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Proteasome inhibitors (PI), a novel class of anticancer drugs, are relatively well tolerated and have recently been introduced into the clinic for the treatment of multiple myeloma. The tumor selectivity and low toxicity of PIs are surprising, given the crucial role of the ubiquitin/proteasome system in a multitude of cellular processes. Here, we show that systemic administration of PIs specifically impairs the ubiquitin/proteasome system in growth plate chondrocytes. Importantly, young mice displayed severe growth retardation during treatment as well as 45 days after the cessation of treatment with clinically relevant amounts of MG262 (0.2 μmol/kg body weight/injection) or bortezomib (1.0 mg/kg body weight/injection). Dysfunction of the ubiquitin/proteasome system was accompanied by the induction of apoptosis of stem-like and proliferative chondrocytes in the growth plate. These results were recapitulated in cultured fetal rat metatarsal bones and chondrocytic cell lines (rat, human). Apoptosis was associated with up-regulation of the proapoptotic molecules, p53 and apoptosis-inducing factor (AIF), both in vitro and in vivo. In addition to the observation that AIF is expressed in the growth plate, we also provide evidence that AIF serves as a direct target protein for ubiquitin, thus explaining its prominent up-regulation upon proteasome inhibition. Suppression of p53 or AIF expression with small interfering RNAs partly rescued chondrocytes from proteasome inhibition–induced apoptosis (35% and 41%, respectively). Our observations show that proteasome inhibition may selectively target essential cell populations in the growth plate causing significant growth failure. These findings could have important implications for the use of proteasome inhibitors in the treatment of childhood cancer. [Cancer Res 2007;67(20):10078–86]

Supplementary Figure Legends 1-7 from Proteasome Inhibition Up-regulates p53 and Apoptosis-Inducing Factor in Chondrocytes Causing Severe Growth Retardation in Mice

Supplementary Figures 1-7 from Proteasome Inhibition Up-regulates p53 and Apoptosis-Inducing Factor in Chondrocytes Causing Severe Growth Retardation in Mice

The drug delivery system (DDS) often causes toxicity, triggering undesired cellular injuries. Thus, developing supramolecules used as DDS with tunable self-assembly and nontoxic behavior is highly desired. To address this, we aimed to develop a tunable amphiphilic ABA-type triblock copolymer that is nontoxic to human blood cells but also capable of self-assembling, binding and releasing the clinically used drug dexamethasone. We synthesized an ABA-type amphiphilic triblock copolymer (P2L) by incorporating tetra(aniline) TANI as a hydrophobic and redox active segment along with monomethoxy end-capped polyethylene glycol (mPEG2k; Mw = 2000 g mol−1) as biocompatible, flexible and hydrophilic part. Cell cytotoxicity was measured in whole human blood in vitro and lung cancer cells. Polymer-drug interactions were investigated by UV–Vis spectroscopy and computational analysis. Our synthesized copolymer P2L exhibited tuned self-assembly behavior with and without external stimuli and showed no toxicity in human blood samples. Computational analysis showed that P2L can encapsulate the clinically used drug dexamethasone and that drug uptake or release can also be triggered under oxidation or low pH conditions. In conclusion, copolymer P2L is nontoxic to human blood cells with the potential to carry and release anticancer/anti-inflammatory drug dexamethasone. These findings may open up further investigations into implantable drug delivery systems/devices with precise drug administration and controlled release at specific locations.

