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A new role for wilms tumor protein 1: differential activities of + KTS and -KTS variants to regulate LHβ transcription.

Bagchi D, Andrade J, Shupnik MA - PLoS ONE (2015)

Bottom Line: WT1 mRNAs and protein were decreased approximately 50% by GnRH treatment, under conditions where Egr1 mRNA and protein, and LHβ transcription, were stimulated.Decreasing expression of mRNA for WT1 (-KTS) decreased stimulation of LHβ and Egr1 by GnRH, whereas decreasing both WT1 (-KTS) and (+KTS) increased endogenous LHβ transcription, and prevented LHβ but not Egr1 stimulation by GnRH, suggesting differing biological activities for the WT1 isoforms.Our data suggest that WT1 can modulate LHβ transcription, with differential roles for the two WT1 variants; WT1 (-KTS) enhances and WT1 (+KTS) suppresses transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America.

ABSTRACT
Luteinizing hormone (LH) is synthesized and secreted throughout the reproductive cycle from gonadotrope cells in the anterior pituitary, and is required for steroidogenesis and ovulation. LH contains an α-subunit common with FSH, and a unique LHβ subunit that defines biological activity. Basal LHβ transcription is low and stimulated by hypothalamic GnRH, which induces synthesis of early growth response protein-1 (Egr1), and stimulates binding of transcription factors Egr1 and steroidogenic factor-1 (SF1) on the promoter. WT1 (Wilms tumor protein1) is a zinc finger transcription factor with an essential role in urogenital system development, and which regulates several reproductive genes via interactions with SF1 or binding to GC-rich elements such as Egr1 binding sites. We investigated a potential role for WT1 in LHβ transcription in clonal mouse gonadotrope LβT2 cells. WT1 was present in LβT2 and mouse pituitary cells, and protein bound to the endogenous LHβ promoter. Interestingly, mRNAs for WT1(+KTS), which contains a three amino-acid insertion between the 3rd and 4th zinc fingers, and the WT1 (-KTS) variant were both expressed at significant levels. WT1 mRNAs and protein were decreased approximately 50% by GnRH treatment, under conditions where Egr1 mRNA and protein, and LHβ transcription, were stimulated. Decreasing expression of mRNA for WT1 (-KTS) decreased stimulation of LHβ and Egr1 by GnRH, whereas decreasing both WT1 (-KTS) and (+KTS) increased endogenous LHβ transcription, and prevented LHβ but not Egr1 stimulation by GnRH, suggesting differing biological activities for the WT1 isoforms. Overexpression of WT1 showed that WT1(-KTS) enhanced LHβ promoter GnRH stimulation 2-to-3-fold and required the 3'Egr1 site, but WT1(+KTS) repressed both basal and GnRH-stimulated LHβ promoter activity by approximately 70%. Our data suggest that WT1 can modulate LHβ transcription, with differential roles for the two WT1 variants; WT1 (-KTS) enhances and WT1 (+KTS) suppresses transcription.

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WT1 (+KTS) decreases basal and GnRH-stimulated LHβ promoter activity.LβT2 cells were transfected with either A) A luciferase reporter construct driven by the rat LHβ promoter (-617 to +41 bp) including both distal and proximal GnRH responsive promoter regions, or B) A luciferase reporter construct driven by the rat LHβ promoter (-245 to +44 bp), including only the proximal GnRH response region of the promoter. Constructs were cotransfected with or without 0.5, 0.7,1 μg of WT1 (+KTS) plasmid or control plasmid to normalize DNA. At 48 h post-transfection, cells were treated with 50nM GnRH for 6 h and collected in lysis buffer. Luciferase activity was measured, and data expressed as average ± SE for 6 samples; the experiment was performed 3 times each. Statistical significance was determined using ANOVA (confidence interval determined by the Bonferroni multiple comparison test). * p<.05 -GnRH vs +GnRH, # = p<.05 control, -GnRH vs –GnRH+WT1, a = p<.05 control, +GnRH vs +GnRH +WT1.
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pone.0116825.g006: WT1 (+KTS) decreases basal and GnRH-stimulated LHβ promoter activity.LβT2 cells were transfected with either A) A luciferase reporter construct driven by the rat LHβ promoter (-617 to +41 bp) including both distal and proximal GnRH responsive promoter regions, or B) A luciferase reporter construct driven by the rat LHβ promoter (-245 to +44 bp), including only the proximal GnRH response region of the promoter. Constructs were cotransfected with or without 0.5, 0.7,1 μg of WT1 (+KTS) plasmid or control plasmid to normalize DNA. At 48 h post-transfection, cells were treated with 50nM GnRH for 6 h and collected in lysis buffer. Luciferase activity was measured, and data expressed as average ± SE for 6 samples; the experiment was performed 3 times each. Statistical significance was determined using ANOVA (confidence interval determined by the Bonferroni multiple comparison test). * p<.05 -GnRH vs +GnRH, # = p<.05 control, -GnRH vs –GnRH+WT1, a = p<.05 control, +GnRH vs +GnRH +WT1.

