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Posttranscriptional regulation of IL-6 has been largely uncharacterized, with the exception of the ribonuclease Regnase-1, which prevents autoimmunity by destabilizing IL-6 mRNA. Here, we identified AT-rich interactive domain-containing protein 5A (Arid5a) as a unique RNA binding protein, which stabilizes IL-6 but not TNF-a mRNA through binding to the 3' untranslated region of IL-6 mRNA. Arid5a was enhanced in macrophages in response to LPS, IL-1β, and IL-6. Arid5a deficiency inhibited elevation of IL-6 serum level in LPS-treated mice and suppressed IL-6 levels and the development of TH17 cells in experimental autoimmune encephalomyelitis. Importantly, Arid5a inhibited the destabilizing effect of Regnase-1 on IL-6 mRNA. These results indicate that Arid5a plays an important role in promotion of inflammatory processes and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Differentiation of ameloblasts from undifferentiated epithelial cells is controlled by diverse growth factors, as well as interactions between epithelium and mesenchyme. However, there is a considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. We found that the expression level of carbonic anhydrase II (CAII) is strongly up-regulated in parallel with differentiation of enamel epithelium tissues, while the enzyme activity of CA was also increased along with differentiation in ameloblast primary cultures. The expression level of amelogenin, a marker of secretory-stage ameloblasts, was enhanced by ethoxzolamide (EZA), a CA inhibitor, as well as CAII antisense (CAIIAS), whereas the expression of enamel matrix serine proteinase-1 (EMSP-1), a marker for maturation-stage ameloblasts, was suppressed by both. These agents also promoted ameloblast proliferation. In addition, inhibition of ameloblast differentiation by EZA and CAIIAS was confirmed using tooth germ organ cultures. Furthermore, EZA and CAIIAS elevated intracellular pH in ameloblasts, while experimental decreases in intracellular pH abolished the effect of CAIIAS on ameloblasts and triggered the activation of c-Jun N-terminal kinase (JNK). SP600125, a JNK inhibitor, abrogated the response of ameloblasts to an experimental decrease in intracellular pH, while the inhibition of JNK also impaired ameloblast differentiation. These results suggest a novel role for CAII during amelogenesis, that is, controlling the differentiation of ameloblasts. Regulation of intracellular pH, followed by activation of the JNK signaling pathway, may be responsible for the effects of CAII on ameloblasts. J. Cell. Physiol. 225: 709-719, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The article examines the role of RANKL-induced activation of MEKK1/JNK/c-jun signaling in the regulation of apoptosis. The scientists' study suggests that c-fos plays a role as a partner of activator protein-1 factor, c-jun, during the regulation of apoptosis in osteoclasts. Also discussed is the abrogation of sRANKL.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce. [ABSTRACT FROM AUTHOR]

ABSTRACT The growth plate is the primary site of longitudinal bone growth with chondrocytes playing a pivotal role in endochondral bone development. Chondrocytes undergo a series of differentiation steps, resulting in the formation of a unique hierarchical columnar structure comprising round, proliferating, pre‐hypertrophic, and hypertrophic chondrocytes. Pre‐hypertrophic chondrocytes, which exist in the transitional stage between proliferating and hypertrophic stages, are a critical cell population in the growth plate. However, the molecular basis of pre‐hypertrophic chondrocytes remains largely undefined. Here, we employed scRNA‐seq analysis on fluorescently labeled growth plate chondrocytes for their molecular characterization. Serine incorporator 5 (Serinc5) was identified as a marker gene for pre‐hypertrophic chondrocytes. Histological analysis revealed that Serinc5 is specifically expressed in pre‐hypertrophic chondrocytes, overlapping with Indian hedgehog (Ihh). Serinc5 represses cell proliferation and Col2a1 and Acan expression by inhibiting the transcriptional activity of Sox9 in primary chondrocytes. Chromatin profiling using ChIP‐seq and ATAC‐seq revealed an active enhancer of Serinc5 located in intron 1, with its chromatin status progressively activated during chondrocyte differentiation. Collectively, our findings suggest that Serinc5 regulates sequential chondrocyte differentiation from proliferation to hypertrophy by inhibiting Sox9 function in pre‐hypertrophic chondrocytes, providing novel insights into the mechanisms underlying chondrocyte differentiation in growth plates. [ABSTRACT FROM AUTHOR]

Investigates the importance of bone morphogenetic protein (BMP) receptor signaling in murine postnatal bone formation. Impairment of bone growth and reduction of bone mineral density in truncated BMP receptor; Role of the receptor in osteoblast differentiation; Stimulation of osteoblast proliferation by BMP.

