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)

Signal amplification is fundamental to the normal operation of the preovulatory LH surge and is achieved through processes such as GnRH self-priming and augmentation of stimulated LH secretion by progesterone. We have proposed a model for GnRH self-priming that requires cross-communication between a GnRH receptor-activated protein kinase A pathway and the progesterone receptor (PR) to achieve amplification of the GnRH signal. We found that a pulse of GnRH administered to gonadotrope-enriched pituitary cells cultured in medium containing charcoal-treated serum plus estradiol (E2) potentiated the LH secretory response to subsequent GnRH pulses, and this potentiation could be blocked by a PR antagonist, RU486, in the absence of progesterone. Similarly, exposure of gonadotrope-enriched cultures to forskolin augmented the response to a pulse of GnRH, and the augmentation due to cAMP elevation could be reduced by RU486 in the absence of progesterone. To directly test whether stimulation with either GnRH or a cAMP analog results in transactivation of the endogenous PR, we used rat anterior pituitary cells cultured in the presence of E2 and transfected with reporter plasmids containing progesterone-responsive elements (PRE) and either a E1b or a thymidine kinase (tk) promoter linked to the chloramphenicol acetyltransferase (CAT) gene. For pituitary cells transfected with the PRE-E1b-CAT plasmid, exposure to either progesterone, GnRH, or 8-bromo-cAMP (8BrcAMP) for 6 h resulted in an induction of CAT activity which could be suppressed by coincubation with RU486. RU486 by itself had no effect on CAT activity. Similar results were obtained when a plasmid containing a different promoter (PRE-tk-CAT) was used. For cells transfected with a construct lacking a PRE (pSV2CAT), 8BrcAMP was without effect on CAT expression. When cells were made PR-deficient by omission of E2 from the incubation medium and transfected with PRE-E1b-CAT, neither progesterone, GnRH, nor 8BrcAMP was able to induce CAT activity. In summary we found that either GnRH or 8BrcAMP is able to stimulate transcription of reporter genes linked to two different PRE-containing promoters in anterior pituitary cells that contain endogenous PR; this occurred in the absence of progesterone and was suppressed by a PR antagonist. A simple interpretation of these data is that a GnRH-triggered signaling cascade can result in progesterone-independent transactivation of the PR. We propose that, in the normal operation of the preovulatory LH surge, the pathways for GnRH self-priming and progesterone augmentation converge at the PR and that the pathways serve as physiological redundancies to ensure the LH surge.
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PMID:Activation of the progesterone receptor by the gonadotropin-releasing hormone self-priming signaling pathway. 798 48

The molecular mechanisms regulating restricted expression of GnRH receptor and gonadotropin subunit genes to gonadotrope cells have been the focus of intense interest. Using deletion and mutational analysis we have identified a tripartite enhancer that regulates cell-specific expression of the GnRH receptor gene in the gonadotrope-derived alphaT3-1 cell line. Individual elements of this enhancer include binding sites for steroidogenic factor-1; activator protein 1 (AP-1); and a novel element referred to as the GnRH receptor activating sequence (GRAS). Mutation of each element alone results in loss of approximately 60% of promoter activity. Combinatorial mutations of any two elements decreases promoter activity by approximately 80%. Finally, mutation of all three elements reduces promoter activity to a level not different from promoterless vector. Using 2-bp mutations, we have defined the functional requirements for transcriptional activation by GRAS. The core motif of GRAS is at -391 to -380 bp relative to the start site of translation and has the sequence 5'-CTAGTCACAACA-3'. Three copies of GRAS or GRAS with a 2-bp mutation (muGRAS) were cloned into a luciferase expression vector immediately upstream of the thymidine kinase minimal promoter (TK) and tested for expression in alphaT3-1 cells. When compared with TK promoter alone, activity of 3xGRAS-TKLUC was increased by more than 5-fold while activity of 3xmuGRAS-TKLUC was unchanged. When 3xGRAS-TKLUC was transfected into a variety of nongo-nadotrope cell lines, it did not increase activity of the TK promoter. We propose that basal activity of the GnRH receptor gene is regulated by a tripartite enhancer, and the key component of this enhancer is an element, GRAS, that activates transcription in a cell-specific fashion.
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PMID:The tripartite basal enhancer of the gonadotropin-releasing hormone (GnRH) receptor gene promoter regulates cell-specific expression through a novel GnRH receptor activating sequence. 936 49

Previous deletion analysis of the 5'-flanking region of human GnRH receptor (GnRHR) gene has revealed a powerful negative regulatory element (NRE) located between nucleotide -1017 and -771. In the present study, we demonstrated that this NRE could repress the homologous promoter, irrespective of its position and completely abolish the activity of a heterologous thymidine kinase promoter in an orientation-dependent manner. Progressive 3'-deletion analysis revealed that most of the silencing activity of the NRE resided in a putative octamer regulatory sequence (5'AAGCAAACT3'), which alone could repress the promoter activities by 69-90% in ovarian OVCAR-3, placental JEG-3, and gonadotrope-derived alphaT3-1 cells. Mutation of the AAAC residues of the octamer sequence completely removed its silencing activity. Interestingly, conversion of the octamer sequence into that of the rodent GnRHR promoter (5'AAGCAAAGT3') did not attenuate its silencing effect, indicating that the repressive role of the octamer sequence is evolutionarily conserved. EMSAs showed that common DNA-protein complexes of the same mobility were formed with nuclear extracts from the reproductive cells and gonadotropes, and a consensus octamer transcription factor-1 (Oct-1) binding sequence could dose dependently inhibit the complex formation. Antibody supershift and Southwestern blot assays confirmed that the protein binding to the octamer sequence was the ubiquitously expressed transcription factor Oct-1. Overexpression of Oct-1 augmented the silencing activity of the octamer sequence in alphaT3-1 cells. Taken together, our results clearly indicate a role of Oct-1 in the transcriptional repression of the human GnRHR gene.
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PMID:Oct-1 is involved in the transcriptional repression of the gonadotropin-releasing hormone receptor gene. 1244 97

A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.
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PMID:[Synthesis of GnRH analogs and their application in targeted gene delivery systems]. 2279 3