Charles A. O'Brien

Nongenotropic, Sex-Nonspecific Signaling through the Estrogen or Androgen Receptors: Dissociation from Transcriptional Activity

The relationship of the classical receptors and their transcriptional activity to nongenotropic effects of steroid hormones is unknown. We demonstrate herein a novel paradigm of sex steroid action on osteoblasts, osteocytes, embryonic fibroblasts, and HeLa cells involving activation of a Src/Shc/ERK signaling pathway and attenuating apoptosis. This action is mediated by the ligand binding domain and eliminated by nuclear targeting of the receptor protein; ERalpha, ERbeta, or AR can transmit it with similar efficiency irrespective of whether the ligand is an estrogen or an androgen. This antiapoptotic action can be dissociated from the transcriptional activity of the receptor with synthetic ligands, providing proof of principle for the development of function-specific-as opposed to tissue-selective-and gender-neutral pharmacotherapeutics.

Matrix-embedded cells control osteoclast formation

Osteoclasts resorb the mineralized matrices formed by chondrocytes or osteoblasts. The cytokine receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast formation and thought to be supplied by osteoblasts or their precursors, thereby linking bone formation to resorption. However, RANKL is expressed by a variety of cell types, and it is unclear which of them are essential sources for osteoclast formation. Here we have used a mouse strain in which RANKL can be conditionally deleted and a series of Cre-deleter strains to demonstrate that hypertrophic chondrocytes and osteocytes, both of which are embedded in matrix, are essential sources of the RANKL that controls mineralized cartilage resorption and bone remodeling, respectively. Moreover, osteocyte RANKL is responsible for the bone loss associated with unloading. Contrary to the current paradigm, RANKL produced by osteoblasts or their progenitors does not contribute to adult bone remodeling. These results suggest that the rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself.

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.

Proteasomal degradation of Runx2 shortens parathyroid hormone-induced anti-apoptotic signaling in osteoblasts. A putative explanation for why intermittent administration is needed for bone anabolism.

It is unknown why sustained elevation of parathyroid hormone (PTH) stimulates bone resorption, whereas intermittent administration stimulates bone formation. We show in mice that daily injections of PTH attenuate osteoblast apoptosis, thereby increasing osteoblast number, bone formation rate, and bone mass, but do not affect osteoclast number. In contrast, sustained elevation of PTH, achieved either by infusion or by raising endogenous hormone secretion with a calcium-deficient diet, does not affect osteoblast apoptosis but increases osteoclast number. Attenuation of apoptosis by PTH in cultured osteoblastic cells requires protein kinase A-mediated phosphorylation and inactivation of the pro-apoptotic protein Bad as well as transcription of survival genes, like Bcl-2, mediated by CREB (cAMP response element-binding protein) and Runx2. But, PTH also increases proteasomal proteolysis of Runx2. Moreover, the anti-apoptotic effect of PTH is prolonged by inhibition of proteasomal activity, by overexpressing a dominant negative form of the E3 ligase (ubiquitin-protein isopeptide ligase) that targets Runx2 for degradation (Smurf1), or by overexpressing Runx2 itself. The duration of the anti-apoptotic effect of PTH, thus, depends on the level of Runx2, which in turn is decreased by PTH via Smurf1-mediated proteasomal proteolysis. The self-limiting nature of PTH-induced survival signaling might explain why intermittent administration of the hormone is required for bone anabolism.

Oxidative Stress Antagonizes Wnt Signaling in Osteoblast Precursors by Diverting β-Catenin from T Cell Factor- to Forkhead Box O-mediated Transcription

We have elucidated that oxidative stress is a pivotal pathogenetic factor of age-related bone loss and strength in mice, leading to, among other changes, a decrease in osteoblast number and bone formation. To gain insight into the molecular mechanism by which oxidative stress exerts such adverse effects, we have tested the hypothesis that induction of the Forkhead box O (FoxO) transcription factors by reactive oxygen species may antagonize Wnt signaling, an essential stimulus for osteoblastogenesis. In support of this hypothesis, we report herein that the expression of FoxO target genes increases, whereas the expression of Wnt target genes decreases, with increasing age in C57BL/6 mice. Moreover, we show that in osteoblastic cell models, oxidative stress (exemplified by H2O2) promotes the association of FoxOs with β-catenin, β-catenin is required for the stimulation of FoxO target genes by H2O2, and H2O2 promotes FoxO-mediated transcription at the expense of Wnt-/T-cell factor-mediated transcription and osteoblast differentiation. Furthermore, β-catenin overexpression is sufficient to prevent FoxO-mediated suppression of T-cell factor transcription. These results demonstrate that diversion of the limited pool of β-catenin from T-cell factor- to FoxO-mediated transcription in osteoblastic cells may account, at least in part, for the attenuation of osteoblastogenesis and bone formation by the age-dependent increase in oxidative stress.

Control of bone mass and remodeling by PTH receptor signaling in osteocytes

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.

Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: antagonism by noggin.

