Elizabeth W. Bradley

TGF-beta coordinately activates TAK1/MEK/AKT/NFkB and SMAD pathways to promote osteoclast survival.

To better understand the roles of TGF-beta in bone metabolism, we investigated osteoclast survival in response TGF-beta and found that TGF-beta inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-beta receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-beta treatment. Since osteoclast survival involves MEK, AKT, and NFkappaB activation, we examined TGF-beta effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IkappaB, and NFkappaB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFkappaB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFkappaB repressed TGF-beta-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-beta-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclX(L) expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-beta-induced NFkappaB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-beta to support of osteoclast survival.

The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas

A cancer-promoting histone protein Mutations in the chromatin protein histone H3 are found in a number of pediatric cancers. The lysine-36–to–methionine (K36M) “oncohistone” mutation is seen in almost all chondroblastomas. Fang et al. show that the K36M mutant histones inhibit the normal methylation of this same residue in wild-type H3 histones. They do so by interfering with the enzymes that normally methylate this residue. The altered chromatin methylation patterns alter the expression of known cancer-related genes and impart cancer-related characteristics to the chondrocyte cells. Science, this issue p. 1344 The lysine-36–to–methionine mutation in histone H3 interferes with inhibitory chromatin marks and promotes cancer. More than 90% of chondroblastomas contain a heterozygous mutation replacing lysine-36 with methionine-36 (K36M) in the histone H3 variant H3.3. Here we show that H3K36 methylation is reduced globally in human chondroblastomas and in chondrocytes harboring the same genetic mutation, due to inhibition of at least two H3K36 methyltransferases, MMSET and SETD2, by the H3.3K36M mutant proteins. Genes with altered expression as well as H3K36 di- and trimethylation in H3.3K36M cells are enriched in cancer pathways. In addition, H3.3K36M chondrocytes exhibit several hallmarks of cancer cells, including increased ability to form colonies, resistance to apoptosis, and defects in differentiation. Thus, H3.3K36M proteins reprogram the H3K36 methylation landscape and contribute to tumorigenesis, in part through altering the expression of cancer-associated genes.

Histone deacetylase 3 is required for maintenance of bone mass during aging

Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKO(OCN) mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKO(OCN) mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKO(OCN) mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKO(OCN) mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging.

Osteoclast culture and resorption assays.

Bone homeostasis depends on balanced bone deposition and bone resorption, which are mediated by osteoblasts and osteoclasts, respectively. The process of bone turnover requires the coordination of these cells. Changes in the ability of either cell type to perform its function results in pathological conditions such as osteoporosis and tumor-induced bone loss (osteolysis). The number of osteoclasts present at the site of bone remodeling as well as the activity of those osteoclasts the control amount of bone resorbed (1). Therefore, factors affecting overall numbers of osteoclasts and osteoclast activation are key to regulating bone loss. Osteoclast numbers are in part controlled by osteoclast differentiation from bone marrow precursors of the monocyte/macrophage lineage (2). Differentiation of these hematopoietic precursors into osteoclasts is supported by bone marrow stromal cell production of two cytokines, receptor activator of NF-kappaB ligand (RANKL) and macrophage colony stimulating factor (M-CSF), which are both necessary and sufficient to mediate osteoclast differentiation (3, 4). Although RANKL production by the stroma supports osteoclast differentiation, this process is antagonized by osteoprotogerin (OPG) production, which acts as a soluble decoy receptor for RANKL (5, 6). Mechanistic studies to elucidate the factors influencing bone metabolism necessitate in vitro studies of osteoclast differentiation, activation and survival. There are a number of in vitro methods used to culture and study osteoclasts, some of which are described in this chapter.

Epigenetic control of skeletal development by the histone methyltransferase Ezh2

Background: Osteogenic differentiation is initiated by transcriptional and post-transcriptional epigenetic mechanisms. Results: Inhibition of H3K27 methyltransferase EZH2 enhances osteogenic commitment of human mesenchymal progenitors, and its depletion in mouse mesenchymal cells causes multiple skeletal abnormalities. Conclusion: EZH2 is required for skeletal patterning and bone formation. Significance: EZH2-dependent epigenetic mechanisms control osteogenesis both in vitro and in vivo. Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Pathway crosstalk between Ras/Raf and PI3K in promotion of M‐CSF‐induced MEK/ERK‐mediated osteoclast survival

While M‐CSF‐mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M‐CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M‐CSF‐promoted MEK/ERK activation and osteoclast survival was investigated. M‐CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M‐CSF‐mediated Ras activation. As further support for Ras‐mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K‐selective or Raf‐selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M‐CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K‐selective Ras‐mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK‐promoted osteoclast survival induced by M‐CSF. J. Cell. Biochem. 104: 1439–1451, 2008.