Purpose: Lymph node metastasis is a key prognostic determinant in cancer patients. To understand the molecular basis underlying lymph node metastasis, we studied the SAS oral squamous cancer cells which metastasized to the regional lymph nodes following tongue inoculation. Material and methods: In our previous study, highly metastatic SAS cells (SAS-LM8) were established by repeated in vivo inoculation and FACS selection. Lymphangiogenesis was determined by immunohistochemistry using anti-LYVE1 antibody, well-established marker for lymphatic vessels. Results: SAS-LM8 tumors showed increased lymphangiogenesis and elevated expression of VEGF-C, a potent stimulator of lymphangiogenesis, compared to parental SAS tumors. A metastasis real-time PCR array demonstrated that 6 genes including fibronectin1 (FN1) were at least 4-fold upregulated in SAS-LM8 compared with SAS. FN1 is one of the epithelial mesenchymal transition (EMT) makers. Further analyses revealed that this cell line exhibited phenotypic and molecular alterations consistent with EMT. Importantly, knockdown of FN1 using shRNA decreased VEGF-C expression and lymphangiogenesis. Consistent with these results, immunohistochemical analysis of human oral cancer tissues revealed that the expression of FN1 and VEGF-C significantly correlates with the lymph node metastasis. Conclusions: SAS-LM8 was subject to EMT and mediated by increased FN1. Our results suggest that VEGF-C expression under the control of FN1 plays a critical role in lymph node metastases and that suppression of FN1 and VEGF-C may be a feasible therapeutic approach for lymph node metastasis. [Copyright &y& Elsevier]

Background: Embryonic craniofacial development involves several cellular and molecular events that are evolutionarily conserved among vertebrates. Vertebrate models such as mice and zebrafish have been used to investigate the molecular and cellular etiologies underlying human craniofacial disorders, including orofacial clefts. However, the molecular mechanisms underlying embryonic development in these two species are unknown. Therefore, elucidating the shared mechanisms of craniofacial development between disease models is crucial to understanding the underlying mechanisms of phenotypes in individual species., Results: We selected mice and zebrafish as model organisms to compare various events during embryonic craniofacial development. We identified genes (Sox9, Zfhx3 and 4, Cjun, and Six1) exhibiting similar temporal expression patterns between these species through comprehensive and stage-matched gene expression analyses. Expression analysis revealed similar gene expression in hypothetically corresponding tissues, such as the mice palate and zebrafish ethmoid plate. Furthermore, loss-of-function analysis of Zfhx4/zfhx4, a causative gene of human craniofacial anomalies including orofacial cleft, in both species resulted in deformed skeletal elements such as the palatine and ethmoid plate in mice and zebrafish, respectively., Conclusions: These results demonstrate that these disease models share common molecular mechanisms, highlighting their usefulness in modeling craniofacial defects in humans., (© 2024 Kao Corporation and The Author(s). Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

