A Role for Bone Morphogenetic Protein-4 in Adipocyte Development

Obesity is characterized by increases in the number of mature adipocytes. Nascent adipocytes arise from mesenchymal stem cells (MSCs) by a multi-step process — MSCs are recruited to the adipocyte lineage forming determined preadipocytes, these committed progenitors proliferate, undergo growth arrest, and finally differentiate into mature adipocytes. Although the genetic mechanisms that control the differentiation of preadipocytes into mature adipocytes are understood to a large extent, the earliest events in adipogenesis — especially the commitment of MSCs into preadipocytes — are largely unknown. Recently, bone morphogenetic protein-4 (BMP-4) has been implicated in the commitment of pluripotent MSCs to the adipocyte lineage by two independent lines of investigation. First, growth-arrested 10T1/2 cells do not normally respond to a hormonal cocktail that causes various growth-arrested preadipocyte cell lines to differentiate into adipocytes, but if 10T1/2 cells are first treated with BMP-4 they will respond to these hormonal inducers by undergoing terminal adipocyte differentiation. Second, a preadipocyte cell line, A33 cells, derived from 10T1/2 cells after exposing the cells to the DNA methyltransferase inhibitor 5-azacytidine was shown to express BMP-4, and this endogenous BMP-4 expression is required for acquisition of the preadipocyte phenotype of these cells. A role for the BMP-4 signaling pathway in adipogenesis is discussed.     

Involvement of PTP-RQ in differentiation during adipogenesis of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are self-renewable multipotent progenitor cells with the capacity to differentiate into several distinct mesenchymal lineages. While MSCs display significant potential in tissue engineering and therapeutic applications, the regulatory mechanisms underlying the differentiation of these cells are yet to be established. Phosphorylation is a post-translational modification that plays a significant role in diverse biological phenomena. In this study, to mine the protein tyrosine phosphatases (PTPs) involved in adipogenesis of human MSCs, differential expression of human PTPs was examined using RT-PCR analysis. Among the 107 human PTPs, PTP-RQ was dramatically downregulated during the early phase of adipogenesis. PTP-RQ is classified as a receptor-type III PTP with phosphatidylinositol phosphatase (PIPase) activity. Overexpression of PTP-RQ consistently led to reduced differentiation of MSCs into adipocytes via decreasing the phosphatidyl inositol phosphate level in cells, and consequently downregulating Akt/PKB phosphorylation. Our results collectively suggest that PTP-RQ is a useful target protein for regulating the differentiation of MSCs into adipocytes, and may be used to develop novel drugs for the treatment of obesity.     

Strontium ranelate rebalances bone marrow adipogenesis and osteoblastogenesis in senescent osteopenic mice through NFATc/Maf and Wnt signaling

With aging, bone marrow mesenchymal stromal cell (MSC) osteoblast differentiation decreases whereas MSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. Here, we investigated whether activation of cell signaling by strontium ranelate (SrRan) can reverse the excessive adipogenic differentiation associated with aging. In murine MSC cultures, SrRan increased Runx2 expression and matrix mineralization and decreased PPARγ2 expression and adipogenesis. This effect was associated with increased expression of the Wnt noncanonical representative Wnt5a and adipogenic modulator Maf and was abrogated by Wnt- and nuclear factor of activated T-cells (NFAT)c antagonists, implying a role for Wnt and NFATc/Maf signaling in the switch in osteoblastogenesis to adipogenesis induced by SrRan. To confirm this finding, we investigated the effect of SrRan in SAMP6 senescent mice, which exhibit decreased osteoblastogenesis, increased adipogenesis, and osteopenia. SrRan administration at a clinically relevant dose level increased bone mineral density, bone volume, trabecular thickness and number, as shown by densitometric, microscanning, and histomorphometric analyses in long bones and vertebrae. This attenuation of bone loss was related to increased osteoblast surface and bone formation rate and decreased bone marrow adipocyte volume and size. The restoration of osteoblast and adipocyte balance induced by SrRan was linked to increased Wnt5a and Maf expression in the bone marrow. The results indicate that SrRan acts on lineage allocation of MSCs by antagonizing the age-related switch in osteoblast to adipocyte differentiation via mechanisms involving NFATc/Maf and Wnt signaling, resulting in increased bone formation and attenuation of bone loss in senescent osteopenic mice.     

