Heterogeneous Nuclear Ribonucleoprotein K and Nucleolin as Transcriptional Activators of the Vascular Endothelial Growth Factor Promoter through Interaction with Secondary DNA Structures

Abstract

The human vascular endothelial growth factor (VEGF) promoter contains a polypurine/polypyrimidine (pPu/pPy) tract that is known to play a critical role in its transcriptional regulation. This pPu/pPy tract undergoes a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We studied the interaction of the cytosine-rich (C-rich) and guanine-rich (G-rich) strands of this tract with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of VEGF. Using chromatin immunoprecipitation (ChIP) assay for our in vivo studies and electrophoretic mobility shift assay (EMSA) for our in vitro studies, we demonstrated that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the VEGF promoter. The small interfering RNA (siRNA)-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within the pPu/pPy tract will provide new insight into mechanisms of transcriptional regulation of the VEGF gene.

RNA-binding protein nucleolin in disease

“…For instance, nucleolin transcriptionally promotes expression of the vascular endothelial growth factor (VEGF) and the interferon regulatory factor-2 (IRF-2), other proteins that are highly expressed in cancers and are potent regulators of cancer cell growth. [127][128][129] In addition, as explained above, cell-surface nucleolin functions as a receptor for tumorigenic factors like the carcinogenic factor released from Helicobacter pylori Tipα (the TNFα-inducing protein); nucleolin shuttles Tipα from the cell membrane to the cytosol and the nucleus, where it induces the expression of TNFα and chemokines and promotes inflammation. 130 …”

“…25 In addition, nucleolin can affect mRNA transcription directly by interacting with DNA by interacting with DNA. 26 Nucleolin's influence on ribosome biogenesis has also been studied in detail. Nucleolin binds to a stem-loop structure found in nascent pre-rRNA and plays a chaperone function by facilitating the proper folding of pre-rRNA.…”

Withanone-Rich Combination of Ashwagandha Withanolides Restricts Metastasis and Angiogenesis through hnRNP-K

“…MMPs, critical regulators of extracellular matrix, metastasis, and angiogenesis (20), are regulated by cytokines, growth factors via interlinked signaling pathways (19,21,22). hnRNP-K is a multifunctional protein that regulates ERK1/2, MMPs, and VEGF and contributes to cell migration, invasion, and ascites formation (19,(23)(24)(25). ERK1/2 and VEGF have been connected by positive autocrine feedback loop (26)(27)(28).…”

The Requirement of c-Jun N-terminal Kinase 2 in Regulation of Hypoxia-inducing Factor-1α mRNA Stability

“…Nucleolin has been found to bind to the mRNA of several important genes, including p53 (24), bcl-2 (25), and bcl-xl (26), leading to regulation on mRNA turnover or translation. Moreover, nucleolin has been shown to have binding activity to a number of G-rich oligonucleotides, like the G-quadruplex structure in the promoter region of c-myc gene (27) and vegf gene (28). Gonzalea et al showed, in an in vitro filter binding assay, that nucleolin bound to the G-quadruplex structure in 5Ј-UTR of hif-1␣ mRNA (27).…”