Mentions: To directly test the role of the WT1 +KTS and –KTS splice variants in LHβ transcription, basal and GnRH-stimulated activity of the LHβ promoter (-617 to +44 bp) was measured in the absence or presence of increasing amounts of WT1(+KTS) or WT1(–KTS) expression vectors. WT1(-KTS) overexpression significantly increased the GnRH stimulation of LHβ promoter activity by approximately 3- to 4-fold at the highest concentrations (Fig. 5A). The -617LHβ promoter contains two regions that might be influenced by WT1, including the distal enhancer that contains Sp1 binding sites, and the proximal enhancer containing Egr1 binding sites; both are required for effective GnRH-stimulated transcription to occur [6, 7]. We thus assessed whether WT1 was able to influence LHβ transcription through the proximal region by testing the LHβ luciferase (-245 to +44 bp) construct containing only proximal GnRH response elements. As shown in Fig. 5B, WT1(-KTS) overexpression significantly increased the basal (up to 3-fold) and GnRH stimulated-LHβ promoter activity of the shorter construct up to 4-fold. In contrast, WT1(+KTS) overexpression significantly decreased the basal and GnRH-stimulated LHβ promoter activity of both the -617 LHβ (Fig. 6A) and the shorter -245 bp constructs (Fig. 6B), by up to 70% at the highest WT1 concentrations.


A new role for wilms tumor protein 1: differential activities of + KTS and -KTS variants to regulate LHβ transcription.

Bagchi D, Andrade J, Shupnik MA - PLoS ONE (2015)

WT1 (+KTS) decreases basal and GnRH-stimulated LHβ promoter activity.LβT2 cells were transfected with either A) A luciferase reporter construct driven by the rat LHβ promoter (-617 to +41 bp) including both distal and proximal GnRH responsive promoter regions, or B) A luciferase reporter construct driven by the rat LHβ promoter (-245 to +44 bp), including only the proximal GnRH response region of the promoter. Constructs were cotransfected with or without 0.5, 0.7,1 μg of WT1 (+KTS) plasmid or control plasmid to normalize DNA. At 48 h post-transfection, cells were treated with 50nM GnRH for 6 h and collected in lysis buffer. Luciferase activity was measured, and data expressed as average ± SE for 6 samples; the experiment was performed 3 times each. Statistical significance was determined using ANOVA (confidence interval determined by the Bonferroni multiple comparison test). * p<.05 -GnRH vs +GnRH, # = p<.05 control, -GnRH vs –GnRH+WT1, a = p<.05 control, +GnRH vs +GnRH +WT1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4305298&req=5

