| Parathyroid hormone (PTH) plays a major role in regulating calcium homeostasis through its actions on bone and kidney. Activation of different signaling pathways by PTH leads to the induction of primary response genes (PRGs). PRG products, in turn, regulate a range of cellular functions, including transcription and signal transduction. One of the primary response genes we identified was Nurr1. Since Nurr1 is a primary response gene that functions as a transcription factor, we hypothesized that it effects osteoblast differentiation and function through modulating the transcription of its target genes. To that end, we attempted to identify genes commonly induced by PTH and Nurr1 through the use of gene microarrays.;Microarray data revealed Tachykinin 1 (Tac1) as a potential gene target of PTH. Tachykinin 1 codes for Substance P, a neuropeptide with stimulatory hematopoietic properties. Interestingly, osteoblasts support the hematopoietic stem cell niche (HSC), while PTH has been shown to increase the HSC/colony forming unit (CFU) pool size. Our data indicate that Tac1 is a PRG induced by PTH in mouse osteoblasts (MOB) and that PTH regulates Tac1 gene expression through the cAMP-PKA pathway using CRE and AP-1 response elements found on its promoter. Fibrinogen Like-2 (fgl2) was also a gene target identified by the microarray. Our data revealed that fgl2 is a common target of both Nurr1 and PTH in primary MOB cells and that PTH regulates fgl2 gene expression through the cAMP-PKA pathway. Moreover, we identified a consensus NBRE response element -3200 base pairs upstream of the fgl2 transcription start site and showed that PTH induced nuclear extracts as well as Nurr1 in vitro synthesized protein can bind this site.;We attempted to study the effects of Nurr1 overexpression and functional inhibition in vivo by the creation of a transgenic mouse model overexpressing Nurr1 or dominant negative Nur77 (dnNur77) protein, and in vitro by developing a Nurr1 or dnNur77 overexpressing lentiviral vectors. We were unable to obtain positive Nurr1 overexpressing founder mice, and we observed compromised transduction efficiency of the Nurr1 overexpressing lentivirus, suggesting that Nurr1 might have deleterious effects on cell survival. To address these problems, we propose alternative experimental strategies. |