Role of Wnt10b and C/EBPalpha in spontaneous adipogenesis of 243 cells

This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPalpha and PPARgamma. The 243 cells express C/EBPalpha early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPalpha is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPalpha, results in induction of PPARgamma and spontaneous adipogenesis of 243 cells.     

Mechanical stretch inhibits myoblast-to-adipocyte differentiation through Wnt signaling

Myoblasts are able to differentiate into other mesenchymal lineages including adipocytes and osteoblasts. However, it is not known how this differentiation is normally regulated in intact animals and humans. Here, we subjected cultured C2C12 myoblasts to cyclic mechanical stretch (20% elongation) during differentiation into adipocytes. Mechanical stretch inhibited the myoblast-to-adipocyte differentiation significantly, concurrent with an enhanced expression of Wnt10b mRNA. Inhibition of the Wnt signaling by incubation of the myoblasts with a soluble Wnt ligand, sFRP-2, abolished the inhibitory function of mechanical stretch on adipogenesis. These findings provide evidence that mechanical stretch inhibits myoblast-to-adipocyte differentiation possibly through Wnt signaling.     

An Adenosine Receptor-Krüppel-like Factor 4 Protein Axis Inhibits Adipogenesis

Adipogenesis represents a key process in adipose tissue development and remodeling, including during obesity. Exploring the regulation of adipogenesis by extracellular ligands is fundamental to our understanding of this process. Adenosine, an extracellular nucleoside signaling molecule found in adipose tissue depots, acts on adenosine receptors. Here we report that, among these receptors, the A2b adenosine receptor (A2bAR) is highly expressed in adipocyte progenitors. Activation of the A2bAR potently inhibits differentiation of mouse stromal vascular cells into adipocytes, whereas A2bAR knockdown stimulates adipogenesis. The A2bAR inhibits differentiation through a novel signaling cascade involving sustained expression of Krüppel-like factor 4 (KLF4), a regulator of stem cell maintenance. Knockdown of KLF4 ablates the ability of the A2bAR to inhibit differentiation. A2bAR activation also inhibits adipogenesis in a human primary preadipocyte culture system. We analyzed the A2bAR-KLF4 axis in adipose tissue of obese subjects and, intriguingly, found a strong correlation between A2bAR and KLF4 expression in both subcutaneous and visceral human fat. Hence, our study implicates the A2bAR as a regulator of adipocyte differentiation and the A2bAR-KLF4 axis as a potentially significant modulator of adipose biology.     

Osteoblastic cells: differentiation and trans-differentiation

The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast differentiation from MSC is a central topic in bone biology that can provide insight into mechanisms of bone maintenance and also novel pharmacological targets to increase osteoblast differentiation and consequently bone formation.     

Expression of p107 and p130 during human adipose-derived stem cell adipogenesis

Within the first 24 h of hormonally stimulated adipocyte differentiation, murine 3T3-L1 preadipocytes undergo a mitotic expansion phase prior to terminal differentiation. During this time, the cell cycle regulatory proteins, p130 and p107 undergo dramatic differential expression and the transient increase in expression of p107 appears to be required for terminal differentiation. Recently, human adipose-derived human stem cells (hASC) of mesenchymal origin have been used as a model of human adipocyte differentiation and we sought to determine if differentiating hASC undergo clonal expansion and if the regulated expression of p130/p107 was similar to that observed during 3T3-L1 adipogenesis. Results indicate that differentiating hASC, unlike 3T3-L1 cells do not undergo clonal expansion and p130 expression gradually diminishes across differentiation. However, p107 expression is transiently increased during hASC differentiation in a manner analogous to 3T3-L1 cells suggesting a similar role for p107 in terminal differentiation in human adipocytes.     

