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.

The mammalian skeleton is continuously remodeled throughout life by the actions of osteoclasts, which resorb bone, and osteoblasts, which replace the bone matrix 1 . During the bone formation process, some of the osteoblasts are buried within the bone matrix and become osteocytes 2 . Osteocytes reside in lacunae and remain connected to one another, and with cells on the bone surface, via cellular projections that are contained within tubular cavities known as canaliculi. This network of osteocytes connected via their projections creates and maintains the lacunocanalicular network, which is thought to act as a mechanosensing system 3 . Recent studies have demonstrated that osteocytes also perform numerous non-mechanosensing functions including the control of bone remodeling and phosphate homeostasis [4][5][6] .
Macroautophagy, hereafter referred to as autophagy, is a stress-activated process that maintains cell function and homeostasis by recycling damaged organelles and macromolecules 7 . This process is thought to promote the health of long-lived cell types such as neurons and myocytes and a decline in autophagy in such cell types may contribute to the detrimental effects of aging 8 . Consistent with this, suppression of autophagy in neurons or myocytes mimics the effects of aging on the nervous system or muscle tissue, respectively 9,10 .
We have reported previously that genetic suppression of autophagy in osteocytes using a Dmp1-Cre transgene recapitulates many of the effects of aging on the skeleton, identifying autophagy as an important determinant of bone homeostasis and a decline in autophagy as a possible contributor to skeletal aging 11 . However, it remains unclear whether autophagy also plays a role in the differentiation or function of osteoblasts. Moreover, even though osteocyte formation was unaffected by deletion of Atg7 using the Dmp1-Cre transgene, it remains possible that loss of autophagy at a stage of osteoblast differentiation earlier than that targeted by Dmp1-Cre might affect the transition of osteoblasts to osteocytes and thereby proper formation of the osteocyte lacunocanalicular network.
To address these questions, we deleted the Atg7 gene, which is essential for autophagy, using an Osterix 1 (Osx1)-Cre transgene, which targets cells at the earliest stages of commitment to the osteoblast lineage 12 . This maneuver caused a low bone mass phenotype that was more pronounced than that obtained using the Dmp1-Cre transgene and was associated with fractures. In addition, the lack of autophagy early in the osteoblast lineage Skeletal analysis and histomorphometry. Bone mineral density (BMD) was measured in live mice by dual-energy x-ray absorptiometry with a PIXImus Mouse Densitometer (GE Lunar Corp., Madison, WI) using the manufacturer's software as described previously 18 . Growth curves were obtained by sequential BMD measurement of the same animals from 8 weeks to 24 weeks of age. Micro-CT analysis of cortical and trabecular architecture was performed in femurs and fourth lumbar vertebrae (L4), as previously described 19 . Biomechanical properties of femurs and L4 vertebrae were measured by 3-point bending and compression testing respectively, as previously described 20 . L1-L3 lumbar vertebrae were fixed, embedded undecalcified in methylmethacrylate, and histomorphometric examination was done on longitudinal sections with a digitizer tablet (OsteoMetrics, Inc., Decatur, GA) interfaced to a Zeiss Axioscope (Carl Zeiss, Thornwood, NY) with a drawing tube attachment, as previously described 21 . Terminology recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research was used in this study 22 . In vitro cell culture. Bone marrow cells were harvested from long bones and used to measure osteoblast differentiation of bone marrow precursors. 5 × 10 6 bone marrow cells per well were plated in 12-well plates in α -MEM containing 15% fetal bovine serum, 1% penicillin/streptomycin/glutamine, 1% ascorbic acid, and 10 mM β -glycerolphosphate. Half of the culture medium was changed every 3 days. After 21 days, the cultures were fixed with phosphate buffered 10% formalin and then stained with an aqueous solution of 40 mM alizarin red. Messenger RNA and protein lysates were produced from parallel cultures lacking β -glycerolphosphate and harvested after 15 days, to evaluate respectively, osteoblast specific gene expression and LC3 conversion by western blot as previously described 18 . Catalase activity was also measured in primary bone marrow cells cultured under osteogenic conditions using the OxiSelect Catalase Activity Assay Kit, following the manufacturer's instructions (Cell Biolabs). The ability of bone marrow cells to support osteoclast differentiation was evaluated by plating bone marrow cells as described above and adding vehicle or 10 −7 M parathyroid hormone (PTH) for 12 days followed by RNA extraction and quantification of osteoclast-specific genes.
