Growth Factor-Mediated Stabilization of Amyloid Precursor Protein mRNA Is Mediated by a Conserved 29-Nucleotide Sequence in the 3′-Untranslated Region

Authors


  • Abbreviations used:βA4, β-amyloid peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; bFGF, basic fibroblast growth factor; DS, Down’s syndrome; EBM, endothelial cell basal medium; EGF, epidermal growth factor; EGM, endothelial cell growth medium; FBS, fetal bovine serum; GAP-43, growth-associated protein-43; Gl-APP, globin-APP; Gl-APPmut, mutated globin-APP; Gl-APPwt, wildtype globin-APP; HCHWA-D, hereditary cerebral hemorrhage with amyloidosis, Dutch type; HUVEC, human umbilical vein endothelial cells; IGF-1, insulin-like growth factor-1; IL-1, interleukin-1; KPI, Kunitz-type protease inhibitor; TGF-β, transforming growth factor-β; UTR, untranslated region; VEGF, vascular endothelial growth factor.

Address correspondence and reprint requests to Dr. J. S. Malter at Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI 53792, U.S.A. E-mail: js.malter@hosp.wisc.edu

Abstract

Abstract: Using a cell-free translation system, we previously demonstrated that the turnover and translation of amyloid precursor protein (APP) mRNA was regulated by a 29-nucleotide instability element, located 200 nucleotides downstream from the stop codon. Here we have examined the regulatory role of this element in primary human capillary endothelial cells under different nutritional conditions. Optimal proliferation required a growth medium (endothelial cell growth medium) supplemented with epidermal, basic fibroblast, insulin-like, and vascular endothelial growth factors. In vitro transcribed mRNAs with the 5′-untranslated region (UTR) and coding region of β-globin and the entire 3′-UTR of APP 751 were transfected into cells cultured in endothelial cell growth medium. Wild-type globin-APP mRNA containing an intact APP 3′-UTR and mutant globin-APP mRNA containing a mutated 29-nucleotide element decayed with identical half-lives (t1/2 = 60 min). Removal of all supplemental growth factors from the culture medium significantly accelerated the decay of transfected wild-type mRNA (t1/2 = 10 min), but caused only a moderate decrease in the half-life of transfected mutant mRNA (t1/2 = 40 min). We therefore conclude that the 29-nucleotide 3′-UTR element is an mRNA destabilizer whose function can be inhibited by inclusion of the aforementioned mixture of growth factors in the culture medium.

Ancillary