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ZBTB20 is required for anterior pituitary development and lactotrope specification.

Cao D, Ma X, Cai J, Luan J, Liu AJ, Yang R, Cao Y, Zhu X, Zhang H, Chen YX, Shi Y, Shi GX, Zou D, Cao X, Grusby MJ, Xie Z, Zhang WJ - Nat Commun (2016)

Bottom Line: Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes.Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ.In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.

ABSTRACT
The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20- mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.

No MeSH data available.


Related in: MedlinePlus

ZBTB20 regulates Prl expression in a cell-autonomous fashion.(a–c) ZBTB20 overexpression in GH3 cells increases PRL expression and secretion without significant effect on GH expression. GH3 cells were infected with Ad-ZBTB20 or Ad-EGFP at MOI of 400. Three days later, GH3 cells were subjected to gene expression analyses at mRNA levels by RT–PCR (a) and at protein levels by western blotting (b), with the expression levels presented as fold change relative to Ad-EGFP control, while PRL secretion was measured in the culture supernatants by ELISA (c). n=4. Values represent mean±s.e.m. **P<0.01 versus Ad-EGFP (Student's t-test). (d–f) ZBTB20 knockdown decreases PRL expression and secretion in MMQ but not GH3 cells. Control or ZBTB20 siRNA was introduced into GH3 or MMQ cells by electroporation, respectively. Three days after transfection, the cells were harvested for western blotting (d) and quantitative RT–PCR analyses (e), and the culture supernatants were measured for PRL levels by ELISA (f). n=4. Values represent mean±s.e.m. *P<0.05 versus control siRNA (Student's t-test). (g) ChIP assay showed the binding of ZBTB20 to rat Prl promoter in MMQ rather than GH3 cells. n=3. Values represent mean±s.e.m. **P<0.01 versus control IgG (Student's t-test). (h) ZBTB20 knockdown decreases the transcriptional activity of Prl promoter in MMQ cells. MMQ cells were co-transfected with siRNA and expression plasmids by electroporation. Luciferase activity was measured at 48-h post transfection, and normalized with internal control. n=3. Values represent mean±s.e.m. ** P<0.01 versus control siRNA (Student's t-test). (i) ZBTB20 overexpression enhances the transcriptional activity of the Prl promoters in 293T cells in the presence of Pit-1. Rat Prl promoters are 0.5, 1.7 kb, or 2.5 kb in length. n=3. Values represent mean±s.e.m. ** P<0.01 versus Pit-1 group (Student's t-test).
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f8: ZBTB20 regulates Prl expression in a cell-autonomous fashion.(a–c) ZBTB20 overexpression in GH3 cells increases PRL expression and secretion without significant effect on GH expression. GH3 cells were infected with Ad-ZBTB20 or Ad-EGFP at MOI of 400. Three days later, GH3 cells were subjected to gene expression analyses at mRNA levels by RT–PCR (a) and at protein levels by western blotting (b), with the expression levels presented as fold change relative to Ad-EGFP control, while PRL secretion was measured in the culture supernatants by ELISA (c). n=4. Values represent mean±s.e.m. **P<0.01 versus Ad-EGFP (Student's t-test). (d–f) ZBTB20 knockdown decreases PRL expression and secretion in MMQ but not GH3 cells. Control or ZBTB20 siRNA was introduced into GH3 or MMQ cells by electroporation, respectively. Three days after transfection, the cells were harvested for western blotting (d) and quantitative RT–PCR analyses (e), and the culture supernatants were measured for PRL levels by ELISA (f). n=4. Values represent mean±s.e.m. *P<0.05 versus control siRNA (Student's t-test). (g) ChIP assay showed the binding of ZBTB20 to rat Prl promoter in MMQ rather than GH3 cells. n=3. Values represent mean±s.e.m. **P<0.01 versus control IgG (Student's t-test). (h) ZBTB20 knockdown decreases the transcriptional activity of Prl promoter in MMQ cells. MMQ cells were co-transfected with siRNA and expression plasmids by electroporation. Luciferase activity was measured at 48-h post transfection, and normalized with internal control. n=3. Values represent mean±s.e.m. ** P<0.01 versus control siRNA (Student's t-test). (i) ZBTB20 overexpression enhances the transcriptional activity of the Prl promoters in 293T cells in the presence of Pit-1. Rat Prl promoters are 0.5, 1.7 kb, or 2.5 kb in length. n=3. Values represent mean±s.e.m. ** P<0.01 versus Pit-1 group (Student's t-test).

