Osteoblast Lifespan Research Articles

Effect of olive oil administration on the level of transforming growth factor β1 during orthodontic tooth movement in old and young guinea pigs.

Background: Orthodontic tooth movement occurs due to continuous pressure on the teeth, causing the remodeling of the alveolar bone. The tissue will respond to bone growth factors, such as transforming growth factor β1 (TGF-β1), fibroblasts, and bone morphogenetic proteins, for new bone formation. The remodeling process is balanced in young adults, but there is an imbalance in older individuals due to decreased differentiation, activity, and life span of osteoblasts, and increased osteoclasts. Olive oil contains lots of antioxidants and can slow down the aging process. This study aims to study the differences in TGF-β1 levels between old and young guinea pigs, and the difference between olive oil administration on TGF-β1 levels in old and young guinea pigs with orthodontic tooth movement. Methods: 12 guinea pigs divided into 4 groups: young guinea pigs (4-5 months) not given olive oil; young guinea pigs given olive oil; old guinea pigs (30-31 months) given olive oil; old guinea pigs given olive oil. The teeth were moved using an open coil spring mounted on the brackets on both lower incisors. Gingival sulcus fluid samples were taken on days 0, 7 and 14 of the movement of the teeth. TGF-β1 levels were analyzed using ELISA. Results: Three-way ANOVA and post hoc statistical tests showed that TGF-β1 levels in young guinea pigs were significantly higher than old guinea pigs on days 0, 7 and 14 (p<0.05). TGF-β1 levels in both young and old guinea pigs who were given olive oil was significantly higher than those not given olive oil on days 0, 7 and 14 (p<0.05). Conclusions: TGF-β1 levels in the younger age guinea pigs were higher than the older age, and olive oil could increase TGF-β1 levels in the older age guinea pigs.

Effect of olive oil administration on the level of transforming growth factor β1 during orthodontic tooth movement in old and young guinea pigs

Background: Orthodontic tooth movement occurs due to continuous pressure on the teeth, causing the remodeling of the alveolar bone. The tissue will respond to bone growth factors, such as transforming growth factor β1 (TGF-β1), fibroblasts, and bone morphogenetic proteins, for new bone formation. The remodeling process is balanced in young adults, but there is an imbalance in older individuals due to decreased differentiation, activity, and life span of osteoblasts, and increased osteoclasts. Olive oil contains lots of antioxidants and can slow down the aging process. This study aims to study the differences in TGF-β1 levels between old and young guinea pigs, and the difference between olive oil administration on TGF-β1 levels in old and young guinea pigs with orthodontic tooth movement. Methods: 12 guinea pigs divided into 4 groups: young guinea pigs (4-5 months) not given olive oil; young guinea pigs given olive oil; old guinea pigs (30-31 months) given olive oil; old guinea pigs given olive oil. The teeth were moved using an open coil spring mounted on the brackets on both lower incisors. Gingival sulcus fluid samples were taken on days 0, 7 and 14 of the movement of the teeth. TGF-β1 levels were analyzed using ELISA. Results: Three-way ANOVA and post hoc statistical tests showed that TGF-β1 levels in young guinea pigs were significantly higher than old guinea pigs on days 0, 7 and 14 (p<0.05). TGF-β1 levels in both young and old guinea pigs who were given olive oil was significantly higher than those not given olive oil on days 0, 7 and 14 (p<0.05). Conclusions: TGF-β1 levels in the younger age guinea pigs were higher than the older age, and olive oil could increase TGF-β1 levels in the older age guinea pigs.

Effect of olive oil administration on the level of transforming growth factor β1 during orthodontic tooth movement in old and young guinea pigs.

Background: Orthodontic tooth movement occurs due to continuous pressure on the teeth, causing the remodeling of the alveolar bone. The tissue will respond to bone growth factors, such as transforming growth factor β1 (TGF-β1), fibroblasts, and bone morphogenetic proteins, for new bone formation. The remodeling process is balanced in young adults, but there is an imbalance in older individuals due to decreased differentiation, activity, and life span of osteoblasts, and increased osteoclasts. Olive oil contains lots of antioxidants and can slow down the aging process. This study aims to study the differences in TGF-β1 levels between old and young guinea pigs, and the difference between olive oil administration on TGF-β1 levels in old and young guinea pigs with orthodontic tooth movement. Methods: 12 guinea pigs divided into 4 groups: young guinea pigs (4-5 months) not given olive oil; young guinea pigs given olive oil; old guinea pigs (30-31 months) given olive oil; old guinea pigs given olive oil. The teeth were moved using an open coil spring mounted on the brackets on both lower incisors. Gingival sulcus fluid samples were taken on days 0, 7 and 14 of the movement of the teeth. TGF-β1 levels were analyzed using ELISA. Results: Three-way ANOVA and post hoc statistical tests showed that TGF-β1 levels in young guinea pigs were significantly higher than old guinea pigs on days 0, 7 and 14 (p<0.05). TGF-β1 levels in both young and old guinea pigs who were given olive oil was significantly higher than those not given olive oil on days 0, 7 and 14 (p<0.05). Conclusions: TGF-β1 levels in the younger age guinea pigs were higher than the older age, and olive oil could increase TGF-β1 levels in the older age guinea pigs.

