Babatunde O. Oyajobi

Smurf1 Inhibits Osteoblast Differentiation and Bone Formation in Vitro and in Vivo

Bone morphogenetic proteins (BMPs) are required for normal postnatal bone formation and osteoblast differentiation. There is evidence from recent studies that BMP signaling in osteoblasts is controlled by an ubiquitin-proteasome regulatory mechanism involving a cascade of enzymatic reactions. The specificity of protein ubiquitination is determined by E3 ubiquitin ligases, which play a crucial role in defining substrate specificity and subsequent protein degradation by 26S proteasomes. We have examined the role of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1), a member of the Hect domain family of E3 ubiquitin ligases in osteoblast function. Smurf1 has been found to interact with BMP-activated Smad1 and -5 and to mediate degradation of these Smad proteins. Recently we have found that Smurf1 mediates the protein degradation of the osteoblast-specific transcription factor Runx2/Cbfa1. To determine the role of Smurf1 in osteoblast differentiation, in the present studies we transfected a Smurf1 expression plasmid into 2T3 osteoblast precursor cells and found that Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation. To further investigate the role of Smurf1 in bone formation in vivo, we generated transgenic mice in which expression of the epitope-tagged Smurf1 transgene was targeted to osteoblasts using the murine 2.3-kb osteoblast-specific type I collagen promoter. In these transgenic mice, bone formation was significantly reduced during postnatal life. Our results demonstrate for the first time that Smurf1 plays a specific role in osteoblast differentiation and bone formation in vivo.

TGF-β promotion of Gli2-induced expression of parathyroid hormone-related protein, an important osteolytic factor in bone metastasis, is independent of canonical Hedgehog signaling

Breast cancer frequently metastasizes to bone, in which tumor cells receive signals from the bone marrow microenvironment. One relevant factor is TGF-β, which upregulates expression of the Hedgehog (Hh) signaling molecule, Gli2, which in turn increases secretion of important osteolytic factors such as parathyroid hormone-related protein (PTHrP). PTHrP inhibition can prevent tumor-induced bone destruction, whereas Gli2 overexpression in tumor cells can promote osteolysis. In this study, we tested the hypothesis that Hh inhibition in bone metastatic breast cancer would decrease PTHrP expression and therefore osteolytic bone destruction. However, when mice engrafted with human MDA-MB-231 breast cancer cells were treated with the Hh receptor antagonist cyclopamine, we observed no effect on tumor burden or bone destruction. In vitro analyses revealed that osteolytic tumor cells lack expression of the Hh receptor, Smoothened, suggesting an Hh-independent mechanism of Gli2 regulation. Blocking Gli signaling in metastatic breast cancer cells with a Gli2-repressor gene (Gli2-rep) reduced endogenous and TGF-β-stimulated PTHrP mRNA expression, but did not alter tumor cell proliferation. Furthermore, mice inoculated with Gli2-Rep-expressing cells exhibited a decrease in osteolysis, suggesting that Gli2 inhibition may block TGF-β propagation of a vicious osteolytic cycle in this MDA-MB-231 model of bone metastasis. Accordingly, in the absence of TGF-β signaling, Gli2 expression was downregulated in cells, whereas enforced overexpression of Gli2 restored PTHrP activity. Taken together, our findings suggest that Gli2 is required for TGF-β to stimulate PTHrP expression and that blocking Hh-independent Gli2 activity will inhibit tumor-induced bone destruction.

Wnt/β-catenin signaling activates bone morphogenetic protein 2 expression in osteoblasts

The BMP and Wnt/β-catenin signaling pathways cooperatively regulate osteoblast differentiation and bone formation. Although BMP signaling regulates gene expression of the Wnt pathway, much less is known about whether Wnt signaling modulates BMP expression in osteoblasts. Given the presence of putative Tcf/Lef response elements that bind β-catenin/TCF transcription complex in the BMP2 promoter, we hypothesized that the Wnt/β-catenin pathway stimulates BMP2 expression in osteogenic cells. In this study, we showed that Wnt/β-catenin signaling is active in various osteoblast or osteoblast precursor cell lines, including MC3T3-E1, 2T3, C2C12, and C3H10T1/2 cells. Furthermore, crosstalk between the BMP and Wnt pathways affected BMP signaling activity, osteoblast differentiation, and bone formation, suggesting Wnt signaling is an upstream regulator of BMP signaling. Activation of Wnt signaling by Wnt3a or overexpression of β-catenin/TCF4 both stimulated BMP2 transcription at promoter and mRNA levels. In contrast, transcription of BMP2 in osteogenic cells was decreased by either blocking the Wnt pathway with DKK1 and sFRP4, or inhibiting β-catenin/TCF4 activity with FWD1/β-TrCP, ICAT, or ΔTCF4. Using a site-directed mutagenesis approach, we confirmed that Wnt/β-catenin transactivation of BMP2 transcription is directly mediated through the Tcf/Lef response elements in the BMP2 promoter. These results, which demonstrate that the Wnt/β-catenin signaling pathway is an upstream activator of BMP2 expression in osteoblasts, provide novel insights into the nature of functional cross talk integrating the BMP and Wnt/β-catenin pathways in osteoblastic differentiation and maintenance of skeletal homeostasis.

