Adipogenesis Of Mesenchymal Stem Cells Research Articles

Notch Signaling in Mesenchymal Stem Cells Harvested From Geriatric Mice

Morbidity associated with geriatric fractures may be attributed, in part, to compromised mesenchymal stem cell (MSC) function within the fracture callus. The Notch signaling pathway is important for the healing of nonskeletal tissues in an age-dependent manner, but the effect of Notch on age-dependent fracture healing and MSC dysfunction has not been substantiated. The objective of this study was to examine Notch signaling in MSCs obtained from young and geriatric mice. Marrow-derived MSCs were harvested from the femora of 5- and 25-month-old C57BL/6 mice. We assessed in vivo MSC number using CFU-F, proliferation using an Alamar Blue assay, osteoblast differentiation by Alizarin Red S staining, and adipogenic differentiation using Oil Red O staining. Notch receptor and ligand expression was assessed using quantitative PCR, and Notch signaling was assessed by evaluating Notch target gene expression (Hey and HES) under basal conditions and when cells were plated to Jagged-1 ligand. MSC from geriatric mice exhibit reduced MSC number (CFU-F), proliferation, adipogenesis, and inconsistent osteogenesis. The highest expressed Notch receptor is Notch 2, and the highest expressed ligand is Jagged-1, but there were no differences in ligand and receptor gene expression between young and old MSCs. Interestingly, geriatric MSCs show decreased basal Notch signaling activity but are fully responsive to Jagged-1 stimulation. These data suggest that therapeutic targeting of Notch signaling should be explored in clinical therapies to improve geriatric fracture healing.

Reversine increases the plasticity of lineage-committed preadipocytes to osteogenesis by inhibiting adipogenesis through induction of TGF-β pathway in vitro

Reversine has been reported to reverse differentiation of lineage-committed cells to mesenchymal stem cells (MSCs), which then enables them to be differentiated into other various lineages. Both adipocytes and osteoblasts are known to originate from common MSCs, and the balance between adipogenesis and osteogenesis in MSCs is reported to modulate the progression of various human diseases, such as obesity and osteoporosis. However, the role of reversine in modulating the adipogenic potential of lineage-committed preadipocytes and their plasticity to osteogenesis is unclear. Here we report that reversine has an anti-adipogenic function in 3T3-L1 preadipocytes in vitro and alters cell morphology and viability. The transforming growth factor-β (TGF-β) pathway appears to be required for the anti-adipogenic effect of reversine, due to reversine-induced expression of genes involved in TGF-β pathway and reversal of reversine-inhibited adipogenesis by inhibition of TGF-β pathway. We show that treatment with reversine transformed 3T3-L1 preadipocytes into MSC-like cells, as evidenced by the expression of MSCs marker genes. This, in turn, allowed differentiation of lineage-committed 3T3-L1 preadipocytes to osteoblasts under the osteogenic condition in vitro. Collectively, these findings reveal a new function of reversine in reversing lineage-committed preadipocytes to osteogenesis in vitro, and provide new insights into adipose tissue-based regeneration of osteoblasts.

ICAM-1 regulates differentiation of MSC to adipocytes via activating MAPK pathway

This study was aimed to explore the molecular mechanism of the regulatory effects of ICAM-1 on the differentiation of mesenchymal stem cells (MSC) to adipocytes. The murine MSC cell line C3H10T 1/2 was treated with the supernatants contained plasmid MIGR1-ICAM-1 and MIGR1-ICAM-1/MSC (high expression of ICAM-1), the activation of the pathway was detected by Western blot. The ICAM-1 modified MSC and its control cells named MIGR1/MSC were cultured in adipocyte medium with or without the inhibitors of the ERK, P38, and JNK pathway. Oil-red-O staining was used to detect the lipid accumulation, and the expression of C/EBPα and PPARγ in differentiation of MSC to adipocyte were examined by real-time-PCR. The results showed that the overexpression of ICAM-1 stably activated the ERK, P38, and JNK pathway in MSC. Inhibiting of the activation of ERK pathways by chemical inhibitors up-regulated the mRNA expression level of C/EBPα and PPARγ in MIGR1-ICAM-1/MSC while inhibiting of P38 pathway resulted in lower mRNA expression of the transcription factors. Consistent with the mRNA expression, the lipid droplets were getting smaller and number of adipocytes increased when P38 pathway was inhibited, while bigger lipid droplet and increased quantity of adipocytes were identified in MIGR1-ICAM-1/MSC with the addition of ERK pathway inhibitor. It is concluded that ICAM-1 may suppress MSC differentiate into adipocyte via activating ERK pathway, while it can maintain the adipogenesis of MSC though P38 pathway.

