Regulation of GTP cyclohydrolase I expression by orphan receptor Nurr1 in cell culture and in vivo.

Nurr1 is an orphan nuclear transcription factor essential for the terminal differentiation of dopamine (DA) neurons in the ventral midbrain (VM). To identify the Nurr1-target genes, we carried out microarray and quantitative real-time PCR analyses of Nurr1 null and wild-type mice in VM at embryonic day (E) 12.5 and shortly after birth (P0). In addition to the absence of mRNAs of DA synthesizing enzymes, the guanosine 5'-triphosphate (GTP) cyclohydrolase I (GTPCH) was also substantially reduced in the VM of Nurr1-null mice. GTPCH is the first enzyme in the synthesis pathway of tetrahydrobiopterin (BH4), an essential cofactor for tyrosine hydroxylase in DA synthesis. In the mouse, Nurr1 and GTPCH mRNA were first detected at E10.5, and GTPCH transcription paralleled that of Nurr1. Small interfering RNA targeted against Nurr1 decreases GTPCH expression in MC3T3-E1 osteoblasts in cell culture. Cotransfection of Nurr1 and the GTPCH-luciferase (luc) reporter increased the luc activity by about threefold in N2A cells. Additional analysis using 5'-deletions and mutants revealed that Nurr1 activates GTPCH transcription indirectly through the proximal promoter region, in the absence of the nerve growth factor-induced clone B (NGFI-B) responsive element-like sites, similarly, as recently reported for DA transporter regulation by Nurr1.

Reduced tyrosine hydroxylase and GTP cyclohydrolase mRNA expression, tyrosine hydroxylase activity, and associated neurochemical alterations in Nurr1-null heterozygous mice.

The nuclear receptor Nurr1 is essential for the development of midbrain dopamine neurons and appears to be an important regulator of dopamine levels as adult Nurr1-null heterozygous (+/-) mice have reduced mesolimbic/mesocortical dopamine levels. The mechanism(s) through which reduced Nurr1 expression affects dopamine levels has not been determined. Quantitative real-time PCR revealed a significant reduction in tyrosine hydroxylase (TH) and GTP cyclohydrolase (GTPCH) mRNA in ventral midbrain of +/- mice as compared to wild-type mice (+/+). The effect on TH expression was only observed at birth, while reduced GTP cyclohydrolase was also observed in the adult ventral tegemental area. No differences in dopamine transporter, vesicular monoamine transporter, dopamine D2 receptor or aromatic amino acid decarboxylase were observed. Since TH and GTPCH are both involved in dopamine synthesis, regulation of in vivo TH activity was measured in these mice. In vivo TH activity was reduced in nucleus accumbens and striatum of the +/- mice (24.7% and 15.7% reduction, respectively). In the striatum, gamma-butyrolactone exacerbated differences on +/- striatal TH activity (29.8% reduction) while haloperidol equalized TH activity between the +/+ and +/-. TH activity in the nucleus accumbens was significantly reduced in all conditions measured. Furthermore, dopamine levels in the striatum of +/- mice were significantly reduced after inhibition of dopamine synthesis or after haloperidol treatment but not under basal conditions while dopamine levels in the nucleus accumbens were reduced under basal conditions. Based on these data the +/- genotype results in changes in gene expression and impairs dopamine synthesis which can affect the maintenance of dopamine levels, although with differential effects between mesolimbic/mesocortical and nigrostriatal dopamine neurons. Together, these data suggest that Nurr1 may function to modify TH and GTPCH expression and dopamine synthesis.

Regulation of osteoblast differentiation by Nurr1 in MC3T3-E1 cell line and mouse calvarial osteoblasts.

The orphan nuclear receptor Nurr1 is primarily expressed in the central nervous system. It has been shown that Nurr1 is necessary for terminal differentiation of dopaminergic (DA) neurons in ventral midbrain. The receptor, however, is also expressed in other organs including bone, even though the role of Nurr1 is not yet understood. Therefore, we investigated the role of Nurr1 in osteoblast differentiation in MC3T3-E1 cells and calvarial osteoblasts derived from Nurr1 null newborn pups. Our results revealed that reduced Nurr1 expression, using Nurr1 siRNA in MC3T3-E1 cells, affected the expression of osteoblast differentiation marker genes, osteocalcin (OCN) and collagen type I alpha 1 (COL1A1), as measured by quantitative real-time PCR. The activity of alkaline phosphatase (ALP), another osteoblast differentiation marker gene, was also decreased in Nurr1 siRNA-treated MC3T3-E1 cells. In addition, Nurr1 overexpression increased OCN and COL1A1 expression. Furthermore, consistent with these results, during osteoblast differentiation, the expression of osteoblast marker genes was decreased in primary cultured mouse calvarial osteoblasts derived from Nurr1 null mice. Collectively, our results suggest that Nurr1 is important for osteoblast differentiation.

Differential role of ERK in cAMP-induced Nurr1 expression in N2A and C6 cells.

We investigated the signal pathway related to induction of Nurr1, transcription factor, by cAMP in neuroblastoma N2A and C6 glioma cell lines. Nurr1 expression was induced by forskolin, an adenylate cyclase activator, via activation of CREB in both N2A and C6 cells. The effect of forskolin on ERK, however, was cell specific. ERK phosphorylation was stimulated by forskolin in N2A cells whereas it was inhibited in C6 cells. Pretreatment with H89, a PKA inhibitor, blocked the forskolin-induced Nurr1 expression in both N2A and C6 cells. Interestingly, pretreatment with PD98059, an MEK inhibitor, showed differential effects. Pretreatment with PD98059 inhibited the forskolin-induced Nurr1 expression in N2A cells, however, in C6 cells, Nurr1 expression was further increased. Our results suggest that ERK pathway plays a differential role in cAMP-induced Nurr1 expression in N2A and C6 cells.

Erythropoietin receptor signalling is required for normal brain development.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor(-/-) mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor(-/-) cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor(+/+) rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.