Insulin-transferrin-sodium Selenite Research Articles

Elevated levels of expression of cytochrome P450 3A4 in a human liver epithelial cell line in differentiation-inducing conditions.

Cytochrome P450 (CYP) enzymes, especially CYP3A4 play a major role in the metabolism of xenobiotics in human liver. CYP3A4-expressing human liver or hepatoma cell lines may be good cell substitutes of human hepatocytes for drug metabolism studies. However, there are only a few cell lines expressing high levels of CYP3A4. The aim of this study is to investigate the expression of CYP3A4 and its mechanism in an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b in differentiation-inducing conditions. When THLE-5b cells were cultivated in culture medium supplemented with hepatocytic differentiation-inducing factors, they showed hepatocytic morphology. In addition, elevated levels of expression not only of α1-antitrypsin (AAT) and albumin (ALB) mRNAs, but also of CYP3A4 mRNA, which are functional hepatocyte markers, were observed compared with the control. Among hepatocytic differentiation-inducing factors, dexamethasone (DEX) and insulin-transferrin-sodium selenite (ITS) seemed to be involved in elevation of expression of CYP3A4 mRNA. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 or the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced CYP3A4 mRNA levels of THLE-5b cells. Furthermore, the CpG site of the CYP3A4 promoter region in THLE-5b cells was found to be unmethylated, although in low CYP3A4-expressing HepG2 cells, the site was methylated. In conclusion, THLE-5b cells, which are unmethylated at the CpG site of the CYP3A4 promoter region, express CYP3A4 mRNA through the MEK/ERK1/2 and PI3K/Akt signaling pathways and acquire hepatocytic functions in differentiation-inducing conditions. Thus, THLE-5b cells could be a useful cell system for the study of drug metabolism.

Supplementation of insulin-transferrin-sodium selenite in culture medium improves the hypothermic storage of bovine embryos produced in vitro

Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.

A combination of bovine serum albumin with insulin-transferrin-sodium selenite and/or epidermal growth factor as alternatives to fetal bovine serum in culture medium improves bovine embryo quality and trophoblast invasion by induction of matrix metalloproteinases.

This study investigated the use of bovine serum albumin (BSA) plus insulin-transferrin-sodium selenite (ITS) and/or epidermal growth factor (EGF) as alternatives to fetal bovine serum (FBS) in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, gene expression and cryotolerance, as well as the invasion ability of trophoblasts. The percentage of embryos that underwent cleavage and formed a blastocyst was higher (P<0.01) in medium containing ITS plus EGF and BSA than in medium containing FBS. Culture with ITS plus EGF and BSA also increased the hatching ability of blastocysts and the total cell number per blastocyst. Furthermore, the beneficial effects of BAS plus ITS and EGF on embryos were associated with a significantly reduced intracellular lipid content, which increased their cryotolerance. An invasion assay confirmed that culture with ITS plus EGF and BSA significantly improved the invasion ability of trophoblasts. Real-time quantitative polymerase chain reaction analysis showed that the mRNA levels of matrix metalloproteinase-2 (MMP2) and MMP9, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain and hydroxymethylglutaryl-CoA reductase significantly increased upon culture with ITS plus EGF and BSA. Moreover, protein expression levels of matrix metalloproteinase-2 and -9 increased (P<0.01) in medium supplemented with ITS plus EGF and BSA compared with medium supplemented with FBS. Taken together, these data suggest that supplementation of medium with ITS plus EGF and BSA improves invitro bovine embryo production, cryotolerance and invasion ability of trophoblasts.

Morphogenesis and Mucus Production of Epithelial Tissues of Three Major Salivary Glands of Embryonic Mouse in 3D Culture.

Embryonic mouse submandibular epithelia initiate branching morphogenesis within two days when embedded in Matrigel and stimulated by members of the epidermal growth factor family. However, it is unknown whether the end buds further branch over longer culture periods, and whether saliva-producing cells differentiate there. In the present study, we cultivated three major (submandibular, sublingual and parotid) salivary epithelia from 13-day embryos for 14 days in mesenchyme-free cultures. All epithelia continued to grow and branch to form numerous acinus-like structures in medium supplemented with neuregulin 1, fibroblast growth factor 1, and insulintransferrin-sodium selenite. Alcian blue staining to detect mucous cells showed that each epithelium differentiated via three distinct modes, as seen in normal development, although the staining intensities were weaker than in normal development. RT-PCR analysis of the amylase gene showed that no epithelia expressed amylase after 14 days of culture, which is inconsistent with the fact that only parotid epithelium does so at postnatal day 7 during normal development. These results suggest that cytodifferentiation progresses to a lesser extent in mesenchyme-free cultures than in vivo.

Dexamethasone inhibits chondrocyte differentiation by suppression of Wnt/β-catenin signaling in the chondrogenic cell line ATDC5.

