Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

Abstract

The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3 untranslated regions. The cellular proteins binding to these RNA sequences (ARE uPA/uPAR ) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an ARE uPA -containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a ␤-globin reporter mRNA containing the ARE uPA . RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steadystate levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the ARE uPA , which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.

Distinct Domains of AU-Rich Elements Exert Different Functions in mRNA Destabilization and Stabilization by p38 Mitogen-Activated Protein Kinase or HuR

“…Taken together, these results suggest that HuR and p38/MK2 activation can induce mRNA stabilization by two independent mechanisms. This does not exclude the possibility that-as an additional mode of stabilization-p38/ MK2 activation might also stabilize indirectly through HuR by inducing its cytoplasmic translocation (41).…”

“…Furthermore, suppression of HuR amounts by antisense RNA or small interfering RNA approaches inhibited stabilization induced by hypoxia (23), cytokines (35), or UV light (43) or during cell cycle progression (42). Most recently, constitutively active MK2 (41), as well as stimuli known to activate the p38/MK2 pathway (H 2 O 2 [41] and TNF-␣ [1]), was found to induce cytoplasmic accumulation of HuR. Thus, HuR could play a role in p38/MK2-induced stabilization.…”

Cytoplasmic Expression of HuR is Related to Cyclooxygenase-2 Expression in Colon Cancer

“…There are some other mRNAs, such as the mRNAs for tumor necrosis factor , cyclin A, cyclin B1, MARCKS, uPA and uPA receptor that are stabilized by HuR (23)(24)(25). Based on the known functions of HuR, we believe that HuR might play an important role in cell cycle regulation, apoptosis, angiogenesis, inflammation, and tumor growth.…”

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

“…NMR spectra were recorded at 298 K on a Bruker Avance III 800 MHz spectrometer with a [ 1 H, 13 C, 15 deposited in BioMagResBank with entry IDs 19494, 19499 and 19500 for the WT, W261E and S318D variants, respectively. Structural models were built based on the measured chemical shifts using the CS23D server.…”