Estrogen enhances human osteoblast survival and function via promotion of autophagy

Estrogen increases bone formation by promoting mineralization and prolonging the lifespan of osteoblasts. To understand the underlying molecular mechanism/s, we identified estrogen-regulated proteins at different stages of human osteoblast differentiation using differential proteomics approach. Among the identified proteins, we observed that estrogen upregulated RAB3GAP1 on day 1 and 5 of differentiation. RAB3GAP1 is critically involved in the process of autophagy, a eukaryotic degradative pathway essential for cell survival. We, therefore, investigated the effect of estrogen on autophagy in differentiating human osteoblasts and their precursors, the mesenchymal stem cells (MSCs). MSCs exhibited high autophagic flux which declined during osteoblast differentiation, resulting in high basal apoptosis in osteoblasts. Estrogen reduced apoptosis in differentiating osteoblasts by promoting autophagy, thus contributing towards their longer lifespan. Further, MSCs were resistant against starvation-induced apoptosis, whereas, differentiating osteoblasts showed significant susceptibility towards it. Estrogen, in addition to promoting mineralization, protected differentiating osteoblasts from starvation-induced apoptosis by increasing autophagic flux. Autophagic flux in RAB3GAP1 knockdown osteoblasts appeared diminished, and showed increased apoptosis even in nutrient-rich conditions, and exhibited significantly impaired mineralization. However, irrespective of the presence of estrogen, starvation further enhanced apoptosis in these cells. Furthermore, estrogen failed to promote mineralization in these osteoblasts. Our study illustrates that autophagy is essential for human osteoblast survival and mineralization, and osteoblasts are susceptible to apoptosis due to reduced autophagy during differentiation. Estrogen, via upregulation of RAB3GAP1, promotes autophagy in osteoblasts during differentiation thereby increasing their survival and mineralization capacity. Our study demonstrates the positive role of autophagy in bone homeostasis.

Glucose-loading reduces bone remodeling in women and osteoblast function invitro.

Aging is associated with a reduction in osteoblast life span and the volume of bone formed by each basic multicellular unit. Each time bone is resorbed, less is deposited producing microstructural deterioration. Aging is also associated with insulin resistance and hyperglycemia, either of which may cause, or be the result of, a decline in undercarboxylated osteocalcin (ucOC), a protein produced by osteoblasts that increases insulin sensitivity. We examined whether glucose-loading reduces bone remodeling and ucOC invivo and osteoblast function invitro, and so compromises bone formation. We administered an oral glucose tolerance test (OGTT) to 18 pre and postmenopausal, nondiabetic women at rest and following exercise and measured serum levels of bone remodeling markers (BRMs) and ucOC. We also assessed whether increasing glucose concentrations with or without insulin reduced survival and activity of cultured human osteoblasts. Glucose-loading at rest and following exercise reduced BRMs in pre and postmenopausal women and reduced ucOC in postmenopausal women. Higher glucose correlated negatively, whereas insulin correlated positively, with baseline BRMs and ucOC. The increase in serum glucose following resting OGTT was associated with the reduction in bone formation markers. D-glucose (>10mmolL(-1)) increased osteoblast apoptosis, reduced cell activity and osteocalcin expression compared with 5mmolL(-1). Insulin had a protective effect on these parameters. Collagen expression invitro was not affected in this time course. In conclusion, glucose exposure reduces BRMs in women and exercise failed to attenuate this suppression effect. The suppressive effect of glucose on BRMs may be due to impaired osteoblast work and longevity. Whether glucose influences material composition and microstructure remains to be determined.

