Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes

Mesenchymal cells can differentiate into osteoblasts, adipocytes, myoblasts, or chondroblasts. Whether mesenchymal cells that have initiated differentiation along one lineage can transdifferentiate into another is largely unknown. Using 3T3-F442A preadipocytes, we explored whether extracellular signals could redirect their differentiation from adipocyte into osteoblast. 3T3-F442A cells expressed receptors and Smads required for bone morphogenetic protein (BMP) signaling. BMP-2 increased proliferation and induced the early osteoblast differentiation marker alkaline phosphatase, yet only mildly affected adipogenic differentiation. Retinoic acid inhibited adipose conversion and cooperated with BMP-2 to enhance proliferation, inhibit adipogenesis, and promote early osteoblastic differentiation. Expression of BMP-RII together with BMP-RIA or BMP-RIB suppressed adipogenesis of 3T3-F442A cells and promoted full osteoblastic differentiation in response to retinoic acid. Osteoblastic differentiation was characterized by induction of cbfa1, osteocalcin, and collagen I expression, and extracellular matrix calcification. These results indicate that 3T3-F442A preadipocytes can be converted into fully differentiated osteoblasts in response to extracellular signaling cues. Furthermore, BMP and retinoic acid signaling cooperate to stimulate cell proliferation, repress adipogenesis, and promote osteoblast differentiation. Finally, BMP-RIA and BMP-RIB induced osteoblast differentiation and repressed adipocytic differentiation to a similar extent.     

Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor γ agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-β1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.     

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.     

Heparin-binding epidermal growth factor-like growth factor inhibits adipocyte differentiation at commitment and early induction stages

Abstract Adipocytokines, bioactive molecules secreted from adipose tissues, play important roles in physiology, development, and disease. Recently, heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as an adipocytokine whose expression correlates with obesity. However, the biological role of fat-secreted HB-EGF is still unclear. In this study, we investigated the effects of HB-EGF on the adipocyte differentiation of C3H10T1/2 pluripotent mesenchymal cells. Upon adipogenic conversion of C3H10T1/2 cells, HB-EGF displayed dynamic changes in expression where an initial decrease was followed by increased levels of expression at later stages. HB-EGF treatment during adipogenic induction inhibited lipid accumulation and decreased the expression of adipocyte molecular markers (fatty acid-binding protein, peroxisome proliferator-activated receptor γ, and CAAT enhancer-binding protein α) and lipogenic genes (glucose transporter, fatty acid synthetase, and lipoprotein lipase). Therefore, HB-EGF has an inhibitory effect on adipocyte differentiation. Administration of HB-EGF at various intervals during adipocyte differentiation revealed that HB-EGF acts during the early stages of adipocyte differentiation, but not at the later stages of differentiation. Furthermore, HB-EGF was able to block the commitment of pluripotent mesenchymal cells to the adipocyte lineage triggered by bone morphogenic protein 4 treatment. These data suggest that HB-EGF acts as a negative regulator of adipogenesis by inhibiting the commitment and early differentiation of the adipose lineage. The inhibitory role of HB-EGF on adipocyte differentiation of pluripotent mesenchymal cells sheds light on potential mechanisms that control adipose tissue homeostasis.     

Expression regulation and function of Pref-1 during adipogenesis of human mesenchymal stem cells (MSCs)

Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARγ (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARγ expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.     

Propyl Gallate Inhibits Adipogenesis by Stimulating Extracellular Signal-Related Kinases in Human Adipose Tissue-Derived Mesenchymal Stem Cells

Propyl gallate (PG) used as an additive in various foods has antioxidant and anti-inflammatory effects. Although the functional roles of PG in various cell types are well characterized, it is unknown whether PG has effect on stem cell differentiation. In this study, we demonstrated that PG could inhibit adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells (hAMSCs) by decreasing the accumulation of intracellular lipid droplets. In addition, PG significantly reduced the expression of adipocyte-specific markers including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein 2 (aP2). PG inhibited adipogenesis in hAMSCs through extracellular regulated kinase (ERK) pathway. Decreased adipogenesis following PG treatment was recovered in response to ERK blocking. Taken together, these results suggest a novel effect of PG on adipocyte differentiation in hAMSCs, supporting a negative role of ERK1/2 pathway in adipogenic differentiation.     

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.     

3,5-dicaffeoyl‑epi-quinic acid from Atriplex gmelinii enhances the osteoblast differentiation of bone marrow-derived human mesenchymal stromal cells via WnT/BMP signaling and suppresses adipocyte differentiation via AMPK activation

BackgroundImpaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment.PurposeIn this context, 3,5-dicaffeoyl‑epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro.MethodsAnti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways.ResultsAt 10 μM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of β-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes.ConclusionDCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.     

Asiatic Acid Inhibits Adipogenic Differentiation of Bone Marrow Stromal Cells

Bone marrow mesenchymal stromal cells (BMSCs) are the common precursors for both osteoblasts and adipocytes. With aging, BMSC osteoblast differentiation decreases whereas BMSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. In the present study, we investigated the effect of asiatic acid (AA) on adipocytic differentiation of BMSCs. AA inhibited the adipogenic induction of lipid accumulation, activity of glycerol-3-phosphate dehydrogenase, and expression of marker genes in adipogenesis: peroxisome proliferation-activated receptor (PPAR)γ, adipocyte fatty acid-binding protein (ap) 2, and adipsin. Further, we found that AA did not alter clonal expansion rate and expression of C/EBPβ, upstream key regulator of PPARγ, and binding activity of C/EBPβ to PPARγ promoter was not affected by AA as well. These findings suggest that AA may modulate differentiation of BMSCs to cause a lineage shift away from the adipocytes, and inhibition of PPARγ by AA is through C/EBPβ-independent mechanisms. Thus, AA could be a potential candidate for a novel drug against osteoporosis.     

The effects of retinoic acid on reversing the adipocyte differentiation into an osteoblastic tendency in ST2 cells, a murine bone marrow-derived stromal cell line

Although the mouse bone marrow stromal cell line ST2 has been known to be differentiated into osteoblasts, the differentiation characteristics of the cell into adipocyte and the concerned relationship between its adipogenesis and osteogenesis remains unknown. The adipogenic induction medium which is made up of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine(IBMX), stimulated the expression of n early adipogenic marker PPAR γ and a late marker GPDH in ST2 cells. The triglyceride accumulation and lipid stain level generated by the induction medium in ST2 cells was inhibited by RA with IC₅₀ at about 1 nM. The induction medium up-regulated expression of PPARγ and GPDH was also inhibited by RA whereas RA (30 nM) exterted no effect on the cell growth. Interestingly, treatment of the cells with induction medium in the presense of RA caused a 3- or 10-fold higher in ALP activity respectively as compared to those treated with RA or the induction medium alone. RT-PCR analysis showed that such a synergistic effect of RA and the induction medium paralleled the process of inhibition on adipogenesis. Additional experiments showed that IBMX played a key role in increasing the effect of RA and ALP activity. Our results suggested that the relationship between adipogenesis and osteogenesis in ST2 cells was reciprocally interrelated and the process of adipogenesis could be potentially reversed into an osteoblastogenic tendency. This is the first report demonstrating that RA transforms adipogenic potential into an osteoblastic tendency. This revised version was published online in July 2006 with corrections to the Cover Date.     