BACKGROUND Substantial evidence exists to show the positive effects of radialextracorporeal shock wave therapy (ESWT) on bone formation. However, it is unknown whether rESWT can act locally at the growth plate level to stimulate linear bone growth. One way to achieve this is to stimulate chondrogenesis in the growth plate without depending on circulating systemic growth factors. We wished to see whether rESWT would stimulate metatarsal rat growth plates in the absence of vascularity and associated systemic growth factors. QUESTIONS/PURPOSES To study the direct effects of rESWT on growth plate chondrogenesis, we asked: (1) Does rESWT stimulate longitudinal bone growth of ex vivo cultured bones? (2) Does rESWT cause any morphological changes in the growth plate? (3) Does rESWT locally activate proteins specific to growth plate chondrogenesis? METHODS Metatarsal bones from rat fetuses were untreated (controls: n = 15) or exposed to a single application of rESWT at a low dose (500 impulses, 5 Hz, 90 mJ; n = 15), mid-dose (500 impulses, 5 Hz, 120 mJ; n = 14) or high dose (500 impulses, 10 Hz, 180 mJ; n = 34) and cultured for 14 days. Bone lengths were measured on Days 0, 4, 7, and 14. After 14 days of culturing, growth plate morphology was assessed with a histomorphometric analysis in which hypertrophic cell size (> 7 µm) and hypertrophic zone height were measured (n = 6 bones each). Immunostaining for specific regulatory proteins involved in chondrogenesis and corresponding staining were quantitated digitally by a single observer using the automated threshold method in ImageJ software (n = 6 bones per group). A p value < 0.05 indicated a significant difference. RESULTS The bone length in the high-dose rESWT group was increased compared with that in untreated controls (4.46 mm ± 0.75 mm; 95% confidence interval, 3.28-3.71 and control: 3.50 mm ± 0.38 mm; 95% CI, 4.19-4.72; p = 0.01). Mechanistic studies of the growth plate's cartilage revealed that high-dose rESWT increased the number of proliferative chondrocytes compared with untreated control bones (1363 ± 393 immunopositive cells per bone and 500 ± 413 immunopositive cells per bone, respectively; p = 0.04) and increased the diameter of hypertrophic chondrocytes (18 ± 3 µm and 13 ± 3 µm, respectively; p < 0.001). This was accompanied by activation of insulin-like growth factor-1 (1015 ± 322 immunopositive cells per bone and 270 ± 121 immunopositive cells per bone, respectively; p = 0.043) and nuclear factor-kappa beta signaling (1029 ± 262 immunopositive cells per bone and 350 ± 60 immunopositive cells per bone, respectively; p = 0.01) and increased levels of the anti-apoptotic proteins B-cell lymphoma 2 (718 ± 86 immunopositive cells per bone and 35 ± 11 immunopositive cells per bone, respectively; p < 0.001) and B-cell lymphoma-extra-large (107 ± 7 immunopositive cells per bone and 34 ± 6 immunopositive cells per bone, respectively; p < 0.001). CONCLUSION In a model of cultured fetal rat metatarsals, rESWT increased longitudinal bone growth by locally inducing chondrogenesis. To verify whether rESWT can also stimulate bone growth in the presence of systemic circulatory factors, further studies are needed. CLINICAL RELEVANCE This preclinical proof-of-concept study shows that high-dose rESWT can stimulate longitudinal bone growth and growth plate chondrogenesis in cultured fetal rat metatarsal bones. A confirmatory in vivo study in skeletally immature animals must be performed before any clinical studies.

Objective The process of longitudinal bone growth occurs at the growth plate where the chondrocytes undergo apparent structural and molecular changes to promote growth. Recent reports suggest that radial shockwave treatment (rSWT) stimulates bone length in cultured fetal rat metatarsals. Therefore, we investigated if rSWT has similar growth promoting effects on cultured human growth plate fragments and addressed the same in a preclinical in vivo rabbit model by subjecting their growth plates to rSWT. Methods Short-term effects of high-energy rSWT were evaluated in a unique model of cultured human growth plate cartilage (n = 5) wherein samples exposed to rSWT were assessed for chondrogenic markers at 24 h in comparison to unexposed samples obtained from the same limb. Local in vivo effects were studied in six-week-old rabbits who had their distal femurs exposed to four weekly sessions of rSWT at low- and high-energy levels (n = 4 each). At sacrifice, histomorphometric and immunohistochemistry analyses were performed. For effect on longitudinal growth, proximal tibiae of 22-week-old rabbits (n = 12) were asymmetrically exposed to rSWT; the contralateral side served as untreated controls. At sacrifice, the final bone length was measured. Results In the ex vivo model of cultured human growth plate cartilage, rSWT exposure upregulated SOX9 and COL2A1 compared to control. In the immature rabbit model, an increased number of proliferative chondrocytes and column density was seen for both the energy levels. In the adolescent rabbits, an increase in tibial length was observed after the fourth session of high-energy rSWT and until six-weeks after rSWT compared to the untreated limb. Conclusions Our preliminary experimental results suggest that rSWT may serve as a non-invasive treatment and possibly a safe strategy to stimulate longitudinal bone growth. However, further studies are needed to assess the in vivo effects of rSWT in models of disturbed bone growth.