The Sclerostin-Independent Bone Anabolic Activity of Intermittent PTH Treatment Is Mediated by T-Cell–Produced Wnt10b

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T-cell production of the Wnt ligand Wnt10b contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti-Scl antibody (Scl-Ab) to determine the contribution of T-cell-produced Wnt10b to the Scl-independent modalities of action of iPTH. We report that combined treatment with Scl-Ab and iPTH was more potent than either iPTH or Scl-Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10b. In T-cell-null mice and mice lacking T-cell production of Wnt10b, combined treatment increased bone turnover significantly more than iPTH or Scl-Ab alone. However, in these mice, combined treatment with Scl-Ab and iPTH was equally effective as Scl-Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl-independent activity of iPTH on osteoblasts and bone mass is mediated by T-cell-produced Wnt10b. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl-Ab than either alone.

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30min to 5h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1 suggesting that PTH utilized an Extracellular Signal Regulated Kinase (ERK)-independent but p38 dependent pathway to regulate CARP-1 protein expression in osteoblasts. Immunofluorescence staining of differentiated osteoblasts further revealed nuclear to cytoplasmic translocation of CARP-1 protein following PTH treatment. Collectively, our studies identified CARP-1 for the first time in osteoblasts and suggest its potential role in PTH signaling and bone anabolic action.

Osteoblasts Display Different Responsiveness to TRAIL-Induced Apoptosis During Their Differentiation Process

Apoptosis can occur throughout the life span of osteoblasts (OBs), beginning from the early stages of differentiation and continuing throughout all stages of their working life. Here, we investigated the effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on normal human OBs showing for the first time that the expression of TRAIL receptors is modulated during OB differentiation. In particular, the TRAIL receptor ratio was in favor of the deaths because of the low expression of DcR2 in undifferentiated OBs, differently it was shifted toward the decoys in differentiated ones. Undifferentiated OBs treated with TRAIL showed reduced cell viability, whereas differentiated OBs displayed TRAIL resistance. The OB sensitiveness to TRAIL was due to the up-regulation of DR5 and the down-regulation of DcR2. The main death receptor involved in TRAIL-reduced OB viability was DR5 as demonstrated by the rescue of cell viability observed in the presence of anti-DR5 neutralizing antibody. Besides the ratio of TRAIL receptors, the sensitivity of undifferentiated OBs to TRAIL-cytotoxic effect was also associated with low mRNA levels of intracellular anti-apoptotic proteins, such as cFLIP, the activation of caspase-8 and -3, as well as the DNA fragmentation. This study suggests that apoptotic effect exerted by TRAIL/TRAIL-receptor system on normal human OB is strictly dependent upon cell differentiation status.

Role of T cells in ovariectomy induced bone loss—revisited

Role of T cells in ovariectomy induced bone loss—revisited

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

Bezafibrate prevents palmitate-induced apoptosis in osteoblastic MC3T3-E1 cells through the NF-κB signaling pathway

Osteoporosis is a bone condition defined by low bone mass and increase of fracture risk due to imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Low bone mass is likely to be due to the alteration of the osteoclast and osteoblast lifespan through regulated apoptosis. Saturated fatty acid (SFA) intake is negatively associated with bone mineral density (BMD). Furthermore, SFA induces apoptosis in osteoblastic cell lines. Bezafibrate could increase bone mass in intact male rats principally through increasing periosteal bone formation. At present, it is unknown whether bezafibrate attenuates palmitate-induced apoptosis in MC3T3-E1 cells. In the present study, we found that palmitate stimulated the degradation of IκBα and NF-κB translocation, as well as up-regulation of NF-κB-mediated Fas expression in obsteoblastic MC3T3-E1 cells. Furthermore, the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could restore palmitate-induced caspase-3 decrease and inhibit palmitate-induced cleaved caspase-3 increase. We observed that bezafibrate, a dual ligand for the peroxisome proliferator-activated receptors α (PPARα) and PPARδ, significantly attenuated the palmitate-induced cytotoxicity as determined by the MTT assay and inhibited the palmitate-induced apoptosis as determined by a flow cytometry assay using Annexin V-FITC/PI and assessment of the activity of caspase-3. Pre-treatment of bezafibrate prevented palmitate-induced NF-κB activation. Therefore, these findings indicate that bezafibrate inbibits palmitate-induced apoptosis via the NF-κB signaling pathway. Our results point to bezafibrate as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions.

