Region Of Tyrosine Hydroxylase Gene Research Articles

Platelet-derived growth factor (PDGF)-BB protects dopaminergic neurons via activation of Akt/ERK/CREB pathways to upregulate tyrosine hydroxylase.

AimsThe neurotropic growth factor PDGF‐BB was shown to have vital neurorestorative functions in various animal models of Parkinson's disease (PD). Previous studies indicated that the regenerative property of PDGF‐BB contributes to the increased intensity of tyrosine hydroxylase (TH) fibers in vivo. However, whether PDGF‐BB directly modulates the expression of TH, and the underlying mechanism is still unknown. We will carefully examine this in our current study.MethodMPTP‐lesion mice received PDGF‐BB treatment via intracerebroventricular (i.c.v) administration, and the expression of TH in different brain regions was assessed by RT‐PCR, Western blot, and immunohistochemistry staining. The molecular mechanisms of PDGF‐BB‐mediated TH upregulation were examined by RT‐PCR, Western blot, ChIP assay, luciferase reporter assay, and immunocytochemistry.ResultsWe validated a reversal expression of TH in MPTP‐lesion mice upon i.c.v administration of PDGF‐BB for seven days. Similar effects of PDGF‐BB‐mediated TH upregulation were also observed in MPP+‐treated primary neuronal culture and dopaminergic neuronal cell line SH‐SY5Y cells. We next demonstrated that PDGF‐BB rapidly activated the pro‐survival PI3K/Akt and MAPK/ERK signaling pathways, as well as the downstream CREB in SH‐SY5Y cells. We further confirmed the significant induction of p‐CREB in PDGF‐BB‐treated animals in vivo. Using a genetic approach, we demonstrated that the transcription factor CREB is critical for PDGF‐BB‐mediated TH expression. The activation and nucleus translocation of CREB were promoted in PDGF‐BB‐treated SH‐SY5Y cells, and the enrichment of CREB on the promoter region of TH gene was also increased upon PDGF‐BB treatment.ConclusionOur data demonstrated that PDGF‐BB directly regulated the expression of TH via activating the downstream Akt/ERK/CREB signaling pathways. Our finding will further support the therapeutic potential of PDGF‐BB in PD, and provide the possibility that targeting PDGF signaling can be harnessed as an adjunctive therapy in PD in the future.

Replication Protein A Unfolds G-Quadruplex Structures with Varying Degrees of Efficiency

Replication protein A (RPA) is known to interact with guanine- (G-) rich sequences that adopt G-quadruplex (GQ) structures. Most studies reported in the literature were performed on GQ formed by homogeneous sequences, such as the human telomeric repeat, and RPA's ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ-forming DNA sequences (PQSs) to their stability against RPA-mediated unfolding using single-molecule fluorescence resonance energy transfer (FRET) and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQSs in the genome. The three GQ constructs have thermal stabilities that differ significantly. Our measurements showed that the most thermally stable structure (Tm = 86 °C) was also the most stable against RPA-mediated unfolding, although the least thermally stable structure (Tm = 69 °C) had at least an order-of-magnitude higher stability against RPA-mediated unfolding than the structure with intermediate thermal stability (Tm = 78 °C). The significance of this observation becomes more evident when considered within the context of the cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these results, we conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single-molecule stopped-flow method. All three GQ structures were unfolded within Δt ≈ 0.30 ± 0.10 s, a surprising result considering that the unfolding time does not correlate with thermal stability or stability against RPA-mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.

Chromosome workshop: Chromosomes 11, 14, and 15

This report describes linkage data presented at the Workshop on Chromosomes 11, 14, and 15 at the Sixth World Congress of Psychiatric Genetics in Bonn, Germany, together with relevant linkage data submitted to the chair and co-chair, and it is presented in the context of the previous literature concerning these chromosomes. We have attempted to collate current linkage data to provide a guide to potentially interesting findings on chromosomes 11, 14, and 15 for the phenotypes of bipolar disorder, schizophrenia, alcoholism, autism, and spelling and reading disability. We discuss methodological limitations and provide chromosome ideograms and tables summarizing findings to date. The most promising region currently appears to be 15q13-q15 in the region of the alpha 7 nicotinic receptor for the phenotype of schizophrenia (and, perhaps, more generally for functional psychosis). Additionally, 15q11-q13 in the region of GABRB3 holds interest as a potential site of a susceptibility gene for autism. Two regions on chromosome 11, 11p15 in the region of tyrosine hydroxylase gene and 11q22-q23 in the region of DRD2, continue to retain some interest for functional psychosis.

AP1-mediated transcriptional enhancement of the rat tyrosine hydroxylase gene by muscarinic stimulation.