Bone morphogenetic proteins (BMPs) have been heretofore implicated in the induction of osteoblast differentiation from uncommitted progenitors during embryonic skeletogenesis and fracture healing. We have tested the hypothesis that BMPs are also involved in the osteoblastogenesis that takes place in the bone marrow in postnatal life. To do this, we took advantage of the properties of noggin, a recently discovered protein that binds BMP‐2 and −4 and blocks their action. Addition of human recombinant noggin to bone marrow cell cultures from normal adult mice inhibited both osteoblast and osteoclast formation; these effects were reversed by exogenous BMP‐2. Consistent with these findings, BMP‐2 and −4 and BMP‐2/4 receptor transcripts and proteins were detected in these primary cultures, in a bone marrow–derived stromal/osteoblastic cell line, as well as in murine adult whole bone; noggin expression was also documented in all these preparations. Moreover, addition of antinoggin antibody caused an increase in osteoblast progenitor formation. These findings suggest that BMP‐2 and −4 are expressed in the bone marrow in postnatal life and serve to maintain the continuous supply of osteoblasts and osteoclasts; and that, in fact, BMP‐2/4‐induced commitment to the osteoblastic lineage is a prerequisite for osteoclast development. Hence, BMPs, perhaps in balance with noggin and possibly other antagonists, may provide the tonic baseline control of the rate of bone remodeling on which other inputs (e.g., hormonal, biomechanical, etc.) operate.

STAT3 Activation in Stromal/Osteoblastic Cells Is Required for Induction of the Receptor Activator of NF-κB Ligand and Stimulation of Osteoclastogenesis by gp130-utilizing Cytokines or Interleukin-1 but Not 1,25-Dihydroxyvitamin D3 or Parathyroid Hormone

Interleukin (IL)-6-type cytokines stimulate osteoclastogenesis by activating gp130 in stromal/osteoblastic cells and may mediate some of the osteoclastogenic effects of other cytokines and hormones. To determine whether STAT3 is a downstream effector of gp130 in the osteoclast support function of stromal/osteoblastic cells and whether the gp130/STAT3 pathway is utilized by other osteoclastogenic agents, we conditionally expressed dominant negative (dn)-STAT3 or dn-gp130 in a stromal/osteoblastic cell line (UAMS-32) that supports osteoclast formation. Expression of either dominant negative protein abolished osteoclast formation stimulated by IL-6 + soluble IL-6 receptor, oncostatin M, or IL-1 but not by parathyroid hormone or 1,25-dihydroxyvitamin D3. Because previous studies suggested that IL-6-type cytokines may stimulate osteoclastogenesis by inducing expression of the tumor necrosis factor-related protein, receptor activator of NF-κB ligand (RANKL), we conditionally expressed RANKL in UAMS-32 cells and found that this was sufficient to stimulate osteoclastogenesis. Moreover, dn-STAT3 blocked the ability of either IL-6 + soluble IL-6 receptor or oncostatin M to induce RANKL. These results establish that STAT3 is essential for gp130-mediated osteoclast formation and that the target of STAT3 during this process is induction of RANKL. In addition, this study demonstrates that activation of the gp130-STAT3 pathway in stromal/osteoblastic cells mediates the osteoclastogenic effects of IL-1, but not parathyroid hormone or 1,25-dihydroxyvitamin D3.

Oxidative stress antagonizes WNT signaling in osteoblast precursors by diverting B-catenin from TCF- to FOXO-mediated transcription

We have elucidated that oxidative stress is a pivotal pathogenetic factor of age-related bone loss and strength in mice, leading to, among other changes, a decrease in osteoblast number and bone formation. To gain insight into the molecular mechanism by which oxidative stress exerts such adverse effects, we have tested the hypothesis that induction of the Forkhead box O (FoxO) transcription factors by reactive oxygen species may antagonize Wnt signaling, an essential stimulus for osteoblastogenesis. In support of this hypothesis, we report herein that the expression of FoxO target genes increases, whereas the expression of Wnt target genes decreases, with increasing age in C57BL/6 mice. Moreover, we show that in osteoblastic cell models, oxidative stress (exemplified by H2O2) promotes the association of FoxOs with β-catenin, β-catenin is required for the stimulation of FoxO target genes by H2O2, and H2O2 promotes FoxO-mediated transcription at the expense of Wnt-/T-cell factor-mediated transcription and osteoblast differentiation. Furthermore, β-catenin overexpression is sufficient to prevent FoxO-mediated suppression of T-cell factor transcription. These results demonstrate that diversion of the limited pool of β-catenin from T-cell factor- to FoxO-mediated transcription in osteoblastic cells may account, at least in part, for the attenuation of osteoblastogenesis and bone formation by the age-dependent increase in oxidative stress.

Transcriptional Activation of the p21 WAF1,CIP1,SDI1 Gene by Interleukin-6 Type Cytokines A PREREQUISITE FOR THEIR PRO-DIFFERENTIATING AND ANTI-APOPTOTIC EFFECTS ON HUMAN OSTEOBLASTIC CELLS

The cyclin-dependent kinase inhibitor p21 WAF1,CIP1,SDI1 plays a critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells.