A novel PPAR?2 modulator sLZIP controls the balance between adipogenesis and osteogenesis during mesenchymal stem cell differentiation.
ABSTRACT: Mesenchymal stem cells (MSCs), also known as multipotent stromal cells, are used in clinical trials. However, the use of MSCs for medical treatment of patients poses a potential problem due to the possibility of transdifferentiation into unwanted tissues. Disruption of the balance during MSC differentiation leads to obesity, skeletal fragility, and osteoporosis. Differentiation of MSCs into either adipocytes or osteoblasts is transcriptionally regulated by the two key transcription factors PPAR?2 and Runx2. PPAR?2 is highly expressed during adipocyte differentiation and regulates expression of genes involved in adipogenesis. Runx2 induces osteogenic gene expression and, thereby, increases osteoblast differentiation. Although transcriptional modulation of PPAR?2 has been investigated in adipogenesis, the underlying molecular mechanisms to control the balance between adipogenesis and osteogenesis in MSCs remain unclear. In this study, the role of sLZIP in regulation of PPAR?2 transcriptional activation was investigated along with sLZIP's involvement in differentiation of MSCs into adipocytes and osteoblasts. sLZIP interacts with PPAR?2 and functions as a corepressor of PPAR?2. sLZIP enhances formation of the PPAR?2 corepressor complex through specific interaction with HDAC3, resulting in suppression of PPAR?2 transcriptional activity. We found that sLZIP prevents expression of PPAR?2 target genes and adipocyte differentiation both in vitro and in vivo. sLZIP also upregulates Runx2 transcriptional activity via inhibition of PPAR?2 activity, and promotes osteoblast differentiation. sLZIP transgenic mice exhibited enhanced bone mass and density, compared with wild-type mice. These results indicate that sLZIP has a critical role in the regulation of osteogenesis and bone development. However, sLZIP does not affect chondrogenesis and osteoclastogenesis. We propose that sLZIP is a novel PPAR?2 modulator for control of the balance between adipogenesis and osteogenesis during MSC differentiation, and that sLZIP can be used as a therapeutic target molecule for treatment of obesity, osteodystrophy, and osteoporosis.
Project description:Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPAR? regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPAR? expression; however, S-nitrosylation of PPAR? was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPAR? as an S-nitrosylation site and demonstrated that S-nitrosylation of PPAR? inhibits its transcriptional activity, suggesting a feedback regulation of PPAR? transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPAR? alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.
Project description:<h4>Background</h4>Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPAR?2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis.<h4>Methodology</h4>We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPAR?2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a "sentinel" that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPAR?2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs).<h4>Principal findings</h4>Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPAR?2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPAR?2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like.<h4>Conclusions</h4>Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs.
Project description:Mesenchymal stem cell (MSC) is a type of stem cell that is capable of differentiating into osteoblasts and adipocytes. The pathological perturbation of MSC fate determination is well demonstrated by the replacement of bone tissues with fat in those with osteoporosis and osteopenia. Cell fate determination can be regulated by epigenetic and post-transcriptional mechanisms. MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules that mediates the post-transcriptional regulation of genes expression. We hypothesized that miRNA specified to PPAR?, a major transcription factor of adipogenesis, is responsible for the differentiation of MSCs into osteoblasts. Candidate miRNA that is responsible for target gene inhibition was identified from the miRNA database via bioinformatic analyses. In this study, miR-130a and miR-27b were selected for investigation on their role in specifically binding to peroxisome proliferator-activated receptor ? (PPAR?) via in vitro osteogenesis of human MSCs. During osteogenic differentiation of human MSCs, the expression level of miR-130a and miR-27b were found to be upregulated. In the meanwhile, adipogenic marker genes (PPAR? and C/EBP?) were found to decrease, which is in contrary to the increased expression of osteogenic marker genes (RUNX2 and Osterix). MSCs were transfected with mimics and inhibitors of miR-130a and miR-27b during in vitro osteogenesis followed by evaluation for the presence of osteogenic markers via quantitative gene expression, Western blot analysis and alkaline phosphatase activity assay. The overexpression of miR-130a and miR-27b is shown to enhance osteogenesis by increasing the gene expression of RUNX2 and Osterix, the protein expression of RUNX2, COL1A1, and Osterix as well as the alkaline phosphatase activity. Taken altogether, these results suggested that miR-130a and miR-27b could promote osteogenesis in human MSCs by targeting the PPAR?.
Project description:In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPAR?, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPAR?. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPAR? phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPAR?-dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant-negative suppression of activity had the opposite effect. In rescue experiments using Runx2(-/-) mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation-deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation-resistant S112A mutant PPAR? strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg(-/-) MEFs, while S112E mutant PPAR? was less active. Competition between RUNX2 and PPAR? was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPAR? phosphorylation in the control of osteoblast and adipocyte lineages.
Project description:Pkd1 localizes to primary cilia in osteoblasts and osteocytes. Targeted deletion of Pkd1 in osteoblasts results in osteopenia and abnormalities in Runx2-mediated osteoblast development. Kif3a, an intraflagellar transport protein required for cilia function, is also expressed in osteoblasts. To assess the relationship between Pkd1 and primary cilia function on bone development, we crossed heterozygous Pkd1- and Kif3a-deficient mice to create compound Pkd1 and Kif3a-deficient mice. Pkd1 haploinsufficiency (Pkd1(+/?)) resulted in osteopenia, characterized by decreased bone mineral density, trabecular bone volume, and cortical thickness. In addition, deficiency of Pkd1 resulted in impaired osteoblastic differentiation and enhanced adipogenesis in both primary osteoblasts and/or bone marrow stromal cell cultures. These changes were associated with decreased Runx2 expression, increased PPAR? expression, and impaired hedgehog signaling as evidenced by decreased Gli2 expression in bone and osteoblast cultures. In contrast, heterozygous Kif3a(+/?) mice display no abnormalities in skeletal development or osteoblast function, but exhibited decreased adipogenic markers in bone and impaired adipogenesis in vitro in association with decreased PPAR? expression and upregulation of Gli2. Superimposed Kif3a deficiency onto Pkd1(+/?) mice paradoxically corrected the effects of Pkd1 deficiency on bone mass, osteoblastic differentiation, and adipogenesis. In addition, Runx2, PPAR? and Gli2 expression in bone and osteoblasts were normalized in compound double Pkd1(+/?) and Kif3a(+/?) heterozygous mice. The administration of sonic hedgehog, overexpression of Gli2, and the PC1 C-tail construct all increased Gli2 and Runx2-II expression, but decreased PPAR?2 gene expression in C3H10T1/2 cells. Our findings suggest a role for Pkd1 and Kif3a to counterbalance the regulation of osteogenesis and adipogenesis through differential regulation of Runx2 and PPAR? by Gli2.
Project description:A common feature of many skeletal diseases is the accumulation of marrow fat. A reciprocal relationship exists between osteogenesis and adipogenesis in bone marrow that is mediated by the relative activity of PPAR? and RUNX2 transcription factors. The ERK/MAPK pathway is an important inducer of MSC differentiation to osteoblasts and an inhibitor of adipogenesis that functions by phosphorylating RUNX2 and PPAR?. To begin to assess the importance of this regulation in vivo, we examined the consequences of blocking one arm of this pathway, PPAR? S112 phosphorylation, by evaluating the bone phenotype of PPAR? S112A mutant mice. This mutation prevents MAPK phosphorylation and inhibition of PPAR? transcriptional activity. Both male and female PPAR? S112A mice had decreased tibial and vertebral BV/TV and decreased trabecular bone relative to wild type littermates. These results were explained by a decrease in bone formation and osteoblast activity in the absence of changes in resorption. In contrast, marrow adipose tissue, adipocyte markers and serum adiponectin were all dramatically increased. Bone marrow stromal cells isolated from PPAR? S112A mice had elevated PPAR? and preferentially differentiated to adipocytes while total and phosphorylated RUNX2 and osteoblastogenesis were inhibited, indicating that the PPAR? S112A mutation affects bone in a cell autonomous manner. Changes in osteoblast/adipocyte lineage allocation in MSC cultures were also seen where CFU-OBs were reduced with a parallel increase in CFU-AD. This study emphasizes the importance of PPAR? phosphorylation in controlling bone mass and marrow adiposity and demonstrates how a regulatory mutation in PPAR? previously associated with peripheral fat metabolism can have broader effects on bone homeostasis that may in turn affect whole body energy metabolism.
Project description:Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-? (PPAR?), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPAR? suppression (P<0.01). Canonical Wnt-?-catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt-?-catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.
Project description:Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific (Runx1f/fTwist2-Cre) and osteoblast-specific (Runx1f/fCol1?1-Cre) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/?-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7, Alk3, and Atf4, and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1-deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1-deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2-/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebp? and Ppar? and largely increased adipogenesis. Co-culture of Runx1-deficient and wild-type cells demonstrated that Runx1 regulates osteoblast-adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/?-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.
Project description:People with Type 2 diabetes mellitus (T2DM) have reduced bone mineral density and an increased risk of fractures due to altered mesenchymal stem cell (MSC) differentiation in the bone marrow. This leads to a shift in the balance of differentiation away from bone formation (osteogenesis) in favour of fat cell development (adipogenesis). The commonly used anti-diabetic drug, metformin, activates the osteogenic transcription factor Runt-related transcription factor 2 (Runx2), which may suppress adipogenesis, leading to improved bone health. Here we investigate the involvement of the metabolic enzyme, AMP-activated protein kinase (AMPK), in these protective actions of metformin. The anti-adipogenic actions of metformin were observed in multipotent C3H10T1/2 MSCs, in which metformin exerted reciprocal control over the activities of Runx2 and the adipogenic transcription factor, PPAR?, leading to suppression of adipogenesis. These effects appeared to be independent of AMPK activation but rather through the suppression of the mTOR/p70<sup>S6K</sup> signalling pathway. Basal AMPK and mTOR/p70<sup>S6K</sup> activity did appear to be required for adipogenesis, as demonstrated by the use of the AMPK inhibitor, compound C. This observation was further supported by using AMPK knockout mouse embryo fibroblasts (MEFs) where adipogenesis, as assessed by reduced lipid accumulation and expression of the adipogeneic transcription factor, C/EBP?, was found to display an absolute requirement for AMPK. Further activation of AMPK in wild type MEFS, with either metformin or the AMPK-specific activator, A769662, was also associated with suppression of adipogenesis. It appears, therefore, that basal AMPK activity is required for adipogenesis and that metformin can inhibit adipogenesis through AMPK-dependent or -independent mechanisms, depending on the cellular context.