Osteocyte network analysis and immunofluorescence. Phalloidin staining of actin was performed on tibia. First, tibia were fixed in buffered 10% formalin for 24 hours, decalcified in 14% EDTA pH 7.1 for one week, stored in 30% sucrose solution, and then embedded in Cryo-Gel (Electron Microscopy Sciences, Hatfield, PA) for frozen sectioning. 20 μm thick frozen sections were cut and rinsed 3 times in PBS for 10 minutes. Sections were incubated in 0.2% Trition-X100 for 20 minutes with agitation, followed by washing in PBS. Sections were then incubated in 2% BSA for 30 minutes and incubated with Alexafluor 488-phalloidin (Molecular Probes) in 0.5% BSA for 48 hours at 4˚C. At the end of the incubation, the sections were rinsed with PBS and cover-slipped with Vectashield mounting medium containing DAPI (VectorLaboratories).
Fluorescein isothiocyanate (FITC) staining of the lacunocanalicular network was performed on femurs as previously described 24 . Briefly, femurs were harvested, placed in 10% phosphate buffered formalin solution for 24 hours, and then dehydrated in a series of ascending graded ethanol solutions. After removal of the distal end to allow efficient penetration of the staining solution, femurs were stained en block for 4 hours in 1% filtered FITC solution (fluorescein isothiocyanate isomer I, Sigma) with gentle agitation. Bones were then washed for 30 minutes in 100% ethanol to remove excess FITC solution and rinsed three times in methyl salicylate for 30, 45, and 60 minutes. After a quick rinse in xylene, femurs were embedded in methyl methacrylate and cross sections of 100 μm were obtained using a diamond saw, mounted and coverslipped using Eukitt mounting medium (Sigma).
Images of phalloidin-or FITC-stained bone sections were acquired using a Zeiss LSM 510 Meta/AxioVert 200 confocal microscope using a 40 × or 63 × oil objective and z-stacks were obtained using ZEN 2009 software. Measurements of osteocyte dendrites, osteocyte cell body, nuclear diameter, and osteocyte lacuna diameter were performed on flattened z-stacks (19 μm for tibia and 10 μm for femur) using ImageJ software. To quantify osteocyte dendrites, first a region of interest (ROI) containing only cortical bone was drawn. Then osteocyte cell bodies were excluded from the ROI. Finally, the total dendrite fluorescence inside the ROI was calculated by subtracting the background fluorescence in a region lacking osteocytes or dendrites from the integrated intensity of the entire region. This value was then divided by the area of the region of ROI. Three fields of cortical bone from each sample were quantified and 3-4 samples per genotype were used.
For endoplasmic reticulum staining, frozen tibial sections were permeabilized in PBS containing 0.4% Triton X-100 for 1 hour at RT with agitation and blocking was performed in 1% BSA/5% normal goat serum (Sigma) in PBS containing 0.4% Triton X-100 at 4 °C overnight. After rinsing in PBS, the sections were incubated with a primary antibody against KDEL (#12223, Abcam) diluted 1:250 in blocking buffer for 1 hour at RT. Sections were washed 3 times for 10 minutes with PBS containing 0.4% Triton X-100 and incubated with Alexa Fluor ® 594 AffiniPure Goat Anti-Mouse IgG (#115-585-003, Jackson Immunoresearch) diluted 1:1000 in blocking buffer for 1 hour at RT. Sections were rinsed in PBS and mounted in Vectashield with DAPI and imaged with confocal microscope using a 40× or 63× oil lens objectiveS. Z-stacks from 45 planes with 0.34 μm Z plane separation were obtained using ZEN 2009 software.
Statistics. Data were analysed using two-way analysis of variance, one-way analysis of variance, nested analysis of variance, or Student's t-test to detect statistically significant effects, after determining that the data were normally distributed and exhibited equivalent variances. In some cases, data were transformed to obtain normally-distributed data. Multiple comparisons were evaluated with Tukey or Holm-Sidak post hoc tests. P-values less than 0.05 were considered as significant. All values are reported as the mean ± S.D. Data that did not pass the normality test after transformation were evaluated using the Mann-Whitney Rank Sum Test. Female Atg7 ΔOb mice and their control littermates, which included wild-type (wt), Atg7-f/f, or Osx1-Cre, were subjected to serial BMD measurements from 8 to 24 weeks of age and then euthanized to perform further skeletal analysis. Femoral, vertebral, and total BMD were low in Atg7 ΔOb mice compared to control groups at all time-points (Fig. 1a-c). There were no differences in body weight between Atg7 ΔOb and Osx1-Cre (Supplementary   Fig. S3).

Deletion of
MicroCT analysis revealed that cancellous bone volume was low in the spine and femur of female conditional knockout mice compared to controls (Fig. 1d-l). Femoral cortical thickness was also low compared with Osx1-Cre mice, and this was associated with a reduction in the outer cortical perimeter (Fig. 1m-o). These cortical bone changes were in addition to the effect of the Osx1-Cre transgene alone, which also caused low cortical thickness compared to Atg7-f/f littermates. In line with these results, biomechanical strength of femurs and isolated vertebrae was low, as revealed by 3-point bending and compression testing ( Supplementary Fig. S4). Moreover, approximately half of the Atg7 ΔOb mice (11 of 23), but none of the control mice (0 of 12), displayed healed or healing fracture calluses in the tibia, indicative of fractures and a profound loss of strength in appendicular bones (Fig. 1p).
Loss of autophagy suppresses bone remodeling. Static and dynamic histomorphometry of cancellous bone of the spine revealed a low bone remodeling phenotype in Atg7 ΔOb mice, with reduced osteoblast and osteoclast number and surface (Fig. 2a-d). Quantification of tetracycline labeling showed reduced mineralized surface in the conditional knockout mice compared to the Osx1-Cre control, which resulted in a low bone formation rate (Fig. 2e-g). The differentiation capacity of osteoblast progenitors in the bone marrow was not different between conditional knockout mice and Osx1-Cre littermates as assessed by osteoblast-specific gene expression and mineralization (Supplementary Fig. S5). Similarly, osteoclast formation in bone marrow cultures was similar between these genotypes (Supplementary Fig. S5). Thus, the low osteoclast and osteoblast numbers in vivo do not appear to be due to cell intrinsic defects in the differentiation of either of these two cell types. Osteocytes produce a number of factors that control bone remodeling and mineralization 2 . There were no significant changes in the expression level of many of these factors, including RANKL, OPG, Sost, Dmp1 and Mepe, as measured in mRNA isolated from L5 vertebra (Fig. 2h-l).

Expression of mitochondria-targeted catalase does not prevent low bone mass. Suppression
of autophagy in other cells types often results in reduced recycling of mitochondria leading to accumulation of damaged mitochondria and oxidative stress. Consistent with this, impairment of autophagy in the entire osteoblast lineage was associated with elevated levels of reactive oxygen species (ROS) in the bone marrow and increased phosphorylation of the redox sensitive protein p66 shc measured in bone protein lysate ( Supplementary  Fig. S6). To determine whether this increase in oxidative stress contributes to the low bone mass caused by Atg7 deletion, Atg7 ΔOb mice were crossed with mice harboring a conditionally-activated transgene expressing mitochondrial-targeted catalase (mCAT), an antioxidant enzyme 15 . Thus, in the offspring of this cross, the Osx1-Cre transgene simultaneously deletes Atg7 and activates the catalase transgene in the same cell populations (osteoblasts and osteocytes).
The catalase transgene was highly expressed in the bone of mCAT;Atg7 ΔOb mice and catalase activity, measured in bone marrow stromal cells cultured in osteogenic medium, was greatly elevated compared to control littermates, confirming activation of the transgene (Fig. 3a,b). In this experiment, ROS levels measured in the bone marrow were not different between genotypes but phosphorylation of p66shc protein was increased in Atg7 ΔOb (e-i) μCT measurements of L4 vertebra from Osx1-Cre (n = 6), mCAT;Osx1-Cre (n = 6), Atg7 ΔOb (n = 6), and mCAT;Atg7 ΔOb (n = 6) mice. BV/TV, bone volume per total tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation. (j-n) μCT measurements of femurs from the same mice described in e, Osx1-Cre (n = 6), mCAT;Osx1-Cre (n = 6), Atg7 ΔOb (n = 6), and mCAT;Atg7 ΔOb (n = 6) mice. All measurements were performed in 6-month-old female littermates. Values are the mean ± sd. *P < 0.05 by one-way ANOVA. mice (Fig. 3c,d). However, this increase was not affected by mCAT expression (Fig. 3d). Thus, mitochondrial expression of catalase in osteoblasts and osteocytes was not sufficient to block the increase in oxidative stress, measured in whole bone, caused by suppression of autophagy.
Serial BMD analysis up to 24 weeks of age confirmed low bone mass in the femur and spine of Atg7 ΔOb mice compared to Osx1-Cre control littermates (Supplementary Fig. S7). Expression of mCAT in Atg7 ΔOb mice did not alter the low bone mass caused by suppression of autophagy ( Supplementary Fig. S7). Analysis of vertebral and femoral architecture confirmed low trabecular bone volume and cortical thickness in Atg7 ΔOb mice, and expression of mCAT did not prevent any of these changes (Fig. 3e-n). Consistent with this, the prevalence of fractures in tibias of conditional knockout mice was similar in conditional knockout mice expressing catalase (Supplementary  Table S1). Together these results suggest that autophagy exerts its positive effects on the skeleton by mechanisms other than suppression of H 2 O 2 levels in the mitochondria of osteoblasts and osteocytes.
Suppression of autophagy alters morphology of the osteocyte network. Autophagy plays a key role in the differentiation process of several cell types including erythrocytes, adipocytes, and lymphocytes [26][27][28][29][30] . Moreover, autophagy in erythrocyte progenitors contributes to the removal of cytoplasmic organelles, such as mitochondria, that is associated with maturation of this cell type 26,31,32 . A similar reduction in cytoplasmic organelles occurs as osteocytes differentiate from osteoblasts 33 . Therefore, we measured different features of osteocyte biology to determine whether loss of autophagy in osteoblasts altered osteocyte formation or function.
The density of osteocytes per bone area was not different between Atg7 ΔOb mice and Osx1-Cre littermates (Fig. 4a). However, the number of empty lacunae, which is an indicator of osteocyte death 34 , was higher in the bones of conditional knockout mice (Fig. 4b). Next we examined the density of osteocyte cell projections, which are formed during the process of embedding into bone matrix, by staining with fluorescently-tagged phalloidin 35 . The average intensity of osteocyte projections normalized to bone area, which likely reflects the number and size of osteocyte cellular projections, was significantly lower in the conditional knockout mice compared with controls (Fig. 4c,d). These results suggest that formation or maintenance of the osteocyte network was disrupted by deletion of Atg7 in osteoblasts.
We noted previously that deletion of Atg7 using a Dmp1-Cre transgene did not alter osteocyte maturation or formation of the osteocyte network 11,18 . However, the latter observation was limited to qualitative evaluation of phalloidin-stained images. Therefore, we quantified the density of osteocyte projections in Dmp1-Cre;Atg7-f/f mice and their littermate controls using the same approach as we used in the current study. Although there was a trend towards reduced area, this difference was not significant (Supplementary Fig. S8). Thus, loss of autophagy in osteoblasts, but not in osteocytes, altered osteocyte projections.
We also noted that the shape of osteocyte nuclei appeared more round and off-centre in conditional knockout mice compared with controls (Fig. 5a). Quantification of this phenomenon revealed that osteocyte cell bodies were larger, but nuclei were smaller, in conditional knockout mice compared to controls (Fig. 5b). One possible cause for this phenomenon could be delay or prevention of the reduction of cytoplasmic components normally associated with osteocyte maturation. Consistent with this idea, the extent of endoplasmic reticulum (ER), as measured by anti-KDEL staining, was greatly expanded in Atg7 ΔOb mice (Fig. 6a-c). Similarly the amount of mitochondrial DNA present in osteocyte-enriched bone preparations was higher in conditional knockout mice compared with controls (Fig. 6d). Together this evidence suggests that the reduction in cytoplasmic components that normally occurs during osteocyte formation was delayed or prevented by loss of autophagy in osteoblasts.
