Glucocorticoids Are Not Always Deleterious for Bone

CLINICAL VIGNETTE JBMR Glucocorticoids Are Not Always Deleterious for Bone

Antoon HJM van Lierop, Neveen AT Hamdy, and Socrates E Papapoulos Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands

ABSTRACT A 23-year-old man with the rare sclerosing bone disorder van Buchem disease presented with progressively worsening headaches that eventually became persistent and associated with papilledema. Increased intracranial pressure was diagnosed, and the patient had a ventriculoperitoneal drain inserted as well as simultaneously receiving treatment with prednisone. Before starting treatment, there was biochemical evidence for increased bone turnover and for steady increases in bone mineral density (BMD) at the spine and total hip despite the patient having reached his peak height of 197 cm at the age of 19 years. Treatment with prednisone for 2 years resulted in biochemical and histologic suppression of bone formation as well as of bone resorption and arrest of further bone accumulation. Our data suggest that glucocorticoids (GCs) may represent an attractive alternative to the high-risk surgical approaches used in the management of patients with progressive sclerosing bone disorders. Our findings also suggest that whereas sclerostin may not be required for the action of GCs on bone formation, it may well be important for the action of GCs on bone resorption. The exact mechanism by which sclerostin may be involved in the regulation of bone resorption is as yet to be explored. ß 2010 American Society for Bone and Mineral Research.

KEY WORDS: BONE RESORPTION; BONE FORMATION; VAN BUCHEM DISEASE; PREDNISONE; SCLEROSTIN

Introduction We present here sequential observations of a patient with van Buchem disease with life-threatening increased intracranial an Buchem disease is a rare bone sclerosing disorder pressure who was treated successfully with prednisone. Vdescribed for the first time in 1955.(1) It belongs to the group of craniotubular hyperostoses and is characterized by progressive generalized osteosclerosis, particularly of the mandible and Case Report the skull, owing to excessive bone formation.(2) It is caused by a 52-kb deletion 35 kb downstream of the SOST gene, which The patient first came under our care at the age of 10 years encodes sclerostin, on chromosome 17q12-21.(3,4) This protein is with an established diagnosis of van Buchem disease. The produced in the skeleton exclusively by the osteocytes and disease was diagnosed clinically and radiologically in infancy inhibits bone formation by antagonizing the Wnt signaling and later confirmed genetically by the finding of a 52-kb pathway.(5) Clinical manifestations of the disease are due to homozygous deletion 35 kb downstream the SOST gene on entrapment of cranial nerves often associated with facial palsy chromosome 17q12-q21 [the patient was briefly described and loss of hearing and smell.(2) Van Buchem disease is thought (patient 15) by Staeling-Hampton and colleagues(3)]. The to have milder clinical manifestations than sclerosteosis, a parents are consanguineous and were both confirmed to be craniotubular hyperostosis with similar phenotype owing to heterozygotes for the disease. There were 3 phenotypically inactivating mutations of the SOST gene.(6,7) Management of the normal sisters in whom no genetic testing has been so far complications of both these sclerosing dysplasias is surgical, undertaken. aiming at removal of the excess of bone, a technically difficult As described in this disorder, clinical manifestations started and sometimes dangerous procedure.(8–10) No medical treat- early in childhood. The patient had a facial palsy at the age of 3 ment is available for either sclerosing disease. Glucocorticoids years and developed progressive deafness requiring the use of a (GCs) are known inhibitors of bone formation,(11,12) and we hearing aid by the age of 10 years, followed by bilateral bone- hypothesized that administration of these agents to patients anchored hearing aids. He has otherwise been well with normal with complications due to bone overgrowth may arrest its growth development along the 95th centile, reaching a final further progress. height of 197 cm by the age of 19 years. He completed his

Received in original form March 24, 2010; revised form April 21, 2010; accepted June 4, 2010. Published online June 14, 2010. Address correspondence to: Socrates E Papapoulos, Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail: [email protected] The December 2010 issue of Journal of Bone and Mineral Research Journal of Bone and Mineral Research, Vol. 25, No. 12, December 2010, pp 2796–2800 was published online on 23 Nov 2010. A pagination error was DOI: 10.1002/jbmr.151 subsequently identified. This notice is included to indicate that the ß 2010 American Society for Bone and Mineral Research pagination is now correct and authoritative [20 January 2011].

2796 secondary education and is employed as office assistant Methods manager. He married at the age of 20 years, and he is the father of 3 healthy children. The biochemical markers of bone turnover procollagen type I N The patient demonstrated the typical clinical and radiologic propeptide (P1NP) and C-terminal telopeptide of type I collagen features of van Buchem disease, with enlarged head and (b-CTX) were measured in serum at regular intervals using the mandible and no syndactyly or other digit malformations. During E-170 system (Roche BV, Woerden, Holland). BMD was measured the 15-year duration of follow-up, there were no other clinical by dual-energy X-ray absorptiometry (DXA; Hologic QDR 4500, signs or symptoms, and blood pressure was normal. Hematologic Waltham, MA, USA). An iliac crest biopsy was obtained after in and biochemical parameters, including those of mineral vivo labeling with two courses of tetracycline separated by metabolism, demonstrated no abnormalities over the years. 12 days. Bone histomorphometry was performed on undecalci- Skeletal radiographs showed thickening of the calvarium, base of fied histologic bone sections by Dr Pascale Chavassieux (INSERM the skull, and long bones and sclerosis of the vertebrae (Fig. 1). Unit 831, University of Lyon, Faculty of Medicine R Laennec, Bone mineral density (BMD) values of the spine and hip were Lyon, France). Immunohistochemical staining for sclerostin was markedly increased at presentation (Z-score þ6.2) and continued performed in our laboratory using a previously described to increase in parallel with that of his healthy peers without, technique.(13) however, attaining a peak (highest Z-score being 7.7). Biochemical markers of bone turnover always were increased compared with normal values for age but followed a normal Results pattern of change with a further increase during the growth Biochemical markers of bone turnover spurt and a progressive decline thereafter, although never reaching the normal range (Fig. 2). The changes in serum P1NP and b-CTX before and during At the age 23 years, the patient complained of progressive prednisone treatment are depicted in Fig. 3. Before treatment, headaches that eventually became persistent and were values of both markers of bone turnover were elevated and associated with dizziness and signs of increased intracranial decreased to within the normal reference adult range within pressure in the form of papilledema. The diagnosis was 4 weeks of starting treatment with prednisone. The effect of confirmed radiologically, and a ventricular-peritoneal drain prednisone on bone turnover depended on the dose adminis- was implanted, and the patient was concomitantly started on tered, and attempts to reduce the dose below 5 mg/day were prednisone 30 mg/day that was reduced to 10 mg/day within associated with increases in serum markers of bone turnover. It 1 month. In the following 2 years he received different doses of was notable that during treatment, there was a close relationship prednisone, as depicted in Fig. 3, but no calcium or vitamin D between serum b-CTX and P1NP values, with the two markers supplements. These interventions were followed by rapid demonstrating parallel changes during adjustments of the dose improvement of his symptoms, and the improvement was of prednisone, suggesting a tight coupling of bone resorption sustained during the follow-up period. There were no and bone formation. There was a highly significant correlation appreciable changes in metabolic parameters with treatment between the two markers throughout the 2-year period of follow 2 (highest values of serum cholesterol and glucose were 5.6 and up (R ¼ 0.765). 6.1 mmol/L, respectively, and of urinary calcium excretion 7.8 mmol/24 hours). BMD The changes in BMD measured at the spine and hip for the 6 years preceding the start of prednisone treatment and for 2 years thereafter are shown in Table 1. Despite high baseline values, BMD continued to increase steadily during adulthood by about 4% every 2 years, demonstrating no further increase after 2 years of treatment with prednisone.

Bone histology On an iliac crest biopsy taken 2 years after the start of prednisone treatment, there was sclerosis and no evidence of active bone remodeling. Cancellous bone volume was clearly increased, and bone trabeculae were thick and well connected. The extent of eroded surfaces was very low (0.4%; normal 3.1% 1.1%), and Howship’s lacunae were devoid of osteoclasts. In addition, no osteoid seams were seen, and there was no tetracycline Fig. 1. Radiographs of the skull showing thickening of the calvarium and uptake on examination under fluorescent light. As expected (14) the base of the skull and of the hand illustrating the absence of and described previously, osteocytes did not stain for syndactyly or other malformations. sclerostin.

GLUCOCORTICOIDS IN VAN BUCHEM DISEASE Journal of Bone and Mineral Research 2797 Fig. 2. Sequential measurement of serum alkaline phosphate activity (AP) in units/L, urinary hydroxyproline/creatinine ratio (OHP/Cr) in mmol/mmol, and height (cm) in a patient with van Buchem disease over a 10-year period. Interrupted lines indicate the upper limit of normal range.

Discussion carriers, being above the upper limit of the normal range in 3 of them.(15) Urinary cross-linked N-telopeptide of type I collagen This case illustrates the beneficial effect of prednisone treatment (NTX) levels were higher in 4 patients with the disease compared on bone metabolism in a patient with van Buchem disease and with carriers. Bone density measured in the phalanges by life-threatening increased intracranial pressure. Treatment radiographic absorptiometry was elevated in all these resulted in a histologically documented dramatic decrease in patients.(15) There are, however, no longitudinal data reported bone formation. Following therapy, there was also no further to date in patients with van Buchem disease. In our patient, at increase in BMD at the spine and hip. Although clinical least up to the age of 23 years, both biochemical markers of manifestations of increased intracranial pressure improved resorption and formation were increased. The clinical progres- significantly, this cannot be attributed solely to treatment with sion of the disease, which was due to bone overgrowth, as also prednisone because the patient had a ventriculoperitoneal drain evidenced by the steady increase in BMD, prompted us to use implanted simultaneously at the time of starting prednisone. GCs in an attempt to arrest the process of bone accumulation. Before prednisone treatment, the patient had an increased The beneficial use of GCs has been reported previously in a rate of bone turnover, as assessed biochemically, associated with patient with craniotubular hyperostosis owing to an unidentified a continuous increase in BMD of the spine and hip. The genetic defect.(16) In this patient, prednisone given for three biochemical markers of bone formation, P1NP and osteocalcin, courses of 10 weeks each reduced serum osteocalcin but had no have been reported previously to be elevated in 6 patients with effect on urinary deoxypyridinoline (DPD) and there were no van Buchem disease compared with their levels in disease reported changes in BMD. In a few patients with progressive diaphyseal dysplasia, a craniotubular hyperostotic disorder distinct from van Buchem disease, which is due to mutations of the gene encoding transforming growth factor b (TGF-b), prednisone treatment during childhood and adolescence led to clinical(17,18) and in one case radiologic improvement.(19)

Table 1. Bone Mineral Density Measurements and Height of a Patient With van Buchem Disease Before and After 2 Years of Prednisone Treatment (Date: Month/Year)

LS Change TH Change Date Height BMD (%) BMD (%) 2-2001 193.9 1.634 — 1.410 — 4-2003 197.0 1.787 9.4 1.741 23.5 1-2005 197.0 1.855 3.8 1.820 4.5 2-2007 197.0 1.934 4.3 1.888 3.7 5-2007 Start prednisone Fig. 3. Biochemical markers of bone formation and resorption before 6-2009 197.0 1.921 –0.7 1.895 0.4 and during treatment with prednisone. P1NP ¼ diamonds and solid line; Note: Height in cm, LS BMD ¼ lumbar spine BMD in g/cm2,TH b-CTX ¼ closed circles and interrupted line. BMD ¼ total hip BMD in g/cm2.

2798 Journal of Bone and Mineral Research VAN LIEROP ET AL. GCs have a deleterious effect on the skeleton, increasing bone complication-associated surgical treatments of such patients. fragility by systemic and local actions.(11) Their main action on The results suggest further that sclerostin may be involved in the bone metabolism is to decrease bone formation by inhibiting the regulation of bone resorption by a mechanism that needs to be proliferation and differentiation of osteoblasts and increasing explored further. their rate of apoptosis.(12,20) CCs also have been reported to increase bone resorption, particularly during the early phase of Disclosures treatment, by stimulating osteoclastic activity and survival (21–23) through an effect on the RANKL/OPG signaling pathway. All the authors state that they have no conflicts of interest. Consistent with these findings, studies in animals(24) and in humans(25–33) have shown that administration of GCs significantly reduce biochemical markers of bone formation but have Acknowledgments no effect or even increase those of bone resorption. Remarkably, administration of prednisone to our patient decreased not only Special thanks to Dr Pascale Chavassieux for performing the bone formation but also bone resorption within 4 weeks of histomorphometric analysis of the bone biopsy. This work was starting of treatment. Serum P1NP and b-CTX decreased and funded by EU FP7 (TALOS:Health-F2-2008-201099). increased concurrently during alterations of prednisone dose, suggesting a tight coupling of bone resorption and formation References during treatment. This was further supported by the strong correlation between the two biochemical markers of bone 1. Van Buchem FS, Hadders HN, Ubbens R. 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NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Bone. 2013 June ; 54(2): 279–284. doi:10.1016/j.bone.2013.01.034.

