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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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Non-transcriptional Hh-responsiveness of the generated iMEFs. A. Example of a migration assay using wild type (Gli3+/+) iMEFs in a Transwell system with varying concentrations of Shh as chemoattractant. Fluorescence was read every two minutes and expressed as relative fluorescence unit (RFU). B. Example of a migration assay using wild type (Gli3+/+), Gli1-/-2-/- and Gli2-/-3-/- iMEFs with 2 μg/ml Shh as chemoattractant. No-attractant condition was subtracted and migration starting points were set to t = 0. Robust migration was observed for each cell line. C. Total migration data from several experiments as performed for B, pooled and expressed as fraction of wild type iMEF migration (Gli3+/+, set to 1, n = 5). To determine whether the migration response was significantly different relative to wild type, a 95% confidence interval was calculated based on the mean and standard deviation of the observations. Reported significant differences thus have a P value of < 0.05.
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Figure 3: Non-transcriptional Hh-responsiveness of the generated iMEFs. A. Example of a migration assay using wild type (Gli3+/+) iMEFs in a Transwell system with varying concentrations of Shh as chemoattractant. Fluorescence was read every two minutes and expressed as relative fluorescence unit (RFU). B. Example of a migration assay using wild type (Gli3+/+), Gli1-/-2-/- and Gli2-/-3-/- iMEFs with 2 μg/ml Shh as chemoattractant. No-attractant condition was subtracted and migration starting points were set to t = 0. Robust migration was observed for each cell line. C. Total migration data from several experiments as performed for B, pooled and expressed as fraction of wild type iMEF migration (Gli3+/+, set to 1, n = 5). To determine whether the migration response was significantly different relative to wild type, a 95% confidence interval was calculated based on the mean and standard deviation of the observations. Reported significant differences thus have a P value of < 0.05.

Mentions: While Hh signaling effects are thought to be exerted primarily through transcriptional regulation, a novel pathway was recently identified which is Smo-dependent but does not require transcription [2,3]. This alternative pathway triggers cytoskeletal rearrangement, driving a cellular migratory response toward Hh ligand. When activation of this pathway was investigated in wild type iMEF cells, a dose-dependent migratory response to recombinant Shh was observed (Figure 3A). In the absence of Shh ligand (no-attractant control), a low level of baseline migration was observed and subsequently subtracted from the migratory responses in all other experiments.


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)

Non-transcriptional Hh-responsiveness of the generated iMEFs. A. Example of a migration assay using wild type (Gli3+/+) iMEFs in a Transwell system with varying concentrations of Shh as chemoattractant. Fluorescence was read every two minutes and expressed as relative fluorescence unit (RFU). B. Example of a migration assay using wild type (Gli3+/+), Gli1-/-2-/- and Gli2-/-3-/- iMEFs with 2 μg/ml Shh as chemoattractant. No-attractant condition was subtracted and migration starting points were set to t = 0. Robust migration was observed for each cell line. C. Total migration data from several experiments as performed for B, pooled and expressed as fraction of wild type iMEF migration (Gli3+/+, set to 1, n = 5). To determine whether the migration response was significantly different relative to wild type, a 95% confidence interval was calculated based on the mean and standard deviation of the observations. Reported significant differences thus have a P value of < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Non-transcriptional Hh-responsiveness of the generated iMEFs. A. Example of a migration assay using wild type (Gli3+/+) iMEFs in a Transwell system with varying concentrations of Shh as chemoattractant. Fluorescence was read every two minutes and expressed as relative fluorescence unit (RFU). B. Example of a migration assay using wild type (Gli3+/+), Gli1-/-2-/- and Gli2-/-3-/- iMEFs with 2 μg/ml Shh as chemoattractant. No-attractant condition was subtracted and migration starting points were set to t = 0. Robust migration was observed for each cell line. C. Total migration data from several experiments as performed for B, pooled and expressed as fraction of wild type iMEF migration (Gli3+/+, set to 1, n = 5). To determine whether the migration response was significantly different relative to wild type, a 95% confidence interval was calculated based on the mean and standard deviation of the observations. Reported significant differences thus have a P value of < 0.05.
Mentions: While Hh signaling effects are thought to be exerted primarily through transcriptional regulation, a novel pathway was recently identified which is Smo-dependent but does not require transcription [2,3]. This alternative pathway triggers cytoskeletal rearrangement, driving a cellular migratory response toward Hh ligand. When activation of this pathway was investigated in wild type iMEF cells, a dose-dependent migratory response to recombinant Shh was observed (Figure 3A). In the absence of Shh ligand (no-attractant control), a low level of baseline migration was observed and subsequently subtracted from the migratory responses in all other experiments.

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