Yuuki Imai

Estrogen Prevents Bone Loss via Estrogen Receptor α and Induction of Fas Ligand in Osteoclasts

Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.

Cathepsin K-Dependent Toll-Like Receptor 9 Signaling Revealed in Experimental Arthritis

Cathepsin K was originally identified as an osteoclast-specific lysosomal protease, the inhibitor of which has been considered might have therapeutic potential. We show that inhibition of cathepsin K could potently suppress autoimmune inflammation of the joints as well as osteoclastic bone resorption in autoimmune arthritis. Furthermore, cathepsin K–/– mice were resistant to experimental autoimmune encephalomyelitis. Pharmacological inhibition or targeted disruption of cathepsin K resulted in defective Toll-like receptor 9 signaling in dendritic cells in response to unmethylated CpG DNA, which in turn led to attenuated induction of T helper 17 cells, without affecting the antigen-presenting ability of dendritic cells. These results suggest that cathepsin K plays an important role in the immune system and may serve as a valid therapeutic target in autoimmune diseases.

Targeting androgen receptor in estrogen receptor-negative breast cancer.

Endocrine therapies for breast cancer that target the estrogen receptor (ER) are ineffective in the 25%-30% of cases that are ER negative (ER-). Androgen receptor (AR) is expressed in 60%-70% of breast tumors, independent of ER status. How androgens and AR regulate breast cancer growth remains largely unknown. We find that AR is enriched in ER- breast tumors that overexpress HER2. Through analysis of the AR cistrome and androgen-regulated gene expression in ER-/HER2+ breast cancers we find that AR mediates ligand-dependent activation of Wnt and HER2 signaling pathways through direct transcriptional induction of WNT7B and HER3. Specific targeting of AR, Wnt or HER2 signaling impairs androgen-stimulated tumor cell growth suggesting potential therapeutic approaches for ER-/HER2+ breast cancers.

GlcNAcylation of histone H2B facilitates its monoubiquitination

Chromatin reorganization is governed by multiple post-translational modifications of chromosomal proteins and DNA. These histone modifications are reversible, dynamic events that can regulate DNA-driven cellular processes. However, the molecular mechanisms that coordinate histone modification patterns remain largely unknown. In metazoans, reversible protein modification by O-linked N-acetylglucosamine (GlcNAc) is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). However, the significance of GlcNAcylation in chromatin reorganization remains elusive. Here we report that histone H2B is GlcNAcylated at residue S112 by OGT in vitro and in living cells. Histone GlcNAcylation fluctuated in response to extracellular glucose through the hexosamine biosynthesis pathway (HBP). H2B S112 GlcNAcylation promotes K120 monoubiquitination, in which the GlcNAc moiety can serve as an anchor for a histone H2B ubiquitin ligase. H2B S112 GlcNAc was localized to euchromatic areas on fly polytene chromosomes. In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over chromosomes including transcribed gene loci, with some sites co-localizing with H2B K120 monoubiquitination. These findings suggest that H2B S112 GlcNAcylation is a histone modification that facilitates H2BK120 monoubiquitination, presumably for transcriptional activation.

PKA-dependent regulation of the histone lysine demethylase complex PHF2–ARID5B

Reversible histone methylation and demethylation are highly regulated processes that are crucial for chromatin reorganization and regulation of gene transcription in response to extracellular conditions. However, the mechanisms that regulate histone-modifying enzymes are largely unknown. Here, we characterized a protein kinase A (PKA)-dependent histone lysine demethylase complex, PHF2–ARID5B. PHF2, a jmjC demethylase, is enzymatically inactive by itself, but becomes an active H3K9Me2 demethylase through PKA-mediated phosphorylation. We found that phosphorylated PHF2 then associates with ARID5B, a DNA-binding protein, and induce demethylation of methylated ARID5B. This modification leads to targeting of the PHF2–ARID5B complex to its target promoters, where it removes the repressive H3K9Me2 mark. These findings suggest that the PHF2–ARID5B complex is a signal-sensing modulator of histone methylation and gene transcription, in which phosphorylation of PHF2 enables subsequent formation of a competent and specific histone demethylase complex.

