and real-time polymerase chain reaction for osteoblast-specific genes was performed to measure cell differentiation. RESULTS: Flattened shapes and strong stress fibers were observed on the machined surfaces; cells on the rough surfaces had a spindle shape, with lower cytoskeletal polarization. Cell proliferation was highest on smooth grade 4 titanium surfaces, whereas cells quickly reached a plateau on rough grade 4 titanium; no difference was observed after 72 hours in the grade 5 titanium groups. Calcium phosphate enrichment on grade 4 titanium significantly increased the messenger RNA levels for alkaline phosphatase and osteocalcin. Osteoblastic markers were higher on the grade 5 titanium machined surfaces than on the rough surfaces, and comparable with acid-etched grade 4 titanium. CONCLUSIONS: Although the grade 4 titanium enriched with calcium phosphate had the highest level of differentiation in vitro, the grade 5 titanium machined surfaces supported cell proliferation and matrix synthesis, and induced high expression of early differentiation markers. Increased mechanical resistance of grade 5 titanium makes it a potential candidate for orthodontic mini-implants.

Soluble RANKL contributes to osteoclast formation in adult mice but not ovariectomy-induced bone loss.

Receptor activator of NFkB ligand (RANKL) is a TNF-family cytokine required for osteoclast formation, as well as immune cell and mammary gland development. It is produced as a membrane-bound protein that can be shed to form a soluble protein. We created mice harboring a sheddase-resistant form of RANKL, in which soluble RANKL is undetectable in the circulation. Lack of soluble RANKL does not affect bone mass or structure in growing mice but reduces osteoclast number and increases cancellous bone mass in adult mice. Nonetheless, the bone loss caused by estrogen deficiency is unaffected by the lack of soluble RANKL. Lymphocyte number, lymph node development, and mammary gland development are also unaffected by the absence of soluble RANKL. These results demonstrate that the membrane-bound form of RANKL is sufficient for most functions of this protein but that the soluble form does contribute to physiological bone remodeling in adult mice.

Old age causes de novo intracortical bone remodeling and porosity in mice.

Decreased cortical thickness and increased cortical porosity are the key anatomic changes responsible for osteoporotic fractures in elderly women and men. The cellular basis of these changes is unbalanced endosteal and intracortical osteonal remodeling by the osteoclasts and osteoblasts that comprise the basic multicellular units (BMUs). Like humans, mice lose cortical bone with age, but unlike humans, this loss occurs in the face of sex steroid sufficiency. Mice are therefore an ideal model to dissect age-specific osteoporotic mechanisms. Nevertheless, lack of evidence for endosteal or intracortical remodeling in mice has raised questions about their translational relevance. We show herein that administration of the antiosteoclastogenic cytokine osteoprotegerin to Swiss Webster mice ablated not only osteoclasts, but also endosteal bone formation, demonstrating the occurrence of BMU-based endosteal remodeling. Femoral cortical thickness decreased in aged male and female C57BL/6J mice, as well as F1 hybrids of C57BL/6J and BALB/cBy mice. This decrease was greater in C57BL/6J mice, indicating a genetic influence. Moreover, endosteal remodeling became unbalanced because of increased osteoclast and decreased osteoblast numbers. The porosity of the femoral cortex increased with age but was much higher in females of both strains. Notably, the increased cortical porosity resulted from de novo intracortical remodeling by osteon-like structures. Age-dependent cortical bone loss was associated with increased osteocyte DNA damage, cellular senescence, the senescence-associated secretory phenotype, and increased levels of RANKL. The demonstration of unbalanced endosteal and intracortical remodeling in old mice validates the relevance of this animal model to involutional osteoporosis in humans.

Low bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast lineage.

Autophagy maintains cell function and homeostasis by recycling intracellular components. This process is also required for morphological changes associated with maturation of some cell types. Osteoblasts are bone forming cells some of which become embedded in bone and differentiate into osteocytes. This transformation includes development of long cellular projections and a reduction in endoplasmic reticulum and mitochondria. We examined the role of autophagy in osteoblasts by deleting Atg7 using an Osterix1-Cre transgene, which causes recombination in osteoblast progenitors and their descendants. Mice lacking Atg7 in the entire osteoblast lineage had low bone mass and fractures associated with reduced numbers of osteoclasts and osteoblasts. Suppression of autophagy also reduced the amount of osteocyte cellular projections and led to retention of endoplasmic reticulum and mitochondria in osteocytes. These results demonstrate that autophagy in osteoblasts contributes to skeletal homeostasis and to the morphological changes associated with osteocyte formation.

Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.

The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

Suppression of autophagy in osteocytes does not modify the adverse effects of glucocorticoids on cortical bone.

