SUMMARY Sox9 encodes an essential transcriptional regulator of chondrocyte specification and differentiation. When Sox9 nuclear activity was compared with markers of chromatin organization and transcriptional activity in primary chondrocytes, we identified two distinct categories of target association. Class I sites cluster around the transcriptional start sites of highly expressed genes with no chondrocyte-specific signature. Here, Sox9 association reflects protein-protein association with basal transcriptional components. Class II sites highlight evolutionarily conserved active enhancers directing chondrocyte-related gene activity through direct binding of Sox9 dimer complexes to DNA. Sox9 binds through sites with sub-optimal binding affinity; the number and grouping of enhancers into super-enhancer clusters likely determines the levels of target gene expression. Interestingly, comparison of Sox9 action in distinct chondrocyte lineages points to similar regulatory strategies. In addition to providing insights into Sox family action, our comprehensive identification of the chondrocyte regulatory genome will facilitate study of skeletal development and human disease.

SUMMARY Bone formation in extant species is restricted to vertebrate species. Sp7/Osterix is a key transcriptional determinant of bone-secreting osteoblasts. We performed Sp7 ChIP-seq analysis identifying a large set of predicted osteoblast enhancers and validated a subset of these in cell culture and transgenic mouse assays. Sp-family members bind GC-rich target sequences through their zinc finger domain. Several lines of evidence suggest Sp7 acts differently, engaging osteoblast targets in Dlx-containing regulatory complexes bound to AT-rich motifs. Amino acid differences in the Sp7 zinc finger domain reduce Sp7's affinity for the Sp-family consensus GC-box target; Dlx5 binding maps to this domain of Sp7. The data support a model in which Dlx recruitment of Sp7 to osteoblast enhancers underlies Sp7-directed osteoblast specification. As an Sp7-like zinc finger variant is restricted to vertebrates, the emergence of an Sp7 member within the Sp family was likely closely coupled to evolution of the bone forming vertebrates.

Nrf2 (nuclear factor E2 p45-related factor 2) is believed to be a transcription factor essential for the regulation of many detoxifying and antioxidative genes in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of osteoblastic differentiation. nrf2 mRNA expression was significantly up-regulated in femur isolated from ovariectomized mice, whereas in situ hybridization analysis revealed that up-regulation of nrf2 mRNA was mainly found in osteoblasts attached on cancellous bone in femur of ovariectomized mice. Expression of Nrf2 protein was also seen in osteoblasts in neonatal mouse tibia and calvaria. In osteoblastic MC3T3-E1 cells stably transfected with nrf2 expression vector, significant inhibition was seen in the maturation-dependent increase in alkaline phosphatase activity as well as the mineralized matrix formation. Stable overexpression of nrf2 significantly impaired Runx2 (runt-related transcription factor 2)-dependent stimulation of osteocalcin promoter activity and recruitment of Runx2 on osteocalcin promoter without affecting the expression of runx2 mRNA. Coimmunoprecipitation and mammalian two-hybrid assay revealed a physical interaction between Runx2 and Nrf2, whereas cellular distribution of endogenous Runx2 was not apparently changed by nrf2 overexpression in MC3T3-E1 cells. Alternatively, Nrf2 bound to antioxidant-responsive element-like-2 sequence of osteocalcin promoter. The inhibition by nrf2 on runx2-dependent osteocalcin promoter activity was partially prevented by the introduction of reporter of deletion mutant for ARE-like-2 sequence of osteocalcin promoter. These data suggest that Nrf2 may negatively regulate cellular differentiation through inhibition of the Runx2-dependent transcriptional activity in osteoblasts.In bone tissues, both formation and maintenance are sophisticatedly regulated by bone-forming osteoblasts and bone-resorbing osteoclasts (1-3). The osteoblast lineage is derived from primitive multipotent mesenchymal stem cells with potentiality to differentiate into bone marrow stromal cells, chondrocytes, muscles, and adipocytes (4), whereas osteoclasts are multinucleated cells derived from the fusion of mononuclear hematopoietic precursors (3). The development and differentiation of these two distinct cells are under tight regulation by a number of endogenous substances. These include growth factors, cytokines, and hormones, which are individually secreted through endocrine, paracrine/autocrine, and neurocrine systems essential for the delicate balancing between bone formation and resorption by the two different cells in the bone marrow microenvironment. An imbalance between these two cells leads to pathogenesis and etiology of certain bone metabolic diseases, including osteoporosis and osteopetrosis (5, 6). Ovariectomy has been employed to experimentally establish model animals with bone loss seen in postmenopausal osteoporosis caused by estrogen deficiency (7). In ovariectomized animals, osteoblast indices, such as osteoblast numbe...

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SummaryPluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

Organoids generated from pluripotent stem cells are used in the development of organ replacement regenerative therapy by recapitulating the process of organogenesis. These processes are strictly regulated by morphogen signalling and transcriptional networks. However, the precise transcription factors involved in the organogenesis of exocrine glands, including salivary glands, remain unknown. Here, we identify a specific combination of two transcription factors (Sox9 and Foxc1) responsible for the differentiation of mouse embryonic stem cell-derived oral ectoderm into the salivary gland rudiment in an organoid culture system. Following orthotopic transplantation into mice whose salivary glands had been removed, the induced salivary gland rudiment not only showed a similar morphology and gene expression profile to those of the embryonic salivary gland rudiment of normal mice but also exhibited characteristics of mature salivary glands, including saliva secretion. This study suggests that exocrine glands can be induced from pluripotent stem cells for organ replacement regenerative therapy.

Background: Gli1 was thought to be dispensable for normal embryogenesis. Results: Gli1 physiologically acts as an osteogenesis-related Gli activator and mediates the functions of Gli2 and Gli3 in osteogenesis. Conclusion: Gli1 collectively functions with Gli2 and Gli3 in osteogenesis.Significance: This study provides insight into the elaborate molecular framework of a context-dependent divergence of functions of Hedgehog-Gli signaling in development.

Nanoscaled drug-loaded carriers are of particular interest for efficient tumor therapy as numerous studies have shown improved targeting and efficacy. Nevertheless, most of these studies have been performed against allograft and xenograft tumor models, which have altered microenvironment features affecting the accumulation and penetration of nanocarriers. Conversely, the evaluation of nanocarriers on genetically engineered mice, which can gradually develop clinically relevant tumors, permits the validation of their design under normal processes of immunity, angiogenesis, and inflammation. Therefore, considering the poor prognosis of pancreatic cancer, we used the elastase 1-promoted luciferase and Simian virus 40 T and t antigens transgenic mice, which develop spontaneous bioluminescent pancreatic carcinoma, and showed that long circulating micellar nanocarriers, incorporating the parent complex of oxaliplatin, inhibited the tumor growth as a result of their efficient accumulation and penetration in the tumors. The reduction of the photon flux from the endogenous tumor by the micelles correlated with the decrease of serum carbohydrate-associated antigen 19-9 marker. Micelles also reduced the incidence of metastasis and ascites, extending the survival of the transgenic mice.chemotherapy | drug delivery | (1,2-diaminocyclohexane)platinum (II)