The zinc finger transcription factor Gli2 mediates bone morphogenetic protein 2 expression in osteoblasts in response to hedgehog signaling.

ABSTRACT Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation.

The Hedgehog Signaling Molecule Gli2 Induces Parathyroid Hormone-Related Peptide Expression and Osteolysis in Metastatic Human Breast Cancer Cells

Parathyroid hormone-related peptide (PTHrP) is a major factor involved in tumor-induced osteolysis caused by breast cancers that have metastasized to bone. However, the molecular mechanisms that mediate PTHrP production by breast cancer cells are not entirely clear. We hypothesized that Gli2, a downstream transcriptional effector of the Hedgehog (Hh) signaling pathway, regulates PTHrP expression in metastatic breast cancer because the Hh pathway regulates physiologic PTHrP expression in the developing growth plate. Here, we show that Gli2 is expressed in several human cancer cell lines that cause osteolytic lesions in vivo and produce PTHrP (MDA-MB-231, RWGT2, and PC-3) but is not expressed in nonosteolytic cancer cell lines that do not secrete PTHrP (MCF-7, ZR-75, and T47D). Transient expression of Gli2 in MDA-MB-231 and MCF-7 breast cancer cells increased PTHrP promoter-luciferase activity dose dependently. Stable expression of Gli2 in MDA-MB-231 cells resulted in an increase in PTHrP protein in the conditioned medium. Alternatively, MDA-MB-231 cells stably transfected with Gli2-EnR, a repressor of Gli2 activity, exhibited a 72% to 93% decrease in PTHrP mRNA by quantitative real-time PCR when compared with control cells. To examine the effects of Gli2 on breast cancer-mediated osteolysis in vivo, athymic nude mice were inoculated with MDA-MB-231 cells stably expressing Gli2 or the empty vector. Following tumor cell inoculation via the left cardiac ventricle, Gli2-expressing tumors caused significantly more osteolysis. Together, these data suggest that PTHrP expression and osteolysis in vivo in human breast cancer cells is driven at least in part by Gli2.

Isolation and Characterization of Human Clonogenic Osteoblast Progenitors Immunoselected from Fetal Bone Marrow Stroma Using STRO-1 Monoclonal Antibody†

Osteoprogenitor cells present in human fetal bone marrow (BM) stroma have not been characterized. We used density gradient centrifugation, aggregation on binding lectin, and enrichment by magnetic activated cell sorting with STRO‐1 antibody to isolate STRO‐1+ cells from nonadherent human fetal BM stromal cells. Immunoselected STRO‐1+ cells were immortalized using SV‐40 large T antigen and a clone, F/STRO‐1+ A, with weak alkaline phosphatase (ALP) activity was selected. The cloned cells proliferated rapidly but were not tumorigenic. Preconfluent F/STRO‐1+ A cells showed immunoreactivity for osteopontin, α1(I) procollagen, and parathyroid hormone–related peptide, but not for the late osteoblast differentiation markers, osteocalcin (OC), or bone sialoprotein. However, differentiation of F/STRO‐1+ A cells was induced by dexamethasone and 1,25‐dihydroxyvitamin D3, as shown by increased ALP activity. In addition, osteogenesis occurred in F/STRO‐1+ A cells cultured in three‐dimentional aggregates, as assessed morphologically, histologically, and biochemically. Moreover, reverse transcription‐polymerase chain reaction analysis showed that OC expression was silent in exponentially growing cells and occurred when cell–cell contacts were established in monolayer and in aggregates, showing induction of mature osteoblast phenotype by cell–cell contacts. Thus, clonal F/STRO‐1+ A cells immunoselected from human fetal BM stroma display features of immature osteoprogenitor cells which can differentiate into mature osteogenic cells by cell–cell interactions or inducing agents. The generation by immunoselection of an immortalized clonogenic human fetal BM stroma‐derived cell line which behaves like an osteoprogenitor cell provides a novel model system for identifying the signals required for the commitment of osteoprogenitors in the human fetal BM stroma.

