Identification of cellular pathways that regulate the
expression of PAI-1 using SureFIND Transcriptome PCR Arrays
Currently, research-based identification of upstream signaling events that
regulate the expression of the target gene mainly involves extensive
literature searches of known signal transduction networks or relies on
bioinformatics predictive algorithms to identify probable regulators.
Chemical Inhibitors Transcriptome PCR Arrays provide a novel experimental
approach for identifying signal transduction networks that potentially
regulate the endogenous level of the target gene.
Plasminogen activator inhibitor 1 (PAI-1), also called
Serpin E1, is a circulating single-chain glycoprotein that belongs to the
family of serine protease inhibitors called SERPINs . Physiologically,
PAI-1 is the major inhibitor of plasminogen activation, and its activity is
tightly regulated at the transcriptional level largely by transforming
growth factor (TGF)-β . We employed Chemical Inhibitors Transcriptome
PCR Array to perform a focused and fast screening experiment to
simultaneously analyzing the contribution of multiple signaling pathways
kinases, and other regulating enzymes toward PAI-1 transcriptional
regulation in MCF-7 cells.
Materials and Methods
In this study we used Chemical Inhibitors SureFind
Transcriptome PCR Array to identify potential regulators of PAI-1
expression in MCF-7 cells. SYBR Green qPCR assay was performed to quantify
the expression of PAI-1 (gene of interest) and GAPDH (house-keeping
control). Fold change in PAI-1 gene expression as a result of each specific
chemical inhibitor treatment relative to DMSO treated control were
calculated and normalized to GAPDH. Fold changes were converted to log2 and
subjected to MAD analysis for positive hits selection.
Figure1: Cellular targets involved in regulation of PAI-1
Chemical Inhibitor-1 Transcriptome PCR Array was used to run SYBR Green-based
qPCR assays for PAI-1 and GAPDH. PAI-1 gene expression level is expressed as
Log2 fold change based on Ct calculation using GAPDH as house-keeping gene and
DMSO treated sample well (VTC) as negative control.
Our results show that TGF-β positively regulates PAI-1 gene
expression in MCF-7 cells, validating previous findings regarding the strong
connection between PAI-1 and the action of TGF-β (Fig. 1). Inhibition of TGF-β
signaling by an ALK5 inhibitor VII, which specifically targets TGF-β type I
receptor kinase, resulted in greater than 5-fold reduction in the level of PAI-1
mRNA. In addition to TGF-β, inhibition of Notch signaling by DAPT, which targets
gamma-secretase, showed the strongest effect on PAI-1 expression, resulting in
nearly 9-fold reduction in the level of PAI-1 mRNA. These findings serve as a
confirmation of a previously suspected convergence of TGF-β and Notch signaling
pathways  in a novel cellular background.
Chemical Inhibitors Transcriptome PCR Array identified several signaling
pathways that negatively regulate PAI-1 expression including TGF-β and Notch
signaling pathways, validating previous knowledge and confirming novel findings.
1. Binder BR, Christ G, Gruber F, Grubic N, Hufnagl P, Krebs M,
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pathophysiological roles. News Physiol Sci. 2002 Apr;17:56-61.
2. Blokzijl A, Dahlqvist C, Reissmann E, Falk A, Moliner A, Lendahl U, Ibáńez
CF. Cross-talk between the Notch and TGF-beta signaling pathways mediated by
interaction of the Notch intracellular domain with Smad3. J Cell Biol. 2003 Nov
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