Search

The ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) interacts with and dephosphorylates the activated insulin receptor, thus affecting insulin signaling. ENPP1 overexpression is as an early, intrinsic defect of human insulin resistance. Previous data point to the 3'UTR as a modulator of ENPP1 expression through the regulation of mRNA stability. Aim of the study was to investigate whether potential trans-acting cytosolic factors bind to the ENPP1 3'UTR and eventually modulate its expression. The most conserved region of ENPP1 3'UTR (nt 2750 to nt 3176), identified by in silico analysis, was used as DNA template for transcribing RNA probe. Cytoplasmic extract of HEK293 cells were incubated with 32P RNA, subjected to a 5% SDS-PAGE and visualized by autoradiography. REMSA analysis demonstrated the formation of a high molecular mass RNA-protein complex, suggesting the presence of mRNA-binding protein/s in HEK293 cells. High molecular weight complexes were excised, eluted and separated on 10% SDS-PAGE. The identified band was eluted and analyzed with tandem mass spectrometry which identified the heat shock protein 70 (Hsp70), as ENPP1 3'UTR binding protein. This result was confirmed by supershift analysis with two different HSP70 antibodies. ENPP1 mRNA stability was evaluated in both siRNA-based Hsp70 suppressed and control HEK293 cells. After transcription inhibition (by Actinomycin-D treatment) a progressive reduction of ENPP1 mRNA levels was observed in the following 24 hours. This reduction was significantly greater in HSP70 down-regulated cells as compared to control cells (P<0.01), thus indicating a marked reduction of half life ENPP1 mRNA in HSP70 downregulated cells. After ENPP1 cDNA transfection, both ENPP1 mRNA levels and protein content were significantly reduced (90% and 50% respectively p<0.01) in HSP70 downregulated ceils as compared to control cells. This was not the case, in cells transfected with ENPP1 cDNA lacking 3'UTR. Taken all together, our data dearly indicate that, by binding the 3'UTR, HSP70 stabilizes ENPP1 mRNA, and eventually increases ENPP1 transcript and protein levels. Our findings suggest a pathogenic role of Hsp70 in insulin resistance, through the modulation of ENPP1 expression. [ABSTRACT FROM AUTHOR]