Glucocorticoids and Osteocyte Autophagy

Wei Yao1, Weiwei Dai1, Jean X. Jiang2, and Nancy E. Lane1 1Department of Medicine, University of California at Davis Medical Center Sacramento, CA 95818 2Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229

Abstract Glucocorticoids are used for the treatment of inflammatory and autoimmune diseases. While they are effective therapy, bone loss and incident fracture risk is high. While previous studies have found GC effects on both osteoclasts and oteoblasts, our work has focused on the effects of GCs on osteocytes. Osteocytes exposed to low dose GCs undergo autophagy while osteocytes exposed to high doses of GCs or for a prolonged period of time undergo apoptosis. This paper will review the data to support the role of GCs in osteocyte autophagy.

Keywords Glucocorticoids; autophagy; bone fragility

Introduction Glucocorticoids (GCs) are used in clinical medicine as effective therapy for inflammatory/ autoimmune diseases. However, GC use creates rapid bone loss that results in a high incident fracture risk. Epidemiologic studies find 50% of rheumatoid arthritis (RA) patients in the United States today are still treated with chronic GCs and; baseline data from clinical trials in RA patients report a prevalence in vertebral fracture of 30-50% [1-6]. Other studies find that both old and young, men and women and all ethnic groups studied have bone loss with GC treatment, making this an important public health problem [7]. Because patients treated with GCs may require the treatment for a long period of time, there is a high medical need to understand the biology of GC induced bone loss so that clinicians can effectively prevent and treat this disease. Interestingly, the loss of trabecular mass, trabecular architecture, and integral bone mass does not explain the increase in fracture risk from GCs, as individuals treated with GCs frequently experience fractures at higher Bone Mineral Densities (BMDs) than women with postmenopausal osteoporosis [8]. In addition, after withdrawal of GC treatment, there can be some recovery of BMD suggesting maintenance of bone architecture despite a change in bone fragility [8-10]. Recently, atypical fractures have been documented to occur more often in the shaft or subtrochanteric regions of the femur in patients treated with long-term bisphosphonates, especially for those who were treated for 6 months or longer with GCs (Girgis C. et al., ASBMR 2010). Although more epidemiologic and pathophysiologic research is needed to better define the risk, the adverse

© 2012 Elsevier Inc. All rights reserved. Corresponding Author Information: Nancy E. Lane, MD, Center for Musculoskeletal Health, 4625 2nt Avenue, Suite 1002, Sacramento, California 95817, Telephone: 916-734-0758, FAX: 916-734-4773, [email protected]. Publisher©s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Yao et al. Page 2

effects of GCs on the cortical bone quality that may be independent of BMD loss warrant further investigation [11]. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Biology of GC-Induced Bone Loss GC treatment results in changes in bone remodeling [12, 13]. Observations of surface and biochemically-based turnover in clinical studies of GC-induced osteoporosis show a reduction in trabecular bone volume, thickness and bone formation [12, 14-16]. The influence of glucocorticoids (GCs) on bone resorption was thought to be indirect and related in part to reduced calcium absorption and increased renal calcium excretion [17]. However, recent studies have found that GCs act directly on osteoclasts to decrease the apoptosis of mature osteoclasts [18]. Kim et al. found that GCs in vitro inhibited the proliferation of osteoclasts from bone marrow macrophages in a dose-dependent manner. In addition, higher GC doses had no effect on osteoclast maturation but inhibited osteoclasts from reorganizing their cytoskeleton [19]. Therefore, excess GC results in an increase in osteoclast number, but in an apparent inhibition of function with impaired spreading and degradation of mineralized matrix [19].

GCs also alter osteoblast and osteocyte function, which contributes to GC-induced osteoporosis [17]. GCs directly inhibit cellular proliferation and differentiation of osteoblast lineage cells [20], reduce osteoblast maturation and activity [13], and also induce osteoblast and osteocyte apoptosis in vivo [21]. The suppression of osteoblast function by GCs is reported to be associated with alteration of the Wnt signaling pathway [22], a critical pathway for osteoblastogenesis [23, 24]. GCs enhance Dickkopf 1 expression [25], one of the Wnt antagonists that prevents soluble Wnt proteins from binding to their receptor complex [26]. GCs maintain levels of glycogen-synthase kinase-3β [27], a key kinase that phosphorylates β-catenin, thereby preventing the translocation of β-catenin into the nucleus and the initiation of transcription in favor of osteoblastogenesis. GCs may also enhance bone marrow stromal cell development towards the adipocyte lineage rather than towards the osteoblast lineage [24, 28]. Moreover, the loss of osteocytes by GC-induced apoptosis [29] may disrupt the osteocyte-canalicular network, resulting in a failure to direct bone remodeling at the trabecular surface. GC-induced changes in osteocyte function also result in a weakening of the localized material properties around osteocytes as well as in decreased whole bone strength [30].

Mineral Metabolism and Osteocytes GC treatment is known to alter calcium metabolism. Treatment with GCs reduces the gastrointestinal absorption of calcium and increases urinary excretion of calcium, which leads to a calcium deficit [17, 31, 32]. Over time this calcium deficit and low serum ionized calcium levels can stimulate PTH release; PTH then catalyzes 1-α- hydroxylase enzyme production in the kidney, which in turn increases 1,25(OH)2 vitamin D3 levels, and this is followed by gastrointestinal absorption of both calcium and phosphorus. If the calcium deficit continues, gastrointestinal absorption of these minerals continues, resulting in elevation of serum phosphorus that then stimulates the production of fibroblast growth factor 23 (FGF23) by osteocytes in an attempt to lower serum phosphorus. FGF23 is a hormonal factor that is produced primarily by osteocytes and reduces serum phosphorus and 1,25(OH)2 vitamin D3 levels by acting on the kidney through FGF receptors and Klotho [33-35]. The production and circulating levels of FGF23 appear to be tightly regulated but the mechanisms responsible are still under investigation.

The association between FGF23, osteocytes and mineralization has recently been explored [36]. FGF23 serves as a phosphaturic factor synthesized by osteocytes and inhibits 1,25(OH)2 vitamin D3 production by the kidney to maintain the balance between phosphate

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homeostasis and skeletal mineralization [37]. A recent in vitro study demonstrated that overexpression of FGF23 suppressed osteoblast differentiation and matrix mineralization

NIH-PA Author Manuscript NIH-PA Author Manuscript[38]. NIH-PA Author Manuscript Another study evaluated the proteins associated with osteocytes and bone mineralization and found that FGF23 co-localized to the secondary spongiosa of trabecular bone and areas of cortical bone where the osteocyte lacunar system was mature, suggesting that FGF23 produced by osteocytes would then be part of the bone-renal axis that is central to proper mineral metabolism [39, 40]. Elevated levels of serum FGF23 have been found in individuals with autosomal hypophosphatemic rickets with mutations in DMP-1 (dentin matrix protein-1) and other forms of rickets and chronic kidney disease exhibit elevated levels of FGF23 despite normal calciuria [41, 42]. In contrast, mice with deletion of Klotho developed elevated DMP-1, hyperphosphatemia and low FGF23 levels [43]. Overexpression of FGF23 in primary rat calvaria cell cultures suppressed matrix mineralization [38]. In one pilot study, increased FGF23 expression in ovine callus was associated with delayed fracture healing [44]. It appears that changes in the production and local concentration of this phosphaturic factor by the osteocyte may result in a reduction in osteocyte-driven mineral metabolism, thereby compromising local bone strength [45-47]. In GC-treated mice, we have observed a dose-dependent increase in serum FGF23, with a decrease in serum phosphorus and 1,25(OH) vitamin D3, suggesting that GC use may influence mineral metabolism through FGF23 [48]. The altered perilacunar mineralization around GC- treated osteocytes may be secondary to increased FGF23 production. If this was the case, adequate calcium supplementation or restricted phosphate dietary intake may prevent some of the changes in the bone renal axis that occur with GC treatment.

GC induced bone loss clearly does affect the osteocyte Osteocytes are terminally differentiated osteoblasts that lie below the bone surface and are connected both to other osteocytes and the bone surface via dendritic processes that travel through canaliculi [46, 49-53]. Our in vivo mouse studies showed that with GC treatment, a number of the osteocyte lacunae were enlarged as measured by a modified atomic force microscopy/scanning probe microscopy (AFM/SPM). Raman microscopy of the perilacunar area of GC treated osteocytes revealed an enlarged area of demineralization, and AFM/SPM revealed reduced elastic modulus around the enlarged osteocyte lacunae (nearly 40% below the other bone matrix) in a number of the osteocytes [30]. A review of the literature described that we had rediscovered “osteocytic osteolysis” a term initially used to described enlarged lacunae in patients with hyperparathyroidism [54], immobilized rats [55], X-linked hypophosphophatemic rickets, and lactation [56, 57]. Osteocyte lacunar architecture can also be modified by poor mineralization when the bone is being formed, such as with renal osteodystrophy which is distinctly different from “osteocytic osteolysis”. Our observation of the removal of mineral by osteocytes (over weeks or months) would certainly be slower than the bone removal by osteoclasts and may involve a different process. As we also found reduced mineral and elastic modulus surrounding the GC treated osteocyte, we postulated that the osteocyte in the presence of GCs modified the pre-existing mineral of its surrounding matrix creating “osteocyte halos” as initially used by Heuck for the pericanicular demineralization in X-linked hypophophatemic rickets [58].

To try to elucidate how the osteocyte could be changing its perilacunar matrix we performed microarray analysis, RT-PCR and immunohistochemistry on selected genes and found with GC (1.4 mg/kg/d, low dose) exposure for either 28 or 56 days, the expression of genes associated with inhibition of bone formation (Dkk-1, SOST, Wif1), inhibition of mineralization (FGF23) and lysosomes/matrix degradation (MMPs, cathepsin, proteinases) were significantly higher compared to the placebo-control at day 0 (preliminary data). In summary, we determined that GC induced changes in the osteocyte metabolism resulted in a number of the osteocytes developing an increase in osteocyte lacunar size with perilacunar

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demineralization, localized reduction in elastic modulus and production of proteins that inhibit osteoblast formation and bone mineralization. However, we did not find much

NIH-PA Author Manuscript NIH-PA Author Manuscriptevidence NIH-PA Author Manuscript for either pro-apoptotic gene expression, or the presence of apoptotic osteocytes at the low GC dose (1.4 mg/kg/d). In contrast, mice treated for 28 days with a higher GC dose (2.4 mg/kg/d) had apoptotic osteocytes present in the cortical bone. Their changes to the osteocyte in its localized microenvironment with exposure to low dose GC for 28 days suggested to our research group that non-apoptotic programmed cell death, such as autophagy, may also play a role in osteocyte's response to the GC induced stress.

Does autophagy explain the osteocyte response to GCs? The autophagy pathway is one of the most important biologic processes that enable cells to survive stress and helps to maintain cellular homeostasis by degrading damaged organelles [59-62]. Autophagy is defined by the formation of autophagosomes, also known as autophagic vacuoles that are lined by two membranes with the recruitment of microtubule- associated protein light-chain 3 (LC3)-phosphatidylethanolamine conjugate (LC3-II) to the autophagosomal membrane, a characteristic for autophagosome [63]. When the autophagosomes fuse with the lysosomes and form autolysosomes, degradation occurs and the amino acids or other small molecules are delivered to the cytoplasm for energy production or recycling. If the cells are subjected to long periods of time under GC stress, this may result in extensive recycling of damaged organelles that may lead to cell death or apoptosis [60, 64, 65]. Autophagy can be inhibited by chloroquine (CQ) as it accumulates within autophagosomes, and inhibits the fusion with lysosomes thereby preventing the formation of autolysosomes. This reduction by chloroquine in the final phase of autophagy that provides a pathway for the breakdown of proteins and removal of metabolic debris from the cell, may augment apoptosis [66-68] or rescue osteocyte from cell death [69]. Recently Xia et al reported that dexamethasone treatment of an osteocytic cell line, MLO-Y4 cells, increased autophagy markers and the accumulation of autophagosome vacuoles as detected by several standard approaches based on recently published guidelines including fluorescent GFP-LC3 punctate dots, MDC fluorescence, LC3 lipidation and electron microscopy imaging in addition to conventional acridine orange staining [70]. The enhancement of autophagy was also validated in isolated primary osteocytes isolated from embryonic chicks treated with dexamethasone and in vivo from osteocytes in bone from mice chronically treated with prednisolone. In addition, gene microarray analysis of the cortical bone from mice after 28 days of prednisolone treatment showed increased messenger RNA for several autophagy markers including autophagy-related 16 like 2, autophagy-related 7, LC-3α and LC-3β. Conversely, gene markers for pro-apoptosis were not significantly increased until after a longer prednisolone treatment (56 days of chronic GC exposure) [24, 30]. We also observed gene and protein expression for matrix proteolysis, including matrix metalloproteinases, caspases and cathepsins increased in the cortical bones following GC treatment [24]. Because the interior of a lysosome is strongly acidic, as it releases the contents of its vacuole through autophagic flux into the microenvironment of the osteocyte, it may induce matrix proteolysis, and demineralization of bone around the osteocyte that over time may weaken both the localized bone tissue and whole bone strength [30].

We also found that dexamethasone reduced the number of metabolically normal osteocytes and this effect was augmented when autophagy was inhibited [70]. This study implies that autophagy could be an attempt by osteocytes to attenuate the effect of GC on osteocyte. Autophagy is reported to act as a “double-edge sword” involved in both cell protection and cell death [62, 71]. The cell protective function of autophagy is likely to occur under short or moderate stress conditions. Our cell viability study showed that cells under the autophagic state are very much alive and are likely under metabolic stress. Autophagy is a probable mechanism by which osteocytes can repair damaged organelles or cell membranes.

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However, higher, or more prolonged stress may result in an accumulation of autophagosomes and cell death. Interestingly, after 56 days of a treatment with a relatively

NIH-PA Author Manuscript NIH-PA Author Manuscripthigh NIH-PA Author Manuscript dose of prednisolone (5.6mg/kg/d) in mice, we studied the trabecular bone from the vertebral bodies and observed increased apoptotic tunnel-positive labeling [21, 72]. Therefore, these studies demonstrate that low dose GC (less than 2.8 mg/kg/d in mice) treatment resulted in osteocyte autophagy both in vitro and in vivo. During the initial period of GC treatment, gene array studies revealed that the oxidative pathway [73-77] was activated and simultaneously autophagy was activated suggesting that the osteocytes responded with autophagy in an attempt to “save themselves”. However, with the prolonged GC exposure or higher doses of GCs (5.6mg/kg/d), the cell may undergo apoptosis and or necrosis. The outcome may be related to either the duration of GC treatment or the dose of GC or both [78, 79]. It is possible that suppression or the prevention of autophagy may be a promising new target in the prevention of GC induced bone fragility. If we find that low dose GCs induce osteocyte autophagy that does not affect bone formation and whole bone strength, as opposed to higher doses of GCs that induce osteocyte apoptotic induced bone remodeling and increased fragility, this represent a major paradigm shift for the mechanism responsible for GC-induced bone fragility. Treatments for GC-induced osteoporosis would focus on the inhibition or augmentation of autophagy.

Why do GCs induce osteocyte autophagy Chronic GC treatment decreases bone formation and increases bone fragility that resembles an accelerated aging process [12, 13]. We found that there was a dose-dependent decrease in the activation of autophagy and anti-oxidative defense gene expression in the cortical bone of mice. GCs at a lower dose increased anti-oxidative responsive as well as autophagic pathways by an average of 30-fold (Figure 1A). In addition, the DNA damage and antioxidant pathways were significantly increased both at the lower GC dose and within the first days of the GC exposure, suggesting that cells were being “over-activated” in response to the initial GC treatment. Prolonged exposure or higher doses of GCs reduced both the expression of genes encoding proteins that are anti-oxidants and the number of autophagic osteocytes [80], supporting a relationship between the cells anti-oxidant ability and autophagy following GC exposure [81, 82] (Figure 1B). Bone formation, measured by serum osteocalcin and surface based histomorphometry was greatly reduced by chronic or high dose GC treatments. MicroCT evaluation of trabecular structure showed reduced trabecular bone volume and thickness, as compared to control mice [30, 83]. Similar observations of surface and biochemical based turnover in clinical studies of GIOP have been made including the reduction in trabecular bone volume, thickness and reduced bone formation [12, 14-16]. In summary, GC treatment effects on bone formation were very similar to that observed with aging in that GCs reduced the activation of anti-oxidant gene expression, decreased bone marrow osteogenic potential, reduced autophagy and bone formation. Based on these studies, we propose that modulation of the oxidative and autophagic pathways may provide promising new targets for maintaining bone formation in the presence of GCs or aging, which over time may preserve bone mass.

Therefore, these studies demonstrate that low dose GC treatment (1.4mg/kg/d for 28 days) resulted in autophagy in osteocytes both in vitro and in vivo. However, with the continued stress of prolonged GC exposure or higher doses of GCs (5.6mg/kg/d for 28 days), the cell may undergo apoptosis and or necrosis. The outcome may be related to either the duration of GC treatment or the dose of GC or both [78, 79]. Autophagy may provide a promising new target in the prevention of GC induced bone fragility (Figure 2). If we find that low dose GCs induce osteocyte autophagy that does not affect bone formation and whole bone strength, as opposed to higher doses of GCs that induce osteocyte apoptotic induced bone remodeling and increased fragility, this represents a major paradigm shift for the mechanism

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responsible for GC-induced bone fragility. Treatments for GC-induced osteoporosis would focus on the inhibition or augmentation of autophagy. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Acknowledgments

This work was funded by National Institute of Health grants nos. 1K12HD05195801 that are co-funded by National Institute of Child Health and Human Development (NICHD), the Office of Research on Women's Health (ORWH), the Office of Dietary Supplements (ODS) and the National Institute of Aging (NIA); R01 AR043052; K24 AR-048841 and 5R21AR57515.

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76. Komatsu F, Kudoh H, Kagawa Y. Evaluation of oxidative stress and effectiveness of low-dose glucocorticoid therapy on exacerbation of chronic obstructive pulmonary disease. J Gerontol A Biol Sci Med Sci. 2007; 62:459–64. [PubMed: 17452743] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 77. Ong SL, Zhang Y, Whitworth JA. Reactive oxygen species and glucocorticoid-induced hypertension. Clin Exp Pharmacol Physiol. 2008; 35:477–82. [PubMed: 18307745] 78. Planey SL, Abrams MT, Robertson NM, Litwack G. Role of apical caspases and glucocorticoid- regulated genes in glucocorticoid-induced apoptosis of pre-B leukemic cells. Cancer Res. 2003; 63:172–8. [PubMed: 12517795] 79. Bonapace L, Bornhauser BC, Schmitz M, Cario G, Ziegler U, Niggli FK, Schafer BW, Schrappe M, Stanulla M, Bourquin JP. Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance. J Clin Invest. 2010; 120:1310–23. [PubMed: 20200450] 80. Jia J, Yao W, Guan M, Dai W, Shahnazari M, Kar R, Bonewald L, Jiang JX, Lane NE. Glucocorticoid dose determines osteocyte cell fate. FASEB J. 2011; 25:3366–76. [PubMed: 21705669] 81. Maiuri MC, Tasdemir E, Criollo A, Morselli E, Vicencio JM, Carnuccio R, Kroemer G. Control of autophagy by oncogenes and tumor suppressor genes. Cell Death Differ. 2009; 16:87–93. [PubMed: 18806760] 82. Tasdemir E, Maiuri MC, Orhon I, Kepp O, Morselli E, Criollo A, Kroemer G. p53 represses autophagy in a cell cycle-dependent fashion. Cell Cycle. 2008; 7:3006–11. [PubMed: 18838865] 83. Weinstein RS, Jia D, Powers CC, Stewart SA, Jilka RL, Parfitt AM, Manolagas SC. The skeletal effects of glucocorticoid excess override those of orchidectomy in mice. Endocrinology. 2004; 145:1980–7. [PubMed: 14715712]

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Figure 1. RNA was extracted from the tibial cortical bone in mice that were treated with PL or various doses of GC. RT-PCR gene arrays were performed for antioxidant defense (A). Correlations between gene expressions associated with antioxidant and autophagy following GC treatments (B).

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Figure 2. Proposed mechanisms for osteocyte autophagy and glucocorticoid-induced bone fragility

Bone. Author manuscript; available in PMC 2014 June 01. T h e new england journal o f medicine

clinical practice

Glucocorticoid-Induced Bone Disease Robert S. Weinstein, M.D.

This Journal feature begins with a case vignette highlighting a common clinical problem. Evidence supporting various strategies is then presented, followed by a review of formal guidelines, when they exist. The article ends with the author’s clinical recommendations.

A 55-year-old woman with severe, persistent asthma requiring glucocorticoid therapy for the past 3 months presents for care. Her medications include albuterol, inhaled fluticasone with salmeterol, montelukast, and prednisone (at a dose of 10 mg per day). In the past, she received several intermittent courses of prednisone at a dose of 15 mg or more per day. Her weight is 45.5 kg (100 lb), and her height 157.5 cm (62 in.); the body-mass index (the weight in kilograms divided by the square of the height in meters) is 18. Scattered wheezing is heard during expiration. Findings on vertebral percussion and rib-cage compression are unremarkable. How should her case be evaluated and managed to minimize the risk of fractures?

The Clinical Problem

From the Division of Endocrinology and Glucocorticoid therapy is the most common cause of secondary osteoporosis and the Metabolism, the Center for Osteoporosis leading iatrogenic cause of the disease.1-3 Often, the presenting manifestation is and Metabolic Bone Diseases, the Depart- ment of Internal Medicine, and the Central fracture, which occurs in 30 to 50% of patients receiving long-term glucocorticoid Arkansas Veterans Healthcare System at the therapy.4 Glucocorticoid-induced osteoporosis predominantly affects regions of the University of Arkansas for Medical Sciences, skeleton that have abundant cancellous bone, such as the lumbar spine and proximal Little Rock. Address reprint requests to Dr. Weinstein at the Division of Endocrinol- femur. In patients with glucocorticoid-induced osteoporosis, the loss of bone min- ogy and Metabolism, University of Arkansas eral density is biphasic; it occurs rapidly (6 to 12% loss) within the first year and for Medical Sciences, 4301 W. Markham St., more slowly (approximately 3% loss yearly) thereafter.5 However, the risk of fracture Slot 587, Little Rock, AR 72205-7199, or at [email protected]. escalates by as much as 75% within the first 3 months after the initiation of therapy, typically before there is a substantial decline in bone mineral density, suggesting N Engl J Med 2011;365:62-70. that there are adverse effects of glucocorticoids on bone that are not captured by Copyright © 2011 Massachusetts Medical Society. bone densitometry.6 Several large case–control studies have shown strong associa- tions between exposure to glucocorticoids and the risk of fractures.4,6,7 An increase in the risk of vertebral and hip fractures occurs rapidly after the start of treatment An audio version and has been reported to occur with doses as small as 2.5 to 7.5 mg of prednisolone of this article per day (equivalent to 3.1 to 9.3 mg of prednisone per day). In a cohort study involv- is available at ing patients 18 to 64 years of age, continuous treatment with 10 mg of prednisone NEJM.org per day for more than 90 days, for a variety of indications, as compared with no exposure to glucocorticoids, was associated with an increase in hip fractures by a factor of 7 and an increase in vertebral fractures by a factor of 17.7 Furthermore, an increase in the risk of fractures has been reported with the use of inhaled glucocorticoids, as well as with alternate-day and intermittent oral regimens.3

Risk Factors Risk factors associated with glucocorticoid-induced osteoporosis are listed in Table 1. One factor whose importance has been recognized in the past decade is the activity of the 11β-hydroxysteroid dehydrogenase (11β-HSD) system, a prereceptor modula- tor of glucocorticoid action.11 Two isoenzymes, 11β-HSD1 and 11β-HSD2, catalyze

62 n engl j med 365;1 nejm.org july 7, 2011 The New England Journal of Medicine Downloaded from nejm.org on August 3, 2013. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. clinical practice conversion between hormonally active glucocorti- and the risk of fracture in patients with glucocor- coids (e.g., cortisol or prednisolone) and inactive ticoid-induced osteoporosis.3,4 Glucocorticoid ex- glucocorticoids (e.g., cortisone or prednisone). cess also directly reduces osteoclast production, The 11β-HSD1 enzyme is an activator, and the but the lifespan of osteoclasts is prolonged, in 11β-HSD2 enzyme is an inactivator. The increased contrast to the decrease in the lifespan of osteo- risk of fracture with glucocorticoid administra- blasts. Therefore, with long-term therapy, the num- tion in the elderly may be explained in part by the ber of osteoclasts is usually maintained in the increase in 11β-HSD1 that occurs with aging. The normal range, whereas the number of osteoblasts risk of glucocorticoid-induced osteoporosis appears plummets and bone formation is substantially re- to be similar in men and women and among var- duced.14,18 These histologic features contrast with ious ethnic groups.13 the increased bone formation and resorption that are typical of postmenopausal osteoporosis or in- Pathogenesis creased parathyroid hormone secretion and indi- Histomorphometric studies in patients with gluco- cate that, contrary to previous assumptions, hypo- corticoid-induced osteoporosis consistently show gonadism and secondary hyperparathyroidism are fewer osteoblasts and an increased prevalence of not central to the pathogenesis of glucocorticoid- osteocyte apoptosis, as compared with normal con- induced osteoporosis.19-22 trols1,3,4,14,15 (Fig. 1). The increased osteocyte apoptosis is associated with decreases in vascular en- Strategies and Evidence dothelial growth factor, skeletal angiogenesis, bone interstitial fluid, and bone strength.16 Thus, glu- Evaluation cocorticoid-induced apoptosis of osteocytes could Physicians who prescribe glucocorticoids should account for the loss of bone strength that occurs educate their patients about side effects and com- before the loss of bone mineral density17 and the plications, including not only osteoporosis and observed mismatch between bone mineral density osteonecrosis but also cataracts and glaucoma,

Table 1. Risk Factors for Glucocorticoid-Induced Osteoporosis.*

Risk Factor Evidence of a Contribution

Advanced age Patients 60 to 80 years of age receiving glucocorticoid therapy, as compared with patients 18 to 31 years of age, had a relative risk of vertebral fracture of 26 and a shorter interval between initiation of treatment and the occurrence of fracture8 Low body-mass index (<24)† Low body-mass index is a risk factor for glucocorticoid-induced osteoporosis and probably fractures as well9 Underlying disease Rheumatoid arthritis, polymyalgia rheumatica, inflammatory bowel disease, chronic pulmonary disease, and transplantation are independent risk factors4 Prevalent fractures, smoking, excessive All are independent risk factors for osteoporosis but have not been exten- alcohol consumption, frequent sively studied in patients receiving glucocorticoids falls, family history of hip fracture Glucocorticoid receptor genotype Individual glucocorticoid sensitivity may be regulated by polymorphisms in the glucocorticoid receptor gene10 Increased 11β-HSD1 expression 11β-HSD1 expression increases with the age of the patient and with glucocorticoid administration11

High glucocorticoid dose (high current Risk of fracture escalates with increased doses and duration of therapy; or cumulative dose; long duration alternate-day or inhaled therapies also confer risks of glucocorticoid- of therapy) induced osteoporosis4,12

Low bone mineral density Glucocorticoid-induced fractures occur independently of a decline in bone mass, but patients with very low bone mineral density may be at higher risk4,8

* 11β-HSD1 denotes 11β-hydroxysteroid dehydrogenase 1. † The body-mass index is the weight in kilograms divided by the square of the height in meters.

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Glucocorticoid excess

Osteoclasts Osteoblasts

Decreased osteoblastogenesis Decreased osteoclastogenesis Osteocytes Increased apoptosis Early, transient increase in Early and continual decrease in • osteoclast survival Increased apoptosis • cancellous osteoclasts • cancellous osteoblasts • bone resorption • synthetic ability • bone formation

Decreased canalicular circulation

Decreased bone quality

Osteonecrosis Fracture

Figure 1. Direct Effects of Glucocorticoids on Bone Cells. Shown are the adverse skeletal changes that result from an excess of glucocorticoids and lead to osteoporosis and osteonecrosis. The brown, condensed cells are apoptotic osteoblasts and osteocytes. Apoptotic osteocytes disrupt the osteocyte–lacunar–canalicular network.

COLOR FIGURE Version 2 05/16/11 hypokalemia, hyperglycemia, hypertension, hyper- increase in the risk of future fractures.Author Weinstein Laboratory lipidemia, weight gain, fluid retention, suscepti- testing that should be performedFig #before1 treatment Title bility to bruising, decreased resistance to infection, is prescribed includes measurementsME of serum impaired healing, myopathy, adrenal insufficiency, 25-hydroxyvitamin D, creatinine,DE and calciumCS lev- 3 Artist JM and the glucocorticoid withdrawal syndrome. Pa- els (in addition to glucose, potassium,AUTHOR PLEASEand NOTE:lipid Figure has been redrawn and type has been reset tients receiving long-term glucocorticoid therapy levels). Since bone turnover after long-termPlease check carefullygluco- should wear medication identification jewelry. corticoid therapy is low, testsIssue ofdate biochemical7/7/11 Malpractice suits precipitated by a failure of phy- markers of bone metabolism are usually not help- sicians to document disclosure of the skeletal ful.4,24-27 Measurement of bone mineral density complications to patients are not rare,23 yet these and vertebral morphologic assessment or plain complications are often ignored in clinicians’ films are often recommended to assess the patient discussions with patients about the use of gluco- for vertebral fractures, but the disparity between corticoids.13 bone quantity and bone quality in glucocorticoid- Measurement of the patient’s height is impor- induced osteoporosis makes measurements of bone tant, since loss of height suggests the possibility mineral density insensitive for identifying patients of prevalent vertebral fractures, with an associated at risk.2 However, measurements of bone mineral

64 n engl j med 365;1 nejm.org july 7, 2011 The New England Journal of Medicine Downloaded from nejm.org on August 3, 2013. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. clinical practice density may be useful for follow-up assessments eral density, alendronate and risedronate increased after an intervention. The use of the World Health bone mineral density at the lumbar spine and Organization fracture prevention algorithm (FRAX) femoral neck and reduced the relative risk of glu- is not recommended in the case of patients with cocorticoid-induced vertebral fractures by about glucocorticoid-induced osteoporosis, since it does 40%4,27,32,33; patients in these trials typically had not take into account the current and cumula- been taking 10 to 20 mg of prednisone daily or the tive dose of glucocorticoids and the duration of equivalent for at least 1 year before enrollment, therapy and underestimates the risk of gluco- although the dose range and the duration of treat- corticoid-induced fractures. Furthermore, bone ment varied widely. In another randomized trial mineral density at the femoral neck is used in the involving patients treated with glucocorticoids, algorithm, but vertebral fractures are more com- zoledronic acid was noninferior to risedronate in mon than hip fractures in patients with gluco- increasing bone mineral density at the lumbar corticoid-induced osteoporosis, and the inclusion spine.30 of the common risk factors for postmenopausal Alendronate decreases glucocorticoid-induced osteoporosis in the algorithm may not be applica- apoptosis of osteocytes,34 which may play a role ble to patients with glucocorticoid-induced osteo- in the preservation of bone strength.17 However, porosis.24 glucocorticoids antagonize the effects of nitrogen- In patients treated with glucocorticoids who containing bisphosphonates in inducing apoptosis report persistent hip, knee, or shoulder pain, of osteoclasts and inhibiting bone resorption.35,36 especially pain that occurs with joint movement Perhaps as a consequence, bisphosphonates ap- or that is associated with tenderness or reduced pear to be less effective in the protection of bone range of motion, magnetic resonance imaging mineral density in patients with glucocorticoid- should be performed to rule out osteonecrosis.23 induced osteoporosis than they are in patients The incidence of osteonecrosis among patients with other forms of osteoporosis. The average who take glucocorticoids has been estimated to percentage increase in bone mineral density at be between 5 and 40%; higher doses of glucocor- the lumbar spine and femoral neck in patients ticoids and prolonged treatment are associated with glucocorticoid-induced osteoporosis after with greater risk, although osteonecrosis may also treatment with alendronate at a dose of 10 mg per occur with short-term exposure to high doses, day for 2 years was 3.9% and 0.6%, respectively including those administered intraarticularly (typ- — considerably less than that reported in women ically 40 to 80 mg of methylprednisolone) and in with postmenopausal osteoporosis (about 7% and the absence of osteoporosis. The mechanisms that 3.6%, respectively) or in men with osteoporosis have been postulated for the development of osteo- (7% and 2.5%, respectively), even though the latter necrosis include fat embolism, vascular thrombo- two groups were, on average, 10 years older than sis, and osteocyte apoptosis.28,29 the patients with glucocorticoid-induced osteoporosis.32,37,38 Moreover, the evidence to support Treatment the use of bisphosphonates in the treatment of All patients should receive adequate calcium sup- patients with glucocorticoid-induced osteoporosis plementation (1200 mg per day in divided doses) is not as strong as the evidence for their use in the and adequate vitamin D supplementation (800 to treatment of patients with postmenopausal osteo- 2000 U per day), but these precautions alone are porosis; the primary end point in the trials of not sufficient to prevent fractures.4,24-27 Bisphos- glucocorticoid treatment was bone mineral den- phonates are considered to be the first-line op- sity rather than the occurrence of fractures, and tions for the treatment of glucocorticoid-induced most trials were only 12 to 24 months in duration osteoporosis (Table 2)4; alendronate, risedronate, and were not powered to study hip fractures. and zoledronic acid are approved by the Food and A limitation of oral bisphosphonate therapy is Drug Administration (FDA) for this indication, al- poor adherence to treatment — a well-recognized though there is controversy regarding the doses problem even in the case of agents that are ad- and duration of glucocorticoid treatment that ne- ministered weekly or monthly. Administration of cessitate intervention to reduce the risk of frac- zoledronic acid as a once-yearly infusion avoids tures. In randomized, double-blind, placebo-con- this problem and provides rapid skeletal protec- trolled trials, including patients with a variety of tion. For protection from fractures in patients underlying diseases and irrespective of bone min- who have received prolonged glucocorticoid ther-

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Table 2. Treatment of Glucocorticoid-Induced Osteoporosis.*

Intervention Advantages Disadvantages

Bisphosphonates Alendronate, 10 mg/ Osteoclast inhibition reduces bone loss and re- Antiresorptive agents do not directly address the de- day or 70 mg/wk, duces vertebral fractures in patients with glu- creased bone formation that is characteristic of gluco- taken orally cocorticoid-induced osteoporosis; alendro- corticoid-induced bone disease and have not been nate also prevents glucocorticoid-induced shown to reduce hip fractures; gastrointestinal side osteocyte apoptosis; if glucocorticoid therapy effects may occur; musculoskeletal discomfort, osteo- Risedronate, 5 mg/day is discontinued, these drugs can be stopped necrosis of the jaw, uveitis, and atypical femoral frac- or 35 mg/wk, taken tures have occurred in rare cases; bisphosphonates orally should be avoided in patients with creatinine clearance of ≤30 ml/min; patients have poor adherence to oral therapy; as compared with intravenous therapy, a longer time is required to obtain skeletal protection Zoledronic acid, 5 mg/yr, Osteoclast inhibition reduces bone loss; as Acute-phase reaction (influenza-like syndrome) may occur given intravenously compared with oral treatment, there is in- within 2 to 3 days and last 3 days or less, particularly creased adherence to intravenous treatment after first dose,30 but can be effectively managed with and more rapid onset of skeletal effects; acetaminophen or ibuprofen gastrointestinal side effects are unlikely Teriparatide, 20 µg/day, Teriparatide directly addresses the increase in Costs are greater than with oral or intravenous bisphos- given subcutaneously, osteoblast and osteocyte apoptosis and the phonates; daily injections are required; response is re- for 2 years, followed by decrease in osteoblast number, bone for- duced when teriparatide is given with high-dose gluco- bisphosphonate treat- mation, and bone strength that are charac- corticoids; it has not been studied in patients with ele- ment for as long as teristic of glucocorticoid-induced osteo vated parathyroid hormone levels; adverse effects in- glucocorticoids are porosis and reduces vertebral fractures clude mild hypercalcemia, headache, nausea, leg required cramps, and dizziness; caution must be taken in patients with preexisting nephrolithiasis; serum calcium should be checked at least once 16 hours or more after injection and oral calcium intake adjusted as needed31 Denosumab, 60 mg every Denosumab is a potent inhibitor of osteo- Denosumab does not address the reduced bone forma- 6 mo, subcutaneously clasts, with ease of administration; it can tion caused by glucocorticoid excess; hypocalcemia be stopped if glucocorticoids are discon- and vitamin D deficiency must be treated before the tinued; it can be used in patients with cre- use of denosumab atinine clearance of ≤30 ml/min

* Alendronate, risedronate, zoledronic acid, and teriparatide have been approved by the Food and Drug Administration for the treatment of gluco corticoid-induced osteoporosis. In Europe, only once-daily oral bisphosphonate regimens, zoledronic acid, and teriparatide are approved for the treatment of glucocorticoid-induced osteoporosis.

apy (e.g., 10 mg per day or more of prednisone continued for at least as long as the glucocorti- for longer than 90 days), intravenous bisphospho- coids are prescribed3,4,27,40; drug holidays are not nate therapy may be preferable to oral therapy. On considered to be appropriate for patients who are the basis of estimates that the maximal absorp- being treated with glucocorticoids. tion of alendronate when it is taken orally on an In a 2-year, randomized, controlled, open-label empty stomach is about 0.7% and that the molar trial involving patients with osteonecrosis of the potency of alendronate is lower than that of intra- femoral head, patients who received alendronate venous zoledronic acid by a factor of 10, it is esti- therapy, as compared with those who received no mated that a patient would need 90 days of treat- treatment, had decreased pain and delayed expan- ment with alendronate at a dose of 70 mg per sion of lesions and were less likely to need sur- week to receive a dose equivalent to 5 mg of zole- gery.41 A prospective, observational study showed dronic acid delivered in 15 minutes,39 although that patients with osteonecrosis had a sustained these regimens have not been compared with reduction in pain and improvement in ambulation respect to rates of fracture in patients with gluco- within months after the initiation of alendronate corticoid-induced osteoporosis. Since substantial therapy.42 In both studies, the most common cause loss of bone mineral density has been observed in of osteonecrosis was the use of glucocorticoids. patients who discontinue bisphosphonate therapy Although bisphosphonates are useful in treat- while continuing to take glucocorticoids, it is usu- ing osteonecrosis of the hip, these drugs are ally recommended that bisphosphonate therapy be associated with the development of osteonecro-

66 n engl j med 365;1 nejm.org july 7, 2011 The New England Journal of Medicine Downloaded from nejm.org on August 3, 2013. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. clinical practice sis of the jaw.43 Osteonecrosis of the jaw is char- (RANKL), which is approved by the FDA for the acterized by exposed maxillofacial bone for at prevention of vertebral, nonvertebral, and hip frac- least 8 weeks and typically occurs after a dental tures in women with postmenopausal osteopo- extraction or other invasive procedure.43 Most re- rosis but is not currently approved for the treat- ported cases of osteonecrosis of the jaw have oc- ment of glucocorticoid-induced osteoporosis.48 curred in patients with osteolytic breast cancer Administered as a subcutaneous injection every or multiple myeloma who have received frequent, 6 months, denosumab rapidly decreases bone re- high doses of intravenous bisphosphonates. In pa- sorption. In a subgroup analysis of a 12-month, tients with osteoporosis treated with bisphospho- randomized, placebo-controlled trial of denosu nates, the estimated risk of osteonecrosis of the mab in patients receiving methotrexate treatment jaw is 1 case per 10,000 to 100,000 patient-years.43 for rheumatoid arthritis, patients receiving denos Before prescribing bisphosphonates, the clinician umab, prednisone (≤15 mg per day), and metho- should perform an oral examination and encourage trexate had increases in bone mineral density at the patient to be examined by a dentist. Concur- the lumbar spine and total hip that were similar rent use of bisphosphonates and glucocorticoids to those in patients receiving methotrexate and may slightly increase the risk of osteonecrosis of denosumab alone, and the rate of side effects the jaw. Bisphosphonates may also be associated was similar in these groups.49 Denosumab may with atypical subtrochanteric femoral fractures, have a role in treating patients taking glucocor- but if there is an association, the risk is low (about ticoids who have stable serum calcium levels and 2 cases per 10,000 patient-years).44 who are not candidates for bisphosphonate or An alternative to bisphosphonates is teripara- teriparatide therapy because of side effects or a tide, recombinant human parathyroid hormone history of renal insufficiency, although, as with 1-34, which is approved by the FDA for the treat- other agents, more data are needed regarding ment of glucocorticoid-induced osteoporosis. In the effect of denosumab on the risk of fracture. an 18-month, randomized, double-blind trial com- Vertebroplasty and kyphoplasty are sometimes paring teriparatide with alendronate in patients performed to treat painful vertebral fractures, but with glucocorticoid-induced osteoporosis, teripar in controlled trials, these procedures have not been atide increased spinal bone mineral density over found to be superior to sham procedures, and the a shorter period and to a greater extent than did risks include leakage of the cement and an increase alendronate and also reduced vertebral fractures in the risk of additional fractures in patients re- by 90%.45 Daily subcutaneous administration of ceiving glucocorticoids.50 parathyroid hormone prevents the expected glucocorticoid-induced increase in osteoblast and osteo- Areas of Uncertainty cyte apoptosis and decrease in osteoblast number, bone formation, and bone strength.46 However, the More data are needed to predict the risk of frac- effect of teriparatide is somewhat compromised tures among patients taking glucocorticoids and by high-dose glucocorticoid therapy47; a lesser in- to establish clinical thresholds for intervention.2 crease in bone mineral density at the lumbar spine Effective strategies are required to educate physi- has been noted in patients taking more than 15 mg cians about the importance of counseling pa- of prednisone per day, as compared with those tak- tients who are receiving long-term glucocorticoid ing less than 5 mg per day. In addition, host factors therapy regarding the risk of fractures. Additional (e.g., the underlying illness and associated weight studies are needed to determine the minimum dose loss, medications, reduced renal function, and low of glucocorticoids and duration of therapy that levels of insulin-like growth factor I) may con- warrant interventions to prevent fractures and to tribute to the diminished efficacy of teriparatide better understand the ways in which other risk fac- in patients with glucocorticoid-induced osteopo- tors for bone loss should guide decisions regard- rosis, as compared with patients who have other ing therapy. Some clinicians prescribe drugs for forms of osteoporosis.46 Disadvantages of teripar the prevention of fractures for virtually every pa- atide include the cost and the risk of mild hyper- tient who requires glucocorticoid therapy and dis- calcemia (Table 2).31 continue these drugs only when the glucocorti- Another potential treatment option is denos coids are discontinued, but the benefits, risks, and umab, a humanized monoclonal antibody to the cost-effectiveness of this strategy, as compared receptor activator of nuclear factor-κB ligand with a more selective approach to the initiation of

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Table 3. Guidelines for Management of Glucocorticoid-Induced Osteoporosis.*

American College of National Osteoporosis Royal College of Variable Rheumatology24 Foundation25 Physicians of London26 Belgian Bone Club27 Dose and duration of ≥7.5 mg/day for at least ≥5 mg/day for at least Any oral dose for at least ≥9.3 mg/day for at least glucocorticoid 3 months, but patients 3 months 3 months in patients 3 months treatment warrant- at increased risk require ≥65 years of age and ing pharmacologic treatment with any dose those with a prior fra- intervention† or duration gility fracture BMD threshold for Threshold to be based on T score, −2.5, unless T score, −1.5 T score, −1.0 to −1.5 treatment if dose the FRAX algorithm in patient is at high and duration addition to “higher daily risk on the basis of qualify and cumulative dose, a modified FRAX intravenous usage, and model declining BMD” Yearly BMD testing Yes Yes Yes Yes recommended Prevalent vertebral fractures as justifi- cation for pharma- Yes Yes Yes Yes cologic intervention Calcium and vitamin D 1200–1500 mg of calcium 1200 mg of calcium per Only for patients with low For all patients supplementation per day and 800–1000 day and 2000 units calcium intake (<1 g/ units of vitamin D per of vitamin D per day) or vitamin D defi- day for all patients‡ day for all patients‡ ciency (not defined)‡ Pharmacologic inter- Bisphosphonates; teripara- Bisphosphonates; Bisphosphonates as first- Bisphosphonates vention tide reserved for patients teriparatide only for line options, followed at highest risk patients at high risk by teriparatide

* BMD denotes bone mineral density, and FRAX fracture prevention algorithm. † Glucocorticoid doses are given in prednisone equivalents. ‡ The recommended calcium intake refers to the total daily intake (diet and supplements).

pharmacotherapy, have not been assessed. There the rate of vertebral fracture) with antiresorptive is no evidence that medication to prevent fractures therapy in patients taking prolonged courses of is needed with occasional dose-pack prescriptions, glucocorticoids (mostly at doses greater than 10 annual short-term (e.g., 7 to 10 days) high-dose to 20 mg of prednisone per day) serve as the ba- intravenous or oral therapy (<1 g of cumulative sis of the recommendations. However, high-qual- exposure), or replacement therapy for patients with ity data are lacking to determine the precise risk hypopituitarism, adrenal insufficiency, or congen- of fractures associated with doses of prednisone ital adrenal hyperplasia, provided that the replace- that are less than 5 to 7.5 mg daily or with inter- ment doses are not excessive. mittent regimens of glucocorticoids, as well as the appropriate care of patients who are taking Guidelines these regimens.

Guidelines from the American College of Rheuma- Conclusions and tology, the National Osteoporosis Foundation, the Recommendations Royal College of Physicians, and the Belgium Bone Club vary somewhat in their recommendations The woman described in the vignette, who is (Table 3).24-27 The well-recognized early increase slender, has been taking prednisone at a dose of in the risk of fracture associated with the use of 10 mg daily for 3 months, and previously re- glucocorticoids, the lack of certainty with respect ceived higher doses of glucocorticoids, is at con- to a known minimum dose and duration of gluco- siderable risk for glucocorticoid-induced osteo corticoid therapy that does not increase the risk porosis. Other asthma therapies should be used of fracture, and available trials showing increased as efficiently as possible in an effort to taper the bone density (and in some cases reductions in prednisone. The assessment should include a mea-

68 n engl j med 365;1 nejm.org july 7, 2011 The New England Journal of Medicine Downloaded from nejm.org on August 3, 2013. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. clinical practice surement of bone mineral density, and I would also convenience of administration, and the side effects. recommend a vertebral morphologic assessment The more rapid onset of action with zoledronic acid or plain films to look for vertebral fractures. Ad- or teriparatide, as compared with oral bisphospho- equate intake of calcium and vitamin D should be nates, is a potential advantage of these medica- encouraged. Because of her long-term use of gluco- tions, but that advantage must be weighed against corticoids, her age, and the low body-mass index, the greater costs of those drugs and, in the case if prednisone cannot be discontinued (or if she has of teriparatide, the need for daily injections. low bone mineral density or vertebral fractures), Dr. Weinstein reports holding a patent for in vitro and in vivo she should be advised about therapies to reduce models for screening compounds to prevent glucocorticoid-induced bone destruction. No other potential conflict of interest relevant to her risk of fracture; bisphosphonates (alendronate, this article was reported. risedronate, and zoledronic acid) and teriparatide Disclosure forms provided by the author are available with the are approved by the FDA for these indications and full text of this article at NEJM.org. I thank Drs. Stavros C. Manolagas, Charles A. O’Brien, and should be continued for as long as the patient re- Robert L. Jilka for their helpful discussions; Dr. Paula J. Ander- quires prednisone. In the absence of data from son (Department of Pulmonary and Critical Care Medicine, Uni- trials directly comparing the risk of fracture as- versity of Arkansas for Medical Sciences) for help with the case vignette; and Drs. Paul D. Miller, Nelson B. Watts, Robert A. sociated with the various therapies, the choice of Adler, and Henry G. Bone for reviewing an earlier version of the medication should take into account the cost, the manuscript.

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Arthritis Care Res threshold and other predictors of verte- Glucocorticoids act directly on osteoblasts (Hoboken) 2010;62:1515-26. bral fracture in patients receiving oral and osteocytes to induce their apoptosis 25. National osteoporosis foundation’s cli- glucocorticoid therapy. Arthritis Rheum and reduce bone formation and strength. nician’s guide to the prevention and treat- 2003;48:3224-9. Endocrinology 2004;145:1835-41. ment of osteoporosis. Washington, DC: 7. Steinbuch M, Youket TE, Cohen S. 16. Weinstein RS, Wan C, Liu Q, et al. En- National Osteoporosis Foundation, 2009. Oral glucocorticoid use is associated with dogenous glucocorticoids decrease skeletal 26. Bone and Tooth Society of Great Britain. an increased risk of fracture. Osteoporos angiogenesis, vascularity, hydration, and Guidelines on the prevention and treatment Int 2004;15:323-8. strength in aged mice. Aging Cell 2010;9: of glucocorticoid-induced osteoporosis. 8. Tatsuno I, Sugiyama T, Suzuki S, et al. 147-61. London: Royal Collage of Physicians, 2003. Age dependence of early symptomatic verte- 17. Seeman E, Delmas PD. Bone quality 27. Devogelaer J-P, Goemaere S, Boonen S, bral fracture with high-dose glucocorticoid — the material and structural basis of et al. Evidence-based guidelines for the pretreatment for collagen vascular diseases. bone strength and fragility. N Engl J Med vention and treatment of glucocorticoid- J Clin Endocrinol Metab 2009;94:1671-7. 2006;354:2250-61. induced osteoporosis: a consensus docu- 9. Thompson JM, Modin GW, Arnaud 18. Jia D, O’Brien CA, Stewart SA, Mano- ment of the Belgian Bone Club. Osteoporos CD, Lane NE. Not all postmenopausal lagas SC, Weinstein RS. Glucocorticoids Int 2006;17:8-19. women on chronic steroid and estrogen act directly on osteoclasts to increase their 28. Calder JDF, Buttery L, Revell PA, treatment are osteoporotic: predictors of life span and reduce bone density. Endo- Pearse M, Polak JM. Apoptosis — a sig- bone mineral density. Calcif Tissue Int crinology 2006;147:5592-9. nificant cause of bone cell death in osteo- 1997;61:377-81. 19. Weinstein RS, Jia D, Powers CC, et al. necrosis of the femoral head. J Bone Joint 10. Russcher H, Smit P, van den Akker The skeletal effects of glucocorticoid excess Surg Br 2004;86:1209-13.