Zerumbone Abolishes RANKL-Induced NF-κB Activation, Inhibits Osteoclastogenesis, and Suppresses Human Breast Cancer–Induced Bone Loss in Athymic Nude Mice

Receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) has emerged as a major mediator of bone resorption, commonly associated with cancer and other chronic inflammatory diseases. Inhibitors of RANKL signaling thus have potential in preventing bone loss. In the present report, the potential of zerumbone, a sesquiterpene derived from subtropical ginger, to modulate osteoclastogenesis induced by RANKL and breast cancer was examined. We found that zerumbone inhibited RANKL-induced NF-kappaB activation in mouse monocyte, an osteoclast precursor cell, through inhibition of activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Zerumbone also suppressed RANKL-induced differentiation of these cells to osteoclasts. This sesquiterpene also inhibited the osteoclast formation induced by human breast tumor cells and by multiple myeloma cells. Finally, we examined whether zerumbone could prevent human breast cancer-induced bone loss in animals. We found that zerumbone decreased osteolysis in a dose-dependent manner in MDA-MB-231 breast cancer tumor-bearing athymic nude mice. These results indicate that zerumbone is an effective blocker of RANKL-induced NF-kappaB activation and of osteoclastogenesis induced by RANKL and tumor cells, suggesting its potential as a therapeutic agent for osteoporosis and cancer-associated bone loss.

Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma.

Purpose: Eradication of post-treatment residual myeloma cells is needed to prevent relapses, and immunostimulatory monoclonal antibodies (mAb) such as anti-CD137, CTLA-4, CD40, etc., which enhance the immune response against malignancies, represent a means of achieving this purpose. This study explores anti-CD137 mAbs for multiple myeloma treatment in preclinical models of the disease because they safely augment tumor immunity and are in clinical trials for other cancers. Experimental Design: The antitumor effect of anti-CD137 mAb on mouse plasmacytomas derived from HOPC and NS0 cell lines was studied and compared with that of anti-CTLA-4, anti-CD40, and anti-ICAM-2 mAbs. The antitumor effect of anti-CD137 mAb was also examined in a mouse syngeneic disseminated myeloma (5TGM1) model, which more closely resembles human multiple myeloma. Depletions of specific cell populations and gene-targeted mice were used to unravel the requirements for tumor rejection. Results: Agonistic mAb against CD137 and blocking anti-CTLA-4 mAb showed activity against i.p. HOPC tumors, resulting in extended survival of mice that also became immune to rechallenge. Anti-CD137 mAbs induced complete eradications of established s.c. NS0-derived tumors that were dependent on IFN-γ, natural killer cells, and CD8+ T lymphocytes. Natural killer cells accumulated in tumor draining lymph nodes and showed increased IFN-γ production. Antitumor efficacy of anti-CD137 mAb was preserved in CD28-deficient mice despite the fact that CD28 signaling increases the expression of CD137 on CD8+ T cells. Importantly, anti-CD137 mAb treatment significantly decreased systemic tumor burden in the disseminated 5TGM1 model. Conclusions: The immune-mediated antitumor activity of anti-CD137 mAb in mouse models holds promise for myeloma treatment in humans.

Cytokines and Bone Remodeling

Publisher Summary Bone is continuously remodeled in normal individuals and this is achieved via a finely regulated balance between the processes of bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. This bone remodeling is regulated, in part, by local factors including cytokines generated in the bone microenvironment. The purpose of this chapter is to summarize what is currently known about the role of cytokines and their receptors in bone remodeling. Recent advances in molecular biological techniques have meant that most of the biological activities ascribed to cytokines have now been associated with specific molecules, and their receptors identified and molecularly cloned. Several cytokines and their cognate receptors have been shown to be expressed by bone cells, marrow cells, or accessory cells in the bone microenvironment. Moreover, studies using knockout and transgenic mice have increased the understanding of the complex signal transduction mechanisms utilized by cytokines and are opening up new and exciting areas of study. Cytokines tend to be pleiotropic and multifactorial, and may have overlapping and seemingly redundant biological effects. Some of this redundancy is apparent in the receptor mechanisms and signal transduction pathways used by groups of cytokines. Classic examples that illustrate this vividly are the various cytokines belonging to the interleukin (IL)-6 family, such as IL-6, leukemia inhibitory factor, oncostatin-M, and IL-11, which utilize a common signal transduction protein known as gp130. These cytokines bind to distinct membrane-associated receptors, which form hetero- or homo-dimers upon binding to the ligand. Further, there is now a body of data derived from in vivo studies in animals which show that over- or under-production of certain cytokines cause profound effects on bone. These fundamental observations have the potential of not only increasing the understanding of the pathophysiology of osteoporosis, but also leading to new and better forms of therapy using these molecules as targets for drug discovery programs.

PD-L1/PD-1 presence in the tumor microenvironment and activity of PD-1 blockade in multiple myeloma

PD-L1/PD-1 presence in the tumor microenvironment and activity of PD-1 blockade in multiple myeloma