Differentiation Of Mouse Embryonic Fibroblasts Research Articles

Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3.

The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis. The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors- Gata4, Foxa3 , and Hnf1a -or alternatively, the expression of two transcription factors, Gata4 and Foxa3 . In vivo , we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-CreER ; Cas9fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Activation of only two factors, Gata4 and Foxa3 , via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

IRE1α inhibits osteogenic differentiation of mouse embryonic fibroblasts by limiting Shh signaling.

This study aimed to investigate the effect of endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1α (IRE1α) on the sonic hedgehog N-terminus (N-Shh)-enhanced-osteogenic differentiation process in mouse embryonic fibroblasts (MEFs). Osteogenesis of MEFs was observed by alkaline phosphatase (ALP) staining, alizarin red staining, and Von Kossa staining assays. Activation of unfolded protein response and Shh signaling were examined using real-time quantitative PCR and western blot assays. IRE1α-deficient MEFs were used to explore the effect of IRE1α on N-Shh-driven osteogenesis. N-Shh increased ALP activity, matrix mineralization, and the expression of Alp and Col-I in MEFs under osteogenic conditions; notably, this was reversed when combined with the ER stress activator Tm treatment. Interestingly, the administration of N-Shh decreased the expression of IRE1α. Abrogation of IRE1α increased the expression of Shh pathway factors in osteogenesis-induced MEFs, contributing to the osteogenic effect of N-Shh. Moreover, IRE1α-deficient MEFs exhibited elevated levels of osteogenic markers. Our findings suggest that the IRE1α-mediated unfolded protein response may alleviate the ossification of MEFs by attenuating Shh signaling. Our research has identified a strategy to inhibit excessive ossification, which may have clinical significance in preventing temporomandibular joint bony ankylosis.

MMP13 promotes the osteogenic potential of BMP9 by enhancing Wnt/β-catenin signaling via HIF-1α upregulation in mouse embryonic fibroblasts.

Bone morphogenetic protein 9 (BMP9) has been validated as one of the most potent osteoinduction factors, but its underlying mechanism remains unclear. As a member of the matrix metalloproteinase (MMP) family, MMP13 may be involved in regulating the lineage-specific differentiation of mouse embryonic fibroblasts (MEFs). The goal of this study was to determine whether MMP13 regulates the osteoinduction potential of BMP9 in MEFs, which are multipotent progenitor cells widely used for stem cell biology research. In vitro and in vivo experiments showed that BMP9-induced osteogenic markers and/or bone were enhanced by exogenous MMP13 in MEFs, but were reduced by MMP13 knockdown or inhibition. The expression of hypoxia inducible factor 1 alpha (HIF-1α) was induced by BMP9, which was enhanced by MMP13. The protein expression of β-catenin and phosphorylation level of glycogen synthase kinase-3 beta (GSK-3β) were increased by BMP9 in MEFs, as was the translocation of β-catenin from the cytoplasm to the nucleus; all these effects of BMP9 were enhanced by MMP13. Furthermore, the MMP13 effects of increasing BMP9-induced β-catenin protein expression and GSK-3β phosphorylation level were partially reversed by HIF-1α knockdown. These results suggest that MMP13 can enhance the osteoinduction potential of BMP9, which may be mediated, at least in part, through the HIF-1α/β-catenin axis. Our findings demonstrate a novel role of MMP13 in the lineage decision of progenitor cells and provide a promising strategy to speed up bone regeneration.

LCN2 inhibits the BMP9-induced osteogenic differentiation through reducing Wnt/β-catenin signaling via interacting with LRP6 in mouse embryonic fibroblasts.

Due to its effective osteogenic ability, BMP9 is a promising candidate for bone regeneration medicine. Whereas, BMP9 can also induce adipogenesis simultaneously. LCN2 is a cytokine associated with osteogenesis and adipogenesis. Reducing the adipogenic potential may be a feasible measure to enhance the osteogenic capability of BMP9. The objective of the study was to explore the role of LCN2 in regulating the BMP9-initialized osteogenic and adipogenic differentiation in mouse embryonic fibroblasts (MEFs), and clarify the possible underlying mechanism. Histochemical stain, western blot, real-time PCR, laser confocal, immunoprecipitation, cranial defect repair, and fetal limb culture assays were used to evaluate the effects of LCN2 on BMP9-induced osteogenic and adipogenic differentiation, as well as Wnt/β-catenin signaling. LCN2 was down-regulated by BMP9. The BMP9-induced osteogenic markers were inhibited by LCN2 overexpression, but the adipogenic markers were increased; LCN2 knockdown exhibited opposite effects. Similar results were found in bone defect repair and fetal limb culture tests. The level of β-catenin nucleus translocation was found to be reduced by LCN2 overexpression, but increased by LCN2 knockdown. The inhibitory effect of LCN2 overexpression on the osteogenic capability of BMP9 was reversed by β-catenin overexpression; whereas, the effect of LCN2 knockdown on promoting BMP9 osteogenic potential was almost eliminated by β-catenin knockdown. LCN2 could bind with LRP6 specifically, and the inhibitory effect of LCN2 on the osteogenic potential of BMP9 could not be enhanced by LRP6 knockdown. LCN2 inhibits the BMP9-induced osteogenic differentiation but promotes its adipogenic potential in MEFs, which may be partially mediated by reducing Wnt/β-catenin signaling via binding with LRP6.

In vivo effects of low dose prenatal bisphenol A exposure on adiposity in male and female ICR offspring

BackgroundBisphenol A (BPA) is known to exhibit endocrine disrupting activities and is associated with adiposity. We examined the obesogenic effect of prenatal BPA exposure in the present study. MethodsPregnant ICR mice were exposed to vehicle or BPA via the drinking water at a dose of 0.5 μg/kg·d throughout the gestation. Obesity-related indexes were investigated in the 12-wk-old offspring. Primary mouse embryonic fibroblasts (MEFs) collected from treated embryos were used to test effects of BPA on adipocyte differentiation. ResultsOffspring presented a significantly higher rate of weight gain than the control, with impaired insulin sensitivity and increased adipocyte size. Differentiation of MEFs from BPA-treated mice showed a higher propensity for the adipocyte commitment as well as up-regulation of genes enriched in lipid biosynthesis. TGF-β signaling pathway was found to modulate obesogenic effect of BPA in MEF model, but estrogen signaling pathway had no effect. ConclusionsThe present study provides strong evidence of the association between prenatal exposure to low dose of BPA and a significant increase in body weight in the offspring mice with a critical role played by TGF-β signaling pathway. The potential interactions modulating the binding of BPA and TGF-β that activate its obesogenic effects need to be examined.

Interleukin 6 promotes BMP9-induced osteoblastic differentiation through Stat3/mTORC1 in mouse embryonic fibroblasts.

Interleukin 6 (IL-6) plays a dual role in regulating bone metabolism, although the concrete mechanism is unclear. Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic inducers, and a promising alternative for bone tissue engineering. The relationship between IL-6 and BMP9 in osteogenic differentiation remains to be elucidated, and the osteoblastic potential of BMP9 needs to be enhanced to overcome certain shortcomings of BMP9. In this study, we used real-time PCR, western blot, immunofluorescent stain, fetal limb culture and cranial defects repair model to explore the IL-6 role in BMP9-induced osteogenic differentiation in mouse embryonic fibroblasts (MEFs). We found that the rat serum level of IL-6 was increased in the dexamethasone-induced osteoporosis model, and IL-6 expression was detectable in several progenitor cells and MEFs. BMP9 upregulated IL-6 in MEFs, and the BMP9-induced osteoblastic markers were elevated by IL-6, but reduced by IL-6 knockdown. BMP9 and/or IL-6 both activated mTOR, and the IL-6 effect on BMP9-induced osteoblastic markers and bone formation were reduced greatly by mTOR inhibition. Raptor was up-regulated by IL-6 and/or BMP9 specifically, and the osteoblastic markers induced by IL-6 and/or BMP9 were reduced by Raptor knockdown. Meanwhile, Stat-3 was activated by IL-6 and/or BMP9, and the increase of Raptor or osteoblastic markers by IL-6 and/or BMP9 were reduced by Stat-3 inhibition. The Raptor promoter activity was regulated by p-Stat-3. Our finding suggested that IL-6 can promote the BMP9 osteoblastic potential, which may be mediated through activating Stat-3/mTORC1 pathway.