Glucocorticoid excess decreases bone mass and strength in part by acting directly on osteoblasts and osteocytes, but the mechanisms remain unclear. Macroautophagy (herein referred to as autophagy) is a lysosome-based recycling pathway that promotes the turnover of intracellular components and can promote cell function and survival under stressful conditions. Recent studies have shown that glucocorticoids stimulate autophagy in osteocytes, suggesting that autophagy may oppose the negative actions of glucocorticoids on this cell type. To address this possibility, we compared the impact of prednisolone administration on the skeletons of adult mice in which autophagy was suppressed in osteocytes, via deletion of Atg7 with a Dmp1-Cre transgene, to their control littermates. In control mice, prednisolone increased autophagic flux in osteocyte-enriched bone as measured by LC3 conversion, but this change did not occur in the mice lacking Atg7 in osteocytes. Nonetheless, prednisolone reduced femoral cortical thickness, increased cortical porosity, and reduced bone strength to similar extents in mice with and without autophagy in osteocytes. Prednisolone also suppressed osteoblast number and bone formation in the cancellous bone of control mice. As shown previously, Atg7 deletion in osteocytes reduced osteoblast number and bone formation in cancellous bone, but these parameters were not further reduced by prednisolone administration. In cortical bone, prednisolone elevated osteoclast number to a similar extent in both genotypes. Taken together, these results demonstrate that although glucocorticoids stimulate autophagy in osteocytes, suppression of autophagy in this cell type does not worsen the negative impact of glucocorticoids on the skeleton.

A DNA segment spanning the mouse Tnfsf11 transcription unit and its upstream regulatory domain rescues the pleiotropic biologic phenotype of the RANKL null mouse.

Receptor activator of NF-κB ligand (RANKL) is a TNFα-like cytokine that is produced by a diverse set of lineage-specific cells and is involved in a wide variety of physiological processes that include skeletal remodeling, lymph node organogenesis, mammary gland development, and thermal regulation. Consistent with these diverse functions, control of RANKL expression is accomplished in a cell-specific fashion via a set of at least 10 regulatory enhancers that are located up to 170 kb upstream of the gene's transcriptional start site. Here we examined the in vivo consequence of introducing a contiguous DNA segment containing these components into a genetically deleted RANKL null mouse strain. In contrast to RANKL null littermates, null mice containing the transgene exhibited normalized body size, skeletal development, and bone mass as well as normal bone marrow cavities, normalized spleen weights, and the presence of developed lymph nodes. These mice also manifested normalized reproductive capacity, including the ability to lactate and to produce normal healthy litters. Consistent with this, the transgene restored endogenous-like RANKL transcript levels in several RANKL-expressing tissues. Most importantly, restoration of RANKL expression from this segment of DNA was fully capable of rescuing the complex aberrant skeletal and immune phenotype of the RANKL null mouse. RANKL also restored appropriate levels of B220+ IgM+ and B220+ IgD+ B cells in spleen. Finally, we found that RANKL expression from this transgene was regulated by exogenously administered 1,25(OH)2 D3 , parathyroid hormone (PTH), and lipopolysaccharide (LPS), thus recapitulating the ability of these same factors to regulate the endogenous gene. These findings fully highlight the properties of the Tnfsf11 gene locus predicted through previous in vitro dissection. We conclude that the mouse Tnfsf11 gene locus identified originally through unbiased chromatin immunoprecipitation with DNA microarray (ChIP-chip) analysis contains the necessary genetic information to direct appropriate tissue-specific and factor-regulated RANKL expression in vivo.

RhoA controls Wnt upregulation on microstructured titanium surfaces.

Rough topography enhances the activation of Wnt canonical signaling in vitro, and this mediates its effects on cell differentiation. However, the molecular mechanisms underlying topography-dependent control of Wnt signaling are still poorly understood. As the small GTPase RhoA controls cytoskeletal reorganization and actomyosin-induced tensional forces, we hypothesized that RhoA could affect the activation of Wnt signaling in cells on micropatterned titanium surfaces. G-LISA assay revealed that RhoA activation was higher in C2C12 cells on rough (SLA) surfaces under basal conditions than on smooth (Polished) titanium. Transfection with dominant negative RhoA decreased Wnt activation by normalized TCF-Luc activity on SLA, whilst transfection with constitutively active RhoA increased TCF-Luc activation on Polished titanium. One mM Myosin II inhibitor Blebbistatin increased RhoA activation but decreased Wnt activation on SLA surfaces, indicating that tension-generating structures are required for canonical Wnt modulation on titanium surfaces. Actin inhibitor Cytochalasin markedly enhanced RhoA and TCF-Luc activation on both surfaces and increased the expression of differentiation markers in murine osteoblastic MC3T3 cells. Taken together, these data show that RhoA is upregulated in cells on rough surfaces and it affects the activation of Wnt canonical signaling through Myosin II modulation.

Osteocyte-derived RANKL is a critical mediator of the increased bone resorption caused by dietary calcium deficiency.

Parathyroid hormone (PTH) excess stimulates bone resorption. This effect is associated with increased expression of the osteoclastogenic cytokine receptor activator of nuclear factor κB ligand (RANKL) in bone. However, several different cell types, including bone marrow stromal cells, osteocytes, and T lymphocytes, express both RANKL and the PTH receptor and it is unclear whether RANKL expression by any of these cell types is required for PTH-induced bone loss. Here we have used mice lacking the RANKL gene in osteocytes to determine whether RANKL produced by this cell type is required for the bone loss caused by secondary hyperparathyroidism induced by dietary calcium deficiency in adult mice. Thirty days of dietary calcium deficiency caused bone loss in control mice, but this effect was blunted in mice lacking RANKL in osteocytes. The increase in RANKL expression in bone and the increase in osteoclast number caused by dietary calcium deficiency were also blunted in mice lacking RANKL in osteocytes. These results demonstrate that RANKL produced by osteocytes contributes to the increased bone resorption and the bone loss caused by secondary hyperparathyroidism, strengthening the evidence that osteocytes are an important target cell for hormonal control of bone remodeling.