The RNA-binding Protein HuR Regulates the Expression of Cyclooxygenase-2

Sibani Sengupta,Byeong-Churl Jang,Ming-Tao Wu,Ji-Hye Paik,Henry Furneaux,Timothy Hla

doi:10.1074/jbc.m301813200

Sibani Sengupta, Byeong-Churl Jang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m301813200

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Abstract

The cyclooxygenase-2 (COX-2) gene encodes the inducible prostaglandin synthase enzyme implicated in inflammation, cell growth, and tumorigenesis. Regulation of the COX-2 gene expression at the post-transcriptional level is poorly understood. For example, protein factors that regulate the post-transcriptional mRNA metabolism of COX-2 have not been fully characterized. In this study, we demonstrate that the RNA-binding protein HuR binds to COX-2 mRNA and regulates its expression. We show that there are three binding sites for HuR in the 3'-untranslated region of human COX-2. These sites are located at the following positions in the COX-2 3'-untranslated region: 39-84 nucleotides (nt), 1155-1187 nt, and 1244-1256 nt (hereinafter referred to as Sites I, II and III, respectively). Although all three sites are present in the 4.6-kb COX-2 mRNA, only site I is present in the shorter 2.8-kb isoform. HuR in MDA-MB-231 cell extracts associated with COX-2 mRNA at the identified sites. Further, HuR location in the cytoplasm was induced by serum withdrawal, a stimulus known to induce COX-2 mRNA. Down-regulation of HuR by two independent methods, namely RNA interference as well as antisense RNA expression, significantly attenuated serum withdrawal-induced increase in COX-2 mRNA (both the 4.6- and 2.8-kb isoforms) and protein levels. These data suggest that HuR binding to COX-2 is critical for its post-transcriptional mRNA stabilization.

Highlights

Messenger RNA stability is an important determinant of gene expression [1, 2]
COX-1 is expressed in a constitutive manner, COX-2 levels are low under basal conditions but are highly induced in response to hormones, tumor promoters
Deletion analysis of murine COX-2 3Ј-UTR identified two translational and stability control elements and two stability control elements, as shown in Fig. 1A [13]. These 3Ј-UTR constructs responded to IL-1-dependent COX-2 induction, they failed to respond to dexamethasone, suggesting that elements within the COX-2 3Ј-UTR may not be sufficient for its responsiveness

Summary

PCR primers used

Fragment 1 (F1) Fragment 2 (F2) Fragment 3 (F3 Fragment 4 (F4) Fragment 5 (F5) Fragment 6 (F6). Using a tetracycline-regulated system, a short ϳ1– 123-nt segment of the human COX-2 3Ј-UTR was identified as the minimal element required for regulation of COX-2 mRNA stability by the p38 (stabilizes COX-2) cascade [17] as well as by dexamethasone, which is known to destabilize COX-2 mRNA [16]. HuR, a member of the ELAV (embryonic lethal abnormal vision) family of mRNA-binding proteins, is known to stabilize several inducible mRNAs such as c-Fos and tumor necrosis factor-␣ (19 –23, 27). The elements that regulate the stability of COX-2 mRNA have been defined (Fig. 1A), the cis-acting factors that mediate this regulation have not been identified unequivocally. We investigated whether HuR could bind to and stabilize the COX-2 transcript

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION