Caspase-mediated Cleavage of RNA-binding Protein HuR Regulates c-Myc Protein Expression after Hypoxic Stress

Abstract

Altered expression of RNA-binding proteins modulates gene expression in association with mRNAs encoding many protooncogenes, cytokines, chemokines, and proinflammatory factors. Hu antigen R (HuR), a ubiquitously expressed protein, controls a range of cellular functions such as tumor progression, apoptosis, invasion, and metastasis by stabilizing the AU-rich element located at the 3-untranslated region (UTR) of target mRNAs. Although significant progress has been made in understanding HuR regulation in gene expression, little is known about how HuR undergoes post-translational modifications and recruits target mRNAs during hypoxic stress. Here, we report that during CoCl 2 -induced hypoxic stress, HuR is significantly overexpressed and undergoes caspase-dependent cleavage in head and neck squamous cell carcinoma cells. Unexpectedly, the HuR-cleavage product 1 (HuR-CP1) was found to strongly associate with the 3-UTR of c-myc mRNA and block mRNA translation. The binding efficiency of HuR to the 3-UTR of c-myc mRNA was confirmed using ribonucleoprotein immunoprecipitation and site-directed mutagenesis at the AU-rich element sequences of the c-myc mRNA. Overexpression of a non-cleavable isoform, HuR-D226A, revealed a potent dominant-negative effect, repressing cleavage of endogenous HuR and promoting cell viability. Surprisingly, under hypoxia, siRNA knockdown of HuR elevated c-Myc protein expression. These findings suggest an important role for HuR in hypoxia, and we may have revealed a novel post-transcriptional mechanism that controls c-Myc expression in oral cancer progression.Gene expression is controlled by multiple biological networks, and post-transcriptional gene regulation determines mRNA fate in association with RNA-binding proteins and microRNAs (1, 2). Several RNA-binding proteins associate with the 3Ј-untranslated region (UTR) of target mRNAs and regulate their expression via alteration of the half-life and/or rate of translation of target mRNAs. An RNA-binding protein that increases mRNA stability through this mechanism is the Hu antigen R (HuR, 3 also named ELAVL1) (3). HuR is expressed ubiquitously in most cancers, including head and neck squamous cell carcinoma (HNSCC) (4, 5). HuR associates with AUand U-rich elements (AREs) in the 3Ј-UTR of target mRNAs to control transcript stability and protein translation (6). In addition, HuR has been shown to be involved in various cancer processes such as cellular proliferation, differentiation, invasion, metastasis, apoptosis, and angiogenesis in association with target mRNAs (7,8). HuR also increases the translation of certain mRNAs and represses the translation of other transcripts (9, 10). HuR is predominantly localized to the nucleus and, in response to cellular stress, is exported to the cytoplasm where it elicits its post-transcriptional influence on target mRNAs. During stress, HuR has been shown to undergo several post-translational modifications including phosphorylation (11, 12), methylation (13), and ubiquitination (14) and cleaved by caspase-3 (1...

Apoptotic-induced cleavage shifts HuR from being a promoter of survival to an activator of caspase-mediated apoptosis

“…This is comparable to observations made by Talwar et al 35 that HuR-CP1, and not wtHuR, inhibits the translation of c-myc mRNA. As previous observations indicated that HuR-CPs exercise their function by associating with various protein partners, 18,25 it would be interesting to determine if their capacity to post-transcriptionally regulate gene expression also depends on other ligands.…”

“…and the asterisk indicating that the observed difference in the means is significant messages that bound HuR by our RIP-ChIP approach are involved at different levels in mediating apoptosis, such as Bad, a promoter of cyt c release. 34 As the HuR-CPs seem to influence the progression of apoptosis at different steps, 18,35 it would be valuable to determine if their multiple-target approach may rescue the ability of cancer cells to respond to chemotherapeutic treatments.…”

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition

“…Many of these stress signals, including oxidative stress (4), UV light (7), amino acid starvation (7), and polyamine depletion (8), result in the cytoplasmic accumulation of HuR and the subsequent stabilization of target mRNAs, suggesting the functional role of HuR in adaptive response to environmental changes to maintain cell homeostasis. However, other stress signals, such as heat shock (5), hypoxia (6), and staurosporine (9), were reported to reduce HuR expression through proteasomal degradation or caspase-mediated cleavage. The function of this HuR proteolysis, however, differs in the context of different types of stress.…”

“…HuR is primarily localized in the nucleus and translocates to the cytoplasm in response to various stress stimuli, including oxidative stress (4), heat shock (5), hypoxia (6), UV light (7), amino acid starvation (7), polyamine depletion (8), and staurosporine (9). This nuclear export is integral to the function of HuR to control the stability and translation of mRNA.…”

Physiological regulation of the heat shock response by glutamine: implications for chronic low-grade inflammatory diseases in age-related conditions

“…During caspase-mediated apoptosis, HuR switches its function from pro-survival to pro-apoptotic [203]. Caspases can mediate cleavage of HuR under different situations [204]. In parallel is the observation that HFD induces, in white adipose tissue, the cleavage of SIRT1 by caspase-1 which is, in turn, activated by the diet-induced inflammatory response in adipocytes [205].…”