Maria Almeida

Skeletal Involution by Age-associated Oxidative Stress and Its Acceleration by Loss of Sex Steroids

Both aging and loss of sex steroids have adverse effects on skeletal homeostasis, but whether and how they may influence each others negative impact on bone remains unknown. We report herein that both female and male C57BL/6 mice progressively lost strength (as determined by load-to-failure measurements) and bone mineral density in the spine and femur between the ages of 4 and 31 months. These changes were temporally associated with decreased rate of remodeling as evidenced by decreased osteoblast and osteoclast numbers and decreased bone formation rate; as well as increased osteoblast and osteocyte apoptosis, increased reactive oxygen species levels, and decreased glutathione reductase activity and a corresponding increase in the phosphorylation of p53 and p66shc, two key components of a signaling cascade that are activated by reactive oxygen species and influences apoptosis and lifespan. Exactly the same changes in oxidative stress were acutely reproduced by gonadectomy in 5-month-old females or males and reversed by estrogens or androgens in vivo as well as in vitro.We conclude that the oxidative stress that underlies physiologic organismal aging in mice may be a pivotal pathogenetic mechanism of the age-related bone loss and strength. Loss of estrogens or androgens accelerates the effects of aging on bone by decreasing defense against oxidative stress.

Wnt Proteins Prevent Apoptosis of Both Uncommitted Osteoblast Progenitors and Differentiated Osteoblasts by β-Catenin-dependent and -independent Signaling Cascades Involving Src/ERK and Phosphatidylinositol 3-Kinase/AKT

Genetic studies in humans and mice have revealed an important role of the Wnt signaling pathway in the regulation of bone mass, resulting from potent effects on the control of osteoblast progenitor proliferation, commitment, differentiation, and perhaps osteoblast apoptosis. To establish the linkage between Wnts and osteoblast survival and to elucidate the molecular pathways that link the two, we have utilized three cell models: the uncommitted bipotential C2C12 cells, the pre-osteoblastic cell line MC3T3-E1, and bone marrow-derived OB-6 osteoblasts. Serum withdrawal-induced apoptosis was prevented by the canonical Wnts (Wnt3a and Wnt1) and the noncanonical Wnt5a in all cell types. Wnt3a induced LRP5-independent transient phosphorylation and nuclear accumulation of ERKs and phosphorylation of Src and Akt. The anti-apoptotic effect of Wnt3a was abrogated by inhibitors of canonical Wnt signaling, as well as by inhibitors of MEK, Src, phosphatidylinositol 3-kinase (PI3K), or Akt kinases, or by the addition of cycloheximide to the culture medium. Wnt3a-induced phosphorylation of GSK-3β and downstream activation of β-catenin-mediated transcription required ERK, PI3K, and Akt signaling. Wnt3a increased the expression of the anti-apoptotic protein Bcl-2 in an ERK-dependent manner. β-Catenin-mediated transcription was permissive for the anti-apoptotic actions of Wnt1 and Wnt3a but was dispensable for the anti-apoptotic action of Wnt5a. However, Src, ERKs, PI3K, and Akt kinases were required for the anti-apoptotic effects of Wnt5a. These results demonstrate for the first time that Wnt proteins, irrespective of their ability to stimulate canonical Wnt signaling, prolong the survival of osteoblasts and uncommitted osteoblast progenitors via activation of the Src/ERK and PI3K/Akt signaling cascades.

Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids

It has been found that 4-estren-3alpha,17beta-diol, a synthetic ligand for the estrogen receptor (ER) or androgen receptor (AR), which does not affect classical transcription, reverses bone loss in ovariectomized females or orchidectomized males without affecting the uterus or seminal vesicles, demonstrating that the classical genotropic actions of sex steroid receptors are dispensable for their bone-protective effects, but indispensable for their effects on reproductive organs. We have now investigated the mechanism of action of this compound. We report that, identically to 17beta-estradiol or dihydrotestosterone, but differently from raloxifene, estren alters the activity of Elk-1, CCAAT enhancer binding protein-beta (C/EBPbeta), and cyclic adenosine monophosphate-response element binding protein (CREB), or c-Jun/c-Fos by an extranuclear action of the ER or AR, resulting in activation of the Src/Shc/ERK pathway or downregulation of JNK, respectively. All of these effects are non-sex specific, require only the ligand-binding domain of the receptor, and are indispensable for the antiapoptotic action of these ligands on osteoblastic and HeLa cells. Moreover, administration of 17beta-estradiol or 4-estren-3alpha,17beta-diol to ovariectomized mice induces phosphorylation of ERKs, Elk-1, and C/EBPbeta, downregulates c-Jun, and upregulates the expression of egr-1, an ERK/SRE target gene. Kinase-initiated regulation of commonly used transcription factors offers a molecular explanation for the profound skeletal effects of sex steroid receptor ligands, including synthetic ones that are devoid of classical transcriptional activity.