We investigated transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by muscarinic stimulation in human neuroblastoma SK-N-BE(2)M17 cells. Carbachol treatment increased the levels of intracellular Ca2+ and inositol 1,4,5-trisphosphate (IP3) and enhanced transcription of the TH gene. The muscarinic receptor antagonist atropine completely abolished the carbachol effect on TH gene expression. When cells were loaded with 50 microM 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) to chelate intracellular Ca2+, carbachol still raised intracellular IP3 level and enhanced TH gene expression. Transient transfection analysis of the 5' upstream region of TH gene revealed that the AP1 cis-acting element at -205 to -199 bp was responsible for carbachol stimulation. But carbachol did not enhance TH gene expression in protein kinase C (PKC)-activated or down-regulated cells that had been induced by 5-min or 24-h exposure to phorbol 12-myristate 13-acetate (PMA), respectively. Thus, Ca(2+)-independent PKC may play a role in carbachol-induced TH gene expression. We demonstrated by gel retardation and competition assays that a DNA sequence containing the wild-type AP1 site formed the specific DNA-protein complex. However, treatment with carbachol or PMA did not change the amount of the specific DNA-protein complex. Our results indicate that stimulation of phospholipase C-linked muscarinic receptors leads to elevated TH gene expression via AP1-mediated enhancement in a PKC-dependent pathway.

A 5′-flanking region of the bovine tyrosine hydroxylase gene is involved in cell-specific expression, activation of gene transcription by phorbol ester, and transactivation by c-Fos and c-Jun

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminergic cells and is transcriptionally activated via a protein kinase C (PKC)-dependent pathway. To identify regulatory regions of the TH gene, transfected neural crest-derived catecholaminergic cells [human neuroblastoma SH-SY5Y and bovine adrenal medullary (BAM) cells] or glia-derived SF-763 cells were used to analyze expression of a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5′-flanking sequences of the bovine TH gene. Plasmid pTH(−245/+21)CAT was constructed by fusing the −245 to +21 region of the TH gene to CAT sequences in the promoterless pCAT basic plasmid. Cells transfected with pTH(−245/+21)CAT expressed CAT activity at levels 8- to 100-fold higher than those of cells transfected with pCAT basic. In SH-SY5Y or BAM cells, pTH(−245/+21)CAT supported the expression of CAT at levels similar to or higher than that supported by the tissue nonspecific viral SV40 promoter/enhancer. In glia-like cells, CAT expression from pTH(−245/+2 1)CAT was about 13-fold lower than that under the control of the SV40 promoter. Incubation of neuroblastoma cells with the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), increased expression of CAT from pTH(−245/+2 1)CAT over 6-fold and was accompanied by induction of c-fos and c-jun mRNAs and proteins. The regulation of TH promoter function by c-Fos/c-Jun was corroborated by transactivation of the TH promoter in SH-SY5Y cells engineered to overexpress exogenous c-Fos and c-Jun. TH gene sequences essential for c-Fos/c-Jun transactivation were located in the −245 to −52 region, upstream from a CRE-like element. Gel mobility assays demonstrated binding of c-Fos-antigen(s) to the region of the TH promoter that supports activation by PMA and c-Fos/c-Jun. Temporal patterns of nicotinic agonist-stimulated induction of c-fos and TH mRNA are also consistent with a role for c-Fos in TH gene transcription. Our results demonstrate that the 5′-flanking region of the bovine TH gene operates as an authentic promoter capable (i) of directing cell-specific expression of a bacterial CAT gene and (ii) of conferring responsivity to PKC-stimulation. The results also suggest that the nuclear proteins c-Fos and c-Jun contribute to PKC pathway-mediated activation of the TH gene via direct interaction with the TH gene promoter. The activation of TH gene expression by PKC/c-Fos/c-Jun may serve as an additional or alternative mechanism to activation by the cAMP-CRE pathway.

Cold-induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene.

It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulated expression of the tyrosine hydroxylase gene by membrane depolarization. Identification of the responsive element and possible second messengers.

Prolonged depolarization has been used as a model of adaptive changes in the expression of various proteins, such as ion channels and neurotransmitter biosynthetic enzymes, in response to increased trans-synaptic activity in the nervous system. In depolarized PC12 cells, tyrosine hydroxylase (TH) mRNA levels increased severalfold (Kilbourne, E. J., and Sabban, E. L. (1990) Mol. Brain Res. 8, 121-127). In this study, membrane depolarization caused an increase in the expression of the reporter gene chloramphenicol acetyltransferase (CAT), under transcriptional control of the 5' region of the rat TH gene. These results indicate that membrane depolarization leads to increased transcription of the TH gene. Protein kinase C inhibitors had no effect on the induction of TH mRNA by depolarization, as well as the increase in formation of CAT under control of the upstream region of the TH gene. The depolarization responsive element in the TH gene was mapped to the region containing the cAMP responsive element. This region of the TH gene also increased CAT activity in response to the calcium ionophore, ionomycin. Interestingly, combined treatment with cAMP analogs and membrane depolarization had a greater effect than either alone on TH mRNA levels, as well as on CAT activity in PC12 cells transfected with the plasmid containing the cAMP responsive element.