Phalloidin staining labels actin and can thus detect changes only in cellular structures within the lacunocanalicular network. To directly visualize the lacunocanalicular network itself, we stained bones from conditional knockout mice and controls with FITC 24 . Using this approach we confirmed the increase in the size of osteocyte lacunae in Atg7 ΔOb mice ( Supplementary Fig. S9). However, there was no difference in the appearance or staining intensity of the canaliculi in Atg7 ΔOb mice compared to Osx1-Cre controls ( Supplementary Fig. S9). This latter finding suggests that it is the osteocyte cellular projections themselves, but not canalicular structure, that is altered by suppression of autophagy in osteoblasts.

Discussion
We have shown previously that suppression of autophagy in osteocytes causes low bone mass and that this is associated with low bone remodeling 11,18 . However, the transformation of osteoblasts into osteocytes was not obviously affected by this maneuver. Here we demonstrate that suppression of autophagy early in the osteoblast lineage causes an even greater reduction in bone mass. The reduction in bone mass and strength was so severe that approximately fifty percent of the conditional knockout mice experienced fractures. Coincidently, suppression of autophagy early in the osteoblast lineage also altered the structure of the osteocyte network. Previous studies have suggested that such disruptions can compromise bone strength independently of changes in bone mass and architecture 21 . Therefore the extremely low bone strength of Atg7 ΔOb mice may result from the combination of low bone mass and the changes in the osteocyte network.
The Osx1-Cre transgenic mouse line used in these studies exhibits a basal skeletal phenotype 25,36,37 . For example, these mice are smaller than non-transgenic littermates and have reduced cortical thickness 25 . In addition, they display delayed mineralization of craniofacial bones shortly after birth but this phenotype appears to resolve as the animals mature 37 . More important to the studies described here, a recent analysis revealed fracture calluses in the scapula, ribs, and fibula in approximately 80% of 6 day old Osx1-Cre mice 36 . In the current study, we did not observe any tibial fractures in adult Osx1-Cre littermates. Nonetheless, it remains possible that the susceptibility of Atg7 ΔOb mice to tibial fractures is due in part to an additive effect of the Osx1-Cre basal skeletal phenotype and that caused by suppression of autophagy in osteoblast-lineage cells.
Two major morphological differences were observed between osteocytes in control versus conditional knockout mice. First, the density of osteocyte projections was reduced in cortical bone of Atg7 ΔOb mice. Second, Scientific RepoRts | 6:24262 | DOI: 10.1038/srep24262 osteocyte cell body diameter was elevated in these mice and this was associated with reduced nuclear diameter. A plausible explanation for the latter changes is that reduced degradation of the ER and other cytoplasmic components such as mitochondria, which normally occurs as osteocytes mature, did not occur efficiently in the conditional knockout mice 33 . This idea is supported by the increased amount of mitochondrial DNA and ER in osteocytes from these animals. Moreover, recent studies have demonstrated a critical role for autophagy in ER turnover and that suppression of autophagy results in ER expansion in several cell types, including mouse embryonic fibroblasts, osteosarcoma cells, and lymphocytes 38,39 . The distorted nuclear morphology may be an indirect consequence of retention of the cytoplasmic components. Together these results suggest that the reduction in cytoplasmic components that occurs during osteocyte maturation depends on autophagy. This requirement may be analogous to the requirement of autophagy to remove cytoplasmic organelles during maturation of mammalian reticulocytes and erythrocytes 26,31,32 .
It is unclear why these changes were not observed when Atg7 was deleted using the Dmp1-Cre transgene. The Dmp1-Cre transgene becomes active during the late stages of osteoblast differentiation 40,41 . Therefore, one possible explanation is that even after the Atg7 gene was deleted in mature osteoblasts, sufficient Atg7 protein remained to allow autophagy to continue until osteocytes were fully formed. In other words, residual autophagy may have still been present for a limited time after Cre-mediated recombination. In contrast, deletion of Atg7 at the earliest   Our findings are consistent with the idea that maintenance of osteocyte projections is influenced by autophagy-driven metabolism. In wild-type mice, osteocyte projections form before removal of cytoplasmic components 42 . Therefore, formation of the projections must not depend on material supplied by autophagic digestion of cytoplasm products. In addition, if initial formation of the canalicular network depends on osteocyte projections, then the normal canalicular morphology in the conditional knock out mice suggests that at the time of canalicular formation, osteocyte projections were present in normal numbers in these mice. The reduction in osteocyte projections observed by phalloidin staining could be explained either by retraction or degradation of projections or by loss of actin filaments within such projections. In vitro studies suggest that actin filaments are required to maintain the number and structure of osteocyte projections 43 , arguing against the latter possibility.
