Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Regulation of galanin gene expression in the anterior pituitary (AP) is positively influenced by estrogen in rodents and undetermined in humans. The objective of this study was to investigate the mechanism behind estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within the human galanin promoter that may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 5'-flanking sequence with similarity to the full 13-base ERE consensus previously defined in the vitellogenin gene (vERE). Both sequences were tested in mobility shift assays for the ability to bind nuclear proteins isolated from rat AP tissue or MtTW-10 pituitary tumors. Only the distal sequence at -527 (gERE1) yielded an ERE-specific DNA/protein complex distinguished by mobility and cross-competition with vERE. The gel mobility pattern of the DNA/protein complex was comparable between the pituitary tissue and tumor extracts. However, DNA/protein affinity estimations demonstrated a greater affinity of pituitary proteins for gERE1 over the vERE sequence. Evidence that the human ER (hER) does recognize the gERE1 sequence in the human galanin gene was provided by electrophoretic mobility shift assays (EMSAs) with Sf9 extracts enriched in recombinant hER. In addition, antibodies specific for the hER recognized the gERE1/protein complex in supershift experiments. Enhancer activity by gERE1 was detected in transient transfections of the rat GH3 pituitary cell line, resulting in a 4-fold induction of expression driven by the heterologous thymidine kinase promoter in the presence of estrogen. Evidence for ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibition of enhancement using the specific ER antagonist ICI 164,384; and 2) enhancement in HeLa cells that was dependent upon coexpression with hER. Enhancement by gERE1 was half the magnitude as that from the vERE element and may reflect a difference in affinity or composition of the ER complex between the two sequences. These data demonstrate the presence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.
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PMID:An estrogen receptor binding site within the human galanin gene. 934 90

The activity of estradiol on the LHRH neuronal network is crucial in the regulation of reproduction. In vivo, estradiol induces galanin (GAL) gene expression in LHRH neurons and GAL/LHRH colocalization is sexually dimorphic and neonatally determined by steroid exposure. The effects of estradiol on LHRH neurons, however, are considered to be indirect because estrogen receptors (ER) have not been detected in LHRH neurons in vivo. Using immortalized mouse LHRH neurons (GT1-7 cells), we demonstrated by RT-PCR and Southern blotting that GT1-7 cells express ER messenger RNA (mRNA). Sequencing of the amplification products indicated that GT1-7 ER is of the alpha-subtype (ER alpha). Additionally, estrogen receptors in GT1-7 cells were characterized by competitive radioligand receptor binding and IC50 values for 17beta-estradiol and ICI-182,780 were found to be 0.24 and 4.1 nM, respectively. The ability of endogenous GT1-7 cell ER to regulate transcription was determined in transient transfection studies using a construct that consisted of a luciferase reporter gene that is driven by tandem estrogen response elements (ERE) and a minimal herpes simplex virus thymidine kinase promoter. 17Beta-estradiol was found to enhance luciferase activity by 2.5-fold at physiological concentrations with an ED50 value of 47 pM. This induction was completely inhibited by ICI-182,780 which had an IC50 value of 4.8 nM. Raloxifene, tamoxifen, 4-hydroxytamoxifen, and droloxifene also fully blocked estrogen-mediated luciferase induction with IC50 values of 58.4, 89.2, 33.2, and 49.8 nM, respectively. In addition, GAL mRNA was detected and identified by RT-PCR followed by Southern blotting using a rat GAL complementary DNA (cDNA) probe. The ability of 17beta-estradiol to modulate expression of the endogenous GAL gene in immortalized LHRH neurons was also determined. Quantitative RT-PCR demonstrated that physiological concentrations of estrogen increase GAL gene expression by 2-fold with an ED50 value of 23 pM. ICI-182,780, raloxifene, and droloxifene completely blocked this induction. In summary, our data demonstrate the presence of ER alpha and GAL mRNA in GT1-7 cells. The ER in GT1-7 cells is biologically active because 17beta-estradiol enhances both endogenous GAL gene expression and an ERE-driven reporter gene. These results suggest that estrogenic control of GAL gene expression in immortalized LHRH neurons may be transduced by ER. Thus, hypothalamic-derived LHRH neurons appear to have the capacity to be directly regulated by estrogen.
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PMID:Expression of functional estrogen receptors and galanin messenger ribonucleic acid in immortalized luteinizing hormone-releasing hormone neurons: estrogenic control of galanin gene expression. 949 23