Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation.

In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Here, we report that insulin- and Ras-induced activation of MAPK is not required for the differentiation process and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocked insulin- and Ras-induced MAPK activation but had no effect on or slightly enhanced adipocytic differentiation. Tumor necrosis factor alpha (TNF-alpha), an inhibitor of insulin-stimulated adipogenesis, activated MAPK in 3T3-L1 cells. PD98059 treatment blocked MAPK activation by TNF-alpha and reversed the blockade of adipogenesis mediated by low (1 ng/ml) TNF-alpha concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK displayed impaired adipocytic differentiation. PD98059 treatment also reversed the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells.     

SiRNA against Fabp5 induces 3T3-L1 cells apoptosis during adipocytic induction

Fatty acid-binding protein 5 (Fabp5), exhibits an important role in binding free fatty acids, as well as regulating lipid metabolism and transport. The purpose of this study was to evaluate the role of Fabp5 during adipogenesis. 3T3-L1 preadipocytes were selected as cell differentiation model and short interfering RNAs (siRNA) against Fabp5 (siFabp5) were prepared. Our results showed that two potent siFabp5 specifically inhibited endogenous expression of Fabp5 at both mRNA and protein level. SiFabp5 had little effect on undifferentiated 3T3-L1 fibroblasts. However, during adipocytic induction, 3T3-L1 preadipocytes transfected with siFabp5 significantly reduced cell viability, as well as increased both caspase-3 activity and procaspase-3 cleavage. Furthermore, we illustrated that knockdown Fabp5 inhibited the expression of PPARγ and C/EBPα during adipocytic induction. In conclusion, our data suggests that Fabp5 is crucial in maintaining the viability of preadipocytes during adipogenesis via the activation of Akt cascade, and decreased Fabp5 expression induce differentiating preadipocytes apoptosis via caspase-3 activation.     

BK virus large T antigen: interactions with the retinoblastoma family of tumor suppressor proteins and effects on cellular growth control.

BK virus (BKV) is a polyomavirus which infects a large percentage of the human population. It is a potent transforming agent and is tumorigenic in rodents. BKV DNA has also been found in human brain, pancreatic islet, and urinary tract tumors, implicating this virus in neoplastic processes. BKV T antigen (TAg) is highly homologous to simian virus 40 TAg, particularly in regions required for mitogenic stimulation and binding to tumor suppressor proteins, The experiments presented in this report show that BKV TAg can bind the tumor suppressor protein p53. BKV TAg also has the ability to bind to members of the retinoblastoma (pRb) family of tumor suppressor proteins both in vivo and in vitro. However, these interactions are detected only when large amounts of total protein are used, because the levels of BKV TAg normally produced from viral promoter-enhancer elements are too low to bind a significant amount of the pRb family proteins in the cell. The low levels of BKV TAg produced by the viral promoter elements are sufficient to affect the levels and the phosphorylation patterns of these proteins and to induce serum-independent growth in these cells. Additional events, however, are required for full transformation. These data further support the notion that BKV TAg can affect cellular growth control mechanisms and may in fact be involved in neoplastic processes.     

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.     

The role of Hsp90N, a new member of the Hsp90 family, in signal transduction and neoplastic transformation.

The 90-kDa heat shock protein (Hsp90), the target of the ansamycin class of anti-cancer drugs, is required for the conformational activation of a specific group of signal transducers, including Raf-1. In this report we have identified a 75-kDa Raf-associated protein as Hsp90N, a novel member of the Hsp90 family. Intriguingly, the ansamycin-binding domain is replaced in Hsp90N by a much shorter, hydrophobic sequence, preceded by a putative myristylation signal. We demonstrate that, although much less abundant, Hsp90N binds Raf with a higher affinity than Hsp90. In sharp contrast to Hsp90, Hsp90N does not associate with p50(cdc37), the Hsp90 kinase cofactor. Hsp90N was found to activate Raf in transiently transfected cells, while Rat F111 fibroblasts stably transfected with Hsp90N exhibited elevated activity of the Raf and downstream ERK kinases. This may be due to Raf binding to myristylated Hsp90N, followed by Raf translocation to the membrane. To examine whether Hsp90N could therefore substitute for Ras in Raf recruitment to the cell membrane, Hsp90N was transfected in c-Ras-deficient, 10T1/2-derived preadipocytes. Our results indicate that, as shown before for activated Ras or Raf, the introduction of even low levels of Hsp90N through transfection in c-Ras-deficient preadipocytes causes a dramatic block of differentiation. Higher levels of Hsp90N expression resulted in neoplastic transformation, including interruption of gap junctional, intercellular communication, and anchorage-independent proliferation. These results indicate that the observed activation of Raf by Hsp90N has a profound biological effect, which is largely c-Ras-independent. With the recent finding that p50(cdc37) is tumorigenic in transgenic mice, these results reinforce the intriguing observation that the family of heat shock proteins represents a novel class of molecules with oncogenic potential.     

Regulation of Wnt signaling during adipogenesis

We have identified Wnt10b as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. Here, we demonstrate that a specific inhibitor of glycogen synthase kinase 3, CHIR 99021, mimics Wnt signaling in preadipocytes. CHIR 99021 stabilizes free cytosolic beta-catenin and inhibits adipogenesis by blocking induction of CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. Preadipocyte differentiation is inhibited when 3T3-L1 cells are exposed to CHIR 99021 for any 24 h period during the first 3 days of adipogenesis. Consistent with this time frame of inhibition, expression of Wnt10b mRNA is suppressed upon induction of differentiation, with a 50% decline by 6 h and complete inhibition by 36 h. Of the agents used to induce differentiation, exposure of 3T3-L1 cells to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b mRNA. Inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrate that disruption of extracellular Wnt signaling by expression of secreted Frizzled related proteins causes spontaneous adipocyte conversion.     