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29. Weinstein RS, Nicholas RW, Manola- onism of bisphosphonate-induced osteo- Bisphosphonates and fractures of the sub- gas SC. Apoptosis of osteocytes in gluco- clast apoptosis by glucocorticoids. J Clin trochanteric or diaphyseal femur. N Engl J corticoid-induced osteonecrosis of the hip. Invest 2002;109:1041-8. Med 2010;362:1761-71. J Clin Endocrinol Metab 2000;85:2907-12. 37. Liberman UA, Weiss SR, Bröll J, et al. 45. Saag KG, Shane E, Boonen S, et al. 30. Reid DM, Devogelaer J-P, Saag K, et al. Effect of oral alendronate on bone min- Teriparatide or alendronate in glucocorti- Zoledronic acid and risedronate in the eral density and the incidence of fractures coid-induced osteoporosis. N Engl J Med prevention and treatment of glucocorti- in postmenopausal osteoporosis. N Engl J 2007;357:2028-39. coid-induced osteoporosis (HORIZON): a Med 1995;333:1437-43. 46. Weinstein RS, Jilka RJ, Roberson PK, multicentre, double-blind, double-dummy, 38. Orwoll E, Ettinger M, Weiss S, et al. Manolagas SC. Intermittent parathyroid randomised controlled trial. Lancet 2009; Alendronate for the treatment of osteoporo- hormone administration counteracts the 373:1253-63. sis in men. N Engl J Med 2000;343:604-10. adverse effects of glucocorticoids on os- 31. Miller PD. Safety of parathyroid hor- 39. Fleisch H. Bisphosphonates in bone teoblast and osteocyte viability, bone for- mone for the treatment of osteoporosis. disease: from the laboratory to the patient. mation, and strength in mice. Endocri- Curr Osteoporos Rep 2008;6:12-6. 4th ed. San Diego, CA: Academic Press, nology 2010;151:2641-9. 32. Adachi JD, Saag KG, Delmas PD, et al. 2000:42. 47. Devogelaer J-P, Adler RA, Recknor C, Two-year effects of alendronate on bone 40. Emkey R, Delmas PD, Goemaere S, et al. et al. Baseline glucocorticoid dose and mineral density and vertebral fracture in Changes in bone mineral density following bone mineral density response with teripar patients receiving glucocorticoids: a ran- discontinuation or continuation of alendro- atide or alendronate therapy in patients domized, double-blind, placebo-controlled nate therapy in glucocorticoid-treated pa- with glucocorticoid-induced osteoporosis. extension trial. Arthritis Rheum 2001;44: tients: a retrospective, observational study. J Rheumatol 2010;37:141-8. 202-11. Arthritis Rheum 2003;48:1102-8. 48. Cummings SR, San Martin J, Mc- 33. Reid DM, Hughes RA, Laan RF, et al. 41. Lai K-A, Shen W-J, Yang C-Y, Shao C-J, Clung MR, et al. Denosumab for preven- Efficacy and safety of daily risedronate in Hsu J-T, Lin R-M. The use of alendronate tion of fractures in postmenopausal the treatment of corticosteroid-induced to prevent early collapse of the femoral women with osteoporosis. N Engl J Med osteoporosis in men and women: a ran- head in patients with nontraumatic osteo- 2009;361:756-65. [Erratum, N Engl J Med domized trial. J Bone Miner Res 2000;15: necrosisd: a randomized clinical study. 2009;361:1914.] 1006-13. J Bone Joint Surg Am 2005;87:2155-9. 49. Dore RK, Cohen SB, Lane NE, et al. 34. Plotkin LI, Weinstein RS, Parfitt AM, 42. Agarwala S, Shah S, Joshi VR. The use Effects of denosumab on bone mineral Roberson PK, Manolagas SC, Bellido T. of alendronate in the treatment of avascu- density and bone turnover in patients Prevention of osteocyte and osteoblast lar necrosis of the femoral head: follow- with rheumatoid arthritis receiving con- apoptosis by bisphosphonates and calci- up to eight years. J Bone Joint Surg Br current glucocorticoids or bisphospho- tonin. J Clin Invest 1999;104:1363-74. 2009;91:1013-8. nates. Ann Rheum Dis 2010;69:872-5. 35. Weinstein RS, Roberson PK, Manolagas 43. Khosla S, Burr D, Cauley J, et al. Bis 50. Syed MI, Patel NA, Jan S, Shaikh A, SC. Giant osteoclast formation and long- phosphonate-associated osteonecrosis of Grunden B, Morar K. Symptomatic refrac- term oral bisphosphonate therapy. N Engl J the jaw: report of a task force of the Ameri- tures after vertebroplasty in patients with Med 2009;360:53-62. can Society for Bone and Mineral Research. steroid-induced osteoporosis. AJNR Am J 36. Weinstein RS, Chen JR, Powers CC, et al. J Bone Miner Res 2007;22:1479-91. Neuroradiol 2006;27:1938-43. Promotion of osteoclast survival and antag- 44. Black DM, Kelly MP, Genant HK, et al. Copyright © 2011 Massachusetts Medical Society.

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70 n engl j med 365;1 nejm.org july 7, 2011 The New England Journal of Medicine Downloaded from nejm.org on August 3, 2013. For personal use only. No other uses without permission. Copyright © 2011 Massachusetts Medical Society. All rights reserved. The FASEB Journal • Research Communication

Glucocorticoid dose determines osteocyte cell fate

Junjing Jia,*,1 Wei Yao,*,1 Min Guan,* Weiwei Dai,* Mohammad Shahnazari,* Rekha Kar,† Lynda Bonewald,‡ Jean X. Jiang,† and Nancy E. Lane*,2 *Department of Medicine, University of California at Davis Medical Center, Sacramento, California, USA; †Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, USA; and ‡School of Dentistry, Department of Oral Biology, University of Missouri at Kansas City, Kansas City, Missouri, USA

ABSTRACT In response to cellular insult, several chronic GCs will have an osteoporotic fracture; base- pathways can be activated, including necrosis, apo- line data from randomized clinical trials report the ptosis, and autophagy. Because glucocorticoids prevalence in vertebral fracture is nearly 30% (1–4). (GCs) have been shown to induce both osteocyte Patients treated with GCs may require the treatment apoptosis and autophagy, we sought to determine for a long period of time, thereby increasing their whether osteocyte cell fate in the presence of GCs risk of fractures. Clinical studies of GC-treated sub- was dose dependent by performing in vivo and in jects observed that the initiation of GC treatment is vitro studies. Male Swiss-Webster mice were treated associated with a change in bone metabolism. In with slow-release prednisolone pellets at 1.4, 2.8, and turn, this leads to a rapid reduction in bone mass at 5.6 mg/kg/d for 28 d. An osteocyte cell line, MLO-Y4 sites rich in trabecular bone, e.g., the vertebrae and cells, was treated with various doses of dexametha- femur, with incident vertebral fracture risk elevated sone. We found that GC treatments dose depend- within 1 yr of initiation of GC treatment (5–7). ently decreased activation of antioxidant-, au- However, the loss of trabecular mass and architecture tophagy-, and antiapoptosis-focused RT-PCR gene does not explain the increase in fracture risk in pathways in mouse cortical bone. The activation of individuals treated with GCs, because these GC- antioxidant genes was correlated with autophagy treated subjects frequently experience fractures with gene expression after the GC treatments. The pres- higher bone mineral density values than do women ence of osteocyte autophagy, as detected by immu- with postmenopausal osteoporosis (6). Therefore, a nostaining for LC3, increased ϳ50% at the distal more comprehensive understanding of the biology of femur cortical bone region but not at trabecular bone GC-induced bone loss could empower clinicians to region at the 1.4 and 2.8 mg/kg/d GC dose levels. effectively prevent and treat this disease. The number of apoptotic osteocytes was increased at Osteocytes, the most abundant type of cells in ϳ the cortical bone region by 40% initially observed bone, are buried in the bone matrix and are now at the 2.8 mg/kg/d dose level. In addition, the known to contribute bone mineral homeostasis (8). presence of the osteocyte autophagy was associated Osteocytes are connected to one another and to the with an increased protein level of cathepsin K in vitro bone surface. Osteocyte lacunae have been reported after the GC treatments. In summary, we found that to change size in clinical situations when there is a GC treatment dose-dependently decreased antioxi- calcium deficiency, including with lactation, GC dant gene expression, with lower GC doses activating treatment, hypophosphatemic rickets, and pro- autophagy, whereas a higher dose increased apopto- longed estrogen deficiency (9–12). The increase in sis. These data suggest that autophagy may provide a osteocyte lacunae size in these metabolic states may mechanism for osteocytes to survive the stress after occur because of an insufficient trabecular and en- GC exposure and provide further insight into how docortical bone surface area for osteoclasts to reab- GCs alter bone cell fate.—Jia, J., Yao, W., Guan, M., sorb bone and maintain serum calcium balance when Dai, W., Shahnazari, M., Kar, R., Bonewald, L., Jiang, calcium is in high demand (13). For example, in the J. X., Lane, N. E. Glucocorticoid dose determines case of lactation, osteocyte lacunae are enlarged osteocyte cell fate. FASEB J. 25, 3366–3376 (2011). during lactation and return to the normal size post- www.fasebj.org lactation, presumably after the calcium demand from lactation is diminished (10). Osteocytes synthesize ⅐ ⅐ ⅐ Key Words: osteoporosis apoptosis autophagy antioxidant and secrete a number of “osteocytic” specific pro- ⅐ MLO-Y4 cell ⅐ LC3

1 These authors contributed equally to this work. Glucocorticoids (GCs) are frequently used in 2 Correspondence: Department of Medicine, 4800 2nd clinical medicine to treat noninfectious inﬂamma- Ave., Suite 2600, Sacramento, CA 95817, USA. E-mail: tory diseases. Epidemiological studies show that 50% [email protected] of patients with rheumatoid arthritis treated with doi: 10.1096/fj.11-182519

3366 0892-6638/11/0025-3366 © FASEB teins, such as dentin matrix acidic phosphoprotein, maintained on commercial rodent chow (22/5 Rodent Diet; matrix extracellular phosphoglycoprotein, and fibro- Teklad, Madison, WI, USA) with 0.95% calcium and 0.67% blast growth factor 23, that contribute to the regula- phosphate, available ad libitum. Mice were housed in a room that was maintained at 20°C with a 12-h light-dark cycle. The tion of both calcium and phosphorus metabolism mice were randomly assigned to 4 experimental groups of (14–18), and the synthesis of these proteins by the 6–16 animals/group. Slow-release pellets (Innovative Re- osteocyte has been associated with changes in the search of America, Sarasota, FL, USA) of prednisolone perilacunar mineral around the osteocyte. However, (GC) were implanted as follows: group 1, the control it is unclear whether GC would affect osteocyte cell group, was implanted with a placebo pellet (PL); group 2 fates and would alter localized perilacunar mineral- was implanted with a 2.5 mg/60 d slow-release GC pellet, ization changes around the osteocyte and whole which is equivalent to 1.4 mg/kg/d for 28 d; group 3 was implanted witha5mg/60 d slow-release GC pellet, which bone strength. is equivalent to 2.8 mg/kg/d for 28 d; and group 4 was Aging and glucocorticoid treatments are associated implanted with a 10 mg/60 d slow-release GC pellet, which with accumulations of destroyed proteins, damaged is equivalent to 5.6 mg/kg/d for 28 d. At 48 h before the nucleic acids, and accumulated oxygen radicals (19– mice were sacrificed, a fluorescent-conjugated monoclonal 21). Cells rely on autophagy, the only known intracel- antibody for the autophagy marker, LC3 (LC3-5F10; 30 ␮ lular degradative mechanism, to remove the dysfunc- g/mouse; NanoTools, San Diego, CA, USA) was injected tional organelles and/or oxidized proteins (22–24). into 3 mice in each of the 4 experimental groups to label the autophagic osteocytes. All animals were treated accord- The autophagic process is also activated when the cell is ing to the U.S. Department of Agriculture animal care under stress, as an attempt to survive (25, 26). Once the guidelines with the approval of the University of California autophagic process is initiated, parts of the cytoplasm at Davis Committee on Animal Research. and intracellular organelles are sequestered within autophagic vacuoles, which are eventually delivered to Biochemical methods lysosomes for bulk degradation (27). Although the process of autophagy can preserve cell viability as a Serum levels for cortisol (R&D Systems, Minneapolis, MN, survival strategy, it can also lead to a self-destructive USA), osteocalcin (Biomedical Technology, Stoughton, MA, process, resulting in programmed cell death with ex- USA), and cathepsin K (Alpco, Salem, NH, USA) were cessive activation of this self-degrading system (28, 29). measured in duplicate by ELISA, following the manufactur- Although autophagy may prolong cell survival under ers’ instructions. The within-run variations in our laboratory are between 4 and 6%, and between-run variations are ϳ5%, stressful conditions, it is an inefficient process, and over which allow us to determine true changes between treatment time cells accumulate metabolic debris, which results in groups (43–50). a decline in both cell and organ functions (30). Endog- enous GCs, secreted by the adrenal glands, are essential Osteocyte culture and experiments in the body’s ability to respond to stress. GCs are known to impair the enzymatic antioxidant defenses or directly MLO-Y4 cells were cultured on collagen-coated (rat tail induce oxidative stress in various tissues (31–36) and collagen type I, 0.15 mg/ml) surfaces (BD, Franklin Lakes, are associated with cell fate in a number of disease NJ, USA) and were grown in phenol red-free ␣-modified states. In lymphoid malignancies, Laane et al. (37) essential medium (␣-minimal essential medium) supple- reported that dexamethasone (Dex) induced lymphoid mented with 2.5% FBS and 2.5% bovine calf serum (Invitro- cell death through the induction of autophagy before gen, Carlsbad, CA, USA) and incubated in a 5% CO2 incubator at 37°C as described previously. The cells were treated apoptosis. The induction of osteocyte apoptosis was Ϫ with Dex (Sigma-Aldrich Corp., St. Louis, MO, USA) at 10 8 thought to be the primary mechanism for GC-induced to 10Ϫ5 M for 24 h (19, 51). osteoporosis and changes in bone quality (38–42). We For the cell GFP-LC3 transfection experiments, the cells recently reported that prolonged GC treatment in mice were transfected with the GFP-LC3 vector for 48 h. The cells resulted in osteocyte autophagy. Inhibition of au- were then treated with 10Ϫ8 to 10Ϫ6 M doses of Dex for 24 h, tophagy with 3-methyladenine, an inhibitor of endoge- fixed with 4% paraformaldehyde, and examined under an nous protein degradation, led to osteocyte apoptosis Olympus BX61 motorized reflected fluorescence microscope (19, 43). Based on these data, we hypothesized that (Olympus, Tokyo, Japan) with an AMCA filter (excitation, 350 nm; emission, 460 nm) for DAPI and FITC filter (excita- autophagy is one of the pathways in which osteocytes tion, 480 nm; emission, 535 nm) using SlideBook4.1 software respond to GC exposure. The purpose of this investi- (Intelligent Imaging Innovations, Denver, CO, USA). Au- gation was to further characterize the dose-dependent tophagic cells were quantified by counting cells exhibiting effects of GC-induced osteocyte cell fates in vivo and GFP-LC3 punctate staining. in vitro. To evaluate the colocalization of the LC3 and lysosomes, MLY-O4 cells were plated on coverslips in a 12-well plate at 1 ϫ 104 cells/cm2. After 24 h of plating, the cells were transfected with 1 ␮g of GFP-LC3 using 5 ␮l of lipofectamine MATERIALS AND METHODS in optimum I reduced serum medium (Invitrogen). Both GFP-LC3 and lipofectamine were diluted in optimum I reduced serum medium in two separate tubes to a final volume Animals and experimental procedures of 100 ␮l. After 5 min of incubation, GFP-LC3 and lipofectamine were mixed together in one tube and were incu- Three-month-old male Swiss-Webster mice were obtained bated for 15 min at room temperature. Then the mixture of from Charles River, Inc. (San Jose, CA, USA). The mice were DNA-lipofectamine was added to cells dropwise in 1 ml of