The functional significance of the changes in osteocyte morphology is unclear but they were associated with profound reductions in bone remodeling and mass, as well as an increase in osteocyte death. We found no changes in the ability of progenitors to differentiate into osteoclasts or osteoblasts in vitro, suggesting that the low bone remodeling was not due to an intrinsic defect in either of these two cell types. Osteocytes have been shown to control osteoclast formation via production of RANKL, which is essential for osteoclast formation and a major determinant of osteoclast number 44 . In addition, osteocytes are also able to control osteoblast formation via production of the Wnt antagonist sclerostin 45 . However, mRNA levels of both of these genes were unaffected by deletion of Atg7. Nonetheless, it is possible that the change in osteocyte morphology, in and of itself, may have influenced bone remodeling. RANKL is produced as both membrane-bound and soluble protein and in vitro studies suggest that it is the membrane-bound form that drives osteoclast formation 46,47 . If this were the case in vivo, then osteocytes may present RANKL to osteoclast progenitors only on those projections that extend into the bone marrow cavity or those that have contact with blood vessels 2 . Since the extent of osteocyte projections is reduced in Atg7 ΔOb mice, then the amount of RANKL available to osteoclast progenitors may also be reduced leading to a low osteoclast number.
It is not clear how changes in osteocyte morphology might alter osteoblast formation or function. A previous study reported that suppression of autophagy in osteoblast-lineage cells suppresses osteoblast differentiation 48 . Specifically, deletion of FIP200 using the same Osx1-Cre transgenic line used in our study reduced bone mass as well as osteoblast differentiation as measured by mineralization of osteoblast cultures and osteoblast-specific gene expression 48 . FIP200 is a protein required for the initiation of autophagosome formation and is also involved in the control of several signalling pathways 49 . Deletion of Atg5, another gene essential for autophagy, using a Col1a1-Cre transgene resulted in low bone mass and osteoblastic cells from these mice displayed reduced mineralization in culture 50 . However, in the latter study, osteoblast differentiation, as measured by expression of osteoblast-specific genes, was actually elevated in the conditional knockout mice. Fractures were not observed in mice when either FIP200 or Atg5 genes were deleted from osteoblast-lineage cells. In the present study we observed no change in osteoblast-specific gene expression or mineralization in culture or in bone mineralization in vivo, suggesting that osteoblast differentiation and function were not affected. The reasons for the different outcomes are not clear, but in the case of FIP200 deletion the differences may be due to suppression of processes other than autophagy 49 .
Suppression of autophagy induces oxidative stress in many cell types and this is often associated with increased abundance of damaged mitochondria [51][52][53] . Similarly, we noted markers of increased oxidative stress in the bones of mice lacking Atg7 in the entire osteoblast lineage or just in mature osteoblasts and osteocytes 11 . We have previously used the mCAT transgene to suppress H 2 O 2 in osteoclast-lineage cells and this prevented the bone loss caused by estrogen deficiency 54 . Moreover, this transgene has been used successfully to suppress oxidative damage in a variety of mouse models 15,[55][56][57][58] . Thus, in the present study it is likely that the mCAT transgene suppressed levels of H 2 O 2 in the mitochondria of osteoblast-lineage cells. However, this was not sufficient to alter any of the skeletal effects caused by suppression of autophagy in these cells. Based on this, it appears that mitochondrial oxidative stress is not a contributing factor to the detrimental effects of autophagy suppression on the skeleton.
In summary, we have shown that suppression of autophagy early in the osteoblast lineage leads to a profound reduction in bone mass associated with reduced maturation of osteocyte morphology. Whether the altered morphology of osteocytes contributes to the reductions in bone remodeling or strength will require a more complete understanding of how osteocytes communicate with osteoclast and osteoblast progenitors. For example, whether osteocytes utilize the membrane-bound form of RANKL, its soluble form, or both, will need to be determined. It will also be important to determine whether the failure of osteocyte maturation leads to activation of pathways that impact cell survival or function.