A novel pathway to enhance adipocyte differentiation of 3T3-L1 cells by up-regulation of lipocalin-type prostaglandin D synthase mediated by liver X receptor-activated sterol regulatory element-binding protein-1c

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at -233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at -194 to be cis-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.     

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

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

MiR-125a-5p抑制3T3-L1前体脂肪细胞分化

MicroRNAs(miRNAs) 是一类在脂肪组织发育中发挥重要作用的小非编码RNA. 为探明miR-125a-5p在3T3-L1前体脂肪细胞中的作用,采用实时qPCR检测了miR-125a-5p在小鼠各组织及3T3-L1前体脂肪细胞分化过程中的表达|使用经化学修饰的miR-125a-5p模拟物agomir及抑制剂antagomir转染3T3-L1前体脂肪细胞,采用实时qPCR 和 Western印迹检测成脂标志基因Pparγ和aP2的表达,油红O染色观察脂肪细胞脂质积累. 结果显示,miR-125-5p在小鼠脂肪组织中高丰度表达,在3T3-L1前体脂肪细胞分化过程中表达下降.过表达miR-125a-5p,与对照组相比,成脂标志基因Pparγ和aP2在mRNA和蛋白质水平均明显下降|油红O染色及定量结果显示脂质积累减少. 抑制剂处理结果显示,Pparγ和aP2在mRNA和蛋白质水平均有不同程度上升,但油红O染色及定量结果差异不显著. 以上结果表明,miR-125a-5p在脂肪细胞分化中发挥负调控作用.     

Proteomic analysis of adipocyte differentiation: Evidence that alpha2 macroglobulin is involved in the adipose conversion of 3T3 L1 preadipocytes

Adipogenesis is an important aspect of energy homeostasis. Here we have used a differential proteome mapping strategy to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3 L1 preadipocytes. Two-dimensional gel electrophoresis analysis identified 8 proteins that are induced following hormone-evoked differentiation. In addition, we found that a alpha2 macroglobulin fragment was abundantly present in 3T3 L1 preadipocytes, but was virtually undetectable in fully differentiated adipocytes. Metabolic radiolabeling with (35S)methionine and Northern blot analysis indicated that the intracellular alpha2 macroglobulin fragment in preadipocytes was derived from the extracellular culture medium, not de novo synthesis. Incubation of preadipocytes with an antialpha2 macroglobulin polyclonal antibody caused depletion of the intracellular alpha2 macroglobulin fragments, and also enhanced spontaneous adipose conversion. These results suggest that intracellular alpha2 macroglobulin fragment inhibits adipocyte differentiation, and that hormone treatment induces differentiation at least in part by suppression of intracellular alpha2 macroglobulin activity in 3T3 L1 preadipocytes.     

Transdifferentiation of preadipose cells into smooth muscle-like cells: role of aortic carboxypeptidase-like protein

Adipocyte differentiation involves dramatic cell shape alterations that are accompanied by changes in the expression of cytoskeletal and extracellular matrix (ECM) proteins. Aortic carboxypeptidase-like protein (ACLP) is a secreted protein associated with the extracellular matrix whose expression is induced during smooth muscle (SM) differentiation. We analyzed the expression of ACLP gene during adipocyte differentiation of 3T3-F442A, 3T3-L1, and Ob1771 preadipocytes. Our results show that ACLP mRNA and protein are expressed in growing cells and after commitment. Thereafter, their expression levels decrease, as opposed to that of aP2 and PPARgamma2. Consistent with these observations, ACLP mRNA is expressed in the stromal-vascular fraction of adipose tissue but not in the adipocyte fraction. Overexpression of ACLP in 3T3-F442A preadipocytes inhibits adipocyte differentiation at both morphological and molecular level. However, ACLP overexpression promotes transdifferentiation of preadipocytes into smooth muscle-like cells, which express specific markers such as SM22alpha, SM alpha-actin, SM-MHC, and caldesmon. These findings demonstrate that overexpression of a single extracellular matrix protein is sufficient to induce transdifferentiation and that ACLP may modulate the commitment of mesodermal cells into different lineages depending upon its pattern of expression.     

Chibby promotes adipocyte differentiation through inhibition of beta-catenin signaling

The canonical Wnt/beta-catenin signaling pathway plays diverse roles in embryonic development and disease. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and in mice. Here, we report that the beta-catenin antagonist Chibby (Cby) is required for adipocyte differentiation. Cby is expressed in adipose tissue in mice, and Cby protein levels increase during adipogenic differentiation of 3T3-L1 cells. Ectopic expression of Cby induces spontaneous differentiation of these cells into mature adipocytes to an extent similar to that of dominant-negative Tcf-4. In contrast, depletion of Cby by RNA interference potently blocks adipogenesis of 3T3-L1 and mouse embryonic stem cells. In support of this, embryonic fibroblasts obtained from Cby-deficient embryos display attenuated differentiation to the adipogenic lineage. Mechanistically, Cby promotes adipocyte differentiation, in part by inhibiting beta-catenin, since gain or loss of function of Cby influences beta-catenin signaling in 3T3-L1 cells. Our results therefore establish Cby as a novel proadipogenic factor required for adipocyte differentiation.