Mentions: To explore the mechanisms by which ZBTB20 promotes lactotrope differentiation in a cell-autonomous fashion, we took advantage of a rat somatolactotrope cell line GH3 expressing both GH and PRL, and a rat lactotrope cell line MMQ, which expresses PRL but not GH. At both mRNA and protein levels, MMQ cells expressed much higher levels of Prl and Zbtb20 compared with GH3 cells (Supplementary Fig. 12). Considering that it is necessary for lactotrope differentiation to maintain PRL expression meanwhile extinguishing GH expression, we first examined the potential effect of ZBTB20 overexpression on Prl expression and lactotropic differentiation of GH3 cells. After 72 h of infection with recombinant replication-deficient adenoviruses co-expressing ZBTB20 and EGFP (hereafter Ad-ZBTB20) at the MOI of 400, more than 85% of GH3 cells were green fluorescent protein-positive by flow cytometry assay (Supplementary Fig. 13). Compared with EGFP mock control, ZBTB20 overexpression in GH3 cells led to a marked increase in Prl expression and protein secretion into culture supernatants, but had no effect on GH expression at either mRNA or protein levels (Fig. 8a–c), suggesting that ZBTB20 upregulation alone is not sufficient to drive lactotropic differentiation from somatolactotropes. In addition, the expression of Pit1 or ERα was not affected by ZBTB20 overexpression in GH3 cells.


ZBTB20 is required for anterior pituitary development and lactotrope specification.

Cao D, Ma X, Cai J, Luan J, Liu AJ, Yang R, Cao Y, Zhu X, Zhang H, Chen YX, Shi Y, Shi GX, Zou D, Cao X, Grusby MJ, Xie Z, Zhang WJ - Nat Commun (2016)