The sulfate transporter gene SLC26A2 is crucial for skeletal formation, as evidenced by its role in diastrophic dysplasia, a type of skeletal dysplasia in humans. Although SLC26A2-related chondrodysplasia also affects craniofacial and tooth development, its specific role in these processes remains unclear. In this study, we explored the pivotal roles of SLC26A2-mediated sulfate metabolism during tooth development. We found that Slc26a2 was predominantly expressed in dental tissues, including odontoblasts and ameloblasts. Slc26a2 knockout (Slc26a2-KO-Δexon2) mice exhibited distinct craniofacial abnormalities, such as a retrognathic upper jaw, small upper incisors and upper molar hypoplasia. These mice also showed flattened odontoblasts and loss of nuclear polarity in upper incisors and molars, with significant reductions in odontoblast differentiation markers Dspp and Dmp1. Ex vivo and in vitro studies further revealed dentin matrix hypoplasia, tooth root shortening and downregulation of Wnt signaling in Slc26a2-deficient cells. These findings highlight the crucial role of SLC26A2-mediated sulfate metabolism in tooth development and offer insights into the mechanisms underlying dental abnormalities in patients with SLC26A2-related chondrodysplasias., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Osteoarthritis is a common disease in joint cartilages. Because the molecular pathogenesis of osteoarthritis remains elusive, early diagnostic markers and effective therapeutic agents have not been developed. To understand the molecular mechanisms, we attempted to identify transcription factors involved in the onset of osteoarthritis. Microarray analysis of mouse articular cartilage cells indicated that retinoic acid, a destructive stimulus in articular cartilage, up-regulated expression of sex-determining region Y-box (Sox)4, a SoxC family transcription factor, together with increases in Adamts4 and Adamts5, both of which are aggrecanases of articular cartilages. Overexpression of Sox4 induced a disintegrin-like and metallopeptidase with thrombospondin type 4 and 5 motif (ADAMTS4 and ADAMTS5, respectively) expression in chondrogenic cell lines C3H10T1/2 and SW1353. In addition, luciferase reporter and chromatin immunoprecipitation assays showed that Sox4 up-regulated ADAMTS4 and Adamts5 gene promoter activities by binding to their gene promoters. Another SoxC family member, Sox11, evoked similar effects. To evaluate the roles of Sox4 and Sox11 in articular cartilage destruction, we performed organ culture experiments using mouse femoral head cartilages. Sox4 and Sox11 adenovirus infections caused destruction of articular cartilage associated with increased Adamts5 expression. Finally, SOX4 and SOX11 mRNA expression was increased in cartilage of patients with osteoarthritis compared with nonosteoarthritic subjects. Thus, Sox4, and presumably Sox11, are involved in osteoarthritis onset by up-regulating ADAMTS4 and ADAMTS5.-Takahata, Y., Nakamura, E., Hata, K., Wakabayashi, M., Murakami, T., Wakamori, K., Yoshikawa, H., Matsuda, A., Fukui, N., Nishimura, R. Sox4 is involved in osteoarthritic cartilage deterioration through induction of ADAMTS4 and ADAMTS5.

Wnt plays important roles in regulation of differentiation of osteoblast and chondrocyte and their function. Wnt family members ingeniously utilize canonical Wnt signaling pathway through β-catenin and non-canonical Wnt signaling pathway independent of β-catenin, consequently regulating development, formation and homeostasis of bone and cartilage. Recent studies revealed that canonical Wnt signal activates transcriptional regulator, TAZ, in addition to transcription factors, LEF and TCF. Canonical Wnt signal crosstalks with BMP signal by stimulating complex formation of LEF1, TAZ and Runx2. Although molecular mechanism of non-canonical Wnt signal is getting clearer, the precise role of non-canonical Wnt signal in bone and cartilage seems still elusive.

One of the major topics of ASBMR in this year was progress in research on "Bone and muscle crosstalk". It has been practically and clinically known that appropriate exercise has good effects on bones strength, however the mechanism is still elusive. In this ASBMR meeting, there were many reports that muscle-derived hormones, e.g. Irisin, β-aminoisobutyric acid(BAIBA)or Myostatin interact with skeletal cells. Especially, the report regarding Irisin was impressive. Moreover, several reports regarding new aspects of osteoblasts and bone formation were also interesting. In this report, we would like to introduce these interesting abstracts.

Chondrocytes, which are originated from undifferentiated mesenchymal stem cells, play roles in skeletal development and growth in mammal, and smooth movement of joints. Endochondral ossification is necessary for skeletal development, and the multiple and complex biological events are precisely regulated by several hormones, cytokines, and their downstream signaling and transcriptional regulation. On the other hands, articular chondrocytes physiologically retains their features during a lifetime. Numerous molecules involved in endochondral ossification have been identified and investigation of the molecular mechanisms have amazingly progressed. Although GDF5 and Prg4 were identified as important molecules associated with articular cartilage development and its homeostasis, the molecular mechanisms are very unclear to date.

Chondrocytes are derived from mesenchymal stem cells and play an essential role in endochondral ossification. Transcription factors, Sox9, Runx2, Runx3 and Osterix are critical for endochondral ossification, and regulate differentiation of mesenchymal stem cells into chondrocytes, and proliferation, maturation and apoptosis of chondrocytes. Recent advances in gene cloning approaches utilizing microarray and high-throughput sequencing technologies have revealed functional regulatory mechanisms of these transcription factors and contributed to understanding of molecular mechanisms of complex and harmonious chondrocyte differentiation.

Genetic and biochemical studies have identified transcription factors critical and specific for bone and cartilage development. More recent studies revealed the molecular mechanisms how these transcription factors regulate bone and cartilage development. Especially, we appreciate recent advances in molecular function of the complex assembled by these transcription factors and epigenetic regulation of them. Aging, inflammation, biological stress, and disorder of endocrine system induce several bone and/or cartilage diseases by affecting the transcriptional and epigenetic regulation. In this review, we would like to describe the transcriptional and epigenetic regulation during developmental and pathological stages. In addition, we discuss possible application of these information in regeneration of bone and cartilage.

