Proinflammatory cytokines such as TNF-α induce uPA expression in multiple cell types, including lung epithelial cells. uPA likewise induces its own expression in lung epithelial cells (2
), endothelial and myeloid cells. (13
) LPS induces both TNF-α and uPA expression in multiple cell types including lung epithelial cells.(15
) Stabilization of uPA mRNA at the posttranscriptional level, at least in part in the case of TNF-α (15
) and exclusively in the case of uPA (2
), contributes to increased uPA mRNA and protein expression. Posttranscriptional regulation of uPA involves the interaction of a ~40 kDa cytoplasmic-nuclear shuttling protein with a 66 nt destabilization determinant present in the uPA mRNA 3′UTR. (15
) Here, we extended our earlier study, purified the uPA mRNABp from the lysates of lung epithelial cells, identified it as RRM2 and characterized the role of the RRM2-uPA mRNA 3′UTR in the regulation of uPA mRNA stability and uPA expression by lung epithelial cells.
Expression of RRM2 inhibited uPA protein expression by lung epithelial cells and this response involves the suppression of uPA mRNA expression. LPS and uPA inhibited RRM2 binding to uPA mRNA 3′UTR, suggesting that RRM2 attenuated uPA expression. This is consistent with our earlier observation that TNF-α-mediated induction of uPA in lung epithelial cells is associated with parallel inhibition of the cytoplasmic uPA mRNABp interaction with the 66 nt uPA mRNA 3′UTR sequence due to translocation of uPA mRNABp to the nucleus.(15
) The uPA mRNA destabilizing effect of the RRM2-uPA mRNA interaction was confirmed by the time dependent translocation of uPA mRNA binding activity from cytoplasm to the nucleus with accumulation of RRM2 proteins in the latter compartment after LPS treatment. This is further supported by the destabilization of uPA mRNA in resting as well as LPS-treated lung epithelial cells that overexpressed RRM2.
Ribonucleotide reductase is an important enzyme involved in the synthesis of DNA and responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides. Three subunits of ribonucleotide reductase, RRM1, RRM2 and p53R2, provide a balanced supply of nucleotide precursors for DNA synthesis. RRM2 interacts with RRM1 to form a heterotertramer complex which is catalytically active. We recently reported that tumor suppressor protein, p53 inhibits uPA expression through destabilization of uPA mRNA.(26
) The process involves sequence specific interaction of p53 with a 35 nucleotide destabilization determinant present in the uPA mRNA 3′UTR. Previous studies (27
) suggested that p53 and RRM2 directly interact to control ribonucleotide reductase activity during DNA damage. Interestingly, the RRM2 binding sequence on uPA mRNA 3′UTR resides adjacent but upstream to the p53 binding sequence, supporting their potential for coordinate interaction. Increased translocation of RRM2 from the cytoplasm to the nucleus after UV irradiation (29
) is consistent with our earlier report that uPA mRNABp shuttles to the nucleus following stimulation of lung epithelial cells with TNF-α, leading to stabilization of uPA mRNA and induction of uPA expression. RRM2 and its mode of regulation of uPA expression through cytoplasmic-nuclear shuttling are illustrated in a schematic diagram (Fig 7).
We reported earlier that uPA induces its own expression as well as that of uPAR through obliteration of p53. We therefore inferred that p53 must have contributed to RRM2-mediated inhibition of uPA expression by lung epithelial cells. Cells lacking p53 express a significant amount of uPA and reintroduction of p53 inhibits uPA expression through destabilization of uPA mRNA.(26
) p53 and RRM2 independently interact with unique 3′UTR sequences that contain information for mRNA degradation (12
). p53 interacts with RRM2 without significant changes in RRM2 expression. However, treatment of the cells with LPS inhibits cytosolic RRM2 and p53 interaction due to translocation of RRM2 to the nucleus. These changes are associated with a parallel induction of uPA expression through mRNA stabilization. Based on our present findings and prior reports (15
) it is unlikely that p53 directly interferes with RRM2 binding to uPA mRNA since they independently bind to two destabilization determinants present in the 3′UTR (15
). However, the possibility of p53 being involved in the translocation of RRM2 from the cytoplasm to the nucleus after LPS treatment is not yet ruled out and could be a subject of future study. This observation demonstrates that RRM2 maintains a complex network of cellular functions depending upon the external stimuli, metabolic state of the cell and the localization of RRM2.
This newly recognized paradigm is to our knowledge the first description of RRM2 regulating uPA expression in any cell type. If operative in vivo, this pathway could contribute to the regulation of uPA expression under normal conditions and in pathophysiologic states including various forms of ALI and its most severe form; acute respiratory distress syndrome.