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Sally Radovick - One of the best experts on this subject based on the ideXlab platform.

  • ap 1 antagonizes thyroid hormone receptor action on the Thyrotropin Beta Subunit gene
    Journal of Biological Chemistry, 1993
    Co-Authors: Fredric E Wondisford, Hans Jurgen Steinfelder, Michelle Nations, Sally Radovick
    Abstract:

    Abstract Thyrotropin-releasing hormone (TRH) stimulates and thyroid hormone (T3) inhibits transcription of the Thyrotropin Beta-Subunit gene (TSH-Beta). The first exon contains DNA sequences necessary for both responses and binds both AP-1 and thyroid hormone receptor (T3R). T3 did not inhibit TSH-Beta gene expression in a T3R-deficient cell line. Transfection of a T3R expression vector, however, resulted in a 70% inhibition of expression by T3, which was abolished by cotransfection of c-jun and c-fos expression vectors. Mutations surrounding the transcription initiation site and DNA binding studies demonstrate both a functional and structural interaction between c-jun and T3R. Thus, TRH, acting through AP-1, may alter the set point and magnitude of thyroid hormone negative feedback of the TSH-Beta gene through an interaction between AP-1 and T3R. Other regulatory pathways acting through AP-1 may alter thyroid hormone action in man.

  • hormonal regulation of the Thyrotropin Beta Subunit gene by phosphorylation of the pituitary specific transcription factor pit 1
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Sally Radovick, Hans J Steinfelder, Fredric E Wondisford
    Abstract:

    Abstract The pituitary-specific transcription factor Pit-1 is a cell-specific activator of prolactin and growth hormone gene transcription in the anterior pituitary. Pit-1 has also been shown to mediate both Thyrotropin-releasing hormone (TRH) and cAMP stimulation of the prolactin and Thyrotropin Beta-Subunit (TSH Beta) genes. The molecular mechanism by which Pit-1 mediates these stimulatory effects remains unclear. At least three Pit-1-binding elements within the TSH Beta gene mediate responsiveness to TRH and cAMP. The present studies were designed to test the hypothesis that phosphorylation is an important modulator of Pit-1 interaction with the TSH Beta gene. TSH Beta elements bind less well to nonphosphorylated Pit-1 than to phosphorylated Pit-1 and are weak activators of gene expression, unlike high-affinity Pit-1 binding sites in the prolactin and growth hormone genes. Phosphorylation by protein kinase A or C enhances Pit-1 binding to TSH Beta elements 3- to 8-fold. Conversely, phosphorylation generally reduces binding of Pit-1 to elements within the prolactin and growth hormone genes. A variation within the consensus sequence for Pit-1 binding in TSH Beta gene elements [A(A/T)(A/T)AATNCAT in the TSH Beta gene versus A(A/T)(A/T)TATNCAT in the prolactin and growth hormone genes] could explain these differences. These elements may limit basal activation of the TSH Beta gene by binding less well to nonphosphorylated Pit-1 while conferring hormonal stimulation through enhanced binding of phosphorylated Pit-1.

  • role of a pituitary specific transcription factor pit 1 ghf 1 or a closely related protein in camp regulation of human Thyrotropin Beta Subunit gene expression
    Journal of Clinical Investigation, 1992
    Co-Authors: Hans Jurgen Steinfelder, Sally Radovick, Bruce D Weintraub, Melisa A Mroczynski, J H Mcclaskey, Peter Hauser, F E Wondisford
    Abstract:

    cAMP regulation of the human Thyrotropin-Beta (TSH Beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH Beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH Beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH Beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH Beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH Beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH Beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH Beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH Beta gene.

  • a detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human Thyrotropin Beta Subunit gene
    Journal of Biological Chemistry, 1991
    Co-Authors: D L Bodenner, Bruce D Weintraub, Sally Radovick, Melisa A Mroczynski, F E Wondisford
    Abstract:

    Abstract The first exon of the human Thyrotropin-Beta (hTSH Beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH Beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH Beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-Beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-Beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-Beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH Beta gene. The upstream site (domain 1) binds c-erbA-Beta with higher affinity and is capable of binding c-erbA-Beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-Beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH Beta expression.