Control of Simultaneous Osteogenic and Adipogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are pluripotent cells which can differentiate into several distinct lineages such as osteoblasts and adipocytes. Osteogenesis and adipogenesis of MSCs induced with specific induction factors have been well exploited. However, it is not clear how simultaneous osteogenesis and adipogenesis of MSCs is balanced by the composition of induction medium. In this study, MSCs were cultured in the mixture medium at different ratio of osteogenic medium (OM) and adipogenic medium (AM) to investigate the simultaneous osteogenesis and adipogenesis of MSCs. Differentiation was investigated by histological staining and real-time PCR analysis. The results showed that adipogenesis was dosage dependent on the concentration of adipogenic induction factors, while osteogenesis was not. The balance of osteogenesis and adipogenesis could be control by different ratio of osteogenic and adipogenic media. The results will be useful for controlling stem cell differentiation.

Mechanically activated fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells

Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of β-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.

Enhancement of Osteogenic Differentiation and Proliferation in Human Mesenchymal Stem Cells by a Modified Low Intensity Ultrasound Stimulation under Simulated Microgravity

Adult stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. Lack of mechanical signals due to disuse can inhibit osteogenesis and induce adipogenesis of mesenchymal stem cells (MSCs). Long-term bed rest due to both brain/spinal cord injury and space travel can lead to disuse osteoporosis that is in part caused by a reduced number of osteoblasts. Thus, it is essential to provide proper mechanical stimulation for cellular viability and osteogenesis, particularly under disuse conditions. The objective of this study was to examine the effects of low intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of adipose-derived human stem cells (Ad-hMSC) in simulated microgravity conditions. Cells were cultured in a 1D clinostat to simulate microgravity (SMG) and treated with LIPUS at 30mW/cm2 for 20 min/day. It was hypothesized that the application of LIPUS to SMG cultures would restore osteogenesis in Ad-hMSCs. The results showed significant increases in ALP, OSX, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures of Ad-MSC compared to non-treated cultures. LIPUS also restored OSX, RUNX2 and RANKL expression in osteoblast cells. SMG significantly reduced ALP positive cells by 70% (p<0.01) and ALP activity by 22% (p<0.01), while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix collagen and mineralization was assessed by Sirius red and Alizarin red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50% (p<0.001) and mineralization by 45% (p<0.001) in LIPUS treated-SMG cultures relative to SMG-only cultures. The data suggest that LIPUS treatment can restore normal osteogenic differentiation of MSCs from disuse by daily short duration stimulation.

The role of focal adhesion kinase in BMP4 induction of mesenchymal stem cell adipogenesis

Obesity is characterized by excessive adipocytic number growth and resultant adipose tissue hyperplasia. However, molecular mechanisms of abnormal recruitment of new adipocytes from precursor cells are not fully known. Several studies showed that bone morphogenetic proteins (BMPs) also play a role in inducing mesenchymal stem cells (MSCs) to commit to adipocytes. We tested the hypothesis that focal adhesion kinase (FAK), one of the vital focal adhesion signaling molecules, is required for BMP4 induction of MSC adipogenesis. BMP4 exposure triggered FAK activation at pY397 auto-phosphorylation site in murine C3H10T1/2 MSCs. Interestingly, silencing FAK by small hairpin RNA (shRNA) significantly suppressed BMP4 induction of MSC adipogenic activities, including lipid accumulation and expression of key adipogenic genes (C/EBPα, PPARγ, aP2), as relative to shRNA vector control. As a potential molecular mechanism, BMP4-triggered phosphorylation in Smad1/5/8 and p38 was significantly downregulated by shRNA-FAK. Pharmacological FAK inhibitor 14 provided similar results in BMP4-mediated MSC adipogenesis and Smad/p38 signaling. Our data clearly suggest a link between FAK and BMP4 induction of MSC adipogenesis, and may indicate a potential therapeutic approach targeting FAK for dealing with obesity.