MicroRNA调控动物脂肪细胞分化研究进展

脂肪组织不仅在维持机体能量代谢和稳态上发挥重要作用,同时也是重要的内分泌器官。脂肪细胞分化是由间充质干细胞(Mesenchymal stem cells, MSC)向成熟脂肪细胞分化的复杂生理过程,该过程由大量转录因子、激素、信号通路分子协同调控。miRNA作为内源性非编码RNA,主要通过抑制转录后翻译等机制来调控基因表达。近年来越来越多的证据表明miRNA通过调控脂肪细胞分化相关的转录因子和重要信号分子进而影响动物脂肪细胞的分化和脂肪形成。本文对miRNA影响动物白色、棕色和米色脂肪细胞分化的作用机制及其相关调控通路和关键因子进行了归纳总结,以期为肥胖等代谢性疾病的治疗提供一定的理论指导和新的治疗思路。     

Biochemical changes induced by strontium ranelate in differentiating adipocytes

Low bone formation in osteoporosis is associated with a shift from osteoblastic to adipogenic differentiation of mesenchymal stem cells (MSC) inducing a concomitant lipotoxic milieu within the bone marrow. Strontium ranelate (SrRN), a treatment for osteoporosis, has both anti-resorptive and anabolic effects on bone. The anabolic effect of SrRN has been associated with its effect on both osteoblastogenesis and adipogenesis. However, the effect of SrRN on the potentially lipotoxic factors produced by differentiating marrow adipocytes remains poorly understood. To expand the knowledge on the effect of SrRN treatment on the bone microenvironment, we assessed changes in adipogenic factors and adipokine expression in adipocytic differentiation of MSC in vitro. Primary human MSC were induced to differentiate in adipogenic conditions in the presence or absence of SrRN (1–2 mM). We tested the dose-dependent effects of SrRN on adipocyte differentiation including changes in the expression of adipogenic markers and adipokines. We report that adipogenesis was negatively affected in the presence of SrRN with a concomitant dose-dependent decrease in the expression of adipogenic markers and changes in adipokine profile. Taken together, our data suggests that SrRN induces biochemical changes in differentiating adipocytes that could generate a favorable osteogenic effect within the bone marrow milieu.     

Effects of arachidonic acid on the concentration of hydroxyeicosatetraenoic acids in culture media of mesenchymal stromal cells differentiating into adipocytes or osteoblasts

Metabolites derived from the polyunsaturated fatty acids (PUFA) may modulate the mesenchymal stromal cell (MSC) differentiation. Such cells can differentiate into different cellular types, including adipocytes and osteoblasts. Aging favors the bone marrow MSC differentiation toward the former, causing a loss of bone density associated with pathologies like osteoporosis. The omega-6 arachidonic acid (AA) favors MSC adipogenesis to a greater extent than omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we study the joint action of both PUFA. Thus, not induced and induced to adipocyte or osteoblast MSC were treated with 20 μM of each PUFA (either AA, AA + DHA or AA + EPA). The expression of osteogenic and adipogenic molecular markers, the alox15b lipoxygenase gene expression and the 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acids (HETE) derived from the AA metabolism in the culture media were determined. The results show that the adipogenesis induction of AA is not suppressed by the joint presence of EPA and DHA. In fact, both increased the adipogenic effect of AA on MSC differentiated into osteoblasts. The different HETE concentrations increased in cultures supplemented with AA, albeit such concentrations were lower in the cultures induced to differentiate, mainly at day 21 after the induction. Furthermore, the reduction in the HETE concentration was correlated with a higher expression of the alox15b gene. These results highlight the PUFA metabolism differences between uninduced and induced MSC to differentiate into adipocytes and osteoblasts, besides the relevant role of the lipoxygenase gene expression in adipogenesis induction.     

Differential redox potential profiles during adipogenesis and osteogenesis

Development is an orderly process that requires the timely activation and/or deactivation of specific regulatory elements that control cellular proliferation, differentiation and apoptosis. While many studies have defined factors that control developmental signaling, the role of intracellular reduction/oxidation (redox) status as a means to control differentiation has not been fully studied. Redox states of intracellular couples may play a very important role in regulating redox-sensitive elements that are involved in differentiation signaling into specific phenotypes. In human mesenchymal stem cells (hMSCs), which are capable of differentiating into many different types of phenotypes, including osteoblasts and adipocytes, glutathione (GSH), cysteine (Cys) and thioredoxin-1 (Trx1) redox potentials were measured during adipogenesis and osteogenesis. GSH redox potentials (E_h) during both osteogenesis and adipogenesis became increasingly oxidized as differentiation ensued, but the rate at which this oxidation occurred was unique for each process. During adipogenesis, Cys E_h became oxidized as adipogenesis progressed but during osteogenesis, it became reduced. Interestingly, intracellular Trx1 concentrations appeared to increase in both adipogenesis and osteogenesis, but the E_h was unchanged when compared to undifferentiated hMSCs. These data show that hMSC differentiation into either adipocytes of osteoblasts corresponds to a unique redox state profile, suggesting that differentiation into specific phenotypes are likely regulated by redox states that are permissive to a specific developmental process.     

