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Establishment and characterization of immortalized Gli- mouse embryonic fibroblast cell lines.

Lipinski RJ, Bijlsma MF, Gipp JJ, Podhaizer DJ, Bushman W - BMC Cell Biol. (2008)

Bottom Line: While loss of Gli1 had no effect on target gene induction, Gli2 cells demonstrated reduced target gene induction while Gli3 cells exhibited elevated basal and ligand-induced expression.Target gene response in Gli1-/-2-/-iMEFs was severely reduced while Gli2-/-3-/-iMEFs were incapable of ligand-induced transcriptional response.However, we found that both Gli1-/-2-/- and Gli2-/-3-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA. lipinski@surgery.wisc.edu

ABSTRACT

Background: Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli mice.

Results: Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 cells demonstrated reduced target gene induction while Gli3 cells exhibited elevated basal and ligand-induced expression. Target gene response in Gli1-/-2-/-iMEFs was severely reduced while Gli2-/-3-/-iMEFs were incapable of ligand-induced transcriptional response. However, we found that both Gli1-/-2-/- and Gli2-/-3-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.

Conclusion: This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli- iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.

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Transcriptional Hh-responsiveness of generated iMEFs. Indicated iMEFs were plated at confluence and treated ± Shh ligand. After 24 hrs, expression of Ptc1 was determined by Real-Time RT-PCR. A. Basal and Shh-induced expression of Ptc1. Values represent the mean ± SEM of 3–5 replicate experiments, * indicates P ≤ 0.05 (paired t-test). B. Ptc1 expression plotted as fold induction (Shh/Veh). Values represent the mean ± SEM of three replicate experiments. The letters above the bars denote the groups produced by the ANOVA pair-wise differences. Genotypes sharing a letter are not statistically significant at p ≤ 0.05 (Fisher's LSD).
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Figure 2: Transcriptional Hh-responsiveness of generated iMEFs. Indicated iMEFs were plated at confluence and treated ± Shh ligand. After 24 hrs, expression of Ptc1 was determined by Real-Time RT-PCR. A. Basal and Shh-induced expression of Ptc1. Values represent the mean ± SEM of 3–5 replicate experiments, * indicates P ≤ 0.05 (paired t-test). B. Ptc1 expression plotted as fold induction (Shh/Veh). Values represent the mean ± SEM of three replicate experiments. The letters above the bars denote the groups produced by the ANOVA pair-wise differences. Genotypes sharing a letter are not statistically significant at p ≤ 0.05 (Fisher's LSD).

Mentions: iMEFs were treated ± Shh ligand, and Hh target gene expression was determined by real time RT-PCR. Figure 2A shows the expression of reliable Hh target gene Ptc1 following stimulation with Shh or vehicle and Figure 2B shows the fold induction (Shh/Veh) of Ptc1 expression. Gli3-/- iMEFs demonstrated elevated basal and Shh-induced expression of Ptc1 (p = 0.03 and p = 0.02 respectively) relative to WT cells. Shh ligand stimulation induced Ptc1 expression in each iMEF line except that lacking expression of both Gli2 and Gli3, which are essential for a transcriptional Hh response. While loss of Gli1 alone had no effect on target gene expression, Ptc1 induction was reduced in both Gli2-/- and Gli1-/-2-/- iMEFs relative to WT.


Establishment and characterization of immortalized Gli- mouse embryonic fibroblast cell lines.

Lipinski RJ, Bijlsma MF, Gipp JJ, Podhaizer DJ, Bushman W - BMC Cell Biol. (2008)

Transcriptional Hh-responsiveness of generated iMEFs. Indicated iMEFs were plated at confluence and treated ± Shh ligand. After 24 hrs, expression of Ptc1 was determined by Real-Time RT-PCR. A. Basal and Shh-induced expression of Ptc1. Values represent the mean ± SEM of 3–5 replicate experiments, * indicates P ≤ 0.05 (paired t-test). B. Ptc1 expression plotted as fold induction (Shh/Veh). Values represent the mean ± SEM of three replicate experiments. The letters above the bars denote the groups produced by the ANOVA pair-wise differences. Genotypes sharing a letter are not statistically significant at p ≤ 0.05 (Fisher's LSD).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2542994&req=5

Figure 2: Transcriptional Hh-responsiveness of generated iMEFs. Indicated iMEFs were plated at confluence and treated ± Shh ligand. After 24 hrs, expression of Ptc1 was determined by Real-Time RT-PCR. A. Basal and Shh-induced expression of Ptc1. Values represent the mean ± SEM of 3–5 replicate experiments, * indicates P ≤ 0.05 (paired t-test). B. Ptc1 expression plotted as fold induction (Shh/Veh). Values represent the mean ± SEM of three replicate experiments. The letters above the bars denote the groups produced by the ANOVA pair-wise differences. Genotypes sharing a letter are not statistically significant at p ≤ 0.05 (Fisher's LSD).
Mentions: iMEFs were treated ± Shh ligand, and Hh target gene expression was determined by real time RT-PCR. Figure 2A shows the expression of reliable Hh target gene Ptc1 following stimulation with Shh or vehicle and Figure 2B shows the fold induction (Shh/Veh) of Ptc1 expression. Gli3-/- iMEFs demonstrated elevated basal and Shh-induced expression of Ptc1 (p = 0.03 and p = 0.02 respectively) relative to WT cells. Shh ligand stimulation induced Ptc1 expression in each iMEF line except that lacking expression of both Gli2 and Gli3, which are essential for a transcriptional Hh response. While loss of Gli1 alone had no effect on target gene expression, Ptc1 induction was reduced in both Gli2-/- and Gli1-/-2-/- iMEFs relative to WT.

Bottom Line: While loss of Gli1 had no effect on target gene induction, Gli2 cells demonstrated reduced target gene induction while Gli3 cells exhibited elevated basal and ligand-induced expression.Target gene response in Gli1-/-2-/-iMEFs was severely reduced while Gli2-/-3-/-iMEFs were incapable of ligand-induced transcriptional response.However, we found that both Gli1-/-2-/- and Gli2-/-3-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA. lipinski@surgery.wisc.edu

ABSTRACT

Background: Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli mice.

Results: Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 cells demonstrated reduced target gene induction while Gli3 cells exhibited elevated basal and ligand-induced expression. Target gene response in Gli1-/-2-/-iMEFs was severely reduced while Gli2-/-3-/-iMEFs were incapable of ligand-induced transcriptional response. However, we found that both Gli1-/-2-/- and Gli2-/-3-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.

Conclusion: This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli- iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.

Show MeSH
Related in: MedlinePlus