  • Thyrotropin-releasing hormone regulation of human TSHB expression: role of a pituitary-specific transcription factor (Pit-1/GHF-1) and potential interaction with a thyroid hormone-inhibitory element.
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Hans Jurgen Steinfelder, Sally Radovick, Bruce D Weintraub, Peter C. Hauser, Yuko Nakayama, J H Mcclaskey, Terry Taylor, Fredric E Wondisford
    Abstract:

    Abstract Regulation of human Thyrotropin Beta Subunit gene (TSHB) expression by Thyrotropin-releasing hormone (TRH) was examined in a clonal rat pituitary-cell line (GH3). Transient expression studies were done with various 5'-flanking DNA sequences of TSHB coupled to reporter gene chloramphenicol acetyltransferase. Deletion analysis defined two discrete regions (-128 to -92 base pairs and -28 to +8 base pairs) that each mediated an approximately 2-fold TRH induction. The upstream site contains a DNA sequence with close homology to the DNA-binding site for a pituitary-specific transcriptional factor Pit-1/GHF-1. DNase I footprinting analysis of mouse thyrotropic tumor extract as well as DNA-transfection studies using an expression vector containing an N-terminal deletion of Pit-1/GHF-1 cDNA suggest that Pit-1/GHF-1 or a closely related protein in the thyrotroph mediates TRH responsiveness of this gene. In addition, the downstream site overlaps with the recently characterized thyroid hormone-inhibitory element of TSHB. In fact, deletion of DNA sequences important in thyroid hormone-receptor binding (c-erbAB/c-ERBA2) from +3 to +8 base pairs, significantly reduced (30%) TRH responsiveness. The location of a TRH-stimulatory element near a thyroid hormone-inhibitory element may allow for fine control of TSHB expression in vivo.

Fredric E Wondisford - One of the best experts on this subject based on the ideXlab platform.

  • ap 1 antagonizes thyroid hormone receptor action on the Thyrotropin Beta Subunit gene
    Journal of Biological Chemistry, 1993
    Co-Authors: Fredric E Wondisford, Hans Jurgen Steinfelder, Michelle Nations, Sally Radovick
    Abstract:

    Abstract Thyrotropin-releasing hormone (TRH) stimulates and thyroid hormone (T3) inhibits transcription of the Thyrotropin Beta-Subunit gene (TSH-Beta). The first exon contains DNA sequences necessary for both responses and binds both AP-1 and thyroid hormone receptor (T3R). T3 did not inhibit TSH-Beta gene expression in a T3R-deficient cell line. Transfection of a T3R expression vector, however, resulted in a 70% inhibition of expression by T3, which was abolished by cotransfection of c-jun and c-fos expression vectors. Mutations surrounding the transcription initiation site and DNA binding studies demonstrate both a functional and structural interaction between c-jun and T3R. Thus, TRH, acting through AP-1, may alter the set point and magnitude of thyroid hormone negative feedback of the TSH-Beta gene through an interaction between AP-1 and T3R. Other regulatory pathways acting through AP-1 may alter thyroid hormone action in man.

  • hormonal regulation of the Thyrotropin Beta Subunit gene by phosphorylation of the pituitary specific transcription factor pit 1
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Sally Radovick, Hans J Steinfelder, Fredric E Wondisford
    Abstract:

    Abstract The pituitary-specific transcription factor Pit-1 is a cell-specific activator of prolactin and growth hormone gene transcription in the anterior pituitary. Pit-1 has also been shown to mediate both Thyrotropin-releasing hormone (TRH) and cAMP stimulation of the prolactin and Thyrotropin Beta-Subunit (TSH Beta) genes. The molecular mechanism by which Pit-1 mediates these stimulatory effects remains unclear. At least three Pit-1-binding elements within the TSH Beta gene mediate responsiveness to TRH and cAMP. The present studies were designed to test the hypothesis that phosphorylation is an important modulator of Pit-1 interaction with the TSH Beta gene. TSH Beta elements bind less well to nonphosphorylated Pit-1 than to phosphorylated Pit-1 and are weak activators of gene expression, unlike high-affinity Pit-1 binding sites in the prolactin and growth hormone genes. Phosphorylation by protein kinase A or C enhances Pit-1 binding to TSH Beta elements 3- to 8-fold. Conversely, phosphorylation generally reduces binding of Pit-1 to elements within the prolactin and growth hormone genes. A variation within the consensus sequence for Pit-1 binding in TSH Beta gene elements [A(A/T)(A/T)AATNCAT in the TSH Beta gene versus A(A/T)(A/T)TATNCAT in the prolactin and growth hormone genes] could explain these differences. These elements may limit basal activation of the TSH Beta gene by binding less well to nonphosphorylated Pit-1 while conferring hormonal stimulation through enhanced binding of phosphorylated Pit-1.

