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Multiple actions of lysophosphatidic acid on fibroblasts revealed by transcriptional profiling.

Stortelers C, Kerkhoven R, Moolenaar WH - BMC Genomics (2008)

Bottom Line: The subset of LPA-specific genes was enriched for those associated with cytoskeletal remodeling, in keeping with LPA's ability to regulate cell shape and motility.This study highlights the importance of LPA in programming fibroblasts not only to proliferate and migrate but also to produce many paracrine mediators of tissue remodeling, angiogenesis, inflammation and tumor progression.Furthermore, our results show that G protein-coupled receptors and receptor tyrosine kinases can signal independently to regulate broadly overlapping sets of genes in the same cell type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cellular Biochemistry and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. c.stortelers@gmail.com

ABSTRACT

Background: Lysophosphatidic acid (LPA) is a lipid mediator that acts through specific G protein-coupled receptors to stimulate the proliferation, migration and survival of many cell types. LPA signaling has been implicated in development, wound healing and cancer. While LPA signaling pathways have been studied extensively, it remains unknown how LPA affects global gene expression in its target cells.

Results: We have examined the temporal program of global gene expression in quiescent mouse embryonic fibroblasts stimulated with LPA using 32 k oligonucleotide microarrays. In addition to genes involved in growth stimulation and cytoskeletal reorganization, LPA induced many genes that encode secreted factors, including chemokines, growth factors, cytokines, pro-angiogenic and pro-fibrotic factors, components of the plasminogen activator system and metalloproteases. Strikingly, epidermal growth factor induced a broadly overlapping expression pattern, but some 7% of the genes (105 out of 1508 transcripts) showed differential regulation by LPA. The subset of LPA-specific genes was enriched for those associated with cytoskeletal remodeling, in keeping with LPA's ability to regulate cell shape and motility.

Conclusion: This study highlights the importance of LPA in programming fibroblasts not only to proliferate and migrate but also to produce many paracrine mediators of tissue remodeling, angiogenesis, inflammation and tumor progression. Furthermore, our results show that G protein-coupled receptors and receptor tyrosine kinases can signal independently to regulate broadly overlapping sets of genes in the same cell type.

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Correlation plot of the effect of EGF receptor inhibition. MEFs were treated with a mixture of two EGF receptor inhibitors (AG1478 and PD168393, 250 nM each) or DMSO (control) prior to stimulation with LPA (5 μM) for 4 hrs. Microarray hybridization was performed using the corresponding time-zero control with the same pretreatment. The 2log expression level of 528 LPA-regulated genes after 4 hrs is shown as a dot plot to correlate the effect of drug treatment on the expression level of individual genes. The expression level of 528 LPA-regulated genes in control cells was set at 100%. The overall reduction of LPA-induced expression by the inhibitors was approx. 35%, as inferred from the correlation coefficient.
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Figure 10: Correlation plot of the effect of EGF receptor inhibition. MEFs were treated with a mixture of two EGF receptor inhibitors (AG1478 and PD168393, 250 nM each) or DMSO (control) prior to stimulation with LPA (5 μM) for 4 hrs. Microarray hybridization was performed using the corresponding time-zero control with the same pretreatment. The 2log expression level of 528 LPA-regulated genes after 4 hrs is shown as a dot plot to correlate the effect of drug treatment on the expression level of individual genes. The expression level of 528 LPA-regulated genes in control cells was set at 100%. The overall reduction of LPA-induced expression by the inhibitors was approx. 35%, as inferred from the correlation coefficient.

Mentions: It has long been proposed that GPCR ligands such as LPA signal through 'transactivation' of the EGF receptor [33,34]. According to this model, GPCR agonists rapidly activate the EGF receptor to exploit the tyrosine-posphorylated receptor as a signaling intermediate. However, blocking EGF receptor activity by the selective EGF receptor kinase inhibitor AG1478 (250 nM) had no effect on LPA-induced MAP kinase activation, Ccl2 expression and DNA synthesis, while the responses to EGF were fully inhibited (additional file 10 and results not shown). This is in agreement with a previous study showing that LPA mitogenic signaling in MEFs does not require EGF receptor tyrosine phosphorylation [35]. Figure 10 illustrates that the transcriptional response to LPA was only little affected by EGF receptor inhibitor treatment (expression of 528 genes, reproducibly regulated by LPA at three different concentrations at T = 4 hr). About 15% of the LPA-induced genes (81 out of 528 transcripts) was >70% inhibited after drug treatment. Otherwise, EGF receptor inhibition did not affect the induction of key immediate-early and early genes by LPA, such as transcription factors and paracrine mediators. While it remains formally possible that basal EGF receptor activity has a permissive effect on some LPA-induced signaling events, we conclude that LPA and EGF signal independently to regulate broadly overlapping sets of genes in MEFs. It thus appears that the transcriptional program induced by either LPA-GPCR or EGF-RTK stimulation in fibroblasts is more strongly conserved than previously appreciated.


