Plasminogen activator inhibitor 1: mechanisms of its synergistic regulation by growth factors

Song, Xiaoling
Major Professor
Marit Nilsen-hamilton
Committee Member
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Biochemistry, Biophysics and Molecular Biology

As the major physiological inhibitor of plasminogen activator (PA), the type I plasminogen activator inhibitor (PAI-1) is important for controlling blood clotting and tissue remodeling events such as those that involve cell migration. The changing needs for protease activity under various physiological conditions necessitates a tight cellular control of gene expression to enable rapid changes in the levels of PAI-1 and PA activity. Cooperation between epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) dramatically increase PAI-1 protein level. In this study, we found activators of tyrosine kinase (EGF, FGF, IGF-I and TNF α) or an activator of PKC (PMA) synergize with TGF- β in stimulation of PAI-1. The mechanism by which EGF and TGFyβ synergistically increase PAI-1 expression was explored. TGF-β increases the sensitivity of the cells to EGF, thereby recruiting EGF at suboptimal concentrations to contribute to the synergistic activation of PAI-1. The contribution of EGF to the regulation of PAI involves the MAPK pathway and the synergistic interface with the TGF-β pathway is downstream of MEK and neither involves phosphorylation of Erk1/2 nor Smad2/3. EGF and TGF-β synergistically increase PAI-1 transcription that involves cooperation between transcription factors Smad and AP-1. This increase of PAI-1 mRNA is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. This work demonstrates the existence of a multidimensional cellular mechan€ism by which EGF and TGF-β are able to promote large and rapid changes in PAI-1 expression.

Identification of regions on mRNAs that are accessible for hybridization in the living cell is required for antisense and RNAi technologies and for accurate prediction of authentic targets of microRNAs. Although the sequence of an mRNA holds some useful information, the secondary and tertiary RNA structure determines access for hybridization. Because in vivo assays are very time consuming and expensive, computational and cell culture analysis have been utilized with varying success for identifying effective oligonucleotide inhibitors. With the advent of microarray spotters and readers as standard equipment in many research facilities, this approach to analyze RNA for accessible regions is worthy of investigation for its application to identify antisense and RNAi targets. We have utilized a tiled microarray format to investigate the accessible regions in two mRNAs (Bcl2 and Lcn2) and compared this with cell culture and computational approaches for predicting accessible targets for antisense oligodeoxynucleotides (ODNs) and siRNAs. Our results suggest that a tiled microarray is an effective means of identifying the mRNA regions that are accessible to antisense ODNs and RNAi.