α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

α-Poly-l-lysine (PLL) has been used for various purposes such as cell attachment, immunization, and molecular delivery, and is known to be cytotoxic to several cell lines. Here, we studied the effect of PLL on the adipogenesis of 3T3-L1 cells and investigated the underlying mechanism. Differentiation media containing PLL with a molecular weight (MW) greater than 4 kDa enhanced lipid droplet formation and increased adipogenic marker levels, indicating an increase in adipocyte differentiation. PLL with a molecular weight between 30 and 70 kDa was more effective than PLL of other sizes in 3T3-L1 cell differentiation. Moreover, PLL induced 3T3-L1 adipogenesis in insulin-free adipocyte differentiation medium. Incubation with insulin and PLL exhibited greater adipogenesis than insulin treatment only even at a high concentration. PLL stimulated insulin signaling and augmented the signaling pathway when it was added with insulin. While PLL did not activate the glucocorticoid receptor, which is phosphorylated by dexamethasone (DEX), it showed a positive effect on the cAMP signal pathway when preadipocytes were treated with PLL and 3-isobutyl-1-methylxanthine (IBMX). Consistent with these results, incubation with PLL and DEX without IBMX induced adipocyte differentiation. We also observed that the mitotic clonal expansion phase was the critical stage in adipogenesis for inducing the effects of PLL. These results suggest that PLL functions as an adipogenic inducer in 3T3-L1 preadipocytes and PLL has a direct effect on insulin signaling, one of the main regulatory pathways.

     

The effects of myostatin on adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are mediated through cross-communication between Smad3 and Wnt/beta-catenin signaling pathways

The effects of myostatin on adipogenic differentiation are poorly understood, and the underlying mechanisms are unknown. We determined the effects of human recombinant myostatin protein on adipogenesis of bone marrow-derived human mesenchymal stem cells (hMSCs) and adipose tissue-derived preadipocytes. For both progenitor cell types, differentiation in the presence of myostatin caused a dose-dependent reduction of lipid accumulation and diminished incorporation of exogenous fatty acid into cellular lipids. Myostatin significantly down-regulated the expression of adipocyte markers PPARgamma, C/EBPalpha, leptin, and aP2, but not C/EBPbeta. Overexpression of PPARgamma, but not C/EBPbeta, blocked the inhibitory effects of myostatin on adipogenesis. Myostatin induced phosphorylation of Smad3 in hMSCs; knockdown of Smad3 by RNAi or inhibition of its upstream kinase by an Alk5 inhibitor blocked the inhibitory effect of myostatin on adipogenesis in hMSCs, implying an important role of Smad3 activation in this event. Furthermore, myostatin enhanced nuclear translocation of beta-catenin and formation of the Smad3-beta-catenin-TCF4 complex, together with the altered expression of a number of Wnt/beta-catenin pathway genes in hMSCs. The inhibitory effects of myostatin on adipogenesis were blocked by RNAi silencing of beta-catenin and diminished by overexpression of dominant-negative TCF4. The conclusion is that myostatin inhibited adipogenesis in human bone marrow-derived mesenchymal stem cells and preadipocytes. These effects were mediated, in part, by activation of Smad3 and cross-communication of the TGFbeta/Smad signal to Wnt/beta-catenin/TCF4 pathway, leading to down-regulation of PPARgamma.     

Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells

Wedelolactone is an herbal medicine that is used to treat septic shock, hepatitis and venom poisoning. Although in differentiated and cancer cells, wedelolactone has been identified as anti‐inflammatory, growth inhibitory, and pro‐apoptotic, the effects of wedelolactone on stem cell differentiation remain largely unknown. Here, we report that wedelolactone inhibits the adipogenic differentiation of human adipose tissue‐derived mesenchymal stem cells (hAMSCs). Wedelolactone reduced the formation of lipid droplets and the expression of adipogenesis‐related proteins, such as CCAAT enhancer‐binding protein‐α (C/EBP‐α), peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), lipoprotein lipase (LPL), and adipocyte fatty acid‐binding protein aP2 (aP2). Wedelolactone mediated this process by sustaining ERK activity. In addition, inhibition of ERK activity with PD98059 resulted in reversion of the wedelolactone‐mediated inhibition of adipogenic differentiation. Taken together, these results indicate that wedelolactone inhibits adipogenic differentiation through ERK pathway and suggest a novel inhibitory effect of wedelolactone on adipogenic differentiation in hAMSCs. J. Cell. Biochem. 113: 3436–3445, 2012. © 2012 Wiley Periodicals, Inc.     

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2

Background aimsHuman bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25–81 years old), including patients with or without systemic vascular diseases.MethodsAll stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining.ResultsSystemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes.ConclusionsThe aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell–based therapy for cartilage-related diseases.