GLUCOCORTICOIDS ON OSTEOCYTE AUTOPHAGY 3367 optimum I medium, and the plates were swirled gently and dant, autophagy, or apoptosis pathways, housekeeping genes, incubated in a 37°C incubator. After3hoftransfection, and no primer or cDNA controls. Detailed gene information medium was replaced with full-growth medium. After 24 h of can be found online (http://www.sabiosciences.com/RTPCR. Ϫ8 Ϫ6 Ն transfection, cells were treated with 10 to 10 M Dex in php). We excluded genes that had values of Ct 35 because 1% serum (FBSϩbovine calf serum) phenol red-free medium low expression levels can result in large fold changes, but the for 24 h. Serum starvation was used as a positive control for differences were not signiﬁcant. After the exclusions, we autophagy (52, 53). The cells were then incubated with 1 ␮M reported 80 test genes in the autophagy RT-PCR gene array LysoSensor Blue (Invitrogen) that was added directly to the (see Fig. 2) and genes that were signiﬁcantly different from treatment medium. The medium was removed after 30 min of PL after GC treatments for antioxidant and antiapoptosis incubation with LysoSensor Blue. Cells were washed once (Tables 1 and 2). with fresh medium, and live cell images were taken to visualize the colocalization of GFP-LC3 puncta and the lysosomes. Immunohistochemistry

Real-time RT-PCR The right distal femurs were decalcified in 10% EDTA for 2 wk and embedded in paraffin. Sections (4 ␮m) were prepared for Total RNA was obtained from the tibiae or from MLO-Y4 cell immunohistochemistry using primary antibodies against the cultures. For the tibiae, joint and bone marrow was removed, autophagy protein LC3-phosphatidylethanolamine conjugate and total RNA was isolated using a modified two-step purifi- antibody (LC3B antibody; Cell Signaling Technology, Danvers, cation protocol with homogenization (PRO250 Homoge- MA, USA). LC3 detection was performed using a Vectastain nizer, 10 mmϫ105-mm generator; PRO Scientific Inc., Ox- ABC system (Vector Laboratories, Burlingame, CA, USA). Sec- ford, CT, USA) in TRIzol (Invitrogen) followed by tions were counterstained with methyl green. Apoptosis was purification over an RNeasy column (Qiagen, Valencia, CA, determined using an In Situ Fluorescein Cell Death Detection USA). The antioxidation autophagy and antiapoptosis focus Kit (Roche, Indianapolis, IN, USA) following the manufac- RT-PCR gene pathway arrays were purchased from SABiosci- turer’s instructions. Results are presented as the percentage of ence (Frederick, MD, USA). Each pathway gene array has a the positive stained osteocytes/trabecular or cortical bone vol- preselected panel of 96 genes, which are related to antioxi- ume 0.5 to 3 mm distal to the growth plate at the distal femurs.

TABLE 1. Antioxidant gene expression after GC treatment (fold change from PL)

Symbol Description 1.4 mg 2.8 mg 5.6 mg

Als2 Amyotrophic lateral sclerosis 2 (juvenile) homolog (human) 12.87 8 7.53 Apc Adenomatosis polyposis coli 12.29 4.04 7.11 Apoe Apolipoprotein E 67.28 26.44 29.19 Aqr Aquarius 10.31 8.86 6.58 Cat Catalase 13.32 7.65 5.29 Ctsb Cathepsin B 10.06 8.17 5.76 Cygb Cytoglobin 79.92 26.05 23.79 Fancc Fanconi anemia, complementation group C 12.59 7.14 4.55 Fmo2 Flavin containing monooxygenase 2 84.42 13.15 29.47 Gab1 Growth factor receptor bound protein 2-associated protein 1 23.55 15.79 13.08 Gpx3 Glutathione peroxidase 3 56.67 31.72 21.45 Gpx5 Glutathione peroxidase 5 50.16 42.9 14.53 Hbq1 Hemoglobin, ␪ 1 68.71 67.88 38.25 Ift172 Intraﬂagellar transport 172 homolog (Chlamydomonas) 14.09 10.67 5.79 Il19 Interleukin 19 44.51 17.21 21.33 Il22 Interleukin 22 77.14 37.96 36.59 Kif9 Kinesin family member 9 42.55 40.83 23.56 Lpo Lactoperoxidase 35.09 34.72 27.84 Mb Myoglobin 36.01 29.65 15.8 Noxa1 NADPH oxidase activator 1 33.46 36.66 12.56 Noxo1 NADPH oxidase organizer 1 31.01 16.23 -6.91 Nxn Nucleoredoxin 19.21 9.78 11.74 Park7 Parkinson disease (autosomal recessive, early onset) 7 11.58 8.62 8.41 Prdx1 Peroxiredoxin 1 9.62 3.56 5.98 Prnp Prion protein 9.21 7.12 6.73 Recql4 RecQ protein-like 4 7.69 5.07 4.36 Slc41a3 Solute carrier family 41, member 3 16.12 8.71 8.48 Sod1 Superoxide dismutase 1, soluble 12.59 6.93 9.52 Sod2 Superoxide dismutase 2, mitochondrial 15.07 6.45 6.8 Sod3 Superoxide dismutase 3, extracellular 30.87 23.55 11.67 Tpo Thyroid peroxidase 129.29 59.1 41.94 Txnip Thioredoxin interacting protein 34.52 24 15.81 Txnrd3 Thioredoxin reductase 3 9.39 4.17 3.29 Ucp3 Uncoupling protein 3 (mitochondrial, proton carrier) 42.34 14.71 13.25

All genes expressed signiﬁcantly from PL (PϽ0.05).

3368 Vol. 25 October 2011 The FASEB Journal ⅐ www.fasebj.org JIA ET AL. TABLE 2. Antiapoptotic gene expression after GC treatment (fold change from PL)

Symbol Description 1.4 mg 2.8 mg 5.6 mg

Api5 Apoptosis inhibitor 5 1.37 Ϫ2.33 Ϫ2.15 Bag1 Bcl2-associated athanogene 1 Ϫ1.31 Ϫ1.12 Ϫ3.70 Bag3 Bcl2-associated athanogene 3 1.18 Ϫ1.41 Ϫ1.94 Bcl2 B-cell leukemia/lymphoma 2 1.24 Ϫ1.64 Ϫ3.05 Bcl2l1 Bcl2-like 1 Ϫ1.11 Ϫ1.14 Ϫ3.15 Bcl2l10 Bcl2-like 10 3.79 Ϫ1.60 Ϫ8.97 Bcl2l2 Bcl2-like 2 1.04 Ϫ1.38 Ϫ3.36 Birc2 Baculoviral IAP repeat-containing 2 1.03 Ϫ1.79 Ϫ1.90 Birc3 Baculoviral IAP repeat-containing 3 1.05 1.05 Ϫ1.76 Bnip2 BCL2/adenovirus E1B interacting protein 2 Ϫ1.23 1.11 Ϫ2.18 Bnip3 BCL2/adenovirus E1B interacting protein 3 2.26 Ϫ1.43 Ϫ1.36 Casp2 Caspase 2 Ϫ1.28 Ϫ1.30 Ϫ1.88 Cflar CASP8 and FADD-like apoptosis regulator 3.14 Ϫ1.11 Ϫ3.60 Dad1 Defender against cell death 1 4.22 Ϫ2.59 Ϫ10.18 Tsc22d3 TSC22 domain family, member 3 Ϫ1.16 Ϫ1.50 Ϫ2.27 Fas Fas (TNF receptor superfamily member 6) 3.84 Ϫ2.61 Ϫ2.81 Il10 Interleukin 10 3.53 Ϫ6.28 Ϫ35.84 Lhx4 LIM homeobox protein 4 2.40 Ϫ2.86 Ϫ3.86 Mcl1 Myeloid cell leukemia sequence 1 3.03 Ϫ1.23 1.06 Nfkb1 Nuclear factor of ␬ light polypeptide gene enhancer in B-cells 1, p105 Ϫ1.36 Ϫ1.96 Ϫ5.45 Nme5 Nonmetastatic cells 5, protein expressed in (nucleoside-diphosphate kinase) 5.02 Ϫ2.02 Ϫ5.36 Pak7 P21 (CDKN1A)-activated kinase 7 2.72 Ϫ2.47 Ϫ3.50 Pim2 Proviral integration site 2 2.84 Ϫ1.71 Ϫ5.34 Polb Polymerase (DNA directed), beta 1.02 1.48 Ϫ4.64 Prdx2 Peroxiredoxin 2 Ϫ1.21 1.36 Ϫ3.17 Rnf7 Ring finger protein 7 1.64 Ϫ3.53 Ϫ7.89 Sphk2 Sphingosine kinase 2 Ϫ1.77 1.20 Ϫ19.57 Tnf Tumor necrosis factor 2.45 Ϫ1.12 Ϫ3.93 Cd40lg CD40 ligand 2.02 Ϫ6.15 Ϫ10.40 Zc3hc1 Zinc finger, C3HC type 1 Ϫ1.37 Ϫ1.43 Ϫ2.14

A Bioquant imaging analyzing system (Bioquant, Nashville, TN, RESULTS USA) was used for the measurements. Dose-dependent effects of GC on serum chemistry Western blot values, bone turnover, and strength The cells were lysed in RIPA buffer with homogenization. The bone lysates or cell lysates were resolved on SDS-PAGE The serum cortisol level was not changed at the 1.4 and electrophoretically transferred to polyvinylidene diflu- mg/kg/d GC dose level but increased by Ͼ200% at oride membranes. Membranes were incubated with pri- the 2.8 mg/kg/d dose level and 600% at the 5.6 mary antibodies that included ␤-actin (1:2000; Santa Cruz mg/kg/d dose level after 28 d of treatment. Bone Biotechnology, Santa Cruz, CA, USA), anti-LC3 (1:1000; formation, measured by the serum osteocalcin level Cell Signaling Technology) or anti-cathepsin K (Cell Sig- or surface-based mineralizing surface at the distal naling Technology) followed by species-specific horserad- ϳ ish peroxidase secondary antibody. Anti-LC3 antibody rec- femurs, decreased nonsignificantly by 10–40% at a ognizes both LC3-I, which is cytoplasmic and LC3-II that 1.4 mg/kg/d GC dose level and 40–60% at the two binds to the autophagic membranes. Immunoreactive mate- higher GC dose levels (PϽ0.05). The maximum rials were detected by chemiluminescence (Pierce Laboratories, vertebral compressive stress was significantly lowered Thermo Fisher Scientific, Rockford, IL, USA), imaged with a at the 5.6 mg/kg/d GC dose level compared with that Kodak Gel Logic 100 Digital Imaging System, and quantitated by for PL (Fig. 1). Kodak 1D 3.6 image analysis software (Eastman Kodak, Roches- ter, NY, USA). Excess GC induced an osteocyte autophagy in vivo Statistical analysis To evaluate the dynamic and integrative nature of GCs Group means and sds were calculated for all outcome on osteocyte stress response and cell fate, we obtained variables. The nonparametric Kruskal-Wallis test was used RNA from the long bones (tibiae, nϭ6/group) of mice to determine the differences between the groups. The treated with PL or 3 doses of GCs for 28 d and 2-tailed Spearman correlation test was used to determine the association between the activation (fold changes from performed real time RT-PCR gene arrays for antioxi- PL) for all genes in the antioxidation, autophagy, or dants, autophagy, and antiapoptosis. We found that GC antiapoptosis gene arrays (version 12; SPSS Inc., Chicago, dose-dependently decreased the activation of oxidative IL, USA). stress responsive gene expressions (Table 1). For exam-

GLUCOCORTICOIDS ON OSTEOCYTE AUTOPHAGY 3369 Figure 1. Three-month-old male Swiss-Webster mice were treated with a placebo pellet (PL), or a 2.5 mg/60 d (1.4 mg/kg/d), 5 mg/60 d (2.8 mg/kg/d), or 10 mg/60 d (5.6 mg/kg/d) slow-release prednisolone pellet. A, B) Mice were sacriﬁced at d 28, and serum was collected for cortisol (A) and osteocalcin (B). C, D) Bone histomorphometry was performed on the distal femur; surface-based bone turnover was assessed by mineralizing surface (MS/BS; C), and the sixth lumbar vertebra was tested for compressive strength (max stress; D). n ϭ 6–16/group. aP Ͻ 0.05 vs. PL; bP Ͻ 0.05 vs. GC 1.4 mg; cP Ͻ 0.05 vs. GC 2.8 mg.

