Shun-ichi Harada

THE MISSING BONE

By analogy to myoblast differentiation, additional transcription factors, possibly linked via regulatory feedback loops to CBFA1, may participate in expression of CBFA1 and mutually regulate each other. This is consistent with its expression in thymocytes, liver, and testis, where it probably participates in the regulation of other genes. Indeed, Ducy and Karsenty 1995xDucy, P and Karsenty, G. Mol. Cell. Biol. 1995; 15: 1858–1869Crossref | PubMedSee all ReferencesDucy and Karsenty 1995 reported on a second regulatory region in the osteocalcin promoter, and different transcription factors that confer bone-specific expression to the α1 promoter of type I collagen, via a distinct cis-acting element, were also recently reported (Rossert et al. 1996xRossert, J.A, Chen, S.S, Eberspaecher, H, Smith, C.N, and de Crombrugghe, B. Proc. Natl. Acad. Sci. USA. 1996; 93: 1027–1031Crossref | PubMedSee all ReferencesRossert et al. 1996).The studies presented here deal with very early osteoblast differentiation during embryogenesis, presumably from a multipotent mesenchymal cell. Bone formation in humans and other vertebrates occurs throughout life as part of continuous skeletal remodeling and occasionally during fracture repair. The two processes differ from each other. Fracture repair usually recapitulates the process of endochondral bone formation, whereas remodeling recruits osteoblastic cells from a pool of committed cells present in the bone marrow. It is of interest if the cells participating in each of these processes express CBFA1, if they express it constitutively, or if it is induced prior to osteoblastic differentiation by BMP or other factors, as observed during embryonic osteogenesis. In other words, in the terminology of developmental biology: what is the relationship of CBFA1 to osteoblast commitment and to sustained phenotype expression? Last but not least: how is the expression of CBFA1 regulated, could it be pharmacologically controlled to stimulate bone formation therapeutically, for example in osteoporosis?In conclusion, following the myogenic and adipogenic systems, we now have the beginning of significant molecular insights into the differentiation of the osteogenic lineage (See Figure 1Figure 1). Interestingly, each of these three mesenchyme-derived cells are induced by transcription factors belonging to completely different families, HLH proteins, a nuclear receptor, and now a core binding factor; family relations can be complicated but can be resolved if patiently pursued.Figure 1Differentiation Pathways for Pluripotent Mesenchymal CellsSee text for details.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Receptor Tyrosine Kinases Inhibit Bone Morphogenetic Protein-Smad Responsive Promoter Activity and Differentiation of Murine MC3T3-E1 Osteoblast-like Cells

Growth factors such as fibroblast growth factor‐2 (FGF‐2) and epidermal growth factor (EGF) that activate extracellular signal‐regulated kinases (ERKs) through receptor tyrosine kinases (RTKs) stimulate proliferation but suppress differentiation of osteoblasts. To study the mechanism of this inhibitory action of these growth factors on osteoblastic differentiation, we evaluated Smad1 transactivity in MC3T3‐E1 osteoblast‐like cells by reporters of promoter activity of mouse Smad6, an early response gene to bone morphogenetic proteins (BMPs). FGF‐2 and EGF inhibited alkaline phosphatase activity and Smad6 promoter activity stimulated by BMP‐2. Overexpression of constitutively active MEK by adenovirus mimicked, but that of dominant negative Ras or treatment with a MEK1 inhibitor, PD098059, reversed, the inhibitory effects of these growth factors on both activities. These effects are mediated by BMP‐responsive elements (BMPREs) on Smad6 promoter, because an artificial reporter driven by three tandem BMPREs gave similar results, and these effects were all abolished when the BMPREs were mutated. RTK‐ERK activation inhibited the promoter activity even when BMP signal was mediated by a mutant Smad1, which lacks phosphorylation sites by ERKs, or by a Smad1 fused to Gal4 DNA binding domain, which constitutively localizes in the nucleus. These results show that the RTK‐Ras‐ERK pathway suppresses BMP signal by interfering with Smad1 transactivity. Because direct phosphorylation of Smad1 by ERKs is not required for the inhibition, other transcriptional factors that are phosphorylated by ERKs might be involved in the regulation of osteoblastic differentiation by ERKs.

