Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1(+) cells

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.     

Different roles of protein kinase C-betaI and -delta in the regulation of adipocyte differentiation

Protein kinase C (PKC) is a member of serine/threonine protein kinase family that plays important roles in the control of vast variety of cellular functions. Nevertheless, the regulatory effect of PKC on adipogenesis remained not well understood. In this study, we investigated the effect of several PKC isoforms on the adipogenic conversion of 3T3-L1 preadipocytes induced by dexamethasone, isobutylmethylxanthine and insulin. Treatment of cells with broad-spectrum PKC inhibitor Rö318220 suppressed the adipogenesis. Gö6976, a selective inhibitor for PKC isoforms-, -βI and -μ, also inhibited the adipogenesis of cells. Pretreatment of cells with peroxisomal proliferator activated receptor-γ (PPARγ) agonist troglitazone abolished the inhibitory effect of Gö6976 on adipogenesis. The plasmic membrane translocation of PKC-βI was observed at the first 2 days of differentiation. Whereas no translocation of PKC- and -μ was observed. Overexpression of dominant negative PKC-βI, but not wild-type PKC-βI, blocked adipogenesis. This effect of dominant negative PKC-βI can be reversed by troglitazone, suggesting that PKC-βI is required for the initiation of adipogenesis. In addition, rottlerin, a specific inhibitor of PKC-δ, can reverse the suppression of adipogenesis mediated by 12-O-tetradecanoyl-phorbol-13-acetate, transforming growth factor-β1, and epidermal growth factor. These data suggest that PKC-βI is important in the induction of adipogenesis, while the PKC-δ has an inhibitory role for adipogenesis.     

Cbfa1/Runx2与成骨细胞分化调控

成骨细胞是由间充质干细胞经骨原细胞和前成骨细胞分化而来的。近年来已鉴定转录因子Cbfal(core binding factor α1)是成骨细胞分化和骨形成的关键调控因子。在成骨细胞分化的过程中,Cbfal通过调控成骨细胞特异性细胞外基质蛋白基因的表达和成骨细胞周期参与成骨细胞的分化过程。新近发现Cbfal能通过自身的PST序列区域与Smads结合形成复合物共同参与成骨细胞的分化调控。     

调控褐色脂肪细胞分化的microRNAs

MicroRNA(miRNA)是近年来在真核生物中发现的一类长约22nt的内源性非编码RNA,在动物中主要通过抑制靶mRNA翻译,在转录后水平调控基因表达。动物体内有两种类型的脂肪组织：褐色和白色脂肪,白色脂肪以甘油三脂形式贮存能量,而褐色脂肪利用甘油三酯产生能量。褐色脂肪因其对肥胖的拮抗作用而对研究肥胖等代谢疾病具有重要意义,大量研究表明miRNA在褐色脂肪细胞分化中扮演着重要角色,其自身也受到多种转录因子和环境因子调控,这个复杂的调控网络维持了体内脂肪组织稳态。文章主要综述了miRNA在褐色脂肪细胞分化中的最新研究进展,以期为利用miRNA进行肥胖、糖尿病等相关疾病及其并发症的治疗提供新思路。     

Zinc-induced sodium-dependent vitamin C transporter 2 expression: potent roles in osteoblast differentiation

Zinc is an essential trace element that increases osteoblast numbers and bone formation. However, the mechanisms involved in the Zn-induced differentiation of osteoblasts are poorly understood. We examined the roles of L-ascorbic acid (AA) and its transporter, sodium-dependent vitamin C transporter (SVCT) 2, in the Zn-induced expression of osteoblastic differentiation markers. Zinc time- and dose-dependently induced SVCT2 mRNA expression in the absence or presence of AA. Western blotting and kinetic assays showed that Zn increased functional SVCT2 protein levels and AA transport. In the presence of AA, 50 microM Zn enhanced mRNA expression of the osteoblastic differentiation markers alkaline phosphatase, alpha(1)(I) procollagen, osteopontin (OPN), and osteocalcin (OCN) by 3.9-, 3.8-, 3.3-, and 3.5-fold, respectively; in the absence of AA, the Zn-induced increase was 2.8-, 2.5-, 1.3-, and 1.1-fold, respectively. These findings suggest that AA and SVCT2 mediate Zn-induced OPN and OCN expression and partly regulate Zn-induced osteoblastic differentiation.     

Bioactive compounds from sclerotia extract of Poria cocos that control adipocyte and osteoblast differentiation

Poria cocos Wolf confers edible sclerotia also known as ‘Indian bread’ in North America, that have been used for the treatment of various diseases in Asian countries. As part of our ongoing aim to identify biologically new metabolites from Korean edible mushrooms, we investigated the ethanol (EtOH) extract of the sclerotia of P. cocos by applying a comparative LC/MS- and bioassay-based analysis approach, since the EtOH extract reciprocally regulated adipocyte and osteoblast differentiation in mouse mesenchymal stem cells (MSCs). Bioassay-based analysis of the EtOH extract led to the successful isolation of two sterols, ergosterol peroxide (1) and 9,11-dehydroergosterol peroxide (2); three diterpenes, dehydroabietic acid (3), 7-oxocallitrisic acid, (4) and pimaric acid (5); and two triterpenes, dehydroeburicoic acid monoacetate (6) and eburicoic acid acetate (7) from the active hexane-soluble fraction. The isolated compounds (1–7) were examined for their effects on the regulation of MSC differentiation. The two sterols (1 and 2) were able to suppress MSC differentiation toward adipocytes. In contrast, the three diterpenes (3–5) showed activity to promote osteogenic differentiation of MSC. These findings demonstrate that the EtOH extract of P. cocos sclerotia is worth consideration as a new potential source of bioactive compounds effective in the treatment of osteoporosis in the elderly, since the extract contains sterols that inhibit adipogenic differentiation as well as diterpenes that promote osteogenic differentiation from MSCs.     