Estrogens Maintain Bone Mass by Regulating Expression of Genes Controlling Function and Life Span in Mature Osteoclasts

Estrogens play a key role in regulation of bone mass and strength by controlling activity of bone‐forming osteoblasts and bone‐resorbing osteoclasts. Cellular effects of estrogens are mediated predominantly by the action of estrogen receptor alpha (ERα). In earlier studies, ablation of the ERα gene in mice did not result in osteoporotic phenotypes due to systemic endocrine disturbance and compensatory effects of elevated levels of testosterone. Despite the relatively well‐established effects in osteoblasts, little is known about the direct action of estrogen in osteoclasts. Development in the last decade of more sophisticated genetic manipulation approaches opened new possibilities to explore cell‐specific roles of nuclear receptors in bone tissue. Recently, we have generated osteoclast‐specific ERα gene knockout mice and shown that in vivo estrogens directly regulate the life span of mature osteoclasts by inducing the expression of pro‐apoptotic Fas ligand (FasL). Inhibitory effects of estrogens on osteoclast function were further studied in vitro. We observed sufficiently detectable ERα expression in osteoclasts differentiating from primary bone marrow cells or RAW264 cells, although levels of ERα were decreasing during progression of the differentiation into mature osteoclasts. Treatment with estrogens led to reduction in expression of osteoclast‐specific genes controlling bone resorption activity. However, estrogens did not affect the size of multinucleated osteoclasts or number of nuclei in a mature osteoclast. In conclusion, in osteoclasts, estrogens function to inhibit bone resorption activity and vitality rather than differentiation.

Minireview: Osteoprotective Action of Estrogens Is Mediated by Osteoclastic Estrogen Receptor-α

The osteoprotective action of estrogen in women has drawn considerable attention because estrogen deficiency-induced osteoporosis became one of the most widely spread diseases in developed countries. In men, the significance of estrogen action for bone health maintenance is also apparent from the osteoporotic phenotype seen in male patients with genetically impaired estrogen signaling. Severe bone loss and high bone turnover, including typical osteofeatures seen in postmenopausal women, can also be recapitulated in rodents after ovariectomy. However, the expected osteoporotic phenotype is not observed in female mice deficient in estrogen receptor (ER)-alpha or -beta or both, even though the degenerative defects are clearly seen in other estrogen target tissues together with up-regulated levels of circulating testosterone. It has also been reported that estrogens may attenuate bone remodeling by cell autonomous suppressive effects on osteoblastogenesis and osteoclastogenesis. Hence, the effects of estrogens in bone appear to be complex, and the molecular role of bone estrogen receptors in osteoprotective estrogen action remains unclear. Instead, it has been proposed that estrogens indirectly control bone remodeling. For example, the enhanced production of cytokines under estrogen deficiency induces bone resorption through stimulation of osteoclastogenesis. However, the osteoporotic phenotype without systemic defects has been recapitulated in female (but not in male) mice by osteoclast-specific ablation of the ERalpha, proving that bone cells represent direct targets for estrogen action. An aberrant accumulation of mature osteoclasts in these female mutants indicates that in females, the inhibitory action of estrogens on bone resorption is mediated by the osteoclastic ERalpha through the shortened lifespan of osteoclasts.

ERKs Induce Expression of the Transcriptional Repressor Blimp-1 and Subsequent Plasma Cell Differentiation