Posttranscriptional regulation of IL-6 has been largely uncharacterized, with the exception of the ribonuclease Regnase-1, which prevents autoimmunity by destabilizing IL-6 mRNA. Here, we identified AT-rich interactive domain-containing protein 5A (Arid5a) as a unique RNA binding protein, which stabilizes IL-6 but not TNF-α mRNA through binding to the 3' untranslated region of IL-6 mRNA. Arid5a was enhanced in macrophages in response to LPS, IL-1β, and IL-6. Arid5a deficiency inhibited elevation of IL-6 serum level in LPS-treated mice and suppressed IL-6 levels and the development of T(H)17 cells in experimental autoimmune encephalomyelitis. Importantly, Arid5a inhibited the destabilizing effect of Regnase-1 on IL-6 mRNA. These results indicate that Arid5a plays an important role in promotion of inflammatory processes and autoimmune diseases.

Endochondral ossification plays critical roles in skeletal development and tissue patterning. Disruption of endochondral ossification and cartilage homeostasis results in skeletal dysplasia and cartilage diseases such as osteoarthritis. Several transcription factors regulate chondrocyte differentiation and expression of chondrogenic matrices, and consequently maintains order of endochondral ossification. In this review article, we would like to introduce recent advancement in understanding of role and functional regulation of transcription factors in endochondral ossification.

Differentiation process of osteoblasts, which play a central role in bone formation, is harmoniously controlled by several cytokines and hormones. Particularly, Bone Morphogenetic Protein (BMP) , Indian Hedgehog (Ihh) , and Wnt family proteins are important cytokines for osteoblast differentiation. Understandings of molecular mechanisms by which these cytokines stimulate osteoblast differentiation have been extensively investigated. BMP/Smad signaling, canonical Wnt pathway, non-canonical Wnt pathway and Ihh/Gli signaling play critical role in osteoblast differentiation. Furthermore, biochemical and genetic studies have demonstrated important roles of Runt related transcription factor 2 (Runx2) , Osterix, Activating transcription factor 4 (ATF4) , Msh homeobox 2 (Msx2) and Oasis for osteoblast differentiation. Thus, these milestone studies have dramatically progressed in our understanding of the cellular and molecular mechanisms of osteoblast differentiation for 20 years.

Clinically, fatty marrow, accumulation of adipocytes in bone marrow, is often observed in the patients who manifest bone diseases such as osteoporosis. Since adipocytes and osteoblasts are differentiated from mesenchymal stem cells, it would be clinically and biologically important to understand regulatory mechanisms of the balance between adipogenesis and osteoblastogenesis. Recently, experimental findings indicated the involvement of adipokines including leptin and adiponectin in bone metabolisms. Thus, adipocytes appear to play a role in regulation of bone metabolisms.

Recently, large body of evidences that Wnt plays an important role in regulation of tissue patterning and skeletogenesis including membranous and enchondral ossification is accumulating. Several genetic studies also indicate that low density lipoprotein receptor related protein (LRP) 5, LRP6, beta-catenin and lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) participate in osteogenic action induced by Wnt family members. In contrast, secreted Frizzled-related proteins and dickkopf (Dkk) proteins, natural Wnt inhibitory factors, inhibit Wnt-dependent osteogenesis, presumably functioning as a negative feedback loop. In addition, Wnt signaling interacts with BMP signaling, thereby modulating its osteogenic action. Thus, the network systems of Wnt signaling temporally and spatially controls osteogenic action of Wnt family members.

Bone is a complex tissue which contains osteoclasts, osteoblasts, chondrocytes, adipocytes, hematopoietic cells and immune cells. Since osteoblasts share the same origin with adipocytes in bone marrow cavity, it is assumed that PPAR (peroxisome proliferator-activated receptor) family, which is an important nuclear receptor family for adipocyte differentiation, plays a role in the bone microenvironment. Indeed, recent evidences support the primitive roles of PPAR family in osteoblast differentiation as well as adipocyte differentiation. Furthermore, PPAR family is also implicated in the regulation of differentiation and function of osteoclasts. Here, we summarized the functional roles of PPAR family in bone remodeling and regulation of bone microenvironments. We also discuss the potential mechanisms that regulate expression and function of PPAR family during bone metabolisms.

