Limits...
A novel pathway of TEF regulation mediated by microRNA-125b contributes to the control of actin distribution and cell shape in fibroblasts.

Gutierrez O, Berciano MT, Lafarga M, Fernandez-Luna JL - PLoS ONE (2011)

Bottom Line: Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity).Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53.These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Genetica Molecular, Hospital Universitario Marques de Valdecilla, Instituto de Formacion e Investigacion Marques de Valdecilla, Servicio Cantabro de Salud, Santander, Spain.

ABSTRACT

Background: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF.

Principal findings: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53.

Conclusions/significance: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts.

Show MeSH

Related in: MedlinePlus

Morphological differences between wild type and TEF (โˆ’/โˆ’) fibroblasts.(A) The morphology of wild type (WT) and TEF (โˆ’/โˆ’) fibroblasts in culture was assessed by phase contrast microscopy. Scale bar: 40 ยตm. (B) Both cell populations were labeled red with phalloidin for staining the actin filaments and visualized by confocal microscopy. Scale bars: 20 ยตm. (C) Confluent cultures were mechanically disrupted, leaving an area devoid of cells, and fibroblasts were labeled green to determine the actin distribution at the leading edge. Scale bar: 20 ยตm. (D) The mRNA expression of different Rho GTPases was analyzed by quantitative RT-PCR. The expression levels in TEF (โˆ’/โˆ’) cells were referred to those in wild type cells. Histograms represent the mean ยฑ SD of three independent experiments. (E) Distribution of cell cycle phases in wild type and TEF (โˆ’/โˆ’) fibroblasts was determined by flow cytometry after staining nuclei with propidium iodide. The percentage of cells in the different phases, G0/G1, S, and G2/M, is indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037971&req=5

pone-0017169-g005: Morphological differences between wild type and TEF (โˆ’/โˆ’) fibroblasts.(A) The morphology of wild type (WT) and TEF (โˆ’/โˆ’) fibroblasts in culture was assessed by phase contrast microscopy. Scale bar: 40 ยตm. (B) Both cell populations were labeled red with phalloidin for staining the actin filaments and visualized by confocal microscopy. Scale bars: 20 ยตm. (C) Confluent cultures were mechanically disrupted, leaving an area devoid of cells, and fibroblasts were labeled green to determine the actin distribution at the leading edge. Scale bar: 20 ยตm. (D) The mRNA expression of different Rho GTPases was analyzed by quantitative RT-PCR. The expression levels in TEF (โˆ’/โˆ’) cells were referred to those in wild type cells. Histograms represent the mean ยฑ SD of three independent experiments. (E) Distribution of cell cycle phases in wild type and TEF (โˆ’/โˆ’) fibroblasts was determined by flow cytometry after staining nuclei with propidium iodide. The percentage of cells in the different phases, G0/G1, S, and G2/M, is indicated.

Mentions: Wild type and TEF (โˆ’/โˆ’) fibroblasts exhibit very different morphologies. Wild type cells display an elongated and stellate phenotype, whereas knockout cells appear to be larger and rounded (Figure 5A). The shape changes are generally accepted to be driven by the actin cytoskeleton, which together with accessory proteins make up the cell cortex. Consistently, we found that F-actin filaments were distributed along the periphery of knockout cells forming circumferential bundles (Figure 5B). When cells are attached to a planar substratum and moving in low density fluid cultures, there is little in the way of external forces to resist cell shape changes. We analyzed F-actin distribution in confluent cultures with a mechanically denuded area and found the staining to be localized to protrusions at the leading edge in cultures of wild type fibroblasts, and evenly distributed in filaments that were oriented more parallel to the cell edge in TEF(โˆ’/โˆ’) fibroblasts (Figure 5C). The dynamic organization of the actin cytoskeleton is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42 [21]. Since TEF is a transcription factor, we studied whether the expression of these genes was modified by the presence of TEF. As shown in Figure 5D, the mRNA levels of Rac1, RhoA and Cdc42 were very similar in both wild type and TEF (โˆ’/โˆ’) fibroblasts. Actin cytoskeleton is reorganized during mitosis to form rounded cells, and re-established after cell division allowing cells to regain a more extended shape. Therefore, we analyzed the cell cycle phase distribution in wild type and TEF (โˆ’/โˆ’) fibroblasts (Figure 5E) and found a similar pattern in both cell populations, suggesting that the difference in cell shape was not due to differences in cell division rates.