Histone Deacetylases in Bone Development and Skeletal Disorders

Histone deacetylases (Hdacs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins. Eleven of the 18 Hdacs encoded by the human and mouse genomes depend on Zn(2+) for enzymatic activity, while the other 7, the sirtuins (Sirts), require NAD2(+). Collectively, Hdacs and Sirts regulate numerous cellular and mitochondrial processes including gene transcription, DNA repair, protein stability, cytoskeletal dynamics, and signaling pathways to affect both development and aging. Of clinical relevance, Hdacs inhibitors are United States Food and Drug Administration-approved cancer therapeutics and are candidate therapies for other common diseases including arthritis, diabetes, epilepsy, heart disease, HIV infection, neurodegeneration, and numerous aging-related disorders. Hdacs and Sirts influence skeletal development, maintenance of mineral density and bone strength by affecting intramembranous and endochondral ossification, as well as bone resorption. With few exceptions, inhibition of Hdac or Sirt activity though either loss-of-function mutations or prolonged chemical inhibition has negative and/or toxic effects on skeletal development and bone mineral density. Specifically, Hdac/Sirt suppression causes abnormalities in physiological development such as craniofacial dimorphisms, short stature, and bone fragility that are associated with several human syndromes or diseases. In contrast, activation of Sirts may protect the skeleton from aging and immobilization-related bone loss. This knowledge may prolong healthspan and prevent adverse events caused by epigenetic therapies that are entering the clinical realm at an unprecedented rate. In this review, we summarize the general properties of Hdacs/Sirts and the research that has revealed their essential functions in bone forming cells (e.g., osteoblasts and chondrocytes) and bone resorbing osteoclasts. Finally, we offer predictions on future research in this area and the utility of this knowledge for orthopedic applications and bone tissue engineering.

Transplanted Senescent Cells Induce an Osteoarthritis-Like Condition in Mice

Abstract Osteoarthritis (OA) is the leading form of arthritis in the elderly, causing pain, disability, and immobility. OA has been associated with accumulation of senescent cells in or near joints. However, evidence for a causal link between OA and cellular senescence is lacking. Here, we present a novel senescent cell transplantation model involving injection of small numbers of senescent or nonsenescent cells from the ear cartilage of luciferase-expressing mice into the knee joint area of wild-type mice. By using bioluminescence and 18FDG PET imaging, we could track the injected cells in vivo for more than 10 days. Transplanting senescent cells into the knee region caused leg pain, impaired mobility, and radiographic and histological changes suggestive of OA. Transplanting nonsenescent cells had less of these effects. Thus, senescent cells can induce an OA-like state and targeting senescent cells could be a promising strategy for treating OA.

Transforming Growth Factor-β Coordinately Induces Suppressor of Cytokine Signaling 3 and Leukemia Inhibitory Factor to Suppress Osteoclast Apoptosis

Local release of TGF-beta during times of high bone turnover leads to elevated levels within the bone microenvironment, and we have shown that TGF-beta suppresses osteoclast apoptosis. Therefore, understanding the influences of TGF-beta on bone resorbing osteoclasts is critical to the design of therapies to reduce excess bone loss. Here we investigated the mechanisms by which TGF-beta sustains suppression of osteoclast apoptosis. We found TGF-beta rapidly increased leukemia inhibitory factor (LIF) expression and secretion by phosphorylated mothers against decapentaplegic-dependent and -independent signaling pathways. TGF-beta also induced suppressor of cytokine signaling 3 (SOCS3) expression, which was required for TGF-beta or LIF to promote osteoclast survival by. Blocking LIF or SOCS3 blocked TGF-beta promotion of osteoclast survival, confirming that LIF and SOCS3 expression are necessary for TGF-beta-mediated suppression of osteoclast apoptosis. Investigation of the mechanisms by which LIF promotes osteoclast survival revealed that LIF-induced expression of Bcl-X(L) and repressed Bcl-2 interacting domain expression by activating MAPK kinase, AKT, and nuclear factor-kappaB pathways. Suppression of Janus kinase/signal transducer and activator of transcription signaling further increased Bcl-X(L) expression and enhanced osteoclast survival, supporting that this pathway is not involved in prosurvival effects of TGF-beta and LIF. These data show that TGF-beta coordinately induces LIF and SOCS3 to promote prosurvival signaling. This alters the ratio of prosurvival Bcl2 family member Bcl-X(L) to proapoptotic family member Bcl-2 interacting domain, leading to prolonged osteoclast survival.

Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis

OBJECTIVE Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in OA progression in a murine model. We also assessed PHLPP1 expression and promoter methylation. DESIGN Knee joints of WT and Phlpp1(-/-) mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression were evaluated in human articular cartilage and chondrocyte cell lines. RESULTS Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1(-/-) mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures (FNFxs). Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines. CONCLUSION Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory cytokines promote PHLPP1 expression.