In childhood medulloblastoma patients, the hedgehog antagonist vismodegib is an effective anti-cancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients. We hypothesized that radial shock wave treatment (rSWT) may protect drug-induced growth impairment owing to its osteogenic effects. Fetal rat metatarsal bones were exposed to vismodegib (day 0–5; 100 nM) and/or rSWT (single session); other bones from day 1 were continuously exposed to a Gli1 antagonist (GANT61; 10 µM) and/or rSWT (single session). Control bones were untreated. The bone length was measured at intervals; histomorphometric analysis and immunostaining for PCNA, Gli1, and Ihh were performed on the sectioned bones. Bones treated with vismodegib showed impaired bone growth, reduced height of the resting-proliferative zone and reduced hypertrophic cell size compared to control. In vismodegib treated bones, a single session of rSWT partially rescued bone growth, increased the growth velocity, hypertrophic cell size, and restored growth plate morphology. Bones exposed to GANT61 showed impaired bone growth and disorganized growth plate while when combined with rSWT these effects were partially prevented. Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegib.

Glucocorticoids (GCs) are frequently used to treat chronic disorders in children, including inflammation and cancer. Prolonged treatment with GCs is well known to impair bone growth, an effect linked to increased apoptosis and suppressed proliferation in growth plate chondrocytes. We hypothesized that the endogenous antiapoptotic protein humanin (HN) may prevent these effects. Interestingly, GC-induced bone growth impairment and chondrocyte apoptosis was prevented in HN overexpressing mice, HN-treated wild-type mice, and in HN-treated cultured rat metatarsal bones. GC-induced suppression of chondrocyte proliferation was also prevented by HN. Furthermore, GC treatment reduced Indian Hedgehog expression in growth plates of wild-type mice but not in HN overexpressing mice or HN-treated wild-type animals. A Hedgehog (Hh) antagonist, vismodegib, was found to suppress the growth of cultured rat metatarsal bones, and this effect was also prevented by HN. Importantly, HN did not interfere with the desired anti-inflammatory effects of GCs. We conclude that HN is a novel regulator of Hh signaling preventing GC-induced bone growth impairment without interfering with desired effects of GCs. Our data may open for clinical studies exploring a new possible strategy to prevent GC-induced bone growth impairment by cotreating with HN.-Zaman, F., Zhao, Y., Celvin, B., Mehta, H. H., Wan, J., Chrysis, D., Ohlsson, C., Fadeel, B., Cohen, P., Savendahl, L. Humanin is a novel regulator of Hedgehog signaling and prevents glucocorticoid-induced bone growth impairment.

Glucocorticoids (GCs) are widely used in the treatment of childhood acute lymphoblastic leukemia (ALL), but their long-term use is also associated with bone-related morbidities. Among others, growth deficit, decreased bone mineral density (BMD) and increased fracture rate are well-documented and severely impact quality of life. Unfortunately, no efficient treatment for the management of bone health impairment in patients and survivors is currently available. The overall goal of this review is to discuss the existing data on how GCs impair bone health in pediatric ALL and attempts made to minimize these side effects.