Multiple actions of lysophosphatidic acid on fibroblasts revealed by transcriptional profiling.

Stortelers C, Kerkhoven R, Moolenaar WH - BMC Genomics (2008)

Correlation plot of the effect of EGF receptor inhibition. MEFs were treated with a mixture of two EGF receptor inhibitors (AG1478 and PD168393, 250 nM each) or DMSO (control) prior to stimulation with LPA (5 μM) for 4 hrs. Microarray hybridization was performed using the corresponding time-zero control with the same pretreatment. The 2log expression level of 528 LPA-regulated genes after 4 hrs is shown as a dot plot to correlate the effect of drug treatment on the expression level of individual genes. The expression level of 528 LPA-regulated genes in control cells was set at 100%. The overall reduction of LPA-induced expression by the inhibitors was approx. 35%, as inferred from the correlation coefficient.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2536681&req=5

Figure 10: Correlation plot of the effect of EGF receptor inhibition. MEFs were treated with a mixture of two EGF receptor inhibitors (AG1478 and PD168393, 250 nM each) or DMSO (control) prior to stimulation with LPA (5 μM) for 4 hrs. Microarray hybridization was performed using the corresponding time-zero control with the same pretreatment. The 2log expression level of 528 LPA-regulated genes after 4 hrs is shown as a dot plot to correlate the effect of drug treatment on the expression level of individual genes. The expression level of 528 LPA-regulated genes in control cells was set at 100%. The overall reduction of LPA-induced expression by the inhibitors was approx. 35%, as inferred from the correlation coefficient.
Mentions: It has long been proposed that GPCR ligands such as LPA signal through 'transactivation' of the EGF receptor [33,34]. According to this model, GPCR agonists rapidly activate the EGF receptor to exploit the tyrosine-posphorylated receptor as a signaling intermediate. However, blocking EGF receptor activity by the selective EGF receptor kinase inhibitor AG1478 (250 nM) had no effect on LPA-induced MAP kinase activation, Ccl2 expression and DNA synthesis, while the responses to EGF were fully inhibited (additional file 10 and results not shown). This is in agreement with a previous study showing that LPA mitogenic signaling in MEFs does not require EGF receptor tyrosine phosphorylation [35]. Figure 10 illustrates that the transcriptional response to LPA was only little affected by EGF receptor inhibitor treatment (expression of 528 genes, reproducibly regulated by LPA at three different concentrations at T = 4 hr). About 15% of the LPA-induced genes (81 out of 528 transcripts) was >70% inhibited after drug treatment. Otherwise, EGF receptor inhibition did not affect the induction of key immediate-early and early genes by LPA, such as transcription factors and paracrine mediators. While it remains formally possible that basal EGF receptor activity has a permissive effect on some LPA-induced signaling events, we conclude that LPA and EGF signal independently to regulate broadly overlapping sets of genes in MEFs. It thus appears that the transcriptional program induced by either LPA-GPCR or EGF-RTK stimulation in fibroblasts is more strongly conserved than previously appreciated.

Bottom Line: The subset of LPA-specific genes was enriched for those associated with cytoskeletal remodeling, in keeping with LPA's ability to regulate cell shape and motility.This study highlights the importance of LPA in programming fibroblasts not only to proliferate and migrate but also to produce many paracrine mediators of tissue remodeling, angiogenesis, inflammation and tumor progression.Furthermore, our results show that G protein-coupled receptors and receptor tyrosine kinases can signal independently to regulate broadly overlapping sets of genes in the same cell type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cellular Biochemistry and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. c.stortelers@gmail.com

ABSTRACT

Background: Lysophosphatidic acid (LPA) is a lipid mediator that acts through specific G protein-coupled receptors to stimulate the proliferation, migration and survival of many cell types. LPA signaling has been implicated in development, wound healing and cancer. While LPA signaling pathways have been studied extensively, it remains unknown how LPA affects global gene expression in its target cells.

Results: We have examined the temporal program of global gene expression in quiescent mouse embryonic fibroblasts stimulated with LPA using 32 k oligonucleotide microarrays. In addition to genes involved in growth stimulation and cytoskeletal reorganization, LPA induced many genes that encode secreted factors, including chemokines, growth factors, cytokines, pro-angiogenic and pro-fibrotic factors, components of the plasminogen activator system and metalloproteases. Strikingly, epidermal growth factor induced a broadly overlapping expression pattern, but some 7% of the genes (105 out of 1508 transcripts) showed differential regulation by LPA. The subset of LPA-specific genes was enriched for those associated with cytoskeletal remodeling, in keeping with LPA's ability to regulate cell shape and motility.

Conclusion: This study highlights the importance of LPA in programming fibroblasts not only to proliferate and migrate but also to produce many paracrine mediators of tissue remodeling, angiogenesis, inflammation and tumor progression. Furthermore, our results show that G protein-coupled receptors and receptor tyrosine kinases can signal independently to regulate broadly overlapping sets of genes in the same cell type.

Show MeSH
Related in: MedlinePlus