Glucocorticoids (GCs) regulate proliferation and differentiation in cultured mesenchymal cells through the modulation of various molecules. However, the relationship between growth factor signaling and GCs in differentiating chondrocytes has not been elucidated. In this study, we examined the effects of Wnt/β-catenin signaling on chondrocyte differentiation and the effects of a GC analogue, dexamethasone (Dex), on Wnt/β-catenin signaling activity by using a chondrocyte progenitor cell line ATDC5. Western blot analysis and TCF/LEF-optimized promoter EGFP (TOPEGFP) assay showed that both β-catenin protein levels and TCF/LEF transcription were up-regulated during insulin-transferrin-sodium selenite (ITS)-induced chondrogenic differentiation. Morphological analysis showed that TCF/LEF transcription activity was most prominent in cartilage nodule-like structures. Furthermore, a β-catenin mutant with constitutive transcriptional activity (ΔN90) showed increased Alcian blue staining intensity and mRNA expression of Sox9, Col2a, aggrecan, Col10, and alkaline phosphatase, even in the absence of ITS stimulation. In contrast, Dex suppressed formation of ITS-induced cartilage nodule-like structures, TCF/LEF-mediated transcription, and β-catenin protein levels. Real-time PCR analysis showed that Dex increased the mRNA expression levels of secreted frizzled-related protein 1 (sFRP1) and Axin2. Furthermore, treatment with a sFRP1 inhibitor or the ΔN90 β-catenin mutant transfection attenuated Dex-induced suppression of cartilage matrix production by increasing Sox9 mRNA levels. These results suggest that Dex inhibits chondrocyte differentiation via down-regulation of Wnt/β-catenin signaling, which promotes chondrocyte differentiation in ATDC5 cells.

A5.12 SFRPS in cartilage biology: more than just WNT antagonists

Background and Objectives Wnts play an important role in cartilage development, homeostasis and degeneration. Therefore, exploring the therapeutic potential of factors that can modulate Wnts is of great interest. Secreted...

Inhibition of COG5, a gene from the C7q22 osteoarthritis susceptibility locus, induces glycosylation defects and affects chondrogenesis and osteogenesis by disturbing Wnt signaling

Purpose: Osteoarthritis (OA) is a highly disabling pathology of the bone-cartilage unit, to which both patient-dependent (weight excess, trauma⋯) and genetic factors contribute. Recently, a locus at chromosome 7q22 was linked to knee and hand OA by genome wide association study. In this locus, the Conserved oligomeric Golgi complex subunit (COG) 5 gene encodes a component of Golgi apparatus. Mutations in humans are associated with mild congenital glycosylation disorders, and COG5 expression is higher in 3D than in monolayer cultures of articular chondrocytes. Here we investigated on the role of COG5 during chondrogenesis and osteogenesis. Methods: We used three independent stable clonal colonies of each, controls (non-sliencing vector) and COG5 knocked-down cells (COG5-), established in the chondrogenic ATDC5 and osteogenic MC3T3-E1 cell lines. ATDC5 were cultured as high density micromasses (2x105 cells/10 μl) to mimic the condensation step of developing cartilage. Chondrogenesis was induced by culturing cells for 14 days (D) in DMEM/F12 + 5%FBS, supplemented with ITS (insulin, transferrin, sodium selenite). On D14, cells were switched to mineralization medium (αMEM + 5%FBS, supplemented with ITS, β-glycerophosphate (BGP) and ascorbic acid (AA)) to investigate the mineralization phase of late chondrogenesis. Monolayers of MC3T3-E1 were cultured for 21D in αMEM + 10%FBS, supplemented with BGP and AA. mRNA expression of typical chondrogenesis markers (Aggrecan (Agg), type II (Col2a1) and X (Col10a1)) collagens and osteogenic markers (Osterix (Osx), Osteocalcin (Ocn) and Osteopontin (Opn)) were assessed by quantitative RT-PCR. We also assessed mRNA expression of Wnts (-4, -5a, -5b, -11) and BMPs (-2, -4, -6, -7). Quantification of Alcian Blue, Safranin O, Sirius red and Alizarin red staining were used to evaluate proteoglycans, collagens and mineralized content respectively. Alkaline phosphatase (ALP) activity allowed to monitor osteogenesis. SDS-PAGE was used to assess the global glycosylation status. Western blot allowed to check canonical (β-catenin) and non-canonical (Calmodulin-Kinase II) Wnt signaling, as well as BMP signaling (Smad and MAPK). Results: During early proliferation (D1-7) and late proliferation/pre-hypertrophy phases (D7-14) of chondrogenesis, Col2a1 and Agg mRNA strongly increased, but to a lesser extent in COG5-. Proteoglycan staining intensity was lower in COG5-. BMP-4 levels progressively increased and were significantly higher in COG5-. This was consistent with increased phosphorylation of Smad 1/5/8. Wnt-4, -5a and -5b mRNA were evenly increased in both controls and COG5-. BMP-6 and Col10a1 mRNA were increased at D14, but were significantly lower in COG5-. Wnt-11 mRNA increased in controls, but not in COG5-. In mineralization phase (D14 to D21), Col10a1 mRNA and mineralization were strongly increased, but to a lesser extent in COG5-. During osteogenesis, Opn, Osx and Ocn mRNA were significantly higher in controls, alike Alizarin red staining and ALP activity. Protein analysis suggested differences in global glycosylation status, in particular of Wnt ligands. Conclusions: COG5 defect reduces chondrogenesis and osteogenesis. COG5 particularly affected glycosylation and Wnt signaling, underlining its critical role in the fate of cells from the articular chondrocyte-bone unit in the course of OA.

