Limits...
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.

Show MeSH

Related in: MedlinePlus

Validation of microarray data. Comparison of the relative expression levels of genes selected from different clusters, as determined by microarray (closed symbols) and qPCR (open symbols). qPCR data were normalized to HPRT mRNA concentration and plotted relative to the level at time zero. Data are presented as means ± SD of duplicate experiments. Note that the qPCR assays generally yielded higher mRNA values than the microarray analysis. See also the correlation plot in additional file 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2536681&req=5

Figure 3: Validation of microarray data. Comparison of the relative expression levels of genes selected from different clusters, as determined by microarray (closed symbols) and qPCR (open symbols). qPCR data were normalized to HPRT mRNA concentration and plotted relative to the level at time zero. Data are presented as means ± SD of duplicate experiments. Note that the qPCR assays generally yielded higher mRNA values than the microarray analysis. See also the correlation plot in additional file 6.

Mentions: We examined the temporal program of gene expression in serum-starved MEFs treated with LPA (5 μM, i.e. about the normal concentration in serum [13]). Total RNA was isolated at different time points after LPA stimulation (0–24 hrs). Global transcription profiles were determined using oligonucleotide microarrays containing 31,770 mouse transcripts. Amplified RNA of the treated samples was matched with the untreated control and hybridized in duplicate with reversal of the Cy3 and Cy5 dyes; the normalized Cy5/Cy3 ratios were combined and used for further analysis. We selected genes that were significantly regulated (p < 0.01) at two or more consecutive time points or in replicate measurements, which yielded 1508 LPA-regulated genes (see additional file 1: complete dataset). The entire dataset has been deposited in the EBI/ArrayExpress database (see Methods). We restricted our data set to genes that were induced by >1.7-fold at two or more time points and grouped them according to the temporal profile of gene induction using K-means clustering. This resulted in ten clusters, each containing genes that show similarly shaped waves of transcription (Figure 2). Seven clusters contained genes that were upregulated by LPA (424 transcripts), whereas three other clusters mainly comprised the down-regulated genes (209 transcripts) (Figure 2; for details see additional files 2 and 3). The genes that were most strongly regulated at different time points are listed in additional file 4 (Table 1: upregulated genes; Table 2: downregulated genes). The microarray results were validated by examining the expression of 22 representative genes from different clusters using real-time PCR (Figure 3). The -fold inductions of expression generally were higher in the qPCR assays than in the corresponding microarray experiments, reflecting the different sensitivities of both methods (see additional file 5: correlation plot of all data points). Gene ontology analysis revealed that LPA regulated the expression of genes in multiple functional categories that mostly corresponded to the different gene clusters (see additional file 6).


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

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

Validation of microarray data. Comparison of the relative expression levels of genes selected from different clusters, as determined by microarray (closed symbols) and qPCR (open symbols). qPCR data were normalized to HPRT mRNA concentration and plotted relative to the level at time zero. Data are presented as means ± SD of duplicate experiments. Note that the qPCR assays generally yielded higher mRNA values than the microarray analysis. See also the correlation plot in additional file 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Validation of microarray data. Comparison of the relative expression levels of genes selected from different clusters, as determined by microarray (closed symbols) and qPCR (open symbols). qPCR data were normalized to HPRT mRNA concentration and plotted relative to the level at time zero. Data are presented as means ± SD of duplicate experiments. Note that the qPCR assays generally yielded higher mRNA values than the microarray analysis. See also the correlation plot in additional file 6.
Mentions: We examined the temporal program of gene expression in serum-starved MEFs treated with LPA (5 μM, i.e. about the normal concentration in serum [13]). Total RNA was isolated at different time points after LPA stimulation (0–24 hrs). Global transcription profiles were determined using oligonucleotide microarrays containing 31,770 mouse transcripts. Amplified RNA of the treated samples was matched with the untreated control and hybridized in duplicate with reversal of the Cy3 and Cy5 dyes; the normalized Cy5/Cy3 ratios were combined and used for further analysis. We selected genes that were significantly regulated (p < 0.01) at two or more consecutive time points or in replicate measurements, which yielded 1508 LPA-regulated genes (see additional file 1: complete dataset). The entire dataset has been deposited in the EBI/ArrayExpress database (see Methods). We restricted our data set to genes that were induced by >1.7-fold at two or more time points and grouped them according to the temporal profile of gene induction using K-means clustering. This resulted in ten clusters, each containing genes that show similarly shaped waves of transcription (Figure 2). Seven clusters contained genes that were upregulated by LPA (424 transcripts), whereas three other clusters mainly comprised the down-regulated genes (209 transcripts) (Figure 2; for details see additional files 2 and 3). The genes that were most strongly regulated at different time points are listed in additional file 4 (Table 1: upregulated genes; Table 2: downregulated genes). The microarray results were validated by examining the expression of 22 representative genes from different clusters using real-time PCR (Figure 3). The -fold inductions of expression generally were higher in the qPCR assays than in the corresponding microarray experiments, reflecting the different sensitivities of both methods (see additional file 5: correlation plot of all data points). Gene ontology analysis revealed that LPA regulated the expression of genes in multiple functional categories that mostly corresponded to the different gene clusters (see additional file 6).

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