Prolonged use of glucocorticoids (GCs) for treatment of inflammatory and autoimmune conditions may have several negative side effects, such as impaired bone growth which has been linked to increased apoptosis in growth plate chondrocytes. It has recently been shown that humanin, a small mitochondrial derived peptide, rescues growth plate chondrocytes from GC-induced apoptosis. Our aim was to study if a synthetic analogue of humanin, [Gly14]-HNG (HNG), can be safely used to prevent GC-induced toxicity in growth plate chondrocytes without interfering with the desired anti-inflammatory effect in an in vivo model of arthritis.Arthritis was induced in DBA/1 mice by collagen type II in complete Freund's adjuvant and the animals were treated with Dexamethasone (Dexa) (0.25 mg/kg/day) with or without HNG (100 μg/kg/day) for 14 days. The animals were observed daily for the presence of arthritis including signs of erythema and swelling of the joints. The paws were scored based on the severity of the swelling. After termination, histological scoring was performed of all paws. Chondrocyte apoptosis and proliferation were analysed by TUNEL assay and PCNA staining, respectively.We found that HNG treatment in combination with Dexa protected from Dexa-induced chondrocyte apoptosis in both articular and growth plate cartilage. Furthermore, based on clinical and histology scoring analyses, HNG did not interfere with the desired anti-inflammatory effect of Dexa.Our results suggest that the combination of HNG and GCs may provide a new treatment strategy in conditions of chronic inflammation, which could potentially prevent bone growth impairment.

Under different pathological conditions, high levels of reactive oxygen species (ROS) cause substantial damage to multiple organs. To counter these ROS levels in multiple organs, we have engineered highly potent novel terpolymers. We found that combination of FDA-approved polyethylene glycol, fumaric acid moieties and electroactive tetra(aniline) by varying the content of tetra(aniline) results into a novel drug composition with biologically active and tunable intrinsic antioxidant properties. To test the intrinsic antioxidative properties of these novel terpolymers, we used alloxan to induce diabetes in rats where ROS generation is known to be higher. The systemic administration of terpolymers to the diabetic rats showed strong electroactive antioxidant behavior which not only normalized ROS levels, but also improved the levels of enzymatic antioxidants including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). As a proof-of-principle, we here show TANI based novel drug composition of terpolymers with tunable intrinsic antioxidant properties in multiple organs.

Glucocorticoids (GCs) are effective for the treatment of many chronic conditions, but their use is associated with frequent and wide-ranging adverse effects including osteoporosis and growth retardation. The mechanisms that underlie the undesirable effects of GCs on skeletal development are unclear, and there is no proven effective treatment to combat them. An in vivo model that investigates the development and progression of GC-induced changes in bone is, therefore, important and a well-characterized pre-clinical model is vital for the evaluation of new interventions. Currently, there is no established animal model to investigate GC effects on skeletal development and there are pros and cons to consider with the different protocols used to induce osteoporosis and growth retardation. This review will summarize the literature and highlight the models and techniques employed in experimental studies to date.

Proinsulin C-peptide is internalized into cells, but a function of its intracellular localization has not been established. We now demonstrate that, upon cellular entry, C-peptide is localized to the nucleoli, where it promotes transcription of genes encoding for ribosomal RNA. We find that C-peptide binds to histones and enhances acetylation of lysine residue 16 of histone H4 at the promoter region of genes for ribosomal RNA. In agreement with synchrony of ribosomal RNA synthesis and cell proliferation, we show that C-peptide stimulates proliferation in chondrocytes and HEK-293 cells. This regulation of ribosomal RNA provides a mechanism by which C-peptide can exert transcriptional effects and implies that the peptide has growth factor activity.

Glucocorticoids (GCs) are widely used to treat inflammatory diseases and cancers. A multitude of undesired side effects have been reported in GC-treated patients including decreased linear bone growth. We have previously reported that GCs activate the caspase cascade and trigger Bax-mediated mitochondrial apoptosis in growth plate chondrocytes causing growth retardation in young mice. To further explore the role of mitochondrial apoptosis in GC-induced bone growth retardation, a number of pro- and anti-apoptotic proteins were studied in ex vivo cultures of human growth plate cartilage and human HCS-2/8 proliferative chondrocytes exposed to dexamethasone. Dexamethasone was found to increase the pro-apoptotic proteins Bcl-xS, Bad, and Bak as well as the proteolysis of Bid. Anti-Bid small interfering RNA partially rescued the chondrocytes from dexamethasone-induced apoptosis. Taken together, our data suggest that GC treatment differentially regulates Bcl-2 family member proteins to facilitate mitochondrial apoptosis in proliferative chondrocytes thereby contributing to GC-induced bone growth impairment. Prevention of this imbalance between pro- and anti-apoptotic Bcl-2 family proteins may provide a new strategy to protect from adverse effects of GCs on bone growth.