pone.0116825.g006: WT1 (+KTS) decreases basal and GnRH-stimulated LHβ promoter activity.LβT2 cells were transfected with either A) A luciferase reporter construct driven by the rat LHβ promoter (-617 to +41 bp) including both distal and proximal GnRH responsive promoter regions, or B) A luciferase reporter construct driven by the rat LHβ promoter (-245 to +44 bp), including only the proximal GnRH response region of the promoter. Constructs were cotransfected with or without 0.5, 0.7,1 μg of WT1 (+KTS) plasmid or control plasmid to normalize DNA. At 48 h post-transfection, cells were treated with 50nM GnRH for 6 h and collected in lysis buffer. Luciferase activity was measured, and data expressed as average ± SE for 6 samples; the experiment was performed 3 times each. Statistical significance was determined using ANOVA (confidence interval determined by the Bonferroni multiple comparison test). * p<.05 -GnRH vs +GnRH, # = p<.05 control, -GnRH vs –GnRH+WT1, a = p<.05 control, +GnRH vs +GnRH +WT1.
Mentions: To directly test the role of the WT1 +KTS and –KTS splice variants in LHβ transcription, basal and GnRH-stimulated activity of the LHβ promoter (-617 to +44 bp) was measured in the absence or presence of increasing amounts of WT1(+KTS) or WT1(–KTS) expression vectors. WT1(-KTS) overexpression significantly increased the GnRH stimulation of LHβ promoter activity by approximately 3- to 4-fold at the highest concentrations (Fig. 5A). The -617LHβ promoter contains two regions that might be influenced by WT1, including the distal enhancer that contains Sp1 binding sites, and the proximal enhancer containing Egr1 binding sites; both are required for effective GnRH-stimulated transcription to occur [6, 7]. We thus assessed whether WT1 was able to influence LHβ transcription through the proximal region by testing the LHβ luciferase (-245 to +44 bp) construct containing only proximal GnRH response elements. As shown in Fig. 5B, WT1(-KTS) overexpression significantly increased the basal (up to 3-fold) and GnRH stimulated-LHβ promoter activity of the shorter construct up to 4-fold. In contrast, WT1(+KTS) overexpression significantly decreased the basal and GnRH-stimulated LHβ promoter activity of both the -617 LHβ (Fig. 6A) and the shorter -245 bp constructs (Fig. 6B), by up to 70% at the highest WT1 concentrations.

Bottom Line: WT1 mRNAs and protein were decreased approximately 50% by GnRH treatment, under conditions where Egr1 mRNA and protein, and LHβ transcription, were stimulated.Decreasing expression of mRNA for WT1 (-KTS) decreased stimulation of LHβ and Egr1 by GnRH, whereas decreasing both WT1 (-KTS) and (+KTS) increased endogenous LHβ transcription, and prevented LHβ but not Egr1 stimulation by GnRH, suggesting differing biological activities for the WT1 isoforms.Our data suggest that WT1 can modulate LHβ transcription, with differential roles for the two WT1 variants; WT1 (-KTS) enhances and WT1 (+KTS) suppresses transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America.

ABSTRACT
Luteinizing hormone (LH) is synthesized and secreted throughout the reproductive cycle from gonadotrope cells in the anterior pituitary, and is required for steroidogenesis and ovulation. LH contains an α-subunit common with FSH, and a unique LHβ subunit that defines biological activity. Basal LHβ transcription is low and stimulated by hypothalamic GnRH, which induces synthesis of early growth response protein-1 (Egr1), and stimulates binding of transcription factors Egr1 and steroidogenic factor-1 (SF1) on the promoter. WT1 (Wilms tumor protein1) is a zinc finger transcription factor with an essential role in urogenital system development, and which regulates several reproductive genes via interactions with SF1 or binding to GC-rich elements such as Egr1 binding sites. We investigated a potential role for WT1 in LHβ transcription in clonal mouse gonadotrope LβT2 cells. WT1 was present in LβT2 and mouse pituitary cells, and protein bound to the endogenous LHβ promoter. Interestingly, mRNAs for WT1(+KTS), which contains a three amino-acid insertion between the 3rd and 4th zinc fingers, and the WT1 (-KTS) variant were both expressed at significant levels. WT1 mRNAs and protein were decreased approximately 50% by GnRH treatment, under conditions where Egr1 mRNA and protein, and LHβ transcription, were stimulated. Decreasing expression of mRNA for WT1 (-KTS) decreased stimulation of LHβ and Egr1 by GnRH, whereas decreasing both WT1 (-KTS) and (+KTS) increased endogenous LHβ transcription, and prevented LHβ but not Egr1 stimulation by GnRH, suggesting differing biological activities for the WT1 isoforms. Overexpression of WT1 showed that WT1(-KTS) enhanced LHβ promoter GnRH stimulation 2-to-3-fold and required the 3'Egr1 site, but WT1(+KTS) repressed both basal and GnRH-stimulated LHβ promoter activity by approximately 70%. Our data suggest that WT1 can modulate LHβ transcription, with differential roles for the two WT1 variants; WT1 (-KTS) enhances and WT1 (+KTS) suppresses transcription.

Show MeSH
Related in: MedlinePlus