Ken Yagi

miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation.

Although various microRNAs regulate cell differentiation and proliferation, no miRNA has been reported so far to play an important role in the regulation of osteoblast differentiation. Here we describe the role of miR-125b in osteoblastic differentiation in mouse mesenchymal stem cells, ST2, by regulating cell proliferation. The expression of miR-125b was time-dependently increased in ST2 cells, and the increase in miR-125b expression was attenuated in osteoblastic-differentiated ST2 cells induced by BMP-4. The transfection of exogenous miR-125b inhibited proliferation of ST2 cells and caused inhibition of osteoblastic differentiation. In contrast, when the endogenous miR-125b was blocked by transfection of its antisense RNA molecule, alkaline phosphatase activity after BMP-4 treatment was elevated. These results strongly suggest that miR-125b is involved in osteoblastic differentiation through the regulation of cell proliferation.

miR‐210 promotes osteoblastic differentiation through inhibition of AcvR1b

Although microRNAs (miRNAs) are involved in many biological processes, the mechanisms whereby miRNAs regulate osteoblastic differentiation are poorly understood. Here, we found that BMP‐4‐induced osteoblastic differentiation of bone marrow‐derived ST2 stromal cells was promoted and repressed after transfection of sense and antisense miR‐210, respectively. A reporter assay demonstrated that the activin A receptor type 1B (AcvR1b) gene was a target for miR‐210. Furthermore, inhibition of transforming growth factor‐β (TGF‐β)/activin signaling in ST2 cells with SB431542 promoted osteoblastic differentiation. We conclude that miR‐210 acts as a positive regulator of osteoblastic differentiation by inhibiting the TGF‐β/activin signaling pathway through inhibition of AcvR1b.

Molecular Basis of Constitutive Production of Basement Membrane Components GENE EXPRESSION PROFILES OF ENGELBRETH-HOLM-SWARM TUMOR AND F9 EMBRYONAL CARCINOMA CELLS

Engelbreth-Holm-Swarm (EHS) tumors produce large amounts of basement membrane (BM) components that are widely used as cell culture substrates mimicking BM functions. To delineate the tissue/organ origin of the tumor and the mechanisms operating in the BM overproduction, a genome-wide expression profile of EHS tumor was analyzed using RIKEN cDNA microarrays containing ∼40,000 mouse cDNA clones. Expression profiles of F9 embryonal carcinoma cells that produce laminin-1 and other BM components upon differentiation into parietal endoderm-like cells (designated F9-PE) were also analyzed. Hierarchical clustering analysis showed that the gene expression profiles of EHS and F9-PE were the most similar among 49 mouse tissues/organs in the RIKEN Expression Array Database, suggesting that EHS tumor is parietal endoderm-derived. Quantitative PCR analysis confirmed that not only BM components but also the machineries required for efficient production of BM components, such as enzymes involved in post-translational modification and molecular chaperones, were highly expressed in both EHS and F9-PE. Pairs of similar transcription factor isoforms, such as Gata4/Gata6, Sox7/Sox17, and Cited1/Cited2, were also highly expressed in both EHS tumor and F9-PE. Time course analysis of F9 differentiation showed that up-regulation of the transcription factors was associated with those of BM components, suggesting their involvement in parietal endoderm specification and overproduction of the BM components.

Id4, a New Candidate Gene for Senile Osteoporosis, Acts as a Molecular Switch Promoting Osteoblast Differentiation

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparγ2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparγ2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Identification of novel PPARγ target genes by integrated analysis of ChIP-on-chip and microarray expression data during adipocyte differentiation

PPARgamma (peroxisome proliferator-activated receptor gamma) acts as a key molecule of adipocyte differentiation, and transactivates multiple target genes involved in lipid metabolic pathways. Identification of PPARgamma target genes will facilitate to predict the extent to which the drugs can affect and also to understand the molecular basis of lipid metabolism. Here, we have identified five target genes regulated directly by PPARgamma during adipocyte differentiation in 3T3-L1 cells using integrated analyses of ChIP-on-chip and expression microarray. We have confirmed the direct PPARgamma regulation of five genes by luciferase reporter assay in NIH-3T3 cells. Of these five genes Hp, Tmem143 and 1100001G20Rik are novel PPARgamma targets. We have also detected PPREs (PPAR response elements) sequences in the promoter region of the five genes computationally. Unexpectedly, most of the PPREs detected proved to be atypical, suggesting the existence of more atypical PPREs than previously thought in the promoter region of PPARgamma regulated genes.

Novel homeodomain-interacting protein kinase family member, HIPK4, phosphorylates human p53 at serine 9

We describe here the cloning and characterization of a novel mouse homeodomain‐interacting protein kinase (HIPK)‐like gene, Hipk4. Hipk4 is expressed in lung and in white adipose tissue and encodes a 616 amino acid protein that includes a serine/threonine kinase domain. We demonstrate that HIPK4 could phosphorylate human p53 protein at serine 9, both in vitro and in vivo. Among known p53‐responsive promoters, activity of the human survivin promoter, which is repressed by p53, was decreased by HIPK4 in p53 functional A549 cells. Human BCL2‐associated X protein‐promoter activity was not affected. These findings suggest that phosphorylation of p53 at serine 9 is important for p53 mediated transcriptional repression.

Expression Profiling of Parietal Endoderm Cells and Development of Systems for Large Scale Production of Laminins

Basement membranes support proliferation, survival, and differentiation of epithelial/parenchymal cells through interaction with cell membrane receptors. Laminins, which have a heterotrimeric structure, are the major class of basement membrane glycoproteins. To date, five genes for α subunits, three for β subunits and three for γ subunits have been identified, and combinations of these subunits give rise to at least 12 heterotrimeric laminin isoforms. These isoforms are expressed in a tissue- and developmental stage- specific manner, suggesting specific physiological roles of each laminin isoform. However, studies on such functions of these various isoforms have been hampered by difficulties in the purification of laminins, except for murine laminin-1 (α1β1γl) from the Engelbreth-Holm-Swarm (EHS) tumor.