Translational upregulation of folate receptors is mediated by homocysteine via RNA-heterogeneous nuclear ribonucleoprotein E1 interactions
Aśok C. Antony, … , Hiremagalur N. Jayaram, Sally P. Stabler
Aśok C. Antony, … , Hiremagalur N. Jayaram, Sally P. Stabler
Published January 15, 2004
Citation Information: J Clin Invest. 2004;113(2):285-301. https://doi.org/10.1172/JCI11548.
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Translational upregulation of folate receptors is mediated by homocysteine via RNA-heterogeneous nuclear ribonucleoprotein E1 interactions

Abstract

Cellular acquisition of folate is mediated by folate receptors (FRs) in many malignant and normal human cells. Although FRs are upregulated in folate deficiency and downregulated following folate repletion, the mechanistic basis for this relationship is unclear. Previously we demonstrated that interaction of an 18-base cis-element in the 5′-untranslated region of FR mRNA and a cystolic trans-factor (heterogeneous nuclear ribonucleoprotein E1 [hnRNP E1]) is critical for FR synthesis. However, the molecular mechanisms controlling this interaction, especially within the context of FR regulation and folate status, have remained obscure. Human cervical carcinoma cells exhibited progressively increasing upregulation of FRs after shifting of folate-replete cells to low-folate media, without a proportionate rise in FR mRNA or rise in hnRNP E1. Translational FR upregulation was accompanied by a progressive accumulation of the metabolite homocysteine within cultured cells, which stimulated interaction of the FR mRNA cis-element and hnRNP E1 as well as FR biosynthesis in a dose-dependent manner. Abrupt reversal of folate deficiency also led to a rapid parallel reduction in homocysteine and FR biosynthesis to levels observed in folate-replete cells. Collectively, these results suggest that homocysteine is the key modulator of translational upregulation of FRs and establishes the linkage between perturbed folate metabolism and coordinated upregulation of FRs.

Authors

Aśok C. Antony, Ying-Sheng Tang, Rehana A. Khan, Mangatt P. Biju, Xiangli Xiao, Qing-Jun Li, Xin-Lai Sun, Hiremagalur N. Jayaram, Sally P. Stabler

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Figure 2

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Determination of the locus of upregulation of FR in HeLa-IU1-LF cells. (...
Determination of the locus of upregulation of FR in HeLa-IU1-LF cells. (a) FR gene transcription. Nuclei (5 × 107) from HeLa-IU1-HF and HeLa-IU1-LF cells were used in nuclear run-on transcription assays. Equal amounts of [α-32P]UTP nuclear RNA were hybridized to filters blotted with 5 μg of denatured plasmid DNA containing human β-actin cDNA or human FR cDNA, followed by autoradiography. (b) Analysis of FR mRNA stability in HeLa-IU1-HF (open circles) and HeLa-IU1-LF (filled circles) cells exposed to actinomycin D for the indicated times followed by Northern blot analysis. The curve-fitting analysis of FR mRNA was determined by linear regression. (c–e) Validation that the procedure for immunoprecipitation of the de novo–synthesized FR with anti-FR antiserum includes incompletely glycosylated forms (see Methods for details). (f) Analysis of the rates of FR protein degradation of either six million HeLa-IU1-HF cells (open circles) or HeLa-IU1-LF cells (filled circles) that were pulsed with [35S]cysteine, chased with high-folate media (open circles) or low-folate media (filled circles) for the various times indicated, and immunoprecipitated [35S]FR was determined. The curve-fitting analyses in protein half-life studies were determined by linear regression. (g) Analysis of the rates of FR protein synthesis of HeLa-IU1-HF cells (filled diamonds) and HeLa-IU1-LF cells (filled squares). 6 × 106 cells were pulsed with [35S]cysteine for the indicated time, and immunoprecipitated [35S]FR was determined by liquid scintillation counting. There was less than 15% variation from the mean for each data point of triplicate samples. The data are representative of a typical experiment that was repeated on three different occasions. (h) Comparison of the intensity of immunoprecipitated [35S]FR signals from HeLa-IU1-HF and HeLa-IU1-LF cells after 10% SDS-PAGE, Western blotting, and autoradiography.