Although both osteoblasts and adipocytes have a common origin, i.e., mesenchymal cells, the molecular mechanisms that define the direction of two different lineages are presently unknown. In this study, we investigated the role of a transcription factor, CCAAT/enhancer binding protein beta (C/EBPbeta), and its isoform in the regulation of balance between osteoblast and adipocyte differentiation. We found that C/EBPbeta, which is induced along with osteoblast differentiation, promotes the differentiation of mesenchymal cells into an osteoblast lineage in cooperation with Runx2, an essential transcription factor for osteogenesis. Surprisingly, an isoform of C/EBPbeta, liver-enriched inhibitory protein (LIP), which lacks the transcriptional activation domain, stimulates transcriptional activity and the osteogenic action of Runx2, although LIP inhibits adipogenesis in a dominant-negative fashion. Furthermore, LIP physically associates with Runx2 and binds to the C/EBP binding element present in the osteocalcin gene promoter. These data indicate that LIP functions as a coactivator for Runx2 and preferentially promotes the osteoblast differentiation of mesenchymal cells. Thus, identification of a novel role of the C/EBPbeta isoform provides insight into the molecular basis of the regulation of osteoblast and adipocyte commitment.

The proto-oncogene, c-src, has been implicated in the tumorigenesis in breast cancer. However, the relationship of c-src with distant metastasis is unclear. Moreover, the role of c-src in organ-preferential metastasis of breast cancer is unknown. Because breast cancer has a strong predilection for metastasizing to bone, we examined the role of c-src in bone metastases using an animal model in which inoculation of the MDA-231 human breast cancer cells into the left cardiac ventricle preferentially developed osteolytic bone metastases in female nude mice. A clone of the MDA-231 with the increased capacity of bone metastasis exhibited elevated c-src tyrosine kinase (TK) activity compared with parental cells. MDAsrc527 cells caused significantly increased size of the osteolytic bone metastases with increased number of osteoclasts and mitotic cancer cells compared with MDA-231EV or MDAsrcWT. In contrast, MDAsrc295 cells caused impaired metastases to bone. Of note, mice inoculated with MDAsrc295 cells via tail vein developed reduced lung metastases and prolonged survival compared with mice with MDA-231EV cells, suggesting that c-src TK is unlikely to play a specific role in bone metastases. The growth in vitro and in vivo and production of parathyroid hormone-related protein, a key cytokine in the pathogenesis of osteolytic bone metastases in breast cancer, were promoted in MDAsrc527 and diminished in MDAsrc295. These results suggest that c-src TK is associated with the capacity of breast cancer to metastasize to bone through regulating cell growth and parathyroid hormone-related protein production. Our results together with the fact that c-src is an essential molecule for bone resorption by osteoclasts, which are central players in osteolytic bone metastases, support the notion that c-src TK is a potential target molecule for designing novel therapeutic interventions, especially for bone metastases in breast cancer.

Bone morphogenetic protein 2 (BMP2) promotes the differentiation of undifferentiated mesenchymal cells into adipocytes. To investigate the molecular mechanisms that regulate this differentiation process, we studied the relationship between BMP2 signaling and peroxisome proliferator-activating receptor gamma (PPARgamma) during adipogenesis of mesenchymal cells by using pluripotent mesenchymal cell line C3H10T1/2. In C3H10T1/2 cells, BMP2 induced expression of PPARgamma along with adipogenesis. Overexpression of Smad6, a natural antagonist for Smad1, blocked PPARgamma expression and adipocytic differentiation induced by BMP2. Overexpression of dominant-negative PPARgamma also diminished adipocytic differentiation of C3H10T1/2 cells, suggesting the central role of PPARgamma in BMP2-induced adipocytic differentiation. Specific inhibitors for p38 kinase inhibited BMP2-induced adipocytic differentiation and transcriptional activation of PPARgamma, whereas overexpression of Smad6 had no effect on transcriptional activity of PPARgamma. Furthermore, activation of p38 kinase by overexpression of TAK1 and TAB1, without affecting PPARgamma expression, led the up-regulation of transcriptional activity of PPARgamma. These results suggest that both Smad and p38 kinase signaling are concomitantly activated and responsible for BMP2-induced adipocytic differentiation by inducing and up-regulating PPARgamma, respectively. Thus, BMP2 controls adipocytic differentiation by using two distinct signaling pathways that play differential roles in this process in C3H10T1/2 cells.