The tyrosine kinase receptor c-kit and its interaction with the ligand, stem cell factor (SCF), play an essential role in the developing testis. C-kit is important for the development of the Leydig cells and for the migration, proliferation and survival of spermatogonia. Platelet-derived growth factor (PDGF) and its tyrosine kinase receptor (PDGFR) are important for the development of Leydig cells and myoid cells. The chemotherapeutic agent, imatinib mesylate (STI571, Glivec; Novartis) inhibits both of these tyrosine kinase receptors. Three-day treatment of immature male rats (SD) with imatinib (150 mg/kg) on postnatal days 5-7 delayed the formation of germ-line stem cell pool, reduced proliferation of type A spermatogonia and induced germ cell apoptosis. PDGFR-mediated proliferation of mesenchymal myoid precursors was also decreased and the length of the seminiferous cord was reduced. However, at the age of 11 weeks the exposed animals had normal epididymal sperm counts, whereas plasma levels of luteinizing hormone and follicle stimulating hormone were significantly increased. Imatinib serves as a good tool to study postnatal formation of the male germ-line stem cell pool and factors determining the final testicular size. As development of the human testis is controlled by the same mechanisms, further studies with primate and human models are needed to explore whether imatinib affects the testis in children as well.

Estrogen affects skeletal growth and promotes growth plate fusion in humans. High doses of estrogen have been used to limit growth in girls with predicted extreme tall stature; a treatment which has been associated with severe side effects. Selective estrogen receptor modulators (SERMs) could potentially be used as an alternative treatment. We chose to study the effects of Tamoxifen (Tam), a first generation SERM that has been used in the treatment of pubertal gynecomastia or McCune-Albright syndrome. Cultured fetal rat metatarsal bones were used to study the effects of Tam on longitudinal bone growth. In sectioned bones, chondrocyte apoptosis and proliferation were analyzed by TUNEL assay and BrdU incorporation, respectively. We also used a human chondrocytic cell line, HSC-2/8, to study the effects of Tam on apoptosis (FACS analysis and Cell Death detection ELISA) and caspase activation (caspase substrate cleavage and Western immunoblotting). Tam caused a dose-dependent growth retardation of cultured metatarsal bones. No catch-up growth was observed after Tam was removed from the culture medium. Detailed analysis of sectioned growth plate cartilage revealed increased apoptosis of chondrocytes within the resting and hypertrophic zones. HCS-2/8 cells also underwent apoptosis upon Tam treatment. Tam-induced apoptosis was caspase-dependent and completely abrogated by either caspase-8 or -9 inhibitors. A substrate assay revealed that caspase-8 is first activated followed by caspase-9 and -3. Finally, FasL secretion was stimulated by Tam and blocking of either FasL or Fas decreased Tam-induced apoptosis in chondrocytes. We here describe a novel mechanism of tamoxifen-induced apoptosis in chondrocytes, involving the activation of caspases and the FasL/Fas pathway, which diminishes the potential for bone growth.