Gdf6 induces commitment of pluripotent mesenchymal C3H10T1/2 cells to the adipocyte lineage

Mesenchymal stem cells have the potential to undergo commitment and differentiation into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes. Growth differentiation factor6 (Gdf6) is a member of the transforming growth factorβ superfamily. We have examined the potential role of Gdf6 in adipogenesis of mesenchymal stem cells, and found that over-expression of Gdf6 induced commitment of pluripotent mesenchymal C3H10T1/2 cells to the adipocyte lineage. The typeI receptor Bmpr1a and the typeII receptors Bmpr2 and Acvr2a mediate the Gdf6 signaling pathway. RNAi silencing of Smad4 and p38 MAPK suggested that both Smad and p38 MAPK pathways are involved in this process. The expression of Runx1t1 was down-regulated in committed pre-adipocytes, and forced expression of Runx1t1 blocked the adipocytic commitment. The results demonstrate a role for Gdf6 in adipocytic commitment and differentiation.

Mesenchymal Stem Cell and Chondrocyte Fates in a Multishear Microdevice Are Regulated by Yes-Associated Protein

Mechanical cues exert considerable influence on the fates of stem cells and terminally differentiated chondrocytes. The elucidation of the interactions between cell fate and mechanical cues in nuclear mechanotransduction will provide new clues to modulate tissue homeostasis and regeneration. In this study, we used an integrated microfluidic perfusion device to simultaneously generate multiple-parameter fluid shear stresses to investigate the role of fluid flow stimuli in the regulation of Yes-associated protein (YAP) expression and the fates of mesenchymal stem cells (MSCs) and primary chondrocytes. YAP expression was regulated by the level of fluid flow stimulus in both MSCs and chondrocytes. An increase in the magnitude of stimulation enhanced the expression of YAP, ultimately resulting in an increase in osteogenesis and a decrease in adipogenesis for MSCs, and initiating dedifferentiation for chondrocytes. Cytochalasin D not only repressed nuclear YAP accumulation in the flow state, but also abrogated flow-induced effects on MSC differentiation and the chondrocyte phenotype, resulting in MSC adipogenesis and the maintenance of the chondrocyte phenotype. Our findings reveal the connection between YAP and MSC/chondrocyte fates in a fluid flow-induced mechanical microenvironment and provide new insights into the mechanisms by which mechanical cues regulate the fates of MSCs and chondrocytes.

Endothelium Specific Up Regulation of HO‐1 Gene by Lentiviral Transfection Promotes Release of Positive Regulators of Adipoctye Function

We aimed to characterize the physiological effects of up regulating endothelial heme oxygenase‐1 (HO‐1) on adipogenesis in human mesenchymal stem cells (MSCs). Microvessel endothelial cells (HMEC‐1) were cultured along with a lentiviral (lenti) construct expressing human HO‐1 under the control of endothelium specific promoter vascular endothelium cadherin heme oxygenase (VECAD‐HO‐1) and Lenti‐VECAD‐GFP used as the control. In complementary experiments HMEC‐1 cells were cultured in the presence or absence of tin‐mesoporphrin (5 μM). Conditioned media (CM) was harvested and tested at 5%, 10% and 20%. The 10% CM from HMEC cells was used for ideal effect, along with adipogenic media, to measure paracrine effect on adipogenesis in MSCs. The MSCs exposed to CM from VECAD‐HO‐1 transfected cells demonstrated reduced (10.2 ± 1 at 490nm) adipogenesis (lipid droplets) as compared to MSCs exposed to CM from Lenti‐VECAD‐GFP cells (17.0±2, p&gt;;0.05). CM from HMEC‐1 cells treated with inhibitor of HO‐1 activity promoted higher adipogenesis with increased adipocyte hypertrophy as opposed to HMEC‐1 treated activity transfected with the VECAD‐GFP control ( 24.5±1, p&lt;0.01). These observations suggest that EC under stressed conditions releases a negative regulator as seen in VECAD‐HO‐1 in presence of HO‐1 inhibitor and increases adipocyte dysfunction.