JAZF1 heterozygous knockout mice show altered adipose development and metabolism

BackgroundJuxtaposed with another zinc finger protein 1 (JAZF1) is associated with metabolic disorders, including type 2 diabetes mellitus (T2DM). Several studies showed that JAZF1 and body fat mass are closely related. We attempted to elucidate the JAZF1 functions on adipose development and related metabolism using in vitro and in vivo models.ResultsThe JAZF1 expression was precisely regulated during adipocyte differentiation of 3T3-L1 preadipocyte and mouse embryonic fibroblasts (MEFs). Homozygous JAZF1 deletion (JAZF1-KO) resulted in impaired adipocyte differentiation in MEF. The JAZF1 role in adipocyte differentiation was demonstrated by the regulation of PPARγ—a key regulator of adipocyte differentiation. Heterozygous JAZF1 deletion (JAZF1-Het) mice fed a normal diet (ND) or a high-fat diet (HFD) had less adipose tissue mass and impaired glucose homeostasis than the control (JAZF1-Cont) mice. However, other metabolic organs, such as brown adipose tissue and liver, were negligible effect on JAZF1 deficiency.ConclusionOur findings emphasized the JAZF1 role in adipocyte differentiation and related metabolism through the heterozygous knockout mice. This study provides new insights into the JAZF1 function in adipose development and metabolism, informing strategies for treating obesity and related metabolic disorders.

Differentiation of Mouse Embryonic Fibroblasts (MEFs) into Cardiomyocytes Using Human-Derived Cardiac Inducing RNA (CIR)

The present study explores an RNA we have discovered in human heart that induces differentiation of mouse embryonic stem cells and human induced pluripotent stem cells into cardiomyocytes in vitro. We have designated this RNA as Cardiac Inducing RNA or CIR. We now find that CIR also induces mouse embryonic fibroblasts (MEF) to form cardiomyocytes in vitro. For these studies, human-derived CIR is transfected into MEF using lipofectamine. The CIR-transfected mouse fibroblasts exhibit spindle-shaped cells, characteristic of myocardial cells in culture and express cardiac-specific troponin-T and cardiac tropomyosin. As such, the CIR-induced conversion of the fibroblasts into cardiomyocytes in vitro appears to take place without initial dedifferentiation into pluripotent stem cells. Instead, after CIR transfection using a lipofectamine transfection system, over the next 8 days there appears to be a direct transdifferentiation of ˃80% of the cultured fibroblasts into definitive cardiomyocytes. Fewer than ˂7% of the untreated controls using non-active RNA or lipofectamine by itself show cardiomyocyte characteristics. Thus, discovery of CIR may hold significant potential for future use in repair/regeneration of damaged myocardial tissue in humans after myocardial infarction or other disease processes such that affected patients may be able to return to pre-heart-disease activity levels.

Abstract 15978: Acetyl-coa Catalysis by Atp-citrate Lyase is Essential for Myofibroblast Differentiation and Persistence