The present investigation examines the influence of IGF-I and the role of IGF-I receptor (IGF-IR) in the apoptosis/survival of Leydig cells. Immunohistochemical analysis of the rat testis at different ages revealed that the level of the phosphorylated IGF-IR increases from birth to d 20 of postnatal life, remaining high in the adult testis. Western blotting revealed that this level is higher in Leydig cells isolated from 40-d-old than from 10- or 60-d-old rats. Application of the terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay revealed that IGF-I decreases the level of apoptosis in Leydig cells at all stages of development, and the selective inhibitor of IGF-IR, picropodophyllin, blocks this antiapoptotic effect. The mechanism underlying the antiapoptotic action of IGF-I involves the phosphatidylinositol 3-kinase/Akt pathway, and in immature Leydig cells, this growth factor enhances the expression of Bcl-2 and cellular inhibitor of apoptosis proteins 2, while preventing activation of caspase-3 by cleavage. Furthermore, IGF-II and high concentrations of insulin also evoke phosphorylation of IGF-IR and, like IGF-I, enhance the expression of the steroidogenic acute regulatory protein by Leydig cells. Inhibition of IGF-IR by picropodophyllin decreases the survival of Leydig cells, both in the presence and absence of IGF-I, demonstrating that signaling via the IGF-IR plays an important role in Leydig cell survival.

The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need.

Children with inflammatory diseases usually display abnormal growth patterns as well as delayed puberty. This is a result of several factors related to the disease itself, such as malnutrition, hypercortisolism, and elevated levels of pro-inflammatory cytokines. These factors in combination with glucocorticoid treatment contribute to growth retardation during chronic inflammation by systemically affecting the major regulator of growth, the GH/IGF1 axis. However, recent studies have also shown evidence of a direct effect of these factors at the growth plate level. In conditions of chronic inflammation, pro-inflammatory cytokines are upregulated and released into the circulation. The most abundant of these, tumor necrosis factor α, interleukin 1β (IL1β), and IL6, are all known to directly act on growth plate cartilage to induce apoptosis and thereby suppress bone growth. Both clinical and experimental studies have shown that growth retardation can partly be rescued when these cytokines are blocked. Therefore, therapy modulating the local actions of these cytokines may be effective for preventing growth failure in patients with chronic inflammatory disorders. In this review, we report the current knowledge of inflammatory cytokines and their role in regulating bone growth.

Bortezomib, a novel proteasome inhibitor approved for the treatment of cancer in adults, has recently been introduced in pediatric clinical trials. Any tissue-specific side effects on bone development have to our knowledge not yet been explored. To address this, we experimentally studied the effects of bortezomib in vivo in young mice and in vitro in organ cultures of rat metatarsal bones and human growth plate cartilage, as well as in a rat chondrocytic cell line. We found that bortezomib while efficiently blocking the ubiquitin/proteasome system (UPS) caused significant growth impairment in mice, by increasing resting/stem-like chondrocyte apoptosis. Our data support a local action of bortezomib, directly targeting growth plate chondrocytes leading to decreased bone growth since no suppression of serum levels of insulin-like growth factor-I (IGF-I) was observed. A local effect of bortezomib was confirmed in cultured rat metatarsal bones where bortezomib efficiently caused growth retardation in a dose dependent and irreversible manner, an effect linked to increased chondrocyte apoptosis, mainly of resting/stem-like chondrocytes. The cytotoxicity of bortezomib was also evaluated in a unique model of cultured human growth plate cartilage, which was found to be highly sensitive to bortezomib. Mechanistic studies of apoptotic pathways indicated that bortezomib induced activation of p53 and Bax, as well as cleavage of caspases and poly-ADP-ribose polymerase (PARP) in exposed chondrocytes. Our observations, confirmed in vivo and in vitro, suggest that bone growth could potentially be suppressed in children treated with bortezomib. We therefore propose that longitudinal bone growth should be closely monitored in ongoing clinical pediatric trials of this promising anti-cancer drug.