The role of estrogen and androgen receptors in bone health and disease

Mouse models with cell-specific deletion of the estrogen receptor (ER) α, the androgen receptor (AR) or the receptor activator of nuclear factor κB ligand (RANKL), as well as cascade-selective estrogenic compounds have provided novel insights into the function and signalling of ERα and AR. The studies reveal that the effects of estrogens on trabecular versus cortical bone mass are mediated by direct effects on osteoclasts and osteoblasts, respectively. The protection of cortical bone mass by estrogens is mediated via ERα, using a non-nucleus-initiated mechanism. By contrast, the AR of mature osteoblasts is indispensable for the maintenance of trabecular bone mass in male mammals, but not required for the anabolic effects of androgens on cortical bone. Most unexpectedly, and independently of estrogens, ERα in osteoblast progenitors stimulates Wnt signalling and periosteal bone accrual in response to mechanical strain. RANKL expression in B lymphocytes, but not T lymphocytes, contributes to the loss of trabecular bone caused by estrogen deficiency. In this Review, we summarize this evidence and discuss its implications for understanding the regulation of trabecular and cortical bone mass; the integration of hormonal and mechanical signals; the relative importance of estrogens versus androgens in the male skeleton; and, finally, the pathogenesis and treatment of osteoporosis.

FoxO-mediated defense against oxidative stress in osteoblasts is indispensable for skeletal homeostasis in mice

Aging increases oxidative stress and osteoblast apoptosis and decreases bone mass, whereas forkhead box O (FoxO) transcription factors defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis. Conditional deletion of FoxO1, FoxO3, and FoxO4 in 3-month-old mice resulted in an increase in oxidative stress in bone and osteoblast apoptosis and a decrease in the number of osteoblasts, the rate of bone formation, and bone mass at cancellous and cortical sites. The effect of the deletion on osteoblast apoptosis was cell autonomous and resulted from oxidative stress. Conversely, overexpression of a FoxO3 transgene in mature osteoblasts decreased oxidative stress and osteoblast apoptosis and increased osteoblast number, bone formation rate, and vertebral bone mass. We conclude that FoxO-dependent oxidative defense provides a mechanism to handle the oxygen free radicals constantly generated by the aerobic metabolism of osteoblasts and is thereby indispensable for bone mass homeostasis.

Endogenous glucocorticoids decrease skeletal angiogenesis, vascularity, hydration, and strength in aged mice

Aging or glucocorticoid excess decrease bone strength more than bone mass in humans and mice, but an explanation for this mismatch remains elusive. We report that aging in C57BL/6 mice was associated with an increase in adrenal production of glucocorticoids as well as bone expression of 11β‐hydroxysteroid dehydrogenase (11β‐HSD) type 1, the enzyme that activates glucocorticoids. Aging also decreased the volume of the bone vasculature and solute transport from the peripheral circulation to the lacunar‐canalicular system. The same changes were reproduced by pharmacologic hyperglucocorticoidism. Furthermore, mice in which osteoblasts and osteocytes were shielded from glucocorticoids via cell‐specific transgenic expression of 11β‐HSD type 2, the enzyme that inactivates glucocorticoids, were protected from the adverse effects of aging on osteoblast and osteocyte apoptosis, bone formation rate and microarchitecture, crystallinity, vasculature volume, interstitial fluid, and strength. In addition, glucocorticoids suppressed angiogenesis in fetal metatarsals and hypoxia inducible factor‐1α transcription and vascular endothelial growth factor production in osteoblasts and osteocytes. These results, together with the evidence that dehydration of bone decreases strength, reveal that endogenous glucocorticoids increase skeletal fragility in old age as a result of cell autonomous effects on osteoblasts and osteocytes leading to interconnected decrements in bone angiogenesis, vasculature volume, and osteocyte‐lacunar‐canalicular fluid.

The Estrogen Receptor-α in Osteoclasts Mediates the Protective Effects of Estrogens on Cancellous But Not Cortical Bone

Estrogens attenuate osteoclastogenesis and stimulate osteoclast apoptosis, but the molecular mechanism and contribution of these effects to the overall antiosteoporotic efficacy of estrogens remain controversial. We selectively deleted the estrogen receptor (ER)alpha from the monocyte/macrophage cell lineage in mice (ERalpha(LysM)(-/-)) and found a 2-fold increase in osteoclast progenitors in the marrow and the number of osteoclasts in cancellous bone, along with a decrease in cancellous bone mass. After loss of estrogens these mice failed to exhibit the expected increase in osteoclast progenitors, the number of osteoclasts in bone, and further loss of cancellous bone. However, they lost cortical bone indistinguishably from their littermate controls. Mature osteoclasts from ERalpha(LysM)(-/-) were resistant to the proapoptotic effect of 17beta-estradiol. Nonetheless, the effects of estrogens on osteoclasts were unhindered in mice bearing an ERalpha knock-in mutation that prevented binding to DNA. Moreover, a polymeric form of estrogen that is not capable of stimulating the nuclear-initiated actions of ERalpha was as effective as 17beta-estradiol in inducing osteoclast apoptosis in cells with the wild-type ERalpha. We conclude that estrogens attenuate osteoclast generation and life span via cell autonomous effects mediated by DNA-binding-independent actions of ERalpha. Elimination of these effects is sufficient for loss of bone in the cancellous compartment in which complete perforation of trabeculae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming osteoblasts. However, additional effects of estrogens on osteoblasts, osteocytes, and perhaps other cell types are required for their protective effects on the cortical compartment, which constitutes 80% of the skeleton.

