HuR regulates p21 mRNA stabilization by UV light

Expression of the cyclin-dependent kinase inhibitor p21 is highly induced by many stresses, including exposure to short-wavelength UV light (UVC), which increases p21 mRNA stability. Investigation into the mechanisms underlying this stabilization process revealed that proteins present in cytoplasmic lysates of human RKO colorectal carcinoma cells formed complexes with p21 mRNA that were inducible by treatment with UVC and other stress agents. The ubiquitous Elav-type RNA-binding protein HuR was identified within the p21 mRNA-protein complexes, as antibodies recognizing HuR supershifted these complexes and revealed HuR-immunoreactive proteins complexing with p21 mRNA on Western blots. Lowering of endogenous HuR levels through expression of antisense HuR decreased p21 RNA-protein complexes, greatly reduced the UVC inducibility and half-life of p21 mRNA, and prevented UVC-mediated induction of luciferase activity in p21 3' untranslated region-containing reporter constructs. Our findings indicate that HuR plays a major role in regulating stress-induced p21 expression by enhancing p21 mRNA stability and that these effects are coupled to HuR's elevated presence in the cytoplasm.     

RNA-dependent association with myosin IIA promotes F-actin-guided trafficking of the ELAV-like protein HuR to polysomes

The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase Cδ (PKCδ)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKCδ remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes.     

Delayed mechanism for induction of gamma-glutamylcysteine synthetase heavy subunit mRNA stability by oxidative stress involving p38 mitogen-activated protein kinase signaling

Expression of the gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh), which encodes the rate-limiting enzymes for glutathione biosynthesis, is regulated by many cytotoxic agents. Moreover, gamma-GCSh mRNA expression is elevated in colorectal cancer, but how gamma-GCSh expression is regulated is not completely understood. By using actinomycin D, which inhibits new RNA synthesis, we showed that treatment of human colorectal cancer cells with the prooxidant sulindac increased the half-life of gamma-GCSh mRNA. By using a tetracycline-regulated gamma-GCSh mRNA assay system, we systematically dissected the cis-acting sequence and trans-acting factors that regulate the stability of gamma-GCSh by cytotoxic prooxidants. We demonstrated that a HuR recognition sequence, AUUUA, in the 3'-untranslated region is responsible for the decay of gamma-GCSh mRNA. Oxidative stress enhanced cytoplasmic content of HuR. Overexpression of HuR by transfection stabilized gamma-GCSh mRNA, whereas overexpression of a dominant-negative HuR mutant suppressed the induced stability. Furthermore, prooxidant-induced gamma-GCSh mRNA stabilization and HuR binding were blocked by p38 mitogen-activated protein kinase inhibitors. We provide the first evidence that reduction-oxidation regulation of gamma-GCSh expression, itself a reduction-oxidation sensor and regulator, is mediated at least in part by the p38 mitogen-activated protein kinase signaling through the HuR RNA-binding protein.     

Upregulation of lipopolysaccharide-induced interleukin-10 by prostaglandin A1 in mouse peritoneal macrophages

The cyclopentenone prostaglandins (cyPGs) prostaglandin A1 (PGA1) and 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) have been reported to exhibit antiinflammatory activity in activated monocytes/macrophages. However, the effects of these two cyPGs on the expression of cytokine genes may differ. In this study, we investigated the mechanism of action of PGA1 in lipopolysaccharide (LPS)-induced expression of interleukin (IL)-10 mRNA in mouse peritoneal macrophages. 15d-PGJ2 inhibited expression of LPSinduced IL-10, whereas PGA1 increased LPS-induced IL-10 expression. This synergistic effect of PGA1 on LPS-induced IL-10 expression reached a maximum as early as 2 h after simultaneous PGA1 and LPS treatment (PGA1/LPS), and did not require new protein synthesis. The synergistic effect of PGA1 was inhibited by GW9662, a specific peroxisome proliferator-activated receptor (PPAR) antagonist, and Bay-11-7082, a NF-kappaB inhibitor. The extracellular signalregulated kinases (ERK) inhibitor PD98059 increased the expression of PGA1/LPS-induced IL-10 mRNA, rather than inhibiting the IL-10 expression. Moreover, PGA1 inhibited LPS-induced ERK phosphorylation. The synergistic effect of PGA1 on LPS-induced IL-10 mRNA and protein production was inhibited by p38 inhibitor PD169316, and PGA1 increased LPS-induced p38 phosphorylation. In the case of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), the SAPK/JNK inhibitor SP600125 did not inhibit IL-10 mRNA synthesis but inhibited the production of IL-10 protein remarkably. These results suggest that the synergistic effect of PGA1 on LPS-induced IL-10 expression is NF-kappaB-dependent and mediated by mitogen-activated protein (MAP) kinases, p38, and SAPK/ JNK signaling pathways, and also associated with the PPARgamma pathway. Our data may provide more insight into the diverse mechanisms of PGA1 effects on the expression of cytokine genes.     

热激蛋白Hsp72促进热休克下HuR对p21CIP1的调控作用

RNA结合蛋白HuR可以结合并调控靶标mRNA稳定性与翻译,但影响HuR 结合活性的因素有待探讨。本研究从蛋白质-蛋白质相互作用角度对影响HuR 与RNA结合活性的因素做了探讨。结果发现,热激蛋白Hsp72在细胞浆与HuR相互作用并促进HuR与p21 (KIP1) 3′UTR（3′非翻译区）的结合; 热休克下Hsp72总蛋白质及细胞浆蛋白质水平上调、但HuR总蛋白质及细胞浆蛋白质水平不变|热休克下HuR与p21 3′UTR的相互作用加强、p21蛋白及mRNA水平上调。上述结果提示,Hsp72可通过与HuR相互作用促进后者与p21 mRNA的结合,进而加强热休克下HuR对p21的表达的促进作用。这些结果为进一步解析HuR的生物学作用机制提供了实验依据。     

The p16INK4a tumor suppressor controls p21WAF1 induction in response to ultraviolet light

p16^INK4a and p21^WAF1, two major cyclin-dependent kinase inhibitors, are the products of two tumor suppressor genes that play important roles in various cellular metabolic pathways. p21^WAF1 is up-regulated in response to different DNA damaging agents. While the activation of p21^WAF1 is p53-dependent following γ-rays, the effect of ultraviolet (UV) light on p21^WAF1 protein level is still unclear. In the present report, we show that the level of the p21^WAF1 protein augments in response to low UVC fluences in different mammalian cells. This up-regulation is mediated through the stabilization of p21^WAF1 mRNA in a p16^INK4a-dependent manner in both human and mouse cells. Furthermore, using p16-siRNA treated human skin fibroblast; we have shown that p16 controls the UV-dependent cytoplasmic accumulation of the mRNA binding HuR protein. In addition, HuR immunoprecipitations showed that UV-dependent binding of HuR to p21 mRNA is p16-related. This suggests that p16 induces p21 by enabling the relocalization of HuR from the nucleus to the cytoplasm. Accordingly, we have also shown that p16 is necessary for efficient UV-dependent p53 up-regulation, which also requires HuR. These results indicate that, in addition to its role in cell proliferation, p16^INK4a is also an important regulator of the cellular response to UV damage.