  • Thyrotropin-releasing hormone regulation of human TSHB expression: role of a pituitary-specific transcription factor (Pit-1/GHF-1) and potential interaction with a thyroid hormone-inhibitory element.
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Hans Jurgen Steinfelder, Sally Radovick, Bruce D Weintraub, Peter C. Hauser, Yuko Nakayama, J H Mcclaskey, Terry Taylor, Fredric E Wondisford
    Abstract:

    Abstract Regulation of human Thyrotropin Beta Subunit gene (TSHB) expression by Thyrotropin-releasing hormone (TRH) was examined in a clonal rat pituitary-cell line (GH3). Transient expression studies were done with various 5'-flanking DNA sequences of TSHB coupled to reporter gene chloramphenicol acetyltransferase. Deletion analysis defined two discrete regions (-128 to -92 base pairs and -28 to +8 base pairs) that each mediated an approximately 2-fold TRH induction. The upstream site contains a DNA sequence with close homology to the DNA-binding site for a pituitary-specific transcriptional factor Pit-1/GHF-1. DNase I footprinting analysis of mouse thyrotropic tumor extract as well as DNA-transfection studies using an expression vector containing an N-terminal deletion of Pit-1/GHF-1 cDNA suggest that Pit-1/GHF-1 or a closely related protein in the thyrotroph mediates TRH responsiveness of this gene. In addition, the downstream site overlaps with the recently characterized thyroid hormone-inhibitory element of TSHB. In fact, deletion of DNA sequences important in thyroid hormone-receptor binding (c-erbAB/c-ERBA2) from +3 to +8 base pairs, significantly reduced (30%) TRH responsiveness. The location of a TRH-stimulatory element near a thyroid hormone-inhibitory element may allow for fine control of TSHB expression in vivo.

F E Wondisford - One of the best experts on this subject based on the ideXlab platform.

  • role of a pituitary specific transcription factor pit 1 ghf 1 or a closely related protein in camp regulation of human Thyrotropin Beta Subunit gene expression
    Journal of Clinical Investigation, 1992
    Co-Authors: Hans Jurgen Steinfelder, Sally Radovick, Bruce D Weintraub, Melisa A Mroczynski, J H Mcclaskey, Peter Hauser, F E Wondisford
    Abstract:

    cAMP regulation of the human Thyrotropin-Beta (TSH Beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH Beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH Beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH Beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH Beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH Beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH Beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH Beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH Beta gene.

  • a detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human Thyrotropin Beta Subunit gene
    Journal of Biological Chemistry, 1991
    Co-Authors: D L Bodenner, Bruce D Weintraub, Sally Radovick, Melisa A Mroczynski, F E Wondisford
    Abstract:

    Abstract The first exon of the human Thyrotropin-Beta (hTSH Beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH Beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH Beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-Beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-Beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-Beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH Beta gene. The upstream site (domain 1) binds c-erbA-Beta with higher affinity and is capable of binding c-erbA-Beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-Beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH Beta expression.

Hans J Steinfelder - One of the best experts on this subject based on the ideXlab platform.

  • hormonal regulation of the Thyrotropin Beta Subunit gene by phosphorylation of the pituitary specific transcription factor pit 1
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Sally Radovick, Hans J Steinfelder, Fredric E Wondisford
    Abstract:

    Abstract The pituitary-specific transcription factor Pit-1 is a cell-specific activator of prolactin and growth hormone gene transcription in the anterior pituitary. Pit-1 has also been shown to mediate both Thyrotropin-releasing hormone (TRH) and cAMP stimulation of the prolactin and Thyrotropin Beta-Subunit (TSH Beta) genes. The molecular mechanism by which Pit-1 mediates these stimulatory effects remains unclear. At least three Pit-1-binding elements within the TSH Beta gene mediate responsiveness to TRH and cAMP. The present studies were designed to test the hypothesis that phosphorylation is an important modulator of Pit-1 interaction with the TSH Beta gene. TSH Beta elements bind less well to nonphosphorylated Pit-1 than to phosphorylated Pit-1 and are weak activators of gene expression, unlike high-affinity Pit-1 binding sites in the prolactin and growth hormone genes. Phosphorylation by protein kinase A or C enhances Pit-1 binding to TSH Beta elements 3- to 8-fold. Conversely, phosphorylation generally reduces binding of Pit-1 to elements within the prolactin and growth hormone genes. A variation within the consensus sequence for Pit-1 binding in TSH Beta gene elements [A(A/T)(A/T)AATNCAT in the TSH Beta gene versus A(A/T)(A/T)TATNCAT in the prolactin and growth hormone genes] could explain these differences. These elements may limit basal activation of the TSH Beta gene by binding less well to nonphosphorylated Pit-1 while conferring hormonal stimulation through enhanced binding of phosphorylated Pit-1.