A feature of heart failure (HF) is excessive extracellular matrix deposition and cardiac remodeling by a differentiated fibroblast population known as myofibroblasts. Identifying mechanisms of myofibroblast differentiation in cardiac fibrosis could yield novel therapeutic targets to delay or reverse HF. Recent evidence suggests that myofibroblast differentiation requires metabolic reprogramming for transcriptional activation of the myofibroblast gene program by chromatin-dependent mechanisms. We previously reported that inhibition of histone demethylation blocks myofibroblast formation, however, whether histone acetylation (e.g., H3K27ac, a prominent mark associated with gene transcription) is involved in fibroblast reprogramming remains unclear. ATP-citrate lyase (ACLY) synthesizes acetyl-CoA and therein supplies acetyl-CoA to the nucleus, where it is used as a substrate by histone acetyltransferases (HATs). To define the role of acetyl-CoA metabolism in myofibroblast differentiation, we stimulated differentiation in mouse embryonic fibroblasts (MEFs) and adult mouse cardiac fibroblasts (ACFs) with the pro-fibrotic agonist transforming growth factor β (TGFβ) and treated cells with a pharmacological inhibitor of ACLY. ACLY inhibition decreased myofibroblast gene expression in ACF and MEFs in TGFβ-stimulated myofibroblast differentiation, in addition to decreasing the population of αSMA positive MEFs. Genetic deletion of ACLY in MEFs recapitulated the results observed with pharmacological inhibition. Encouragingly, the ACLY inhibitor was sufficient to revert fully differentiated myofibroblasts under continuous TGFβ stimulation to a quiescent, non-fibrotic phenotype. Altogether, our data indicate that ACLY activity is necessary for myofibroblast differentiation and persistence. We hypothesize that ACLY-dependent acetyl-CoA synthesis is necessary for histone acetylation and transcriptional activation of the myofibroblast gene program. Currently, we are examining mechanisms of ACLY-dependent chromatin remodeling in fibroblasts and the in vivo relevance of this mechanism in mutant mice. In summary, ACLY is a potential target to reverse cardiac fibrosis and lessen HF.

Exosome-Mediated Differentiation of Mouse Embryonic Fibroblasts and Exocrine Cells into β-Like Cells and the Identification of Key miRNAs for Differentiation.

Diabetes is a concerning health malady worldwide. Islet or pancreas transplantation is the only long-term treatment available; however, the scarcity of transplantable tissues hampers this approach. Therefore, new cell sources and differentiation approaches are required. Apart from the genetic- and small molecule-based approaches, exosomes could induce cellular differentiation by means of their cargo, including miRNA. We developed a chemical-based protocol to differentiate mouse embryonic fibroblasts (MEFs) into β-like cells and employed mouse insulinoma (MIN6)-derived exosomes in the presence or absence of specific small molecules to encourage their differentiation into β-like cells. The differentiated β-like cells were functional and expressed pancreatic genes such as Pdx1, Nkx6.1, and insulin 1 and 2. We found that the exosome plus small molecule combination differentiated the MEFs most efficiently. Using miRNA-sequencing, we identified miR-127 and miR-709, and found that individually and in combination, the miRNAs differentiated MEFs into β-like cells similar to the exosome treatment. We also confirmed that exocrine cells can be differentiated into β-like cells by exosomes and the exosome-identified miRNAs. A new differentiation approach based on the use of exosome-identified miRNAs could help people afflicted with diabetes

Tm7sf2 gene promotes adipocyte differentiation of mouse embryonic fibroblasts and improves insulin sensitivity

Adipogenesis is a finely orchestrated program involving a transcriptional cascade coordinated by CEBP and PPAR family members and by hormonally induced signaling pathways. Alterations in any of these factors result into impaired formation of fully differentiated adipocytes. Tm7sf2 gene encodes for a Δ(14)-sterol reductase primarily involved in cholesterol biosynthesis. Furthermore, TM7SF2 modulates the expression of the master gene of adipogenesis PPARγ, suggesting a role in the regulation of adipose tissue homeostasis. We investigated the differentiation of Tm7sf2−/− MEFs into adipocytes, compared to Tm7sf2+/+ MEFs. Tm7sf2 expression was increased at late stage of differentiation in wild type cells, while Tm7sf2−/− MEFs exhibited a reduced capacity to differentiate into mature adipocytes. Indeed, Tm7sf2−/− MEFs had lower neutral lipid accumulation and reduced expression of adipogenic regulators. At early stage, the reduction in C/EBPβ expression impaired mitotic clonal expansion, which is needed by preadipocytes for adipogenesis induction. At late stage, the expression and activity of C/EBPα and PPARγ were inhibited in Tm7sf2−/− cells, leading to the reduced expression of adipocyte genes like Srebp-1c, Fasn, Scd-1, Adipoq, Fabp4, and Glut4. Loss of the acquisition of adipocyte phenotype was accompanied by a reduction in the levels of Irs1, and phosphorylated Akt and ERK1/2, indicating a blunted insulin signaling in differentiating Tm7sf2−/− cells. Moreover, throughout the differentiation process, increased expression of the antiadipogenic Mmp3 was observed in MEFs lacking Tm7sf2. These findings indicate Tm7sf2 as a novel factor influencing adipocyte differentiation that could be relevant to adipose tissue development and maintenance of metabolic health.

