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Krüppel-like factor 6 expression changes during trophoblast syncytialization and transactivates ßhCG and PSG placental genes.

Racca AC, Camolotto SA, Ridano ME, Bocco JL, Genti-Raimondi S, Panzetta-Dutari GM - PLoS ONE (2011)

Bottom Line: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance.Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters.Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT

Background: Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown.

Methodology/principal findings: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human β-chorionic gonadotropin (βhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter.

Conclusions/significance: Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization.

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KLF6 protein expression throughout trophoblast cell differentiation.A- Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). B- Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. C- Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. D- Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.
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pone-0022438-g001: KLF6 protein expression throughout trophoblast cell differentiation.A- Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). B- Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. C- Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. D- Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.

Mentions: KLF6 has been detected in villous trophoblast cells with a nuclear and cytoplasmic signal, [21], [23] however, it is not known whether KLF6 is expressed through the whole syncytialization process and if it maintains the same nucleo-cytoplasmic pattern. Therefore, CTB cells were isolated from human normal-term placenta and cultured, as previously described [7]. KLF6 protein was detected during the entire differentiation process by immunofluorescence assays, as visualized by conventional epifluorescence microscopy (Fig 1A). Confocal immunofluorescence microscopy revealed a cytoplasmic as well as nuclear staining in CTB cells at several time points of the differentiation process (Fig 1B). The presence of KLF6 in the nucleus was clearly observed performing fluorescence intensity profiles where the green peak (KLF6 signal) matches with the red peak (nuclear signal) (Fig 1C). Morphological and biochemical differentiation of CTBs into the STB pathway was corroborated by fluorescence staining for nuclei, desmosomes and PSG proteins (Fig 1D), as well as by the increase in PSG and βhCG mRNA levels (data not shown). In sum, these results established that KLF6 is expressed throughout the differentiation of CTB cells into the syncytial pathway and that it is localized to both the nucleus and the cytoplasm during this process.


Krüppel-like factor 6 expression changes during trophoblast syncytialization and transactivates ßhCG and PSG placental genes.

Racca AC, Camolotto SA, Ridano ME, Bocco JL, Genti-Raimondi S, Panzetta-Dutari GM - PLoS ONE (2011)

KLF6 protein expression throughout trophoblast cell differentiation.A- Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). B- Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. C- Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. D- Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3142166&req=5

pone-0022438-g001: KLF6 protein expression throughout trophoblast cell differentiation.A- Isolated mononuclear villous CTB cells cultured during the indicated hours and stained for KLF6 immunofluorescence detection (middle panels) with the polyclonal R-173 (green) anti-KLF6 antibody. Nuclei were counterstained with Hoechst 33342 dye (blue) and the overlay is shown (right panels). B- Confocal microscopy imaging of KLF6 at the indicated time points of the differentiation process. KLF6 was labelled with the polyclonal R-173 antibody (left panels) and DNA was stained with propidium iodide (IP) (middle panel). Overlay is shown in the right panels. C- Fluorescence intensity profile of KLF6 (green) and IP (red) along the yellow line shown in the confocal microscopy images. D- Morphological and biochemical differentiation of isolated mononuclear CTB cells were confirmed by the disappearance of desmoplakin intercellular staining (red), the appearance of multinucleated structures and the expression of PSG proteins (green). Original magnification, x1000. Scale bar, 10 µm. Immunofluorescence assays were performed with at least three different CTB purifications and representative figures are shown.
Mentions: KLF6 has been detected in villous trophoblast cells with a nuclear and cytoplasmic signal, [21], [23] however, it is not known whether KLF6 is expressed through the whole syncytialization process and if it maintains the same nucleo-cytoplasmic pattern. Therefore, CTB cells were isolated from human normal-term placenta and cultured, as previously described [7]. KLF6 protein was detected during the entire differentiation process by immunofluorescence assays, as visualized by conventional epifluorescence microscopy (Fig 1A). Confocal immunofluorescence microscopy revealed a cytoplasmic as well as nuclear staining in CTB cells at several time points of the differentiation process (Fig 1B). The presence of KLF6 in the nucleus was clearly observed performing fluorescence intensity profiles where the green peak (KLF6 signal) matches with the red peak (nuclear signal) (Fig 1C). Morphological and biochemical differentiation of CTBs into the STB pathway was corroborated by fluorescence staining for nuclei, desmosomes and PSG proteins (Fig 1D), as well as by the increase in PSG and βhCG mRNA levels (data not shown). In sum, these results established that KLF6 is expressed throughout the differentiation of CTB cells into the syncytial pathway and that it is localized to both the nucleus and the cytoplasm during this process.

Bottom Line: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance.Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters.Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT

Background: Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown.

Methodology/principal findings: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human β-chorionic gonadotropin (βhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated βhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter.

Conclusions/significance: Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization.

Show MeSH
Related in: MedlinePlus