Although glucocorticoids are known to induce apoptosis in chondrocytes, the mechanisms for this effect and the potential antiapoptotic role of IGF-I are unknown. To address this, we studied the effects of dexamethasone (Dexa) on apoptosis in the HCS-2/8 chondrocytic cell line. Dexa (25 μm) increased apoptosis (cell death ELISA) by 39% and 45% after 48 and 72 h, respectively (P < 0.01 and P < 0.05, respectively). IGF-I (100 ng/ml) decreased Dexa-induced apoptosis to levels similar to control cells. Apoptosis was associated with cleavage of poly-ADP-ribose polymerase (PARP) and α-fodrin and activation of caspases-8, -9, and -3 (Western), an effect that was counteracted when chondrocytes were cocultured with Dexa + IGF-I. Inhibitors for caspases-8, -9, and -3 (50 μm each) equally suppressed Dexa-induced apoptosis (P < 0.01). Time-response experiments showed that caspase-8 was activated earlier (at 12 h) than caspase-9 (at 36 h). We studied the phosphatidylinositol 3′-kinase (PI3K) pathway to further investigate the mechanisms of Dexa-induced apoptosis. Dexa decreased Akt phosphorylation by 93% (P < 0.001) without affecting total Akt and increased the p85α subunit 4-fold. The Akt inhibitor SH-6 (10 μm) increased apoptosis by 54% (P < 0.001). When combining Dexa with SH-6, apoptosis was not further increased, showing that Dexa-induced apoptosis is mediated through inhibition of the PI3K pathway. Addition of IGF-I to SH-6- or Dexa + SH-6-treated cells decreased apoptosis by 21.2% (P < 0.001) and 20.6% (P < 0.001), respectively. We conclude that Dexa-induced apoptosis is caspase dependent with an early activation of caspase-8. IGF-I can rescue chondrocytes from Dexa-induced apoptosis partially through the activation of other pathways than the PI3K signaling pathway. Based on our in vitro data, we speculate that in vivo treatment with glucocorticoids may diminish longitudinal growth by increasing apoptosis of proliferative growth plate chondrocytes.

We enjoyed the excellent and timely review by Terpos et al.1 in a recent issue of Leukemia on the effects of novel anti-myeloma agents on bone metabolism. In this review, the authors elaborate on the ubiquitin/proteasome system in bone, and the use of proteasome inhibitors (PIs) in myeloma bone disease. The authors emphasize that PIs, including bortezomib, act locally in the bone to stimulate osteoblast differentiation while inhibiting osteoclast formation and making them attractive agents for further investigation, as they may combine anti-myeloma activity with beneficial effects on bone. Indeed, recent in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on the bone formation process could occur during bortezomib treatment.2 Terpos et al. propose prospective studies with specific clinical end points, such as bone mineral density, to be undertaken. However, when designing such studies, we believe that it is important to pay particular attention to the recent observation that systemic administration of PIs specifically impairs the ubiquitin/proteasome system in chondrocytes and growth plate cartilage.3

A recent clinical study in mothers with rheumatoid arthritis (RA) published in this journal reported that high maternal serum levels of interleukin-6 (IL-6) are associated with low birthweight deliveries.1 Low birth weight in turn is associated with impaired growth during childhood.2 Interestingly, overexpression of IL-6 leads to reduced bone growth in mice.3 Although the systemic effects of IL-6 are well understood, any local effects on the growth plate have not yet been clarified. Aiming to study the local actions of IL-6, we isolated fetal rat metatarsal bones and cultured them ex vivo for 12 days while monitoring bone growth. When exposed to IL-6 and its soluble receptor IL-6Rα (10+100 ng/mL), growth was decreased by 21.1% compared with control (p

Dexamethasone (Dexa) is a widely used glucocorticoid to treat inflammatory diseases; however, a multitude of undesired effects have been reported to arise from this treatment including osteoporosis, obesity, and in children decreased longitudinal bone growth. We and others have previously shown that glucocorticoids induce apoptosis in growth plate chondrocytes. Here, we hypothesized that Bax, a pro-apoptotic member of the Bcl-2 family, plays a key role in Dexa-induced chondrocyte apoptosis and bone growth impairment. Indeed, experiments in the human HCS-2/8 chondrocytic cell line demonstrated that silencing of Bax expression using small-interfering (si) RNA efficiently blocked Dexa-induced apoptosis. Furthermore, ablation of Bax in female mice protected against Dexa-induced bone growth impairment. Finally, Bax activation by Dexa was confirmed in human growth plate cartilage specimens cultured ex vivo. Our findings could therefore open the door for new therapeutic approaches to prevent glucocorticoid-induced bone growth impairment through specific targeting of Bax.