ZBTB20 regulates Prl expression in a cell-autonomous fashion.(a–c) ZBTB20 overexpression in GH3 cells increases PRL expression and secretion without significant effect on GH expression. GH3 cells were infected with Ad-ZBTB20 or Ad-EGFP at MOI of 400. Three days later, GH3 cells were subjected to gene expression analyses at mRNA levels by RT–PCR (a) and at protein levels by western blotting (b), with the expression levels presented as fold change relative to Ad-EGFP control, while PRL secretion was measured in the culture supernatants by ELISA (c). n=4. Values represent mean±s.e.m. **P<0.01 versus Ad-EGFP (Student's t-test). (d–f) ZBTB20 knockdown decreases PRL expression and secretion in MMQ but not GH3 cells. Control or ZBTB20 siRNA was introduced into GH3 or MMQ cells by electroporation, respectively. Three days after transfection, the cells were harvested for western blotting (d) and quantitative RT–PCR analyses (e), and the culture supernatants were measured for PRL levels by ELISA (f). n=4. Values represent mean±s.e.m. *P<0.05 versus control siRNA (Student's t-test). (g) ChIP assay showed the binding of ZBTB20 to rat Prl promoter in MMQ rather than GH3 cells. n=3. Values represent mean±s.e.m. **P<0.01 versus control IgG (Student's t-test). (h) ZBTB20 knockdown decreases the transcriptional activity of Prl promoter in MMQ cells. MMQ cells were co-transfected with siRNA and expression plasmids by electroporation. Luciferase activity was measured at 48-h post transfection, and normalized with internal control. n=3. Values represent mean±s.e.m. ** P<0.01 versus control siRNA (Student's t-test). (i) ZBTB20 overexpression enhances the transcriptional activity of the Prl promoters in 293T cells in the presence of Pit-1. Rat Prl promoters are 0.5, 1.7 kb, or 2.5 kb in length. n=3. Values represent mean±s.e.m. ** P<0.01 versus Pit-1 group (Student's t-test).
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f8: ZBTB20 regulates Prl expression in a cell-autonomous fashion.(a–c) ZBTB20 overexpression in GH3 cells increases PRL expression and secretion without significant effect on GH expression. GH3 cells were infected with Ad-ZBTB20 or Ad-EGFP at MOI of 400. Three days later, GH3 cells were subjected to gene expression analyses at mRNA levels by RT–PCR (a) and at protein levels by western blotting (b), with the expression levels presented as fold change relative to Ad-EGFP control, while PRL secretion was measured in the culture supernatants by ELISA (c). n=4. Values represent mean±s.e.m. **P<0.01 versus Ad-EGFP (Student's t-test). (d–f) ZBTB20 knockdown decreases PRL expression and secretion in MMQ but not GH3 cells. Control or ZBTB20 siRNA was introduced into GH3 or MMQ cells by electroporation, respectively. Three days after transfection, the cells were harvested for western blotting (d) and quantitative RT–PCR analyses (e), and the culture supernatants were measured for PRL levels by ELISA (f). n=4. Values represent mean±s.e.m. *P<0.05 versus control siRNA (Student's t-test). (g) ChIP assay showed the binding of ZBTB20 to rat Prl promoter in MMQ rather than GH3 cells. n=3. Values represent mean±s.e.m. **P<0.01 versus control IgG (Student's t-test). (h) ZBTB20 knockdown decreases the transcriptional activity of Prl promoter in MMQ cells. MMQ cells were co-transfected with siRNA and expression plasmids by electroporation. Luciferase activity was measured at 48-h post transfection, and normalized with internal control. n=3. Values represent mean±s.e.m. ** P<0.01 versus control siRNA (Student's t-test). (i) ZBTB20 overexpression enhances the transcriptional activity of the Prl promoters in 293T cells in the presence of Pit-1. Rat Prl promoters are 0.5, 1.7 kb, or 2.5 kb in length. n=3. Values represent mean±s.e.m. ** P<0.01 versus Pit-1 group (Student's t-test).
Mentions: To explore the mechanisms by which ZBTB20 promotes lactotrope differentiation in a cell-autonomous fashion, we took advantage of a rat somatolactotrope cell line GH3 expressing both GH and PRL, and a rat lactotrope cell line MMQ, which expresses PRL but not GH. At both mRNA and protein levels, MMQ cells expressed much higher levels of Prl and Zbtb20 compared with GH3 cells (Supplementary Fig. 12). Considering that it is necessary for lactotrope differentiation to maintain PRL expression meanwhile extinguishing GH expression, we first examined the potential effect of ZBTB20 overexpression on Prl expression and lactotropic differentiation of GH3 cells. After 72 h of infection with recombinant replication-deficient adenoviruses co-expressing ZBTB20 and EGFP (hereafter Ad-ZBTB20) at the MOI of 400, more than 85% of GH3 cells were green fluorescent protein-positive by flow cytometry assay (Supplementary Fig. 13). Compared with EGFP mock control, ZBTB20 overexpression in GH3 cells led to a marked increase in Prl expression and protein secretion into culture supernatants, but had no effect on GH expression at either mRNA or protein levels (Fig. 8a–c), suggesting that ZBTB20 upregulation alone is not sufficient to drive lactotropic differentiation from somatolactotropes. In addition, the expression of Pit1 or ERα was not affected by ZBTB20 overexpression in GH3 cells.

Bottom Line: Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes.Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ.In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.

ABSTRACT
The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20- mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.

No MeSH data available.


Related in: MedlinePlus