Mechanically Activated Fyn Utilizes mTORC2 to Regulate RhoA and Adipogenesis in Mesenchymal Stem Cells

Mechanically Activated Fyn Utilizes mTORC2 to Regulate RhoA and Adipogenesis in Mesenchymal Stem Cells

Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes

Evidence indicates that the balance between osteoblastogenesis and adipogenesis of mesenchymal stem cells (MSCs) is regulated by several hormones, growth factors, and their downstream signaling cascades. Previous studies suggest that retinoic acid (RA) plays a role in osteoblastogenesis and adipogenesis. However, it is unknown whether RA regulates commitment of MSCs into osteoblasts and adipocytes. In this study, we investigated the role of RA in differentiation of MSCs using the C3H10T1/2 cell line. RA stimulated activity and expression of alkaline phosphatase (ALP) and upregulated activity of the ALP gene promoter. The effects of RA were further enhanced by bone morphogenetic protein 2 (BMP2) and resultant Smad signaling. Furthermore, overexpression of Runx2 and Msx2, critical transcription factors for bone formation and BMP2-dependent osteoblastogenesis, enhanced RA-dependent ALP activity. In view of these findings, RA likely stimulates osteoblast differentiation through the BMP2-Smad-Runx2/Msx2 pathway. In contrast, RA markedly inhibited BMP2-induced adipocyte differentiation, suppressing expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP)α and C/EBPδ, and inhibiting adipogenic function of C/EBPβ, C/EBPδ, and PPARγ. In conclusion, our data suggest that RA regulates commitment of MSCs into osteoblasts and adipocytes by controlling transcriptional regulators.

Abstract 73: Selective Inhibition of COX-1 &amp; 2 Results in Reprogramming of Adipocyte Gene Expression and Adipogenesis in Mesenchymal Stem Cells: Role of 20-HETE-derived 20-OH-PGE2

Eicosanoids are increasingly recognized to play a central role in modulating the process of adipogenesis in human bone marrow derived mesenchymal stem cells (MSCs) where prostaglandin E2 (PGE2) has been shown to negatively regulate adipogenesis. 20-Hydroxyeicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress, and in clinical studies, is associated with increased BMI and metabolic syndrome. In this study we sought to characterize the effects of exogenous 20-HETE on MSC-derived adipogenesis. 20-HETE dose-dependently (0.01-1 μM) increased (p&lt;0.05) adipogenesis (0.11±0.001, 0.12±0.007, 0.14±0.015 and 0.16±0.013) in MSCs, an effect not attenuated by concurrent administration of a putative 20-HETE antagonist (DKB-H1-40-32). However, a selective cyclooxygenase-2 (COX-2) inhibitor prevented (0.071±0.004 vs 0.16±0.013) this pro-adipogenic effect of 20-HETE thus raising the possibility that a metabolic product of 20-HETE is mediating its effects on MSCs. We next exposed these MSCs to exogenous 20-OH-PGE2, an endogenous COX-2 derived product of 20-HETE. Treatment with 20-OH-PGE2 also dose-dependently enhanced (0.08±0.02, 0.14±0.01, 0.21±0.01 and 0.4±0.02) adipogenesis and lipid accumulation in MSC-derived adipocytes. In addition, in cells undergoing COX-1 &amp;2 blockade, treatment of MSCs with PGE2 shifted the dose response curve of 20-OH-PGE2 to the right, indicative of a competitive antagonistic interactions of COX-1 derived PGE2 and COX-2-derived 20-OH-PGE2 on the process of adipogenesis. Both 20-HETE and 20-OH-PGE2-induced adipocyte hypertrophy was accompanied with increased expression of adipogenic regulators, PPARδ, C/EBPα and MEST1. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE2 could enhance lipid accumulation and induces adipocyte hypertrophy in MSC undergoing adipogenic differentiation. We also propose that effects of COX-1 derived PGE2, a known inhibitor of adipocyte differentiation, are opposed by endogenous 20-OH-PGE2 production which appears to predominantly COX-2 dependent.

Abstract 357: Targeting Vascular Endothelium with HO-1 Gene Promotes Release of Positive Regulators of Adipocyte Function

Vascular endothelium is a secretory organ which not only regulates vascular function but also secretes bioactive compounds to modulate tissue homeostasis. We aimed to characterize the physiological effects of up regulating endothelial heme oxygenase-1 (HO-1) on adipogenesis in mesenchymal stem cells (hMSCs). Human microvessel endothelial cells (hMEC-1) were cultured along with a lentiviral construct expressing human HO-1 under the control of endothelium specific promoter VE-cadherin (VECAD-HO1) with VECAD-GFP used as a negative control. In complementary experiments hMEC-1 cells were cultured in the presence or absence of siRNA-HO-1 with scrambled siRNA as a negative control. Three days post-transfection, the conditioned media (CM) from hMEC-1 cells was used, along with adipogenic growth factors, to promote adipogenesis in hMSCs. Supporting in vivo experiments were conducted in SD rats injected with either VECAD-HO-1 or VECAD-GFP construct via the tail vein. Transduction efficiency was confirmed in hMEC-1 cells by demonstrating over expression (p&lt;0.05) of HO-1 or GFP in respectively treated cells. hMSCs exposed to CM from VECAD-HO-1 transfected cells demonstrated reduced (p&lt;0.05) adipogenesis (0.16± 0.008 vs 0.12±0.006 ) as compared to hMSCs exposed to CM from VECAD-GFP cells. On the contrary, CM from hMEC-1 cells treated with siRNA for HO-1 promoted higher adipogenesis (p&lt;0.05) with increased adipocyte hypertrophy as opposed to hMSCs treated with CM from hMEC-1 cells transfected with the control siRNA (p&lt;0.05) (0.17±0.008 vs 0.14±0.04). These effects on hMSCs were corroborated by immunoblot analysis demonstrating reduced expression (p&lt;0.05) of positive regulators of adipogenesis, PPARγ, and increased levels (p&lt;0.05) of negative regulators, β-catenin, Wnt 5b and hedgehog in cells treated with CM from hMEC-1 transfected with VECAD-HO1. In SD rats transfected with VECAD-HO-1, plasma adiponectin levels were enhanced (p&lt;0.05) along with increased (p&lt;0.05) adipose tissue levels of pAKT and pAMPK. In conclusion, we show here that a crosstalk between the vascular endothelium and its microenvironment has the potential to regulate adipogenesis and improve adipocyte function via HO-1-depndent pathways.

Aloe‐emodin inhibits adipocyte differentiation and maturation during in vitro human mesenchymal stem cell adipogenesis

In this study, we examined the effects of Aloe-emodin (AE) on the inhibition of adipocyte differentiation during 3-isobutyl-1-methylxanthine (IBMX)-induced adipocyte differentiation in human mesenchymal stem cells (hMSCs). AE treatment (5, 10, and 20 µM) of preadipocyte cells resulted in a significant (p < 0.05) decrease in glycerol phosphate dehydrogenase and triglyceride levels as well as an increase in lactate dehydrogenase activity and attenuated lipid accumulation compared with untreated differentiated adipocytes. Using quantitative reverse transcription polymerase chain reaction, we studied the mRNA expression levels of resistin, adiponectin, aP(2), lipoprotein lipase, PPARγ, and tumor necrosis factor-α in hMSCs undergoing adipocyte differentiation; treatment with AE decreased the expression of these adipogenic genes and decreased adipocyte differentiation. In addition, AE suppresses the differentiation of hMSCs into adipocytes by downregulating PPARγ and C/EBPα expressions. AE significantly inhibited hMSCs proliferation and preadipocyte differentiation within the first 2 days of treatment, indicating that the antiadipogenic effect.