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.     

Characterization of two populations of mesenchymal progenitor cells in umbilical cord blood

Umbilical cord blood (UCB) is a valuable source for hematopoietic progenitor cell therapy. Moreover, it contains another subset of non-hematopoietic population referred to as mesenchymal progenitor cells (MPCs), which can be ex vivo expanded and differentiated into osteoblasts, chondrocytes and adipocytes. In this study, we successfully isolated the clonogenic MPCs from UCB by limiting dilution method. These cells exhibited two different morphologic phenotypes, including flattened fibroblasts (majority) and spindle-shaped fibroblasts (minority). Both types of MPCs shared similar cell surface markers except CD90 and had similar osteogenic and chondrogenic potentials. However, the spindle-shaped clones possessed the positive CD90 expression and showed a greater tendency in adipogenesis, while the flattened clones were CD90 negative cells and showed a lower tendency in adipogenesis. The high number of flattened MPCs might be linked to the less sensitivity of UCB-derived MPCs in adipogenic differentiation.     

The Insulin-like Growth Factor-1 Binding Protein Acid-labile Subunit Alters Mesenchymal Stromal Cell Fate

Age-related osteoporosis is accompanied by an increase in marrow adiposity and a reduction in serum insulin-like growth factor-1 (IGF-1) and the binding proteins that stabilize IGF-1. To determine the relationship between these proteins and bone marrow adiposity, we evaluated the adipogenic potential of marrow-derived mesenchymal stromal cells (MSCs) from mice with decreased serum IGF-1 due to knockdown of IGF-1 production by the liver or knock-out of the binding proteins. We employed 10–16-week-old, liver-specific IGF-1-deficient, IGFBP-3 knock-out (BP3KO) and acid-labile subunit knock-out (ALSKO) mice. We found that expression of the late adipocyte differentiation marker peroxisome proliferator-activated receptor γ was increased in marrow isolated from ALSKO mice. When induced with adipogenic media, MSC cultures from ALSKO mice revealed a significantly greater number of differentiated adipocytes compared with controls. MSCs from ALSKO mice also exhibited decreased alkaline-phosphatase positive colony size in cultures that were stimulated with osteoblast differentiation media. These osteoblast-like cells from ALSKO mice failed to induce osteoclastogenesis of control cells in co-culture assays, indicating that impairment of IGF-1 complex formation with ALS in bone marrow alters cell fate, leading to increased adipogenesis.     

Expression of tumor necrosis factor alpha and its receptors during cellular differentiation

Tumor necrosis factor alpha (TNFalpha) is a potent proinflammatory cytokine also involved in cellular differentiation processes. TNFalpha and both of its receptors (TNFR1 and TNFR2) can be co-expressed on the same cell, allowing for local signaling. This study has examined the expression of all components necessary for autocrine cytokine regulation during human hematopoietic, epithelial, and mesenchymal models of cellular differentiation. Macrophage and dendritic differentiation of human peripheral blood monocytes decreased their TNFalpha and TNFR2 expression while increasing the TNFR1 mRNA. In colon epithelial cell lines (HT-29 and Caco-2) TNFalpha-, TNFR1-, and TNFR2-expression was decreased upon differentiation. No changes, however, were seen during human skin keratinocyte differentiation. TNFR1 expression was unchanged in all three mesenchymal lineages (adipogenesis, chondrogenesis, osteogenesis) tested. Differentiation decreases the TNFalpha message in adipocytes and the TNFR2 mRNA in adipocytes and osteocytes. Our results demonstrate that there is no general principle for TNFalpha signaling during conversion of cells from progenitor to a more differentiated phenotype. Paracrine signaling by TNFalpha to orchestrate different cell types during tissue development and remodeling, therefore, probably overrides the autocrine regulation of differentiation by TNFalpha. Non-signaling TNF-receptors may protect chondrocytes and osteocytes from the anti-differentiation effects of local TNFalpha production.