Dexamethasone Affects FAS-and Serum Deprivation-Induced Cell Death of Human Osteoblastic Cells through Survivin Regulation

Glucocorticoid-induced bone loss is the most prevalent form of secondary osteoporosis. Such loss could be due to the alteration of osteoclast and osteoblast lifespan through regulated apoptosis. The current study investigated the effect of dexamethasone on Fas- and starvation-induced apoptosis of mature osteoblasts and their precursors. Using the human osteoblastic hFOB1.19 and the MG63 osteosarcoma cell lines, we found that sub-lethal doses of dexamethasone act on pre-osteoblasts but not on mature cells by increasing their susceptibility to apoptosis. Apoptosis occurs in a caspase-dependent manner as both DNA fragmentation and mitochondrial transmembrane potential dissipation (ΔΨm) are inhibited by the pan-caspase inhibitor zVAD. The increased susceptibility of osteoblast precursors to apoptosis could be due to dexamethasonemediated down-regulation of survivin expression. Dexamethasone can up-regulate survivin, and to a lesser extent Bcl-2, in mature cells but not in pre-osteoblasts. In addition, it can induce FLIP over-expression in osteosarcoma cells. All these effects are inhibited by the glucocorticoid antagonist RU486, indicating that dexamethasone action is specific and, furthermore, that it depends on glucocorticoid receptor. Finally, we have found that survivin and Bcl-2 are essential for pre- and mature osteoblast survival as their silencing is sufficient to induce spontaneous apoptosis in both cell types. In conclusion, our data outline a new molecular mechanism of glucocorticoid-mediated bone loss due to the enhanced apoptosis of precursors compared to mature osteoblasts. Furthermore, the data suggest a mechanism of dexamethasone-induced resistance of osteosarcoma cells to Fas- and stress-induced apoptosis.

Effects of curcumin on the skeletal system in rats

There is increasing interest in the discovery of natural compounds that could favorably affect the skeletal system. Curcumin is a constituent of turmeric, a plant which has been used for centuries as a dietary spice and a traditional Indian medicine. Curcumin has been reported to affect differentiation, activity and the lifespan of osteoblasts and osteoclasts in vitro. The aim of the present study was to investigate the effects of curcumin on the skeletal system of rats in vivo.Curcumin (10mg/kg, po daily) was administered for four weeks to normal (non-ovariectomized) and bilaterally ovariectomized (estrogen-deficient) three-month-old female Wistar Cmd:(WI)WU rats. Ovariectomy was performed seven days before the start of curcumin administration. Bone mass, mineral and calcium content, macrometric and histomorphometric parameters, as well as the mechanical properties of the bone, were examined. Serum total cholesterol and estradiol levels were also determined. In rats with normal estrogen levels, curcumin decreased serum estradiol level and slightly increased cancellous bone formation, along with decreased mineralization. Estrogen deficiency induced osteoporotic changes in the skeletal system of the ovariectomized control rats. In ovariectomized rats, curcumin decreased body mass gain and serum total cholesterol level, slightly improved some bone histo-morphometric parameters impaired by estrogen deficiency, but did not improve bone mineralization or mechanical properties. In conclusion, the results of the present in vivo study in rats did not support the hypothesis that curcumin, at doses that are readily achievable through dietary intake, could be useful for the prevention or treatment of osteoporosis.

Dependence of bone yield (volume of bone formed per unit of cement surface area) on resorption cavity size during osteonal remodeling in human rib: implications for osteoblast function and the pathogenesis of age-related bone loss

It is both a necessary and a sufficient condition for bone to be lost with age at any surface location that during remodeling the replacement of resorbed bone is incomplete. In both the ilium and the rib, the degree of such focal imbalance is smaller on the intracortical than on the endocortical or cancellous surfaces that are adjacent to bone marrow. The reason for this difference is unknown. To further examine this question, we measured various geometric variables in 1263 osteons in rib cross sections from 65 persons, including both sexes and age ranges 20 to 30 years and 60 to 70 years (four groups). Haversian canal (HC) area did not differ significantly between sexes or age groups. Percent osteonal refilling was close to 95% in all groups and did not differ between sexes but fell slightly with age. There was a very highly significant linear relationship between osteon bone area and (osteon area + HC area) in all groups, with coefficients of determination (r2) greater than 0.98. The regression slopes declined slightly with age in women but not in men. There was a very highly significant quadratic relationship between osteon bone area and osteon perimeter in all groups, with r2 values greater than 0.97. The ratio osteon bone area:osteon perimeter, an index of bone yield—the volume of bone deposited on each unit area of cement surface—was strongly related to osteon area and did not differ between sexes but was slightly less in the older groups. We conclude the following: (1) The high efficiency of intracortical remodeling in the rib is confirmed, with only trivial effects of age. (2) For HC area to be maintained within narrow limits and bone balance preserved, either initial osteoblast density or osteoblast capacity (the two determinants of bone yield) or, most likely, both must increase progressively with the size of the resorption cavity, suggesting that osteoblast recruitment (relative to available surface) and osteoblast lifespan increase with the volume of bone resorbed. (3) Intracortical remodeling in the rib is more efficient than marrow-adjacent remodeling at any site, possibly because of the different relationships to the circulation. In osteonal remodeling, all molecules released from resorbed bone must travel past the sites of osteoblast recruitment and operation, but in hemiosteonal remodeling, some molecules may not be subject to this constraint. (4) If marrow-adjacent remodeling became as efficient as rib intracortical remodeling, age-related bone loss would cease to be an important medical problem. © 2010 American Society for Bone and Mineral Research

