Inhibitory effect of lactoferrin on hypertrophic differentiation of ATDC5 mouse chondroprogenitor cells

The skeleton is formed by two different mechanisms. In intramembranous ossification, osteoblasts form bone directly, whereas in endochondral ossification, chondrocytes develop a cartilage template, prior to osteoblast-mediated skeletogenesis. Lactoferrin is an iron-binding glycoprotein belonging to the transferrin family. It is known to promote the growth and differentiation of osteoblasts. In this study, we investigated the effects of bovine lactoferrin on the chondrogenic differentiation of ATDC5 chondroprogenitor cells. This mouse embryonic carcinoma-derived clonal cell line provides an in vitro model of chondrogenesis. Lactoferrin treatment of differentiating ATDC5 cells promoted cell proliferation in the initial stage of the differentiation process. However, lactoferrin treatment resulted in inhibition of hypertrophic differentiation, characterized by suppression of alkaline phosphatase activity, aggrecan synthesis and N-cadherin expression. This inhibitory effect was accompanied by sustained Sox9 expression, as well as increased Smad2/3 expression and phosphorylation, suggesting that lactoferrin regulates chondrogenic differentiation by up-regulating the Smad2/3-Sox9 signaling pathway.     

Iron overload inhibits calcification and differentiation of ATDC5 cells

There is a little information about the effects of iron overload on cartilage metabolism. In the present study, we examined the effects of excess iron on the differentiation and mineralization of cultured chondrocytes, ATDC5 cells. We used ferric ammonium citrate (FAC) as a ferric ion donor and desferrioxamine (DFO) as a ferric ion chelator. Neither chemical affected the production of proteoglycan, a marker of an early stage of ATDC5 differentiation. In contrast, FAC inhibited the deposition of calcium, a late-stage event in chondrocyte differentiation, by ATDC5 cells in a dose-dependent manner, and DFO accelerated it. Energy dispersive X-ray spectroscopy/scanning electron microscope analysis revealed that the levels of iron and calcium in cells treated with FAC were increased and decreased, respectively. Furthermore, FAC inhibited the expression of matrix metalloproteinase 13 mRNA, another marker of late-stage chondrocyte differentiation. In addition, we found that the heavy and light chains of ferritin were expressed specifically at a late stage of ATDC5 differentiation, and the levels of both proteins were enhanced by the addition of iron. These results suggest that iron overload might give rise to osteopenia and arthritis by inhibiting chondrocyte differentiation and mineralization.     

The effects of Sp7/Osterix gene silencing in the chondroprogenitor cell line, ATDC5

Ubiquitin is a small polypeptide and ubiquitination is the post-translational modification by ubiquitin protein, resulting in degradation of target proteins by the 26S proteasome complex. Here, we found that E3 ubiquitin ligase SINAT5, an Arabidopsis homologue of the Drosophila SINA RING-finger protein, interacts directly with LHY, a component of the circadian oscillator, and DET1, a negative regulator of light-regulated gene expression. We also found that SINAT5 has E3 ubiquitination activity for LHY but not for DET1. Interestingly, LHY ubiquitination by SINAT5 was inhibited by DET1. Late flowering of sinat5 mutants indicates that flowering time can be controlled by DET1 through regulation of LHY stability by SINAT5.     

Transforming growth factor-beta1 (TGF-beta1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression

Regulated splicing of fibronectin (FN) occurs during the mesenchymal to chondrocyte transition and ultimately results in the relative enrichment of an extra domain B (EDB) exon-containing FN isoform with the suggestion that FN isoforms may play a functional role in chondrogenesis. Promotion of chondrogenesis can also be achieved by treatment with transforming growth factor-beta (TGF-beta), which also regulates FN isoform expression. We have examined the effects of TGF-beta treatment on the assumption of the chondrogenic phenotype in the teratoma-derived cell line ATDC5 and tested whether these effects on chondrogenesis are paralleled by appropriate changes in FN isoform expression. ATDC5 cells were maintained in a pre-chondrogenic state and, in this state, treated with 10 ng/ml TGF-beta. The cells started to elaborate a matrix rich in sulfated proteoglycans, such that within the first 12 days of culture, TGF-beta1 treatment appeared to slightly accelerate early acquisition of an Alcian blue-stained matrix, and caused a dose- and time-dependent decrease in collagen type I expression; changes in collagen type II expression were variable. At later times, cells treated with TGF-beta became indistinguishable from those of the controls. Interestingly, TGF-beta treatment caused a significant dose- and time-dependent decrease in the proportion of FN containing the extra domain A (EDA) and the EDB exons. These data suggest that TGF-beta induces the early stages of chondrogenic maturation in this pre-chondrogenic line and that TGF-beta treatment increases expression of FN isoforms that lack the EDA and EDB exons.     

Laminin-5 suppresses chondrogenic differentiation of murine teratocarcinoma cell line ATDC5

Laminin-5 is an important basement membrane protein that regulates cell adhesion and motility. It was previously found that the gamma2 chain of laminin-5 is transiently expressed in embryonic cartilage. This suggests a possible role of laminin-5 in chondrogenesis. Here, we examined this possibility using the murine teratocarcinoma cell line ATDC5. ATDC5 cells transiently and weakly expressed laminin-5 when they were stimulated for differentiation. Exogenous laminin-5 in either insoluble or soluble form strongly inhibited the differentiation phenotypes, i.e. formation of cartilaginous cell aggregates and production of chondrogenic marker proteins through its integrin-binding domain LG3 in the alpha3 chain. Laminin-5 had no effect on cell growth. In addition, we found that the laminin-5 with the 105-kDa, processed gamma2 chain suppressed differentiation more strongly than one with the 150-kDa gamma2 chain. This indicated that the proteolytic processing of gamma2 chain regulated the activity of laminin-5. However, a gamma2 chain short arm fragment had no effect on the chondrogenesis, and it rather suppressed the differentiation at excessive concentrations. These results suggest that laminin-5 and its processing modulate chondrogenic differentiation during development.     