Sigma-1 receptor ablation impedes adipocyte-like differentiation of mouse embryonic fibroblasts

The sigma-1 receptor (Sig1R) is a unique ligand-operated endoplasmic reticulum (ER) protein without any mammalian homolog. It has long been a pharmacological target for intervention of psychiatric disorders, and recently garnered refreshed interest for its neuroprotective potential. Though reported to modulate various intracellular events, its influence on cell identity is little known. We explored a role for Sig1R in adipocyte differentiation.We induced adipogenic differentiation of mouse embryonic fibroblasts (MEFs) with a differentiation medium. MEFs were isolated from Sigmar1−/− and Sigmar1+/+ mice. The induced adipocyte-like phenotype was detected through Western blots of master transcription factors (PPARγ, CEBPA, SREBP1, SREBP2), lipogenic proteins (FABP4, ACC1, ACAT2), and Oil-Red-O staining of lipids. We found that the induced upregulation of these proteins and lipid accumulation were severely mitigated in Sigmar1−/− (vs Sigmar1+/+) MEFs. Sig1R activation with a selective agonist (PRE084) increased Sig1R protein and further enhanced the induced adipocyte-like phenotype in Sigmar1+/+ MEFs. We also determined mouse body weight gain induced by high-fat diet for 6 months, which was impeded in Sigmar1−/− (vs Sigmar1+/+) male mice.In summary, genetic ablation of Sig1R impairs, and agonist activation of Sig1R enhances adipocyte-like phenotype of induced MEFs. In vivo, Sig1R ablation impedes the body weight gain of male mice on high-fat diet. This study warrants further investigation of a previously unrecognized role for Sig1R in adipocyte differentiation.

All-trans retinoic acid and vascular endothelial growth factor induced the directional osteogenic differentiation of mouse embryonic fibroblasts

To investigate the effect of all-trans retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) on the osteogenic differentiation of mouse embryonic fibroblasts (MEFs). The fetal mice in the uterus of NIH pregnant mice (pregnancy 12-15 days) were collected, and the heads and hearts etc. were removed. Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture. HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein (Ad-RFP) and Ad-VEGF by repeatedly freezing and thawing. Alkaline phosphatase (ALP) staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3rd and 5th days. The cultured 3rd to 4th generation MEFs were divided into groups A, B, C, and D, and were cultured with DMSO plus Ad-RFP, ATRA, Ad-VEGF, ATRA plus Ad-VEGF, respectively. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of osteogenic markers including ALP, collagen type Ⅰ, osteopontin (OPN), osteocalcin (OCN), and angiogenic markers including VEGF, angiopoietin 1 (ANGPT1), and endomucin (EMCN) on the 3rd and 7th days. Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3rd, 5th, and 7th days in each group. Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction. Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group. Then MEFs treated with ATRA, Ad-VEGF, and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides, respectively. X-ray observation, gross observation, and histological staining (Masson, HE, and Safranin O-fast green stainings) were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group. MEFs were successfully isolated and cultured. The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50% and 20% transfection rates. ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs ( P<0.05), and ATRA had a stronger effect than Ad-VEGF; and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs ( P<0.05). qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP, OPN, and collagen type I ( P<0.05); the relative mRNA expressions of angiogenesis-related markers VEGF, EMCN, and ANGPT1 increased at 7 days ( P<0.05). Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression, but also increased VEGF protein expression on 7th day. Alizarin red staining showed that the application of ATRA or Ad-VEGF induced weak calcium salt deposition, and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs. The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group, and the bone was larger than that in other groups. Histological staining showed a large amount of collagen and mature bone trabeculae, bone matrix formation, and gray-green collagen bone tissue, indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo. The combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.

Role of Mineralocorticoid Receptor in Adipogenesis and Obesity in Male Mice.

Increased visceral adiposity and hyperglycemia, 2 characteristics of metabolic syndrome, are also present in conditions of excess glucocorticoids (GCs). GCs are hormones thought to act primarily via the glucocorticoid receptor (GR). GCs are commonly prescribed for inflammatory disorders, yet their use is limited due to many adverse metabolic side effects. In addition to GR, GCs also bind the mineralocorticoid receptor (MR), but there are many conflicting studies about the exact role of MR in metabolic disease. Using MR knockout mice (MRKO), we find that both white and brown adipose depots form normally when compared with wild-type mice at P5. We created mice with adipocyte-specific deletion of MR (FMRKO) to better understand the role of MR in metabolic dysfunction. Treatment of mice with excess GCs for 4 weeks, via corticosterone in drinking water, induced increased fat mass and glucose intolerance to similar levels in FMRKO and floxed control mice. Separately, when fed a high-fat diet for 16 weeks, FMRKO mice had reduced body weight, fat mass, and hepatic steatosis, relative to floxed control mice. Decreased adiposity likely resulted from increased energy expenditure since food intake was not different. RNA sequencing analysis revealed decreased enrichment of genes associated with adipogenesis in inguinal white adipose of FMRKO mice. Differentiation of mouse embryonic fibroblasts (MEFs) showed modestly impaired adipogenesis in MRKO MEFs compared with wild type, but this was rescued upon the addition of peroxisome proliferator-activated receptor gamma (PPARγ) agonist or PPARγ overexpression. Collectively, these studies provide further evidence supporting the potential value of MR as a therapeutic target for conditions associated with metabolic syndrome.

IGF-1 reverses the osteogenic inhibitory effect of dexamethasone on BMP9-induced osteogenic differentiation in mouse embryonic fibroblasts via PI3K/AKT/COX-2 pathway

Glucocorticoid-Induced Osteoporosis (GIOP) is a prevalent clinical complication caused by large dose administration of glucocorticoids, such as Dexamethasone (Dex) and Prednisone. GIOP may lead to fractures and even Osteonecrosis of the Femoral Head (ONFH). It has been reported that glucocorticoids inhibit osteogenesis via the suppression of osteogenic differentiation in Mesenchymal Stem Cells (MSCs), but the precise mechanism underlying this suppression awaits further investigation. Meanwhile, novel and efficacious therapies are recommended to cope with GIOP. In this study, we demonstrated that Dex had the inhibitory effect on Bone Morphogenetic Protein 9 (BMP9)-induced ALP activities and matrix mineralization in Mouse Embryonic Fibroblasts (MEFs). In addition, the study confirmed that Dex decreased the expression of osteogenic markers such as Runx2 and OPN. However, the inhibitory effect of Dex on these osteogenic markers can be reversed when combined with insulin-like growth factor 1 (IGF-1). Regarding the inhibitory mechanism, we found that the level of AKT and p-AKT can be decreased by Dex and that Ly294002, the PI3K inhibitor, can block the reversal effect of IGF-1. Moreover, the knockdown or inhibition of COX-2 produced similar results to those of Ly294002. Our findings indicated that IGF-1 may reverse the osteogenic inhibitory effect of Dex via PI3K/AKT pathway, which may be associated with the up-regulation of COX-2. This study may provide new clinical management strategy for GIOP cases.

CCN3 and DLL1 co-regulate osteogenic differentiation of mouse embryonic fibroblasts in a Hey1-dependent manner

Notch signaling pathway is one of the most important pathways to regulate intercellular signal transduction and is crucial in the regulation of bone regeneration. Nephroblastoma overexpressed (NOV or CCN3) serves as a non-canonical secreted ligand of Notch signaling pathway and its role in the process of osteogenic differentiation of mesenchymal stem cells (MSCs) was undefined. Here we conducted a comprehensive study on this issue. In vivo and in vitro studies have shown that CCN3 significantly inhibited the early and late osteogenic differentiation of mouse embryonic fibroblasts (MEFs), the expression of osteogenesis-related factors, and the subcutaneous ectopic osteogenesis of MEFs in nude mice. In mechanism studies, we found that CCN3 significantly inhibited the expression of BMP9 and the activation of BMP/Smad and BMP/MAPK signaling pathways. There was also a mutual inhibition between CCN3 and DLL1, one of the classic membrane protein ligands of Notch signaling pathway. Additionally, we further found that Hey1, the target gene shared by BMP and Notch signaling pathways, partially reversed the inhibitory effect of CCN3 on osteoblastic differentiation of MEFs. In summary, our findings suggested that CCN3 significantly inhibited the osteogenic differentiation of MEFs. The inhibitory effect of CCN3 was mainly through the inhibition of BMP signaling and the mutual inhibition with DLL1, so as to inhibit the expression of Hey1, the target gene shared by BMP and Notch signaling pathways.

Follicle-Stimulating Hormone β-Subunit Potentiates Bone Morphogenetic Protein 9-Induced Osteogenic Differentiation in Mouse Embryonic Fibroblasts.

Postmenopausal osteoporosis (PMOP)-related fractures usually result in morbidity and mortality in aging women, so it remains a global public health concern, and new effective safe treatments are urgently needed recently. Efficient osteogenesis from mesenchymal stem cells (MSCs) would have the clinical application potential in treating multiple osteal disorders. Follicle-stimulating hormone (FSH), a pituitary glycoprotein hormone highly associated with menopausal bone turnover, whose peculiar part of receptor binding is follicle-stimulating hormone β-subunit (FSHβ). Bone morphogenetic protein 9 (BMP9), a potent osteogenic factor, can up-regulate FSHβ in mouse embryonic fibroblasts (MEFs). However, it is unclear, whether extrapituitary FSHβ affects BMP9-induced osteogenesis in MEFs. In this study, we investigated the role of FSHβ in BMP9-induced osteogenesis in MEFs. We found that exogenous expression of FSHβ significantly increased BMP9-induced alkaline phosphatase activity (ALP), the expression of osteogenic transcriptional factors, Runx2 and Osx, and the established late osteogenic markers, osteopontin (OPN) and osteocalcin (OCN), so does the ectopic bone formation. Mechanistically, FSHβ dramatically enhanced BMP9-induced BMP/Smad signal transduction, presenting the augment phosphorylation of Smad1/5/8, whereas treatment with anti-FSHβ antibodies suppressed these effects. An adenylate cyclase inhibitor obviously suppressed ALP and BMP/Smad signal transduction induced by BMP9 or the combination of BMP9 and FSHβ in MEFs. Collectively, our findings suggested that FSHβ may promote BMP9-induced activation of BMP/Smad signaling through a FSH/FSH receptor (FSHR)/cAMP dependent pathway in MEFs partly. J. Cell. Biochem. 118: 1792-1802, 2017. © 2016 Wiley Periodicals, Inc.

Follistatin Targets Distinct Pathways To Promote Brown Adipocyte Characteristics in Brown and White Adipose Tissues.

We previously demonstrated that Fst expression is highest in brown adipose tissue (BAT) and skeletal muscle, but is also present at substantial levels in epididymal and subcutaneous white adipose tissues (WATs). Fst promotes mouse brown preadipocyte differentiation and promotes browning during differentiation of mouse embryonic fibroblasts. Fst-transgenic (Fst-Tg) mice show substantial increases in circulating Fst levels and increased brown adipose mass. BAT of Fst-Tg mice had increased expression of brown adipose-associated markers including uncoupling protein 1 (UCP1), PRDM16, PGC-1α, and Glut4. WATs from Fst-Tg mice show upregulation of brown/beige adipose markers and significantly increased levels of phosphorylated p38 MAPK/ERK1/2 proteins compared with the wild-type (WT) mice. Pharmacological inhibition of pp38 MAPK/pERK1/2 pathway of recombinant mouse Fst (rFst) treated differentiating 3T3-L1 cells led to significant blockade of Fst-induced UCP1 protein expression. On the other hand, BAT from Fst-Tg mice or differentiating mouse BAT cells treated with rFst show dramatic increase in Myf5 protein levels as well as upregulation of Zic1 and Lhx8 gene expression. Myf5 levels were significantly downregulated in Fst knock-out embryos and small inhibitory RNA-mediated inhibition of Myf5 led to significant inhibition of UCP1, Lhx8, and Zic1 gene expression and significant blockade of Fst-induced induction of UCP1 protein expression in mouse BAT cells. Both interscapular BAT and WAT tissues from Fst-Tg mice display enhanced response to CL316,243 treatment and decreased expression of pSmad3 compared with the WT mice. Therefore, our results indicate that Fst promotes brown adipocyte characteristics in both WAT and BAT depots in vivo through distinct mechanisms.

