A RTK-based functional RNAi screen reveals determinants of PTX-3 expression

Abstract

Aim: The aim of the present study was to explore the role of receptor tyrosine kinases (RTKs) in the regulation of expression of PTX-3, a protector in atherosclerosis.

Methods: Human monocytic U937 cells were infected with a shRNA lentiviral vector library targeting human RTKs upon LPS stimuli and PTX-3 expression was determined by ELISA analysis. The involvement of downstream signaling in the regulation of PTX-3 expression was analyzed by both Western blotting and ELISA assay.

Results: We found that knocking down of ERBB2/3, EPHA7, FGFR3 and RET impaired PTX-3 expression without effects on cell growth or viability. Moreover, inhibition of AKT, the downstream effector of ERBB2/3, also reduced PTX-3 expression. Furthermore, we showed that FGFR3 inhibition by anti-cancer drugs attenuated p38 activity, in turn induced a reduction of PTX-3 expression.

Conclusion: Altogether, our study demonstrates the role of RTKs in the regulation of PTX-3 expression and uncovers a potential cardiotoxicity effect of RTK inhibitor treatments in cancer patients who have symptoms of atherosclerosis or are at the risk of atherosclerosis.

Keywords: PTX-3; RNAi screening; RTKs; atherosclerosis; cardiotoxicity; target therapy.

Figure 1

Schematic of RNAi screen. A. U937 cells were treated with 100 ng·ml^-1 of LPS for 6 hrs and PTX-3 expression was determined by qPCR analysis. B. Schematic outline of high-throughput RNAi screen. The human RTKs lentiviral vector library was used to infect U937 cells for 4 days to allow efficient knocking down of RTKs and was then treated with LPS at 100 ng·ml^-1 for 6 hrs. The supernatant media and U937 cells were collected by centrifuged and analyzed by ELSIA and MTS assay, respectively. In total, all human 55 RTKs were analyzed.

Figure 2

RNAi screen for RTKs required for PTX-3 secretion induced by LPS. A. Scatter plot of PTX-3 relative secretion levels for each RTK gene in U937 cells infected by two independent shRNAs per gene. The relative secretion levels of PTX-3 were represented by ratio of the ELISA absorbance to the MTS absorbance. B. Candidate genes with an inhibition rate of >30% were listed.

Figure 3

The downstream signaling involved in RTKs regulating PTX-3 secretion induced by LPS. U937 cells were pretreated with AKT inhibitor MK2206 for 24 hrs and then added with LPS for another 6 hrs. U937 cells were centrifuged and analyzed by MTS assay. The supernatant were collected and PTX-3 secretion was determined by ELISA assay. The ELISA/MTS value was calculated as described in Materials and methods.

Figure 4

FGFR3-p38 signaling regulates PTX expression. A. p38 inhibitor LY2228820 reduced the PTX-3 secretion induced by LPS in U937 cells.( **, p<0.01; ***, p<0.001), Error bars show data ±standard error. Means were derived from three replicates. B. Western blot analysis of p38 phosphorylation in U937 cells treated by RTK inhibitor Linifanib. U937 cells were treated with FGFR3 inhibitor, Linifanib, with different doses for 24 hrs and centrifuged for western blot analysis. Whole cell lysates were loaded for the expression analysis of phosphorylation of p38 and total p38. C. Optical density analysis of western blot in B. PTX-3 secretion and U937 cell viability was detected by ELISA and MTS assay, respectively. The ELISA/MTS value was calculated as described in Materials and methods.