ple, the expression of superoxide dismutase (Sod) 1, activation of antioxidant and antiapoptosis genes. In soluble increased ϳ12-fold at the 1.4 mg/kg/d dose addition, there was no significant correlation be- level and increased 6- and 9-fold, respectively, at the 2.8 tween activation of autophagy and antiapoptosis and 5.6 mg/kg/d GC dose levels, Sod2 (mitochondrial) genes (data not shown). increased 15 fold at the 1.4 mg/kg/d dose level and GC treatment at all three doses did not increase the ϩ increased 6-fold at the 2.8 and 5.6 mg/kg/d GC dose percentage of LC3 osteocytes in the trabecular bone levels; and Sod3 (extracellular) increased 30-fold at the region (Fig. 3A). However, GC treatment increased 1.4 mg/kg/d GC dose level and increased 23- and autophagic osteocytes in the cortical bone region of the 11-fold, respectively, at 2.8 and 5.6 mg/kg/d GC dose distal femurs at the 1.4 and 2.8 mg/kg/d (Fig. 3B) levels. GC at the 1.4 mg/kg/d dose level activated a measured by immunohistochemical staining against number of genes that are associated with autophagy by LC3 (Fig. 3C) or by injecting the fluorescent-conju- an average increase of 20- to 30-fold from the PL gated LC3 antibody (LC3-5F10) into the mice (Fig. treatment and by Յ10-fold at the 2.8 mg/kg/d dose 3D). GC exposure did not significantly change osteo- level and was similar to PL (1-fold) at the 5.6 mg/kg/d cyte apoptosis in the trabecular bone region of the dose level (Fig. 2A). Activation of the antioxidant gene distal femur at either dose level but increased apoptotic pathway was positively correlated with activation of the osteocytes significantly in the cortical bone region at autophagic gene array, especially at the 1.4 mg/kg/d the 2.8 and 5.6 mg/kg/d GC dose levels (Fig. 4). GC dose level (Fig. 2B). For example, the expressions of autophagy-related proteins (Atg) 12 and 7, both of GC increased osteocyte autophagy and cathepsin K which are essential for the formation of double-mem- secretion in vitro brane vesicles and autophagosomes, increased 23- and 42-fold, respectively, at the 1.4 mg/kg/d GC dose level and increased 14- and 11-fold, respectively, at the 2.8 To determine the dose-dependent effect of GCs on mg/kg/d GC dose but did not differ from PL at the 5.6 autophagy in vitro, osteocytic MLO-Y4 cells were trans- mg/kg/d GC dose level. In contrast, the expression of fected by GFP-LC3 for 48 h and then were treated with Ϫ Ϫ genes associated with antiapoptosis increased by Յ5- various concentrations of Dex (10 8 to 10 6 M) for fold from PL at the 1.4 mg/kg/d GC group but were 24 h. GFP-LC3 was diffusely distributed in the cyto- significantly decreased by an average of Ϫ1toϪ6-fold plasm in the absence of Dex (control). In contrast, in the 2.8 mg/kg/d GC dose group and Ϫ2toϪ30-fold treatment with Dex increased the number of GFP-LC3 in the 5.6 mg/kg/d GC dose group (Table 2). For puncta, indicating that LC3 was recruited and aggre- example, the expression of B-cell leukemia/lym- gated in the cytoplasm (Fig. 5A). Increased colocaliza- phoma 2 (Bcl2) was not changed at the 1.4 mg/kg/d tion of GFP-LC3 was seen, and lysosomes were observed GC dose level but decreased 1.6-fold at the 2.8 accumulating in the cytoplasm in Dex-treated cells, Ϫ Ϫ mg/kg/d GC dose and decreased 3-fold at the 5.6 especially in 10 7 to 10 6 M Dex (Fig. 5B). Western mg/kg/d GC dose level; the expression of IL-10 blot confirmed the increase in GFP-LC3-II levels after increased 3-fold at the 1.4 mg/kg/d GC dose level Dex treatment at all doses starting from the lowest dose Ϫ Ϫ but decreased 6-fold at the 2.8 mg/kg/d GC dose and (10 8 M), and the maximal response was seen at 10 7 decreased 35-fold at the 5.6 mg/kg/d GC dose level. M Dex (Fig. 5C, D). The enzyme for matrix metabolism, There was no significant correlation between the cathepsin K, was increased in the MLO-Y4 cells (Fig. 5C,

3370 Vol. 25 October 2011 The FASEB Journal ⅐ www.fasebj.org JIA ET AL. Figure 2. RNA was extracted from the tibial shafts of PL- or GC-treated mice at d 28. RT-PCR gene arrays for antioxidants and autophagy were performed. A) RT-PCR data are expressed as fold changes from the PL-treated mice. Approximately 70% of the autophagic genes expressed were signiﬁcantly different from the those for PL. B) Correlation of fold changes from placebo between antioxidant and autophagy gene expression was performed on the same samples for all three GC doses or at the 1.4 mg/kg/d dose level. n ϭ 6/group.

D) treated with Dex, as well as in the culture medium, ent from placebo treatment. A decreased antiapoptosis Ϫ which was most significant at the 10 7 M dose level response and osteocyte apoptosis were observed at the (Fig. 5E). Interestingly, when the MLO-Y4 cells were cortical bone region after the higher-dose GC treat- Ϫ Ϫ treated with Dex (10 8 to 10 5 M) and evaluated for ment. the antioxidant gene expressions, we did not find that Weinstein et al. (38, 42) reported that reduced bone Dex treatment in vitro activated the antioxidant path- formation in GC-treated mice was associated with in- way significantly (data not shown). creased apoptosis of osteoblasts and osteocytes. Plotkin et al. (54) also reported in vitro evidence of apoptosis of osteocytes exposed to GCs using 3 separate assays DISCUSSION (trypan blue exclusion, nuclear morphology, and an- After 28 d of low-dose GC treatment in mice, there was nexin V/propidium iodine ratios by FACS analysis) to significant activation of the autophagy pathway and accurately confirm that there was osteocyte apoptosis increased osteocyte autophagy in the cortical bone present (54). However, these investigations were per- region of the distal femur that were significantly differ- formed before reagents were available to assess the

GLUCOCORTICOIDS ON OSTEOCYTE AUTOPHAGY 3371 Figure 3. Distal femurs from PL- or GC-treated mice were embedded in paraffin at d 28. A, B) Numbers of LC3ϩ osteocytes present in a defined trabecular bone area (A) or cortical bone area (B) were calculated. C) Immunohistochemical staining was performed using an anti-LC3 antibody (LC3ϩ cells were stained in red; red arrows). D) Autophagic osteocytes were also quantitated by injecting fluorescent-conjugated monoclonal antibody for LC3 (LC3–5F10; green arrows) into mice at 48 h before sacrifice. n ϭ 8/group, 3 sections/animal were analyzed. aP Ͻ 0.05 vs. PL. presence of autophagy. Our study evaluated the dose investigators also demonstrated that the increased os- response of osteocytes to GCs and found that osteocyte teocyte apoptosis was significantly associated with a apoptosis increased after a higher dose of GC at reduction in whole bone strength. the cortical bone region. Our results (43) are similar to In additional to apoptosis, we found that autophagic those reported by Weinstein et al. (38, 42), who also osteocytes were observed in the cortical bone region of reported that in male mice treated with higher doses of the lumbar vertebral bodies in mice that had received GCs nearly 20% of the osteocytes at the cortical bone 56 d of GC treatment (19). The autophagy pathway is region of the tibiae had undergone apoptosis. These one of the most important biological processes that

Figure 4. Presence of apoptosis in the trabecular and cortical bone of the right distal femurs in mice treated with PL or GC. Ap- optosis was determined by in situ ﬂuorescence TUNEL staining. Apoptotic osteocytes (A, yellow arrows) in both the trabecular bone (B) and cortical bone (C) regions were measured. n ϭ 8/group. aP Ͻ 0.05 vs. PL.

3372 Vol. 25 October 2011 The FASEB Journal ⅐ www.fasebj.org JIA ET AL. Figure 5. In vitro assessment of autophagy in ML0-Y4 cells treated with GCs. A) MLO-Y4 cells were cultured on collagen-coated plates and grown in phenol red-free ␣-modiﬁed essential medium supplemented with 2.5% FBS and 2.5% bovine calf serum. Cells were transfected with GFP-LC3 for 48 h before they were treated with Dex at 10Ϫ8 to 10Ϫ6 M and LysoSensor for 24 h. A few autophagosomes (GFP-LC3ϩ dots, yellow arrows) were seen in the cytoplasm in the control (Con) cells. Treatment with Dex increased in GFP-LC3ϩ vacuoles (ϫ100 original view for all images). B) GFP-LC3ϩ vacuoles (stained in green) and lysosomes (stained in blue) were increased in 10Ϫ7 to 10Ϫ6 M Dex dose levels. C) Western blots of LC3 and cathepsin K (CtsK) contents in MLO-Y4 cells treated with DEX, as indicated, for 24 h. D) Arbitrary units of Western blots in C for LC3-II/actin, LC3-II/LC3-I, and cathepsin K (CatK)/actin. LC3-I protein level was not changed by Dex treatment, whereas LC3-II protein level was increased after treatment with 10Ϫ8–10Ϫ6 M Dex. Maximum increase was observed with 10Ϫ7 and 10Ϫ6 M Dex. Cathepsin K levels were also increased after Dex treatment, measured by Western blot in cell lysates. E) LC3ϩ cell numbers. Percentage of LC3ϩ cells was measured by ELISA in the culture medium. F) Cathepsin K levels measured by ELISA in the culture medium. Cathepsin K levels were increased after Dex treatment. Each experiment was repeated 4 times. aP Ͻ 0.05 vs. control; bP Ͻ 0.05 vs. 10Ϫ8 M Dex. enables the cells to survive stress and starvation and 63–65). GC treatment in vitro did not activate the helps to maintain cellular homeostasis by degrading antioxidant pathway but signiﬁcantly increased the damaged organelles (22, 25, 28, 55). The hallmark of overall stress level in vivo in mice by increasing autophagy is the formation of autophagosomes, also the systemic circulating cortisol levels. On the basis of known as autophagic vacuoles that are lined by two our data, it appears that antioxidative responses were membranes with the recruitment of LC3-II to the provoked by GC-induced stress, and the osteocytes may autophagosomal membranes, a characteristic for au- have responded with autophagy. An increased LC3-II tophagosomes, whereas LC3-I remains in the cytoplasm protein level and autophagic osteocytes were observed (56). Aging is associated with an increase in the intra- primarily in the cortical bone regions. This autophagic cellular overabundance of oxidative products, includ- attempt to “survive” the insult from the GC exposure ing reactive oxygen species (e.g., oxygen ions and free may be successful if the dose is low. However, with the radicals) and increased defense mechanisms for oxida- increasing doses of GCs, the cells’ antioxidant defenses tive stress such as glutathione (GSH), thioredoxin, and were overwhelmed, and the ability of the osteocyte to GSH peroxidase, which convert the peroxides to harm- survive was reduced, which could then direct the cells less materials, and the expressions of superoxide dis- to apoptosis. Other investigators have suggested that mutase, the antioxidant enzymes in bone tissues (57– osteocyte cell fate may be related to the dose of GC (66, 62). Likewise, GCs also activate the oxidative pathway 67). Our data clearly demonstrate the dynamics of the and accelerate the aging process in bone tissue (34, 36, osteocyte cell fate with different GC doses within the

GLUCOCORTICOIDS ON OSTEOCYTE AUTOPHAGY 3373 cortical bone. The lack of osteocyte response in the ventions directed to alter autophagy or the inhibition trabecular bone regions may due to the fact that of cathepsin K may be a useful approach to reduce trabecular and cortical bone have different remodeling GC-induced bone fragility. rates, and these two bone compartments have different responses to various stimuli, such as immobilization The authors thank Dr. Frank Chuang (Center for Biopho- and exercise (68–72). The life spans of trabecular bone tonics and Science Technology, University of California at osteocytes and cortical bone osteocytes are also differ- Davis Medical Center) for generously providing GFP-tagged LC3 expression vector and photographing the cells. This ent: cortical bone osteocytes live significantly longer, work was funded by U.S. National Institutes of Health grants and this may help to explain the differing response to 1K12-HD05195801 (cofunded by the National Institute of GC treatment (73). The activation of the autophagic Child Health and Human Development, the Office of Re- gene pathway and osteocyte autophagy was significantly search on Women’s Health, the Office of Dietary Supple- increased when the cells or mice were treated with a low ments, and the National Institute of Aging), R01-AR043052- dose of GC. The higher doses of GC activated the gene 07, K24-AR048841, 5R21-AR57515-2, and P01-AR46798 (to L F.B. and J.X.J), and Welch Foundation grant AQ-1507 (to pathway for apoptosis and osteocyte apoptosis was then J.X.J.). significantly increased. During the initial autophagic process, cells may be able to remain viable during periods of metabolic stress (26, 74, 75). 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