All-trans retinoic acid shifts rosiglitazone-induced adipogenic differentiation to osteogenic differentiation in mouse embryonic fibroblasts.

Rosiglitazone (RSG) is a potent drug used in the treatment of insulin resistance; however, it is associated with marked skeletal toxicity. RSG-induced osteoporosis may contribute to the promotion of adipogenic differentiation at the expense of osteogenic differentiation in bone marrow stromal cells. The aim of this study was to investigate whether RSG-induced bone toxicity can be reversed by combined treatment with all-trans retinoic acid (ATRA). We examined different osteogenic markers in mouse embryonic fibroblasts (MEFs) following treatment with RSG, ATRA, or RSG and ATRA in combination. We examined the effects of RSG and/or ATRA on ectopic bone formation, and dissected the possible molecular mechanisms underlying this process. We found that ATRA or RSG both induced alkaline phosphatase (ALP) activity in the MEFs, and that the ATRA-induced ALP activity was enhanced by RSG and vice versa. However, only the combination of RSG and ATRA increased the expression of osteopontin and osteocalcin, promoted matrix mineralization, and induced ectopic ossification in MEFs. Mechanistically, we found that the osteogenic differentiation induced by the combination of RSG and ATRA may be mediated partly by suppressing RSG-induced adipogenic differentiation and activating bone morphogenetic protein (BMP)/Smad signaling. On the whole, our findings demonstrate that RSG in combination with ATRA promotes the commitment of MEFs to the osteoblast lineage. Thus, the combination of these two agents may prove to be a promising and novel therapeutic regimen for insulin resistance without skeletal toxicity. It may also be a better strategy with which to prevent RSG-induced osteoporosis.

Nerve growth factor potentiates bone morphogenetic protein-9-induced osteogenic differentiation in mouse embryonic fibroblasts

Objective To investigate the effect of nerve growth factor (NGF) on osteogenesis induced by bone morphogenetic protein-9 (BMP-9) in mouse embryonic fibroblasts (MEFs). Methods MEFs were respectively transfected with adenovirus-mediated NGF (NGF group), BMP-9 (BMP-9 group) and NGF + BMP-9 (combined group) and green fluorescence protein (GFP) (control group). Cytochemical staining was used to test the activity of alkaline phosphatase (ALP) 3 d and 5 d after treatment. Level of osteopontin (OPN) mRNA was detected by RT-PCR 9 d after treatment. Level of OPN protein was assayed by Western blot and immunocytochemistry 9 d after treatment. Mineralization was detected by Alizarin red staining 14 d after treatment. Results ALP activity in MEFs was elevated in BMP-9 group rather than in NGF group, but a significant increase in ALP activity was noted in combined group. In control group, BMP-9 group, NGF group and combined group, level of OPN mRNA was 0.92±0.03, 1.28±0.04, 0.94±0.03 and 1.62±0.04 respectively (F=214.60, P<0.01); level of OPN protein was 0.60±0.05, 0.84±0.03, 0.53±0.05 and 1.27±0.05 respectively(F=162.5, P<0.01). In comparison, OPN mRNA and protein were significantly up-regulated in combined group than in BMP-9 group (t=10.569 and 11.778, P<0.05). In control group, BMP-9 group, NGF group and combined group, relative density of OPN protein was 3.63±0.17, 6.27±0.30, 3.86±0.18 and 10.16±0.18 respectively (F=602.6, P<0.01), with a significant higher level in combined group than in BMP-9 group (t=22.280, P<0.05). Level of mineralization was significantly higher in combined group than in BMP-9 or NGF group. Conclusion NGF can potentiate the osteogenesis induced by BMP-9 in MEFs. Key words: Nerve growth factor; Bone morphogenetic proteins; Osteogenesis; Mesenchymal stem cells