Vascular endothelial growth factor regulates osteoblast survival – evidence for an autocrine feedback mechanism

BackgroundApoptosis of osteoblasts and osteoclasts regulates bone homeostasis. Skeletal injury in humans results in 'angiogenic' responses primarily mediated by vascular endothelial growth factor(VEGF), a protein essential for bone repair in animal models. Osteoblasts release VEGF in response to a number of stimuli and express receptors for VEGF in a differentiation dependent manner. This study investigates the putative role of VEGF in regulating the lifespan of primary human osteoblasts(PHOB) in vitro.MethodsPHOB were examined for VEGF receptors. Cultures were supplemented with VEGF(0–50 ng/mL), a neutralising antibody to VEGF, mAB VEGF(0.3 ug/mL) and Placental Growth Factor (PlGF), an Flt-1 receptor-specific VEGF ligand(0–100 ng/mL) to examine their effects on mineralised nodule assay, alkaline phosphatase assay and apoptosis.. The role of the VEGF specific antiapoptotic gene target BCl2 in apoptosis was determined.ResultsPHOB expressed functional VEGF receptors. VEGF 10 and 25 ng/mL increased nodule formation 2.3- and 3.16-fold and alkaline phosphatase release 2.6 and 4.1-fold respectively while 0.3 ug/mL of mAB VEGF resulted in approx 40% reductions in both. PlGF 50 ng/mL had greater effects on alkaline phosphatase release (103% increase) than on nodule formation (57% increase). 10 ng/mL of VEGF inhibited spontaneous and pathological apoptosis by 83.6% and 71% respectively, while PlGF had no significant effect. Pretreatment with mAB VEGF, in the absence of exogenous VEGF resulted in a significant increase in apoptosis (14 vs 3%). VEGF 10 ng/mL increased BCl2 expression 4 fold while mAB VEGF decreased it by over 50%.ConclusionVEGF is a potent regulator of osteoblast life-span in vitro. This autocrine feedback regulates survival of these cells, mediated via a non flt-1 receptor mechanism and expression of BCl2 antiapoptotic gene.

Beta-catenin is not activated by downregulation of PTEN in osteoblasts

Selective knockdown of phosphatase and tensin homolog (PTEN) has been recently shown to increase life long accumulation of bone and its ability to increase osteoblast lifespan. In order to determine how loss of PTEN function affects osteoblast differentiation, we created cell lines with stable knockdown of PTEN expression using short hairpin RNA vectors and characterized several clones. The effect of deregulated PTEN in osteoblasts was studied in relationship to cell proliferation and differentiation. Downregulation of PTEN initially affected the cell's attachment and spreading on plastic but cells recovered after a brief period of time. When cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we noticed a small but significant increase in growth rates with PTEN reduction. The size of individual cells appeared larger when compared to control cells. Differentiation properties of these osteoblasts were increased as evidenced by higher expression of several of the bone markers tested (alkaline phosphatase, osteocalcin, osterix, bone morphogenetic protein 2, Cbfa1, osteoprotegerin, and receptor activator of NF-kappaB ligand) and their mineralization capacity in culture. As stabilization of beta-catenin is known to be responsible for growth deregulation with PTEN loss in other cell types, we investigated the activation of the canonical Wnt pathway in our cell lines. Immunofluorescence staining, protein expression in subcellular fractions for beta-catenin, and assays for activation of the canonical Wnt/beta-catenin signaling were studied in the PTEN downregulated cells. There was an overall decrease in beta-catenin expression in cells with PTEN knockdown. The distribution of beta-catenin was more diffuse within the cell in the PTEN-reduced clones when compared to controls where they were mostly present in cell borders. Signaling through the canonical pathway was also reduced in the PTEN knockdown cells when compared to control. The results of this study suggest that while decreased PTEN augments cell proliferation and positively affects differentiation, there is a decrease in beta-catenin levels and activity in osteoblasts. Therefore, at least in osteoblasts, beta-catenin is not responsible for mediating the activation of osteoblast differentiation with reduction in PTEN function.