Differentiation of neonatal rat epithelial cells from biliary origin into immature hepatic cells by sequential exposure to hepatogenic cytokines and growth factors reflecting liver development

It was investigated whether cryopreserved rat liver epithelial cells (RLEC) from biliary origin are capable of undergoing hepatic differentiation upon sequential exposure to liver-specific factors (fibroblast growth factor (FGF)-4, hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS), dexamethasone (Dex) and oncostatin M (OSM)), reflecting liver embryogenesis in vivo. As differentiation progressed, cells acquired a hepatic morphology (polygonal-to-cuboidal shaped, binucleated cells), corresponding well with the phenotypic changes observed. Biliary cytokeratin (CK)19 and connexin (Cx)43-expression both gradually decreased; CK19-expression disappeared even completely. In contrast, hepatic CK18-expression persisted throughout the culture time. Hepatocyte nuclear factor (HNF)3β, alpha-foetoprotein (AFP), transthyretin (TTR), HNF4, albumin (ALB), HNF1α, multidrug resistance protein (MRP)2 and Cx32 were expressed at specific stages during RLEC-differentiation, thereby showing a progressive hepatic maturation. Indeed, immature AFP and mature ALB were sequentially expressed, in line with the in vivo liver embryogenesis. Expression of the early and mid-late ‘liver-enriched’ transcription factors (LETF) HNF3β and HNF4 declined and translocated to the cytosol, respectively, while the late LETF HNF1α underwent a nuclear upregulation. In conclusion, RLEC are bipotent cells, capable of differentiation into immature hepatocytes in a hepatic-stimulating micro-environment. The robustness of the sequential conditions, developed before for hepatic ‘transdifferentiation’ of rat bone marrow stem cells (rBMSC), was hereby confirmed.

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

BackgroundThe capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated.ResultsExposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity.ConclusionhMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.

Preservation and Expansion of the Primate Keratocyte Phenotype by Downregulating TGF-β Signaling in a Low-Calcium, Serum-Free Medium

To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-beta signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2+] medium. Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco's modified Eagle's medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/10% FBS. TGF-beta signaling was monitored by measuring the promoter activity of TGF-beta1, -beta2, and -beta RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4. In KSFM, monkey keratocytes proliferated while maintaining the expression of keratocan, CD34, and ALDH proteins and keratocan promoter-driven ECFP for at least 15 passages. The nuclear accumulation of Smad2 and Smad4 and the promoter activities of TGF-beta1 and -beta RII were significantly downregulated in KSFM compared with DMEM/10% FBS. In KSFM, an increase of [Ca2+] to 1.8 mM and addition of 10% FBS synergistically downregulated the keratocan promoter activity, facilitated Smad2 and Smad4 nuclear translocation, and upregulated TGF-beta1 and -beta RII promoter activities. The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by downregulating Smad-mediated TGF-beta signaling.

Glyoxalase I Is Critical for Human Retinal Capillary Pericyte Survival under Hyperglycemic Conditions

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.

CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1.

To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation. Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining. Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days. These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.

319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P&amp;lt;0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P&amp;lt;0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media

BIG-3, a novel WD-40 repeat protein, is expressed in the developing growth plate and accelerates chondrocyte differentiation in vitro.

Among the local signaling pathways that regulate the sequential steps of chondrocyte differentiation is the bone morphogenetic protein (BMP) signaling pathway. We have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb) that is expressed in a BMP-regulated fashion in the prechondroblastic cell line MLB13MYC clone 17. BIG-3 is also expressed in proliferating and hypertrophic chondrocytes in the developing growth plate in vivo. We undertook studies to address whether BIG-3 played a functional role in chondrocyte differentiation, using mouse clonal chondrogenic ATDC5 cells. BIG-3 protein levels increased during ITS (insulin, transferrin, sodium selenite)-induced ATDC5 differentiation and in response to BMP-2 treatment. To determine whether stable expression of BIG-3 could alter the program of chondrocytic differentiation, ATDC5 cells were stably transfected with the full-length coding region of BIG-3 (ATDC5-BIG-3) or with the empty vector (ATDC5-EV). Accelerated matrix proteoglycan synthesis was observed in the pooled ATDC5-BIG-3 clones. Alkaline phosphatase and osteopontin mRNA levels were also increased in ATDC5-BIG-3 clones compared with ATDC5-EV clones. Stable expression of BIG-3 also accelerated mineralized matrix formation in both the presence and absence of ITS. These findings, which demonstrate that BIG-3 accelerates chondrocyte differentiation in vitro, combined with the observation that BIG-3 is expressed in the growth plate during embryonic development, suggest that this novel protein is likely to play an in vivo regulatory role in the developing growth plate.