Oxidative Stress Stimulates Apoptosis and Activates NF-κB in Osteoblastic Cells via a PKCβ/p66shc Signaling Cascade: Counter Regulation by Estrogens or Androgens

Aging or acute loss of estrogens or androgens increases the levels of reactive oxygen species, activates nuclear factor-κB (NF-κB), and promotes the phosphorylation of p66(shc), a redox enzyme that amplifies mitochondrial reactive oxygen species generation and stimulates apoptosis. We report that in mesenchymal progenitor and osteoblastic cell models, H(2)O(2) activated a protein kinase C (PKC)β/p66(shc)/NF-κB signaling cascade and that p66(shc) was an essential mediator of the stimulating effects of H(2)O(2) on the apoptosis of osteoblastic cells as well as their ability to activate NF-κB. 17β-Estradiol (E(2)) or the nonaromatizable androgen dihydrotestosterone abrogated the effects of H(2)O(2) on p66(shc) and NF-κB activation by attenuating the phosphorylation of the redox-sensitive cytoplasmic kinase PKCβ. Additionally, both E(2) and dihydrotestosterone prevented H(2)O(2)-induced apoptosis by a mechanism that involved attenuation of p66(shc) resulting from decreased phosphorylation of PKCβ. Consistent with a kinase-mediated mechanism of sex steroid action, the effects of E(2) were reproduced by a polymeric form of estradiol that is not capable of stimulating the nuclear-initiated actions of ERα. These results demonstrate that p66(shc) is an essential mediator of the effects of oxidative stress on osteoblastic cell apoptosis, NF-κB activation, and cytokine production. The ability of either estrogen or androgen to attenuate the effects of oxidative stress on osteoblastic cell apoptosis, NF-κB activation, and cytokine production results from their common property to suppress PKCβ-induced p66(shc) phosphorylation via a mechanism that does not require stimulation of the nuclear-initiated actions of sex steroids.

Stimulation of bone marrow cells and bone formation by nacre: in vivo and in vitro studies

There is frequently a loss of vertebral bone due to disease or aging. Nacre (mother of pearl from the oyster Pinctada maxima) stimulates bone cell differentiation and bone formation in vitro and in vivo. Experimental bone defects were prepared in the vertebrae of sheep and used to test the suitability of nacre as an injectable osteogenic biomaterial for treating vertebral bone loss. Twenty-one cavities were prepared in the first four upper lumbar vertebrae of 11 sheep and filled with nacre powder. The lumbar vertebrae were removed after 1 to 12 weeks, embedded undecalcified in methacrylate, and processed for histological studies. The nacre slowly dissolved and the experimental cavities contained a large active cell population. By 12 weeks, the experimental cavity was occupied by newly matured bone trabeculae in contact with or adjacent to the dissolving nacre. The functional new bone trabeculae were covered with osteoid lined with osteoblasts, indicating continuing bone formation. The in vitro study on rat bone marrow explants cultured with a water-soluble extract of the nacre organic matrix also resulted in the stimulation of osteogenic bone marrow cells with enhanced alkaline phosphatase activity. Thus, both the in vivo and in vitro findings suggest that nacre contains one or more signal molecules capable of activating osteogenic bone marrow cells.

Intermittent Parathyroid Hormone Administration Counteracts the Adverse Effects of Glucocorticoids on Osteoblast and Osteocyte Viability, Bone Formation, and Strength in Mice

Glucocorticoids act directly on bone cells to decrease production of osteoblasts and osteoclasts, increase osteoblast and osteocyte apoptosis, and prolong osteoclast life span. Conversely, daily injections of PTH decrease osteoblast and osteocyte apoptosis and increase bone formation and strength. Using a mouse model, we investigated whether the recently demonstrated efficacy of PTH in glucocorticoid-induced bone disease results from the ability of this therapeutic modality to counteract at least some of the direct effects of glucocorticoids on bone cells. Glucocorticoid administration to 5- to 6-month-old Swiss-Webster mice for 28 d increased the prevalence of osteoblast and osteocyte apoptosis and decreased osteoblast number, activation frequency, and bone formation rate, resulting in reduced osteoid, wall and trabecular width, bone mineral density, and bone strength. In contrast, daily injections of PTH caused a decrease in osteoblast and osteocyte apoptosis and an increase in osteoblast number, activation frequency, bone formation rate, bone mineral density, and bone strength. The decreased osteocyte apoptosis was associated with increased bone strength. When the two agents were combined, all the adverse effects of glucocorticoid excess on bone were prevented. Likewise, in cultured osteoblastic cells, PTH attenuated the adverse effects of glucocorticoids on osteoblast survival and Wnt signaling via an Akt phosphorylation-dependent mechanism. We conclude that intermittent PTH administration directly counteracts the key pathogenetic mechanisms of glucocorticoid excess on bone, thus providing a mechanistic explanation of its efficacy against glucocorticoid-induced osteoporosis.