Glucocorticoid-induced osteoporosis: hope on the HORIZON

Glucocorticoids are used to treat many disorders. Their negative effect on skeletal health is a substantial drawback, such that their use is the most frequent secondary cause of osteoporosis.1 30–50% of patients who take glucocorticoids chronically have a fracture. Glucocorticoids decrease bone formation by reducing the lifespan of osteoblasts and osteocytes (figure).

Recent developments in the management of postmenopausal osteoporosis with bisphosphonates: enhanced efficacy by enhanced compliance

Bisphosphonates are the current mainstay of treatment for postmenopausal osteoporosis. Although daily oral dosing is effective, it is associated with poor compliance, partly because of the pre and postdose fasting and posture requirements. This negatively impacts treatment outcomes, leading to a reduced clinical benefit. Improved, yet still suboptimal adherence has been noticed with less frequent bisphosphonate dosing e.g. once-weekly and once-monthly oral regimens. The recently approved quarterly intravenous (i.v.) injection regimen of ibandronate and yearly i.v. infusion of zoledronic acid are attractive options in the management of postmenopausal osteoporosis. These regimens may assure quarterly and year long compliance.

Perspective: Quantifying Osteoblast and Osteocyte Apoptosis: Challenges and Rewards

Since the initial demonstration of the phenomenon in murine and human bone sections approximately 10 yr ago, appreciation of the biologic significance of osteoblast apoptosis has contributed greatly not only to understanding the regulation of osteoblast number during physiologic bone remodeling, but also the pathogenesis of metabolic bone diseases and the pharmacology of some of the drugs used for their treatment. It is now appreciated that all major regulators of bone metabolism including bone morphogenetic proteins (BMPs), Wnts, other growth factors and cytokines, integrins, estrogens, androgens, glucocorticoids, PTH and PTH-related protein (PTHrP), immobilization, and the oxidative stress associated with aging contribute to the regulation of osteoblast and osteocyte life span by modulating apoptosis. Moreover, osteocyte apoptosis has emerged as an important regulator of remodeling on the bone surface and a critical determinant of bone strength, independently of bone mass. The detection of apoptotic osteoblasts in bone sections remains challenging because apoptosis represents only a tiny fraction of the life span of osteoblasts, not unlike a 6-mo-long terminal illness in the life of a 75-yr-old human. Importantly, the phenomenon is 50 times less common in human bone biopsies because human osteoblasts live longer and are fewer in number. Be that as it may, well-controlled assays of apoptosis can yield accurate and reproducible estimates of the prevalence of the event, particularly in rodents where there is an abundance of osteoblasts for inspection. In this perspective, we focus on the biological significance of the phenomenon for understanding basic bone biology and the pathogenesis and treatment of metabolic bone diseases and discuss limitations of existing techniques for quantifying osteoblast apoptosis in human biopsies and their methodologic pitfalls.

Bone changes in myelofibrosis with myeloid metaplasia: a histomorphometric and microcomputed tomographic study

Myelofibrosis with myeloid metaplasia (MMM) is a clonal disorder of the haematopoietic stem cell which can be associated with marrow fibrosis and/or osteosclerosis. Because bone progenitors and mature bone cells are influenced by the marrow microenvironment, cellular and tissular changes were assessed by histomorphometry in MMM. Thirteen patients, with a clinical proven MMM, had a bone biopsy of the iliac crest with double tetracycline labelling and osteoclast count. Histomorphometry was done at the 2D level (bone volume, osteoid parameters, bone histodynamic parameters and osteoclast count) and 3D level by microcomputed tomography. All patients had clusters of abnormal megakaryocytes in bone marrow. Newly apposed bone packets were observed in 12 patients and corresponded to an increased thickness of some bone units with new lamellae or focal areas of woven bone anchored on the pre-existing trabeculae. Osteoid parameters were unchanged, only bone formation rate appeared considerably increased in seven patients. There was a net tendency for decrease in osteoclast number and conversion of trabecular pillars into plates. An uncoupling of bone remodelling was evidenced with an increased life-span of osteoblasts associated with a normal/reduced osteoclast activity. A very complex network of factors is candidate to explain bone changes observed in MMM.