The Effects of an Antiosteoporosis Herbal Formula Containing Epimedii Herba, Ligustri Lucidi Fructus and Psoraleae Fructus on Density and Structure of Rat Long Bones Under Tail‐Suspension, and its Mechanisms of Action

An innovative anti-osteoporosis herbal formula containing epimedii herba, ligustri lucidi fructus and psoraleae fructus (ELP) has been previously shown its bone protecting effects in ovariectomized osteoporotic rats and also in post-menopausal osteopenic women. This study aimed to investigate the efficacy of ELP against bone loss during physical inactivity or weightlessness. A hindlimb unloading tail-suspended rat model was used for studying the effects of ELP on bone mineral density (BMD) and bone micro-architecture. For in vitro mechanistic studies, rat mesenchymal stem cells (MSCs) and mouse macrophage cells (RAW264.7) were used for studying the effects of ELP on osteogenic/adipogenic differentiations and osteoclastogenesis, respectively. Our data illustrated that ELP had a significant preventive effect against bone loss induced by tail-suspension (TS) at day 28 (p < 0.01) as indicated in the reduction in BMD loss and the preservation of bone micro-architecture. ELP could significantly promote the osteogenesis and suppress the adipogenesis (p < 0.05) in MSCs. Besides, significant inhibition of osteoclast formation (p < 0.01) was found in ELP-treated RAW264.7 cells upon receptor activator of nuclear factor kappa-B ligand induction. Our study presents the first scientific evidence that ELP had a significant preventive effect against bone loss induced by TS through the actions of enhancing osteogenesis, suppressing adipogenesis and osteoclastogenesis.

Molecular characterization and transcriptional analysis of fad24 in pigs

Fad24 is a novel gene involved in initiating adipogenesis. Most of the current information on fad24 is limited to mice, and the transcriptional regulation of fad24 remains unknown. Here we revealed that porcine fad24 encodes a protein structurally homologous to its human and rodent orthologs. Like its mouse counterpart, pig fad24 had positive effect on the adipogenic differentiation of mesenchymal stem cells (MSCs). Distinct fad24 expression patterns were observed during development of skeletal muscle and subcutaneous fat in pigs. The proximal promoter region of porcine fad24 was predicted to contain a number of transcription factor binding motifs related to both adipogenesis and myogenesis. Among them, a putative binding site for krüppel-like factor (KLF)-15 was chosen for further analysis because the role of KLF15 in early adipogenesis is unclear, although its expression in preadipocytes is up-regulated shortly after adipogenic induction. We demonstrated that expression of fad24 and klf15 is concomitantly up-regulated at the onset of adipogenesis in MSCs. Functional KLF15 significantly enhanced the promoter activity of fad24, whereas dominant negative KLF15 had no such effects. Transactivation of fad24 by KLF15 was further confirmed by site-directed mutation and chromatin immunoprecipitation assays. Taken together, our findings provide additional information on fad24 that may contribute to understanding the molecular events involved in early adipocyte differentiation.

Heme induced oxidative stress attenuates sirtuin1 and enhances adipogenesis in mesenchymal stem cells and mouse pre‐adipocytes

