Differential Expression of the Peripheral Benzodiazepine Receptor and Gremlin During Adipogenesis

This study used the mRNA differential display technique to identify differentially expressed genes during the process of adipogenesis in the preadipocyte cell line, 3T3‐L1. 3T3‐L1 cells were treated with dexamethasone, isobutyl‐1‐methylxanthine, and insulin to induce differentiation into mature adipocytes. Cells were collected at three time‐points during differentiation: Day 0 (d0), or nondifferentiated; Day 3 (d3), during differentiation; and Day 10 (d10), >90% of the cells had differentiated into mature adipocytes. Initial studies yielded 18 potentially differentially regulated cDNA candidates (8 down‐regulated and 10 up‐regulated). Reverse Northern and Northern blots confirmed differential expression of six of the candidates. Four of the candidates up‐regulated on d3 and d10 were identified by sequence analysis to be lipoprotein lipase, a well‐known marker of adipocyte differentiation. A fifth candidate that was expressed in d0, but not d3 or d10, was identified as DRM/gremlin, a bone morphogenetic protein antagonist. Finally, a sixth candidate that was increased at d3 and d10 was identified as the peripheral benzodiazepine receptor, which has been implicated in proliferation, differentiation, and cholesterol transport in cells. This study is the first to show that peripheral benzodiazepine receptor and DRM/gremlin are expressed in preadipocyte cell lines and that they are differentially regulated during adipogenesis.     

Sortilin is upregulated during osteoblastic differentiation of mesenchymal stem cells and promotes extracellular matrix mineralization

Osteoblasts and adipocytes are derived from a common precursor in bone marrow, the mesenchymal stem cell (MSC). Factors driving human MSCs (hMSCs) to differentiate down the two lineages play important roles in determining bone density because it has been shown that bone volume loss associated with osteoporosis and aging is accompanied by reduced osteoblastic bone formation and increased marrow adipose tissue. The genes upregulated in hMSCs during osteogenic differentiation were screened using cDNA microarrays and were semi-quantitated by real-time RT-PCR. One of the genes identified was sortilin, which was upregulated one day after osteogenic induction and remained upregulated for a week. The overexpression of sortilin in hMSCs using an adenovirus vector resulted in the acceleration of mineralization during osteogenic differentiation without affecting alkaline phosphatase activity. Lipoprotein lipase (LPL), produced by adipocytes, is bound by sortilin, which may mediate its endocytosis. By adding LPL to osteogenic induction medium, osteoblastic mineralization was inhibited in a dose-dependent manner. Interestingly, sortilin overexpression abolished the LPL-mediated suppression of osteogenic differentiation. hMSCs exist in marrow where LPL-producing adipose cells are abundant and where osteogenesis is negatively regulated by LPL. Sortilin has a counter effect of promoting osteogenesis by acting as a scavenger of LPL.     

Differential redox potential profiles during adipogenesis and osteogenesis

Development is an orderly process that requires the timely activation and/or deactivation of specific regulatory elements that control cellular proliferation, differentiation and apoptosis. While many studies have defined factors that control developmental signaling, the role of intracellular reduction/oxidation (redox) status as a means to control differentiation has not been fully studied. Redox states of intracellular couples may play a very important role in regulating redox-sensitive elements that are involved in differentiation signaling into specific phenotypes. In human mesenchymal stem cells (hMSCs), which are capable of differentiating into many different types of phenotypes, including osteoblasts and adipocytes, glutathione (GSH), cysteine (Cys) and thioredoxin-1 (Trx1) redox potentials were measured during adipogenesis and osteogenesis. GSH redox potentials (E_h) during both osteogenesis and adipogenesis became increasingly oxidized as differentiation ensued, but the rate at which this oxidation occurred was unique for each process. During adipogenesis, Cys E_h became oxidized as adipogenesis progressed but during osteogenesis, it became reduced. Interestingly, intracellular Trx1 concentrations appeared to increase in both adipogenesis and osteogenesis, but the E_h was unchanged when compared to undifferentiated hMSCs. These data show that hMSC differentiation into either adipocytes of osteoblasts corresponds to a unique redox state profile, suggesting that differentiation into specific phenotypes are likely regulated by redox states that are permissive to a specific developmental process.     

Genomic-scale analysis of Pasteurella multocida gene expression during growth within liver tissue of chickens with fowl cholera

We have recently reported the gene expression profile of Pasteurella multocida during growth in the blood of chickens with fowl cholera. Here we report the gene expression profile of P. multocida during growth in the livers of similarly infected chickens. We compared expression profiles of bacteria harvested from the livers of infected chickens with late-stage fowl cholera with those of bacteria grown in rich medium. Independent analysis of bacterial expression profiles from three individual chickens indicated that 93 P. multocida genes were always differentially expressed during growth in liver tissue. Of these 93 genes, 49 were upregulated and 44 downregulated in the host. Many of the upregulated genes were involved in energy production and conversion (9/49) and carbohydrate transport and metabolism (8/49), and a number of these have been shown to be induced under anaerobic conditions in other species. The downregulated genes were generally of unknown or poorly characterised functions (14/44). Comparison of the differentially regulated gene sets identified for growth in liver with those identified previously for growth in blood allowed the identification of a core set of 13 upregulated and 16 downregulated genes that were differentially regulated in at least five of the six infections studied.     

Enhancement of adipogenic and osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells by supplementation with umbilical cord blood serum

Umbilical cord blood serum (UCBS) is a promising replacement for animal sera for the culture of human mesenchymal stem cells (hMSC), the unique serum composition of UCBS appearing to have variable effects on their proliferation and differentiation. Conditioning UCBS with methods such as charcoal stripping assists specific processes such as adipogenesis and osteogenesis in hMSCs. The charcoal stripping of serum removes lipophilic materials such as oestrogens, which are known inhibitors of adipogenesis. hMSC cultures supplemented with charcoal-stripped UCBS (CS-UCBS) show enhanced adipogenesis in adipogenic induction medium (AIM) containing indomethacin, 3-isobutyl-1-methylxanthine and dexamethasone. To obtain efficient adipogenesis without CS-UCBS, we have developed a modified protocol in which cells cultured separately with UCBS and CS-UCBS are constantly treated with minimal doses of insulin (1.1 μg/ml) for 10 days prior to the addition of AIM. hMSC cultures differentiated by using the modified protocol show improved adipogenesis under fetal bovine serum (FBS), UCBS and CS-UCBS conditions, with levels of adipogenesis being highest in UCBS, thereby eliminating the need for charcoal stripping. Furthermore, in each of the three sera, the insulin-pre-treated hMSCs accumulate lipid droplets faster and exhibit improved adipogenesis overall when compared with normal AIM-induced adipogenesis. We have also compared the levels of osteogenesis in hMSCs by using an induction medium devoid of dexamethasone. Maximum calcium deposition has been observed in hMSCs cultured with UCBS, as compared with those cultured with FBS or CS-UCBS. Our newly developed methods with a humanized serum supplement thus enhance the differentiation of cultured hMSCs.     

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

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

The effects of cyclin-dependent kinase inhibitors on adipogenic differentiation of human mesenchymal stem cells

The molecular mechanisms that couple growth arrest and cell differentiation were examined during adipogenesis. Here, to understand the cyclin-dependent kinase inhibitor (CKI) genes involved in the progression of adipogenic differentiation, we examined changes in the protein and mRNA expression levels of CKI genes in vitro. During the onset of growth arrest associated with adipogenic differentiation, two independent families of CKI genes, p27Kip1 and p18INK4c, were significantly increased. The expressions of p27Kip1 and p18INK4c, regulated at the level of protein and mRNA accumulation, were directly coupled to adipogenic differentiation. This finding was supported by the inhibition of adipogenic differentiation caused by short interfering RNA (siRNA). In this study, we investigated the regulatory effects of transforming growth factor beta-1 (TGFβ-1) on CKI genes involved in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Only the up-regulation of p18INK4c during adipogenic differentiation, and not that of the p27Kip1 gene was prevented by treatment with TGFβ-1, one of the factors that inhibit adipogenesis in vitro. This finding indicates a close correlation between adipogenic differentiation and p18INK4c induction in hMSCs. Thus, these data demonstrate a role for the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors in regulating adipogenic differentiation, thereby providing a molecular mechanism that couples growth arrest and differentiation.     

Ex vivo expansion, adipogenesis and neurogenesis of cryopreserved human bone marrow mesenchymal stem cells

This study aimed to investigate the potentials of ex vivo expansion and pluridifferentiation of cryopreserved adult human bone marrow mesenchymal stem cells (hMSCs) into adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced to adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and expression of triglyceride or neuron-specific enolase and nestin was detected. The result showed that the resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and insulin-like growth factor I (IGF-I) showed adipogenesis, and lipid vacuole accumulation was detectable after 21days. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron-specific enolase (NSE), which are special surface markers associated with neural cells at different stages. This study suggested that resuscitated hMSCs should still be a population of pluripotential cells and should be accessible for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.