Limits...
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.

Show MeSH

Related in: MedlinePlus

WT1 siRNA increases the basal and decreases GnRH stimulated expression of endogenous LHβ primary transcript.LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4305298&req=5

pone.0116825.g004: WT1 siRNA increases the basal and decreases GnRH stimulated expression of endogenous LHβ primary transcript.LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.

Mentions: To further investigate a potential role for WT1 on endogenous LHβ gene transcription, we decreased endogenous WT1 expression via targeted knock-down by siRNA. A non-targeting siRNA was used in parallel as a control. After 72 h of siRNA treatment, cells were incubated with GnRH for 90 min, and LHβ primary transcript mRNA was measured. As shown in Fig. 4A, knock-down of WT1(-KTS) mRNA alone reduced endogenous WT1 protein by approximately 50%. Under these conditions, basal LHβ mRNA transcription was not significantly affected, but GnRH-stimulated transcription was suppressed by approximately 50%. When both mRNA isoforms for WT1 were decreased, with endogenous WT1 protein decreased by >90% (Fig. 4B), basal LHβ transcription was significantly increased (approximately 2-fold) compared to siControl, and GnRH treatment resulted in lower LHβ primary transcript levels compared to siControl GnRH, or siWT1 cells treated with vehicle. Because WT1 is a transcriptional regulator, and because Egr1 expression is important for LHβ transcription, we also tested the effects of WT1 variant knock-down on the basal and GnRH-stimulated expression of Egr1 mRNA. Interestingly, knock-down of WT1(-KTS) alone reduced GnRH stimulation of Egr1 mRNA by approximately 60%, similar to the suppression of GnRH-stimulated LHβ transcription. In contrast, knock-down of both WT1 isoforms does not suppress Egr1 expression, and GnRH stimulation of Egr1 occurs even though LHβ transcription is not stimulated by GnRH. These data suggest that there may be different, and perhaps even opposing, biological roles for the two WT1 variants on LHβ transcription.


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 siRNA increases the basal and decreases GnRH stimulated expression of endogenous LHβ primary transcript.LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4305298&req=5

pone.0116825.g004: WT1 siRNA increases the basal and decreases GnRH stimulated expression of endogenous LHβ primary transcript.LβT2 cells were transfected with siRNA against WT1 and a non-targeting siRNA as a control. After 72 h of incubation, cells were treated with or without GnRH for 1.5 h, followed by cell lysate collection, RNA extraction and western blot analysis (30 μg protein) on 10% PAGE-SDS gels. WT1 protein was detected by immunoblotting with specific antibodies for WT1, and normalized for β-actin on the same blot. A. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where the WT1 (-KTS) variant mRNA was reduced. B. Expression of the endogenous LHβ primary transcript, Egr1 primary transcript mRNA, and WT1 protein under conditions where both the WT1 (-KTS) and WT1 (+KTS) variant mRNAs were reduced. For each condition, the experiment was performed twice with 5 replicates. LHβ and Egr1 primary transcript mRNAs were normalized for GAPDH mRNA in the same sample. Control samples contained non-targeting siRNA. * p<.05 -GnRH vs +GnRH, in either Control siRNA or WT1 siRNA treatments. Values are mean ± SEM for 5 replicates. # = p<.05 -GnRH Control siRNA vs –GnRH WT1 siRNA, or p<.05+GnRH Control siRNA vs +GnRH WT1 siRNA.
Mentions: To further investigate a potential role for WT1 on endogenous LHβ gene transcription, we decreased endogenous WT1 expression via targeted knock-down by siRNA. A non-targeting siRNA was used in parallel as a control. After 72 h of siRNA treatment, cells were incubated with GnRH for 90 min, and LHβ primary transcript mRNA was measured. As shown in Fig. 4A, knock-down of WT1(-KTS) mRNA alone reduced endogenous WT1 protein by approximately 50%. Under these conditions, basal LHβ mRNA transcription was not significantly affected, but GnRH-stimulated transcription was suppressed by approximately 50%. When both mRNA isoforms for WT1 were decreased, with endogenous WT1 protein decreased by >90% (Fig. 4B), basal LHβ transcription was significantly increased (approximately 2-fold) compared to siControl, and GnRH treatment resulted in lower LHβ primary transcript levels compared to siControl GnRH, or siWT1 cells treated with vehicle. Because WT1 is a transcriptional regulator, and because Egr1 expression is important for LHβ transcription, we also tested the effects of WT1 variant knock-down on the basal and GnRH-stimulated expression of Egr1 mRNA. Interestingly, knock-down of WT1(-KTS) alone reduced GnRH stimulation of Egr1 mRNA by approximately 60%, similar to the suppression of GnRH-stimulated LHβ transcription. In contrast, knock-down of both WT1 isoforms does not suppress Egr1 expression, and GnRH stimulation of Egr1 occurs even though LHβ transcription is not stimulated by GnRH. These data suggest that there may be different, and perhaps even opposing, biological roles for the two WT1 variants on LHβ transcription.

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