Patho-physiological conditions with high oxidative stress, such as conditions associated with increased denatured heme-proteins, are associated with enhanced adipogenic response. This effect predominantly manifests as adipocyte hypertrophy characterized by dysfunctional, pro-inflammatory adipocytes exhibiting reduced expression of anti-inflammatory hormone, adiponectin. To understand how increased levels of cellular heme, a pro-oxidant molecule, modulates adipogenesis; the following study was designed to evaluate effects of heme on adipogenesis in human mesenchymal stem cells (hMSCs) and mouse pre-adipocytes (3T3L1). Experiments were conducted in the absence and in the presence of a superoxide dismutase (SOD) mimetic (tempol, 100 µM). Heme (10 µM) increased (P<0.05) adipogenesis in hMSCs and mouse pre-adipocytes, where tempol alone (100 µmol/L) attenuated adipogenesis in these cells (P<0.05). Tempol also reversed heme-induced increase in adipogenesis in both hMSCs and mouse pre-adipocytes (P<0.05). In addition, heme exposed 3T3L1 exhibited reduced (P<0.05) expression of transcriptional regulator-sirtuin 1 (Sirt1), along with, increased (P<0.05) expression of adipogenic markers peroxisome proliferators-activated receptor-gamma (PPARγ), C/EBPα, and aP2. These effects of heme were rescued (P<0.05) in cells concurrently treated with heme and tempol (P<0.05) and prevented in cells over-expressing Sirt1. Taken together, our results indicate that heme-induced oxidative stress inhibits Sirt1, thus un-inhibiting adipogenic regulators such as PPARγ and C/EBPα; which in turn induce increased adipogenesis along with adipocyte hypertrophy in pre-adipocytes. Anti-oxidant induced offsetting of these effects of heme supports the role of heme-dependent oxidative stress in mediating such events.

Noggin Is Novel Inducer of Mesenchymal Stem Cell Adipogenesis: IMPLICATIONS FOR BONE HEALTH AND OBESITY

Noggin is a glycosylated-secreted protein known so far for its inhibitory effects on bone morphogenetic protein (BMP) signaling by sequestering the BMP ligand. We report here for the first time a novel mechanism by which noggin directly induces adipogenesis of mesenchymal stem cells independently of major human adipogenic signals through C/EBPδ, C/EBPα and peroxisome proliferator-activated receptor-γ. Evaluation of a possible mechanism for noggin-induced adipogenesis of mesenchymal stem cells identified the role of Pax-1 in mediating such differentiation. The relevance of elevated noggin levels in obesity was confirmed in a preclinical, immunocompetent mouse model of spontaneous obesity and in human patients with higher body mass index. These data clearly provide a novel role for noggin in inducing adipogenesis and possibly obesity and further indicates the potential of noggin as a therapeutic target to control obesity.

Cytochrome P450 Derived Epoxyeicosatrienoic Acid Retard Adipogenesis in Mesenchymal Stem cells by Heme Oxygenase‐ AKT Signaling

Human mesenchymal stem cells (MSCs) expressed substantial levels of CYP2J2, a major CYP450 involved in epoxyeicosatrienoic acid (EET) formation and their metabolites (DiHETs). We examined the effect of 12‐(3‐hexylureido)dodec‐ 8(Z)‐enoic acid, an EET agonist, on MSC‐derived adipocytes and demonstrated an increased number of healthy small adipocytes, attenuated PPARγ, C/EBPα, FAS levels (p&lt;0.01), and lipid accumulation (p&lt;0.05). These effects were accompanied by increased levels of heme oxygenase (HO)‐1 and adiponectin (p&lt;0.05). Inhibition of HO activity or AKT by tin mesoporphyrin (SnMP) and LY2940002, respectively, reversed EET‐induced inhibition of adipogenesis, demonstrated an increase in number of small healthy adipocytes, attenuation of fatty acid synthase (FAS) expression (p&lt;0.01) and reduction in lipid accumulation (p&lt;0.05). These effects were accompanied by increased levels of HO‐1 and adiponectin (p&lt;0.05). These findings suggest that EETs decrease adipocyte differentiation by upregulation of HO‐1‐adiponectin‐ AKT signaling and play essential roles in the regulation of adipogenesis by inhibiting PPARγ, C/EBPαand FAS. These novel observations highlight the seminal role of arachidonic acid metabolism in MSCs and suggest that an EET agonist may have potential therapeutic use in the treatment of dyslipidemia, diabetes, and the metabolic syndrome.