Western analysis of total TIP5 cell lysates. the high-throughput screening for modulators of molecular pathways entails cell-free biochemical assays, or in some cases, highly specialized cell-based phenotypic assays [1]. However, in many cases the optimal target for therapeutic intervention is not known, or the development of a suitable phenotypic read-out is not technically feasible. For example, it is becoming increasingly of interest to modulate the activity of particular transmission transduction pathways, but the components of such pathways are in many cases only partially known. It would therefore be of interest to develop a screening approach that could identify inhibitors of such pathways without first defining the biochemical target of candidate small molecules. Here we demonstrate that it is possible to use mRNA expression levels as a read-out to infer activity of a signal transduction pathway, thus establishing a general approach to screening for modulators of transmission transduction pathways. Endogenous mRNA expression has been previously successfully used as a surrogate of cellular says in high-throughput screening for compounds inducing differentiation of acute myeloid leukemia cells, and for identifying inhibitors of androgen receptor-mediated transcriptional activation in prostate malignancy [2-5]. It is not obvious, however, that gene expression signatures could be used to identify inhibitors of transmission transduction pathways that are regulated at the level of post-translational modification (phosphorylation), as opposed to transcriptional regulation. To test the feasibility of using gene expression-based high-throughput screening (GE-HTS) to identify inhibitors of a signaling pathway, we chose platelet derived growth factor receptor (PDGFR) signaling for any proof-of-concept study, with particular emphasis on downstream activation of the extracellular regulated kinase (ERK) pathway (also known as the p42/p44 mitogen activated protein (MAP) kinase pathway) as a target pathway for the screen. The ERK pathway plays a major role in the control of cell growth, cell differentiation and cell survival [6]. The protein kinase cascade Raf mitogen/extracellular signal-regulated kinase (MEK) ERK, also referred to as the MAP kinase module, is usually activated in mammalian cells through receptor tyrosine kinases, G-protein coupled receptors and integrins [6]. Activated ERKs translocate to the nucleus where they phosphorylate transcription factors. The ERK pathway is usually often upregulated in human tumors [6], and as such is an attractive target for anticancer therapy. Furthermore, because the pathway has been extensively analyzed, many experimental tools are available with which to interrogate the pathway. We demonstrate here that indeed small molecule inhibitors of the PDGFR/ERK pathway can be discovered using the GE-HTS approach. Results Identification of a signature of PDGFR/ERK activity In GE-HTS, a gene expression signature is used as a surrogate of a biological state. In the present context, we sought to define a signature of ERK activation mediated by PDGFR activation. Specifically, we treated SH-SY5Y neuroblastoma cells with the BB homodimer of PDGF (PDGF-BB), which resulted in PDGFR phosphorylation and subsequent ERK activation. We selected PDGFR over PDGFR for our studies because of previous observations that PDGFR might mediate functions of other PDGF isoforms in addition to PDGF-A [7,8]. The activation state of the users of the PDGF pathway can be traced by increase in their phosphorylation levels shortly after introduction of the growth factor [9]. In particular, ERK phosphorylation peaks at Vialinin A about 15-20 moments Vialinin A after induction, and then decreases to background levels some 20-30 moments later [10]. Accordingly, we performed gene expression profiling using Affymetrix U133A arrays 30 minutes following PDGF stimulation, thereby identifying those genes whose expression is usually correlated with PDGFR activity. In order to identify the component of the gene expression signature Vialinin A that was attributable to ERK activation by PDGFR (as opposed to other pathways downstream of PDGFR), we also pretreated the cells with the MEK inhibitor U0126 and the ERK inhibitor apigenin, and repeated the gene expression profiling studies (Physique ?(Figure1a1a). Open in a separate window Physique 1 PDGFR/ERK activation signature.Widlund and V. partially known. It would therefore be of interest to develop a screening approach that could identify inhibitors of such pathways without first defining the biochemical target of candidate small molecules. Here we demonstrate that it is possible to use mRNA expression levels as a read-out to infer activity of a signal transduction pathway, thus establishing a general approach to screening for modulators of transmission transduction pathways. Endogenous mRNA expression has been previously successfully used as a surrogate of cellular says in high-throughput screening for compounds inducing differentiation of acute myeloid leukemia cells, and for identifying inhibitors of androgen receptor-mediated transcriptional activation in prostate malignancy [2-5]. It is not obvious, however, that gene expression signatures could be used to identify inhibitors of transmission transduction pathways that are regulated at the level of post-translational modification (phosphorylation), as opposed to transcriptional regulation. To test the feasibility of using gene expression-based high-throughput screening (GE-HTS) to identify inhibitors of a signaling pathway, we chose platelet derived growth factor receptor (PDGFR) signaling for any proof-of-concept study, with particular emphasis on downstream activation of the extracellular regulated kinase (ERK) pathway (also known as the p42/p44 mitogen activated protein (MAP) kinase pathway) as a target pathway for the screen. The ERK pathway plays a major role in the control of cell growth, cell differentiation and cell survival [6]. The protein kinase cascade Raf mitogen/extracellular signal-regulated kinase (MEK) ERK, also referred to as the MAP kinase module, is usually activated in mammalian cells through receptor tyrosine kinases, G-protein coupled receptors and integrins [6]. Activated ERKs translocate to the nucleus where they phosphorylate transcription factors. The ERK pathway is usually often upregulated in human tumors [6], and as such is an attractive target for anticancer therapy. Furthermore, because the pathway has been extensively analyzed, many experimental tools are available with which to interrogate the pathway. We demonstrate here that indeed small molecule inhibitors of the PDGFR/ERK pathway can be discovered using the GE-HTS approach. Results Identification of a signature of PDGFR/ERK activity In GE-HTS, a gene expression signature is used as a surrogate of a biological state. In the present context, we sought to define a signature of ERK activation mediated by PDGFR activation. Specifically, we treated SH-SY5Y neuroblastoma cells with the BB homodimer of PDGF (PDGF-BB), which resulted in PDGFR phosphorylation and subsequent ERK activation. We selected PDGFR over PDGFR for our studies because of previous observations that PDGFR might mediate functions of other PDGF isoforms in addition to PDGF-A [7,8]. The activation state of the users of the PDGF pathway can be traced by increase in their phosphorylation levels shortly after introduction of the growth factor [9]. In particular, ERK phosphorylation peaks at about 15-20 moments after induction, and then decreases to background levels some 20-30 moments later [10]. Accordingly, we performed gene expression profiling using Affymetrix U133A arrays 30 minutes following PDGF stimulation, thereby identifying those genes whose expression is usually correlated with PDGFR activity. In order to identify the component of the gene expression signature that was attributable to ERK activation by PDGFR (as opposed to other pathways downstream of PDGFR), we also pretreated the cells with the MEK inhibitor U0126 and the ERK inhibitor apigenin, and repeated the gene expression profiling studies (Physique ?(Figure1a1a). Open in a separate window Physique 1 Rabbit Polyclonal to CELSR3 PDGFR/ERK activation signature for high-throughput.