Epidermal Growth Factor Increases the Interaction between Nucleolin and Heterogeneous Nuclear Ribonucleoprotein K/Poly(C) Binding Protein 1 Complex to Regulate the Gastrin mRNA Turnover

Abstract

Gastrin, a gastrointestinal hormone responsible for gastric acid secretion, has been confirmed as a growth factor for gastrointestinal tract malignancies. High expression of gastrin mRNA was observed in pancreatic and colorectal cancer; however, the mechanism is unclear. Epidermal growth factor (EGF) was found to increase gastrin mRNA stability, indicating mRNA turnover regulation mechanism is involved in the control of gastrin mRNA expression. Using biotinlabeled RNA probe pull-down assay combined with mass spectrometry analysis, we identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and poly(C) binding protein 1 (PCBP1) bound with the C-rich region in gastrin mRNA 3 untranslated region. Nucleolin bound with the AGCCCU motif and interacted with hnRNP K were also demonstrated. Under EGF treatment, we observed the amount of nucleolin interacting with hnRNP K and gastrin mRNA increased. Using small interfering RNA technology to define their functional roles, we found hnRNP K, PCBP1, and nucleolin were all responsible for stabilizing gastrin mRNA. Moreover, nucleolin plays a crucial role in mediating the increased gastrin mRNA stability induced by EGF signaling. Besides, we also observed hnRNP K/PCBP1 complex bound with the C-rich region in the gastrin mRNA increased nucleolin binding with gastrin mRNA. Finally, a novel binding model was proposed. INTRODUCTIONRegulation of gene expression is essential for the homeostasis of an organism. Transcriptional regulation is well documented in controlling gene expression; however, it is becoming increasingly clear that regulation of mRNA decay rates is also an important control point in determining the abundance of cellular transcripts (Wilusz et al., 2001;Wilusz and Wilusz, 2004). The aberrant control of mRNA turnover has been implicated in disease states, including cancer, chronic inflammatory responses, and coronary disease (Hollams et al., 2002;Audic and Hartley, 2004). For example, in myeloma and human T-cell leukemia, the c-myc gene is mutated due to a translocation or loss of the 3Ј untranslated regions (UTRs). This renders the c-myc mRNA up to 7 times more stable than the wild type (Hollis et al., 1988;Aghib et al., 1990). mRNA turnover rate is mainly specified by control elements that are usually located within the 3ЈUTR of mRNAs and they are recognized by various RNA-binding proteins. The most well studied specific cis-acting element controlling the half-life of mRNA is the adenylate-and uridylate-rich (AU-rich) element (ARE) found in 3ЈUTR of a variety of short-lived mRNAs, including those encoding cytokines, lymphokines, proto-oncogenes, and growth factors (Chen and Shyu, 1995;Wilusz et al., 2001). A plethora of AREbinding proteins have been identified, characterized, and cloned, including AUF1, HuR, TIA-1, and tristetraprolin, which exerted either negative or positive effects on mRNA stability (Brennan and Steitz, 2001;Bevilacqua et al., 2003). C-rich element has also been reportedly involved in the regulation of mRNA stability. Three discontinuo...

Synergistic and Multidimensional Regulation of Plasminogen Activator Inhibitor Type 1 Expression by Transforming Growth Factor Type β and Epidermal Growth Factor

“…This result is consistent with other reports of EGF stabilizing mRNAs, including those encoding ␤-tubulin, the EGF receptor, p21/CIP1, cyclin D1, TGF␣, amphiregulin, IL-1␣, IL-1␤ and IL-6 leukemia-inhibitory factor, TGF␤RII, and gastrin (11,33,(35)(36)(37)(38)(39)(40)(41). Because PAI-1 mRNA is rapidly degraded, the regulation of its half-life can have a large impact on PAI-1 expression (42).…”

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

“…Previous studies have shown that hnRNP K directly interacts with nucleic acids and regulates gene expression at multiple levels (Bomsztyk et al, 2004;Lee et al, 2007). TP acts as an angiogenic factor and mediates inhibition of tumor cell apoptosis, thereby contributing to tumor progression (reviewed in Liekens et al, 2007).…”

“…Heterogeneous nuclear ribonucleoprotein K (hnRNP K) belongs to the hnRNP family of proteins, which directly interact with DNA and RNA through their K homology domains and regulate gene expression at multiple levels, including transcription, RNA splicing, RNA stability and translation (Bomsztyk et al, 2004;Lee et al, 2007). The expression of hnRNP K is aberrantly increased in numerous cancers, including nasopharyngeal carcinoma (NPC; Pino et al, 2003;Carpenter et al, 2006;Hatakeyama et al, 2006;Roychoudhury and Chaudhuri, 2007;Chen et al, 2008).…”

RNA-binding protein nucleolin in disease

“…46 GAST mRNA is co-regulated by nucleolin, PCBP1 [poly(C)-binding protein 1] and hnRNP K, as the three RBPs are required for stabilizing GAST mRNA in cells treated with epidermal growth factor (EGF). 47 HBB mRNA. Dysregulation of β globin, encoded by HBB mRNA, can result in pathologies such as thalassemia.…”

“…46,47 Indeed, enhanced expression of gastrin by nucleolin is directly relevant to the tumorigenicity of human colon cancer cells and upregulates cyclooxygenase-2 (COX-2), which in turn catalyzes prostaglandin E2 production and promotes tumor cell proliferation. 121,122 Nucleolin may also assist cancer cells in overcoming cellular senescence, as it associates with the enzyme telomerase (TERT) and its RNA template (TERC) in the nucleoplasm.…”