No abstract

Nurr1 is an orphan nuclear receptor best known for its essential role in the development and maintenance of midbrain dopaminergic (DA) neurons. During DA neurogenesis, Nurr1 directly targets human tyrosine hydroxylase (hTH). Here we investigated this targeting to identify the molecular mechanisms by which Nurr1 regulates DA neurogenesis. We previously cloned the hTH promoter and found three consensus elements for Nurr1 binding: NBRE-A, -B, and -C. In the present study, gel retardation and luciferase assays using hTH constructs showed that Nurr1 preferentially bound to NBRE-A, through which it mediated transcriptional activity. Furthermore, Nurr1 displayed dual-function transcriptional activities depending on the cell type. In DA-like SH-SY5Y cells, Nurr1 dose-dependently stimulated hTH-3174 promoter activity by 7- to 11-fold. However, in the human neural stem cell (hNSC) line HB1.F3, Nurr1 strongly repressed transcription from the same promoter. This repression was relieved by mutation of only the NBRE-A element and by nicotinamide [an inhibitor of class III histone deacetylases (HDACs), such as SIRT1], but not by trichostatin A (an inhibitor of class I and II HDACs). SIRT1 was strongly expressed in the nucleus of HB1.F3 cells, while it was localized in the cytoplasm in SH-SY5Y cells. ChIP assays of HB1.F3 cells showed that Nurr1 overexpression significantly increased the SIRT1 occupancy of the NBRE-A hTH promoter region, while low SIRT1 levels were observed in control cells. In contrast, no significant SIRT1 recruitment was observed in SH-SY5Y cells. These results indicate that differential SIRT1 localization may be involved in hTH gene regulation. Overall, our findings suggest that Nurr1 exists in dual transcriptional complexes, including co-repressor complexes that can be remodeled to become co-activators and can fine-tune hTH gene transcription during human DA neurogenesis.

In this study, a heparinized micropattern surface was prepared for the spatial control of human mesenchymal stem cells (hMSCs) that can differentiate into the desired tissues. Poly(styrene-co-vinylbenzyl N,N-diethyldithiocarbamate) (poly(ST-co-VBDC)) was synthesized as a photoreactive polymer; poly(ethylene glycol) methacrylate (PEGMA) was polymerized on the poly(ST-co-VBDC) coated surface by UV irradiation. XPS spectra revealed the residual DC moieties on the PEGMA-grafted surface and the linear chain growth of PEGMA was monitored according to irradiation time. After chemical immobilization of heparin onto this PEGMA surface, surface micropatterning was carried out by additional photopolymerization of PEGMA using a photomask. After incubation for 4 hour, the hMSCs adhered to the heparinized surface, while the hydrophilic PEGMA surface demonstrated no cell adhesion even after basic fibroblast growth factor (bFGF) treatment. Good alignment of hMSCs on the pattern-surface was distinctly observed along micron-sized grooves due to the presence of both heparin and bFGF. This heparinized micropattern surface can be used to study in vitro hMSCs responses with various heparin-binding growth factors in tissue engineering fields as well as cellular array for the spatial control of hMSCs.