Plasminogen activator inhibitor-1 (PAI-1) plays a key role in control of coagulation and tissue remodeling and has been shown to be regulated by a number of cell stimuli, among those hypoxia. In this study we characterize the hypoxia-mediated induction of PAI-1 in human hepatoma cell line HepG2. We found that PAI-1 is tightly regulated in a narrow oxygen gradient. After incubation at oxygen concentrations of 1% to 2%, a 60-fold increase in PAI-1 messenger RNA levels was observed, whereas mild hypoxic conditions of more than 3.5% did not appear to induce transcription. Moreover, increased levels of PAI-1 protein were observed after incubation at low oxygen tensions. Through sequence analysis, several putative hypoxia-response elements (HREs 1-5) were identified in the human PAI-I promoter. Reporter gene assays showed that the HRE-2 (؊194 to ؊187) was necessary and sufficient for the hypoxia-mediated response. By electrophoretic mobility assay we observed hypoxia-dependent binding of a protein complex to the HRE-2 motif. Further analysis demonstrated that HRE-2 was specifically recognized by the hypoxia-inducible transcription factor 1␣-arylhydrocarbon nuclear translocator complex. Taken IntroductionPlasminogen activator inhibitor-1 (PAI-1) plays a central role in the control of physiologically important mechanisms involved in the homeostasis of blood coagulation and remodeling of extracellular matrix (reviewed by Booth 1 ). The effect of PAI-1 is mediated through inhibition of urokinase and tissue type plasminogen activators. The importance of PAI-1 in the regulation of fibrinolytic activity is highlighted by several studies documenting an association between increased levels of PAI-1 and the risk of developing a cardiovascular disease. [2][3][4] Furthermore, numerous clinical studies of different types of cancer have identified high levels of plasma PAI-1 as a strong prognostic factor for more metastatic forms of cancer, concomitant with a poorer clinical outcome (reviewed by Harbeck et al 5 ).PAI-1 is produced by a variety of cell types in vitro, such as hepatocytes, 6 platelets, 7 smooth muscle cells, 8 and endothelial cells. 9 The sources of PAI-1 in vivo have not as yet been identified. However, studies in rabbits indicate that the liver and endothelial cells are the most important producers. 10 Several agents induce PAI-1 at the transcriptional level, including phorbol esters, 11 inflammatory cytokines, 12 transforming growth factor ␤, 13 and hypoxia. 14 For most of these stimuli, the signal transduction pathways have been identified, including target transcription factors and the corresponding specific transcriptional control elements within the regulated target genes. 15,16 Recently, it has been found that a 300-base pair stretch in the promoter region of the human PAI-1 gene is necessary for the responses to hypoxia and that these responses were mediated by the hypoxia-inducible transcription factor, HIF-1. 17,18 There is, however, no information available about molecular mechanisms involved in the cont...

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Background: For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained.

Human mesenchymal stem cells (hMSCs) have the capacity to differentiate along several pathways to form bone, cartilage, tendon, muscle, and adipose tissues. The adult hMSCs reside in vivo in the bone marrow in niches where oxygen concentration is far below the ambient air, which is the most commonly encountered laboratory condition. The study reported here was designed to determine whether oxygen has a role in the differentiation of hMSCs into adipocytes. Indeed, when exposed to atmosphere containing only 1% of oxygen, the formation of adipocytelike phenotype with cytoplasmic lipid inclusions was observed. The effect of hypoxia on the expression of adipocyte-specific genes was determined by real-time reverse transcription polymerase chain reaction. Interestingly, neither of the two central regulators of adipogenesis-the transcription factors peroxisome proliferatoractivated receptor γ2 (PPAR-γ2) and ADD1/SREBP1c-was induced. Furthermore, hypoxia did not have any effect on the transcription of early (lipoprotein lipase) or late (aP2) marker genes. By the same token, neither of the mature adipocyte-specific genes-leptin and adipophilinwas found responsive to the treatment. High level of induction, however, was observed with the PPAR-γ-induced angiopoietin-related gene, PGAR. The lack of an adipocyte-specific transcription pattern thus indicates that despite accumulation of the lipid, true adipogenic differentiation did not take place. In conclusion, hypoxia appears to exert a potent lipogenic effect independent of PPAR-γ2 maturation pathway.