A novel pathway of TEF regulation mediated by microRNA-125b contributes to the control of actin distribution and cell shape in fibroblasts.

Gutierrez O, Berciano MT, Lafarga M, Fernandez-Luna JL - PLoS ONE (2011)

Morphological differences between wild type and TEF (โˆ’/โˆ’) fibroblasts.(A) The morphology of wild type (WT) and TEF (โˆ’/โˆ’) fibroblasts in culture was assessed by phase contrast microscopy. Scale bar: 40 ยตm. (B) Both cell populations were labeled red with phalloidin for staining the actin filaments and visualized by confocal microscopy. Scale bars: 20 ยตm. (C) Confluent cultures were mechanically disrupted, leaving an area devoid of cells, and fibroblasts were labeled green to determine the actin distribution at the leading edge. Scale bar: 20 ยตm. (D) The mRNA expression of different Rho GTPases was analyzed by quantitative RT-PCR. The expression levels in TEF (โˆ’/โˆ’) cells were referred to those in wild type cells. Histograms represent the mean ยฑ SD of three independent experiments. (E) Distribution of cell cycle phases in wild type and TEF (โˆ’/โˆ’) fibroblasts was determined by flow cytometry after staining nuclei with propidium iodide. The percentage of cells in the different phases, G0/G1, S, and G2/M, is indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037971&req=5

pone-0017169-g005: Morphological differences between wild type and TEF (โˆ’/โˆ’) fibroblasts.(A) The morphology of wild type (WT) and TEF (โˆ’/โˆ’) fibroblasts in culture was assessed by phase contrast microscopy. Scale bar: 40 ยตm. (B) Both cell populations were labeled red with phalloidin for staining the actin filaments and visualized by confocal microscopy. Scale bars: 20 ยตm. (C) Confluent cultures were mechanically disrupted, leaving an area devoid of cells, and fibroblasts were labeled green to determine the actin distribution at the leading edge. Scale bar: 20 ยตm. (D) The mRNA expression of different Rho GTPases was analyzed by quantitative RT-PCR. The expression levels in TEF (โˆ’/โˆ’) cells were referred to those in wild type cells. Histograms represent the mean ยฑ SD of three independent experiments. (E) Distribution of cell cycle phases in wild type and TEF (โˆ’/โˆ’) fibroblasts was determined by flow cytometry after staining nuclei with propidium iodide. The percentage of cells in the different phases, G0/G1, S, and G2/M, is indicated.
Mentions: Wild type and TEF (โˆ’/โˆ’) fibroblasts exhibit very different morphologies. Wild type cells display an elongated and stellate phenotype, whereas knockout cells appear to be larger and rounded (Figure 5A). The shape changes are generally accepted to be driven by the actin cytoskeleton, which together with accessory proteins make up the cell cortex. Consistently, we found that F-actin filaments were distributed along the periphery of knockout cells forming circumferential bundles (Figure 5B). When cells are attached to a planar substratum and moving in low density fluid cultures, there is little in the way of external forces to resist cell shape changes. We analyzed F-actin distribution in confluent cultures with a mechanically denuded area and found the staining to be localized to protrusions at the leading edge in cultures of wild type fibroblasts, and evenly distributed in filaments that were oriented more parallel to the cell edge in TEF(โˆ’/โˆ’) fibroblasts (Figure 5C). The dynamic organization of the actin cytoskeleton is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42 [21]. Since TEF is a transcription factor, we studied whether the expression of these genes was modified by the presence of TEF. As shown in Figure 5D, the mRNA levels of Rac1, RhoA and Cdc42 were very similar in both wild type and TEF (โˆ’/โˆ’) fibroblasts. Actin cytoskeleton is reorganized during mitosis to form rounded cells, and re-established after cell division allowing cells to regain a more extended shape. Therefore, we analyzed the cell cycle phase distribution in wild type and TEF (โˆ’/โˆ’) fibroblasts (Figure 5E) and found a similar pattern in both cell populations, suggesting that the difference in cell shape was not due to differences in cell division rates.

Bottom Line: Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity).Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53.These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Genetica Molecular, Hospital Universitario Marques de Valdecilla, Instituto de Formacion e Investigacion Marques de Valdecilla, Servicio Cantabro de Salud, Santander, Spain.

ABSTRACT

Background: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF.

Principal findings: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53.

Conclusions/significance: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts.

Show MeSH
Related in: MedlinePlus