Rab5 regulates motility of early endosomes on microtubules

The small GTPase Rab5 regulates membrane docking and fusion in the early endocytic pathway. Here we reveal a new role for Rab5 in the regulation of endosome interactions with the microtubule network. Using Rab5 fused to green fluorescent protein we show that Rab5-positive endosomes move on microtubules in vivo. In vitro, Rab5 stimulates both association of early endosomes with microtubules and early-endosome motility towards the minus ends of microtubules. Moreover, similarly to endosome membrane docking and fusion, Rab5-dependent endosome movement depends on the phosphatidylinositol-3-OH kinase hVPS34. Thus, Rab5 functionally links regulation of membrane transport, motility and intracellular distribution of early endosomes.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Activation of genes for growth factor and cytokine pathways late in chondrogenic differentiation of ATDC5 cells

The mouse embryonal carcinoma cell line ATDC5 provides an excellent model system for chondrogenesis in vitro. To understand better the molecular mechanisms of endochondral bone formation, we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold. These genes were classified into five clusters based on their expression patterns. Genes for growth factor and cytokine pathways were significantly enriched in the cluster characterized by increases in expression during late stages of chondrocyte differentiation. mRNAs for decorin and osteoglycin, which have been shown to bind to transforming growth factors-beta and bone morphogenetic proteins, respectively, were found in this cluster and were detected in hypertrophic chondrocytes of developing mouse bones by in situ hybridization analysis. Taken together with assigned functions of individual genes in the cluster, interdigitated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage for autocrine and paracrine regulation among chondrocytes, as well as for chemoattraction and stimulation of progenitor cells of other lineages.     

Leptin regulates estrogen receptor gene expression in ATDC5 cells through the extracellular signal regulated kinase signaling pathway

Both estrogen and leptin play an important role in the regulation of physiological processes of endochondral bone formation in linear growth. Estrogen receptors (ERα and ERβ) are known as members of the superfamily of nuclear steroid hormone receptors and are detected in all zones of growth plate chondrocytes. They can be regulated in a ligand-independent manner. Whether leptin regulates ERs in the growth plate is still not clear. To explore this issue, chondrogenic ATDC5 cells were used in the present study. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting. We found that both ERα and ERβ were dynamically expressed during the ATDC5 cell differentiation for 21 days. Leptin (50 ng/ml) significantly upregulated ERα and ERβ mRNA and protein levels 48 h after leptin stimulation (P<0.05) at day 14. The up-regulation of ERα and ERβ mRNA by leptin was shown in a dose-dependent manner, but the most effective dose of leptin was different (100 and 1,000 ng/ml, respectively). Furthermore, we confirmed that leptin augmented the phosphorylation of ERK1/2 in a time-dependent manner. A maximum eightfold change was observed at 15 min. Finally, a specific ERK1/2 inhibitor, UO126, blocked leptin-induced ERs regulation in ATDC5 cells, indicating that ERK1/2 mediates, partly, the effects of leptin on ERs. These data demonstrate, for the first time, that leptin regulates the expression of ERs in growth plate chondrocytes via ERK signaling pathway, thereby suggesting a crosstalk between leptin and estrogen receptors in the regulation of bone formation.     

Rab23 研究进展

Rab23为小GTP结合蛋白,是Ras超家族Rab家族成员,其突变可引起小鼠开脑综合征(open brain syndrome),因此又称为opb基因.它是Sonic hedgehog信号通路的负调控因子,在不同肿瘤中发挥不同作用,并参与机体组织器官的发育和分化.它可能通过转运细胞内蛋白发挥相关作用.     

The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.     

Rab23研究进展

Rab23为小GTP结合蛋白,是Ras超家族Rab家族成员,其突变可引起小鼠开脑综合征（openbrain syndrome）,因此又称为opb基因。它是Sonic hedgehog信号通路的负调控因子,在不同肿瘤中发挥不同作用,并参与机体组织器官的发育和分化。它可能通过转运细胞内蛋白发挥相关作用。     

Rab14 regulates apical targeting in polarized epithelial cells

Epithelial cells display distinct apical and basolateral membrane domains, and maintenance of this asymmetry is essential to the function of epithelial tissues. Polarized delivery of apical and basolateral membrane proteins from the trans Golgi network (TGN) and/or endosomes to the correct domain requires specific cytoplasmic machinery to control the sorting, budding and fission of vesicles. However, the molecular machinery that regulates polarized delivery of apical proteins remains poorly understood. In this study, we show that the small guanosine triphosphatase Rab14 is involved in the apical targeting pathway. Using yeast two-hybrid analysis and glutathione S-transferase pull down, we show that Rab14 interacts with apical membrane proteins and localizes to the TGN and apical endosomes. Overexpression of the GDP mutant form of Rab14 (S25N) induces an enlargement of the TGN and vesicle accumulation around Golgi membranes. Moreover, expression of Rab14-S25N results in mislocalization of the apical raft-associated protein vasoactive intestinal peptide/MAL to the basolateral domain but does not disrupt basolateral targeting or recycling. These data suggest that Rab14 specifically regulates delivery of cargo from the TGN to the apical domain.