The differentiation of B cells into antibody-producing plasma cells requires extracellular signal–regulated kinases. Differential Role for ERKs Given that cellular differentiation is associated with cell cycle arrest, the processes of differentiation and proliferation are thought to be mutually exclusive, and the transcriptional programs underlying these events are frequently stimulated by distinct extracellular cues. The extracellular signal–regulated kinase (ERK) signaling pathway is responsible for driving proliferation in many cell types, including immune cells, and thus is thought to be involved only in dividing cells (see the Perspective by Allman and Cancro). In B cells, the transcriptional program of proliferating cells is suppressed by the transcriptional repressor Blimp-1, which is required for the differentiation of B cells into antibody-presenting plasma cells, a terminally differentiated cell type. Through experiments with mice deficient in both ERK1 and ERK2 in B cells, Yasuda et al. provide evidence of a critical role for the ERKs in driving the differentiation of plasma cells and show that ERK signaling is required for the expression of Blimp-1. Together, these data expand the role of ERK signaling in B cells and should prompt investigation of the potential involvement of ERK proteins in the differentiation of other cell types. In immune cells, the positive role of the extracellular signal–regulated kinase (ERK) signaling pathway in cell cycle progression and survival is well established; however, it is unclear whether ERK signaling plays a role in cell differentiation. Here, we report that ERKs are essential for the differentiation of B cells into antibody-producing plasma cells and that ERKs induce the expression of Prdm1, which encodes Blimp-1, a transcriptional repressor and “master regulator” of plasma cell differentiation. Transgenic mice with conditional deletion of both ERK1 and ERK2 in germinal center (GC) B cells lacked plasma cells differentiated after GC formation, and memory B cells from these mice failed to differentiate into plasma cells. In addition, ERK1- and ERK2-deficient B cells exhibited impaired Prdm1 expression upon stimulation with antibody against CD40 in the presence of interleukin-4; conversely, enforced expression of Prdm1 in ERK1- and ERK2-deficient B cells restored the generation of plasma cells. Thus, our study suggests that cytokines stimulate ERKs to induce the production of Blimp-1 and that ERKs thereby contribute to the process of cellular differentiation.

Local delivery of siRNA using a biodegradable polymer application to enhance BMP-induced bone formation.

Small interfering RNA (siRNA) is useful tool for specific and efficient knockdown of disease-related genes. However, in vivo applications of siRNA are limited due to difficulty in its efficient delivery to target cells. In this study, we investigated the efficacy of a biodegradable hydrogel, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block co-polymer (PLA-DX-PEG), as a siRNA carrier. PLA-DX-PEG pellets with or without fluorescein-labeled dsRNA were implanted into mouse dosal muscle pouches. The cellular uptake of dsRNA surround the polymer was confirmed by fluorescent microscopy. The fluorescence intensity was dose-dependent of the dsRNA, and exhibited a time-dependent decrease. To investigate its biological efficiency, noggin (antagonoist to BMPs) gene-silencing with siRNA (siRNA/Noggin) was examined by the amount of suppression of BMP-2-induced noggin expression and the level of performance of BMP, indicated by ectopic bone formation. Noggin gene expression induced by BMP-2 was suppressed by addition of siRNA/Noggin to the implant, and the ectopic bone formation induced by implants with both BMP-2 and siRNA/Noggin was significantly greater than those induced by implants with BMP-2 alone. These results indicate the efficacy of local delivery of siRNAs by PLA-DX-PEG polymer, which intensified bone-inducing effects of BMP and promoted new bone formation by suppressing gene expression of Noggin.

Nuclear Receptors in Bone Physiology and Diseases

During the last decade, our view on the skeleton as a mere solid physical support structure has been transformed, as bone emerged as a dynamic, constantly remodeling tissue with systemic regulatory functions including those of an endocrine organ. Reflecting this remarkable functional complexity, distinct classes of humoral and intracellular regulatory factors have been shown to control vital processes in the bone. Among these regulators, nuclear receptors (NRs) play fundamental roles in bone development, growth, and maintenance. NRs are DNA-binding transcription factors that act as intracellular transducers of the respective ligand signaling pathways through modulation of expression of specific sets of cognate target genes. Aberrant NR signaling caused by receptor or ligand deficiency may profoundly affect bone health and compromise skeletal functions. Ligand dependency of NR action underlies a major strategy of therapeutic intervention to correct aberrant NR signaling, and significant efforts have been made to design novel synthetic NR ligands with enhanced beneficial properties and reduced potential negative side effects. As an example, estrogen deficiency causes bone loss and leads to development of osteoporosis, the most prevalent skeletal disorder in postmenopausal women. Since administration of natural estrogens for the treatment of osteoporosis often associates with undesirable side effects, several synthetic estrogen receptor ligands have been developed with higher therapeutic efficacy and specificity. This review presents current progress in our understanding of the roles of various nuclear receptor-mediated signaling pathways in bone physiology and disease, and in development of advanced NR ligands for treatment of common skeletal disorders.