Sox8 is a critical regulator of adult Sertoli cell function and male fertility

Sox8 encodes a high-mobility group transcription factor that is widely expressed during development. Sox8, -9 and -10 form group E of the Sox gene family which has been implicated in several human developmental disorders. In contrast to other SoxE genes, the role of Sox8 is unclear and Sox8 mouse mutants reportedly showed only idiopathic weight loss and reduced bone density. The careful analysis of our Sox8 null mice, however, revealed a progressive male infertility phenotype. Sox8 null males only sporadically produced litters of reduced size at young ages. We have shown that SOX8 protein is a product of adult Sertoli cells and its elimination results in an age-dependent deregulation of spermatogenesis, characterized by sloughing of spermatocytes and round spermatids, spermiation failure and a progressive disorganization of the spermatogenic cycle, which resulted in the inappropriate placement and juxtaposition of germ cell types within the epithelium. Those sperm that did enter the epididymides displayed abnormal motility. These data show that SOX8 is a critical regulator of adult Sertoli cell function and is required for both its cytoarchitectural and paracrine interactions with germ cells.

Expression of the prostaglandin E2 (PGE2) receptor subtype EP4 and its regulation by PGE2 in osteoblastic cell lines and adult rat bone tissue1

Abstract Prostaglandins E (especially PGE 2 ) stimulate bone formation and increase bone mass in several species including man. The mechanism for this effect, the target cells, and the receptors involved are not known. Specific cell-surface receptors for PGE 2 (EP 1–4 ) have been cloned and characterized. EP 4 was reported to be the major receptor in embryonic and neonatal bone tissue in mice, especially in preosteoblasts; however, no data are available regarding its expression in adult bone. This study examines the expression of EP 4 in bone tissue of young adult rats, in which PGE 2 is markedly anabolic, and in various osteoblastic cell lines. Using northern blot analysis, we found that osteoblastic cell lines RCT-1, RCT-3, TRAB-11, and RP-1, primary osteoblastic cells harvested from fetal rat calvaria, as well as tibiae and calvariae of 5-week-old rats express 3.8 kb EP 4 messenger RNA (mRNA). Treatment of periosteal cells (RP-1) in vitro with 10 −6 mol/L PGE 2 increased the levels of both EP 4 mRNA and EP 4 protein, peaking at 1–2 h. Similarly, systemic administration of an anabolic dose of PGE 2 (3–6 mg/kg) to young adult rats upregulated the expression of EP 4 in the tibia and calvaria, also peaking at 1–2 h. Using in situ hybridization, we found increased expression of EP 4 in bone marrow cells of the tibial metaphysis in response to systemic PGE 2 treatment. The preosteoblastic nature of these EP 4 -expressing cells was suggested by the fact that dexamethasone-treated bone marrow stromal cells in culture express EP 4 mRNA, which is upregulated by PGE 2 . Northern blot analysis failed to detect both basal and PGE 2 -induced EP 2 mRNA in the bone samples or cell lines tested. Taken together, these data implicate EP 4 as the major cyclic AMP-related PGE 2 receptor subtype expressed in bone tissue and osteoblastic cells and indicate that this receptor is upregulated by its ligand, PGE 2 .

A Regulatory Circuit Mediating Convergence between Nurr1 Transcriptional Regulation and Wnt Signaling

ABSTRACT The orphan nuclear receptor Nurr1 is essential for the development and maintenance of midbrain dopaminergic neurons, the cells that degenerate during Parkinsons disease, by promoting the transcription of genes involved in dopaminergic neurotransmission. Since Nurr1 lacks a classical ligand-binding pocket, it is not clear which factors regulate its activity and how these factors are affected during disease pathogenesis. Since Wnt signaling via β-catenin promotes the differentiation of Nurr1+ dopaminergic precursors in vitro, we tested for functional interactions between these systems. We found that β-catenin and Nurr1 functionally interact at multiple levels. In the absence of β-catenin, Nurr1 is associated with Lef-1 in corepressor complexes. β-Catenin binds Nurr1 and disrupts these corepressor complexes, leading to coactivator recruitment and induction of Wnt- and Nurr1-responsive genes. We then identified KCNIP4/calsenilin-like protein as being responsive to concurrent activation by Nurr1 and β-catenin. Since KCNIP4 interacts with presenilins, the Alzheimers disease-associated proteins that promote β-catenin degradation, we tested the possibility that KCNIP4 induction regulates β-catenin signaling. KCNIP4 induction limited β-catenin activity in a presenilin-dependent manner, thereby serving as a negative feedback loop; furthermore, Nurr1 inhibition of β-catenin activity was absent in PS1−/− cells or in the presence of small interfering RNAs specific to KCNIP4. These data describe regulatory convergence between Nurr1 and β-catenin, providing a mechanism by which Nurr1 could be regulated by Wnt signaling.

Discovery of the Selective Androgen Receptor Modulator MK-0773 Using a Rational Development Strategy Based on Differential Transcriptional Requirements for Androgenic Anabolism Versus Reproductive Physiology

Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40–80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs.

Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC50, 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5α-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands.

The Role of Prostaglandins in Bone Formation

Prostaglandins of the E series have been shown to be effective inducers of bone formation in vivo. In this study, the effects of PGE2 were evaluated in vivo using subcutaneous administration (3 mg/kg/d for 25 days) to ovariectomized rats or local application in the marrow cavity of tibiae of rats using biodegradable implants (0.13, 1.4 and 32 microg released over 8 days). Systemic treatment of rats with PGE2 stimulated cancellous bone formation in the metaphysis of the proximal tibiae as well as endocortical bone formation and de novo trabecular bone formation in the marrow cavity. Local delivery of PGE2 increased cancellous bone volume in the secondary spongiosa and cortical thickness (at 32 microg). Comparisons of prostanoid effects in vitro, in a bone-derived cell line, showed that PGF2alpha was a better stimulator of DNA synthesis than PGE2. PGF2alpha increased the steady state levels of IGF-I receptor mRNA while PGE2 increased IGF-I expression. Although the mechanism of bone formation by PGE2 is not known at this time, it is clear that PGE2 has powerful local anabolic effects on bone formation in vivo possibly by mediating responses to signals such as changes in mechanical force.

Suppression of Adjuvant-Induced Arthritic Bone Destruction by Cyclooxygenase-2 Selective Agents With and Without Inhibitory Potency Against Carbonic Anhydrase II†

In vitro assays revealed that COX‐2 inhibitors with CA II inhibitory potency suppressed both differentiation and activity of osteoclasts, whereas that without the potency reduced only osteoclast differentiation. However, all COX‐2 inhibitors similarly suppressed bone destruction in adjuvant‐induced arthritic rats, indicating that suppression of osteoclast differentiation is more effective than that of osteoclast activity for the treatment.

Downstream Genes of Sox8 That Would Affect Adult Male Fertility

Sertoli cells provide nutritional and physical support to germ cells during spermatogenesis. Sox8 encodes a high mobility group transcription factor closely related to Sox9 and Sox10. Sertoli cells produceSOX8 protein, and its elimination results in an age-dependent deregulation of spermatogenesis resulting in male infertility. This suggests that Sox8 is a critical regulator of Sertoli cell function for the maintenance of male fertility beyond the first wave of spermatogenesis. To better understand the roles of Sox8 in testicular development and maintenance of male fertility, we have performed a detailed analysis to explore its downstream genes. We have used mRNA expression profiling to identify affected genes in Sertoli cells in the mutant testes of 2-month-old mice. Expression profiling of the heterozygous and homozygous Sox8 mutant testes indicates alterations in several important spermatogenesis and blood-testis barrier genes, providing insight into the molecular basis of the defects in Sox8–/– testes beyond the first wave of spermatogenesis.