Hans Jurgen Steinfelder - One of the best experts on this subject based on the ideXlab platform.

  • ap 1 antagonizes thyroid hormone receptor action on the Thyrotropin Beta Subunit gene
    Journal of Biological Chemistry, 1993
    Co-Authors: Fredric E Wondisford, Hans Jurgen Steinfelder, Michelle Nations, Sally Radovick
    Abstract:

    Abstract Thyrotropin-releasing hormone (TRH) stimulates and thyroid hormone (T3) inhibits transcription of the Thyrotropin Beta-Subunit gene (TSH-Beta). The first exon contains DNA sequences necessary for both responses and binds both AP-1 and thyroid hormone receptor (T3R). T3 did not inhibit TSH-Beta gene expression in a T3R-deficient cell line. Transfection of a T3R expression vector, however, resulted in a 70% inhibition of expression by T3, which was abolished by cotransfection of c-jun and c-fos expression vectors. Mutations surrounding the transcription initiation site and DNA binding studies demonstrate both a functional and structural interaction between c-jun and T3R. Thus, TRH, acting through AP-1, may alter the set point and magnitude of thyroid hormone negative feedback of the TSH-Beta gene through an interaction between AP-1 and T3R. Other regulatory pathways acting through AP-1 may alter thyroid hormone action in man.

  • role of a pituitary specific transcription factor pit 1 ghf 1 or a closely related protein in camp regulation of human Thyrotropin Beta Subunit gene expression
    Journal of Clinical Investigation, 1992
    Co-Authors: Hans Jurgen Steinfelder, Sally Radovick, Bruce D Weintraub, Melisa A Mroczynski, J H Mcclaskey, Peter Hauser, F E Wondisford
    Abstract:

    cAMP regulation of the human Thyrotropin-Beta (TSH Beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH Beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH Beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH Beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH Beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH Beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH Beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH Beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH Beta gene.

  • Thyrotropin-releasing hormone regulation of human TSHB expression: role of a pituitary-specific transcription factor (Pit-1/GHF-1) and potential interaction with a thyroid hormone-inhibitory element.
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Hans Jurgen Steinfelder, Sally Radovick, Bruce D Weintraub, Peter C. Hauser, Yuko Nakayama, J H Mcclaskey, Terry Taylor, Fredric E Wondisford
    Abstract:

    Abstract Regulation of human Thyrotropin Beta Subunit gene (TSHB) expression by Thyrotropin-releasing hormone (TRH) was examined in a clonal rat pituitary-cell line (GH3). Transient expression studies were done with various 5'-flanking DNA sequences of TSHB coupled to reporter gene chloramphenicol acetyltransferase. Deletion analysis defined two discrete regions (-128 to -92 base pairs and -28 to +8 base pairs) that each mediated an approximately 2-fold TRH induction. The upstream site contains a DNA sequence with close homology to the DNA-binding site for a pituitary-specific transcriptional factor Pit-1/GHF-1. DNase I footprinting analysis of mouse thyrotropic tumor extract as well as DNA-transfection studies using an expression vector containing an N-terminal deletion of Pit-1/GHF-1 cDNA suggest that Pit-1/GHF-1 or a closely related protein in the thyrotroph mediates TRH responsiveness of this gene. In addition, the downstream site overlaps with the recently characterized thyroid hormone-inhibitory element of TSHB. In fact, deletion of DNA sequences important in thyroid hormone-receptor binding (c-erbAB/c-ERBA2) from +3 to +8 base pairs, significantly reduced (30%) TRH responsiveness. The location of a TRH-stimulatory element near a thyroid hormone-inhibitory element may allow for fine control of TSHB expression in vivo.