Crucial roles of D-type cyclins in the early stage of adipocyte differentiation

Cyclin D2 was isolated as one of the genes expressed early in adipogenesis. The expression of cyclin D2 increased temporarily early on and then again late in the differentiation process. The expression of cyclin D1 and cyclin D3, the other D-type cyclins, was also transiently induced early during adipocyte differentiation. RNAi (RNA interference)-mediated knockdown of cyclin D1, D2, or D3 inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Moreover, the knockdown of cyclin D1 or D3 significantly inhibited mitotic clonal expansion (MCE), while silencing of the cyclin D2 gene had a milder effect on MCE. Each of the D-type cyclins seems to play a crucial role in adipocyte differentiation by regulating MCE.     

Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenic cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice.     

Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

Background  

It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.     

Chromatin remodeling and epigenetic regulation of the CrabpI gene in adipocyte differentiation

Retinoic acid (RA) acts by binding to nuclear RA receptors (RARs) to regulate a broad spectrum of downstream target genes in most cell types examined. In cytoplasm, RA binds specifically to cellular retinoic acid binding proteins I (CRABPI), and II. Although the function of CRABPI in animals remains the subject of debate, it is believed that CRABPI binding facilitates RA metabolism, thereby modulating the concentration of RA and the type of RA metabolites in cells. The basal promoter of the CrabpI gene is a housekeeping promoter that can be regulated by thyroid hormones (T3), DNA methylation, sphinganine, and ethanol acting on its upstream regulatory region. T3 regulation of CrabpI is mediated by the binding of thyroid hormone receptor (TR) to a TR response element (TRE) approximately 1 kb upstream of the basal promoter. Specifically, in the adipocyte differentiation process, T3 regulation is bimodal and closely associated with the cellular differentiation status: T3 activates CrabpI in predifferentiated cells (e.g., mesenchymal precursors or fibroblasts), but suppresses this gene once cells are committed to adipocyte differentiation. These disparate effects are functions of T3-triggered differential recruitment of coregulatory complexes in conjunction with chromatin looping/folding that alters the configuration of this genomic locus along adipocyte differentiation. Subsequent sliding, disassembly and reassembly of nucleosomes occur, resulting in specific changes in the conformation of the basal promoter chromatin at different stages of differentiation. This chapter summarizes studies illustrating the epigenetic regulation of CrabpI expression during adipocyte differentiation. Understanding the pathways regulating CrabpI in this specific context might help to illuminate the physiological role of CRABPI in vivo. This article is part of a special issue entitled: Retinoid and Lipid Metabolism.     

Regulation of osteoblast and adipocyte differentiation from human mesenchymal stem cells by conjugated linoleic acid

Conjugated linoleic acid (CLA) describes a group of isomers of linoleic acid and has variable effects on bone formation and adiposity in vivo and in vitro. The variability may be due to individual effects of the predominant bioactive 9cis,11trans (9,11) and 10trans,12cis (10,12) CLA isomers. Osteoblasts and adipocytes are derived from mesenchymal stem cells (MSCs), and bone loss is accompanied by an increase in marrow adiposity. Osteoblast differentiation from MSCs requires activation of Wnt/β-catenin signaling by Wnt10b, which inhibits adipocyte differentiation by suppressing CCAAT/enhancer-binding protein (C/EBP) α. The objective of this study was to determine if 9,11 and 10,12 CLA affect osteoblast and adipocyte differentiation from MSCs and to determine whether any effects are associated with changes in Wnt10b and C/EBPα expression. Osteoblast differentiation was assessed by calcium deposition, alkaline phosphatase (ALP) activity, and the expression of Wnt10b, runx2 and osteocalcin. Adipocyte differentiation was assessed by oil red O staining and C/EBPα, PPARγ and FABP4 expression. Compared to vehicle, 9,11 CLA decreased calcium deposition (～15%), increased oil red O staining (～21-28%) and increased FABP4 (AP2) expression (～58-75%). In contrast, 10,12 CLA increased calcium deposition (～12-60%), ALP activity (～2.1-fold) and the expression of Wnt10b (～60-80%) and osteocalcin (～90%), but decreased oil red O staining (～30%) and the expression of C/EBPα (～24-38%) and PPARγ (～60%) (P<.05). Thus, our findings demonstrate isomer-specific effects of CLA on MSC differentiation, and suggest that 10,12 CLA may be a useful therapeutic agent to promote osteoblast differentiation from MSCs.     

Role of diabetes- and obesity-related protein in the regulation of osteoblast differentiation

Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men.     

Gene expression regulation of adipocyte differentiation: cell cycle and hormones

The adipose conversion of cultured preadipose cells involves the activation of numerous genes and is controlled by various adipogenic and mitogenic factors. The differentiation program can be divided into early and late events. Early events are triggered by growth arrest at the G1/S boundary and characterized by the activation of a set of genes (pOb24, lipoprotein lipase, etc.). The expression of the terminal differentiation-related genes takes place after a limited growth resumption of early markers containing cells and requires the presence of permissive hormones (growth hormone and triiodothyronine). Insulin acts solely as a modulator in the expression of the terminal differentiation-related genes. In vivo studies suggest that the acquisition of new adipocytes might result from terminal differentiation of dormant, already committed (pOb24 positive) cells when exposed to appropriate mitogenic or adipogenic stimuli.