Regulation of Prion Gene Expression by Transcription Factors SP1 and Metal Transcription Factor-1

Shayne A Bellingham,Louise A Coleman,Colin L Masters,James Camakaris,Andrew F Hill

doi:10.1074/jbc.m804755200

Abstract

Prion diseases are associated with the conformational conversion of the host-encoded cellular prion protein into an abnormal pathogenic isoform. Reduction in prion protein levels has potential as a therapeutic approach in treating these diseases. Key targets for this goal are factors that affect the regulation of the prion protein gene. Recent in vivo and in vitro studies have suggested a role for prion protein in copper homeostasis. Copper can also induce prion gene expression in rat neurons. However, the mechanism involved in this regulation remains to be determined. We hypothesized that transcription factors SP1 and metal transcription factor-1 (MTF-1) may be involved in copper-mediated regulation of human prion gene. To test the hypothesis, we utilized human fibroblasts that are deleted or overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. Menkes deletion fibroblasts have high intracellular copper, whereas Menkes overexpressed fibroblasts have severely depleted intracellular copper. We have utilized this system previously to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor protein. Here we demonstrate that copper depletion in MNK overexpressed fibroblasts decreases cellular prion protein and PRNP gene levels. Conversely, expression of transcription factors SP1 and/or MTF-1 significantly increases prion protein levels and up-regulates prion gene expression in copper-replete MNK deletion cells. Furthermore, siRNA "knockdown" of SP1 or MTF-1 in MNK deletion cells decreases prion protein levels and down-regulates prion gene expression. These data support a novel mechanism whereby SP1 and MTF-1 act as copper-sensing transcriptional activators to regulate human prion gene expression and further support a role for the prion protein to function in copper homeostasis. Expression of the prion protein is a vital component for the propagation of prion diseases; thus SP1 and MTF-1 represent new targets in the development of key therapeutics toward modulating the expression of the cellular prion protein and ultimately the prevention of prion disease.

Highlights

Prion diseases, traditionally known as transmissible spongiform encephalopathies, are invariably fatal, transmissible neurodegenerative disorders that include Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle
Because copper homeostasis from yeast to mammals is regulated by several cellular mechanisms including copperdependent transcriptional regulation [27, 28] and the PRNP promoter region is highly conserved among several species [29],we propose that copper regulation of the human PRNP gene is controlled by metal-regulated or metal-responsive transcription factors via putative metal response element (MRE) sequences
Immortalized human fibroblasts isolated from a Menkes disease patient [43], referred to as Menkes protein (MNK)(Del) cells, and MNK(Del) cells stably transfected and overexpressed with the MNK efflux protein or the empty mammalian expression vector [43], referred to as MNK(ϩϩ) and MNK(v/o) cells, respectively, represent powerful tools to manipulate intracellular copper concentrations

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Establishment and characterization of human skin fibroblast cells, MNK(Del), MNK(v/o), and MNK(ϩϩ) has been reported previously [43]. 48 h post-transfection the cells were washed with ice-cold PBS several times, lysed, and extracted for total protein or total RNA using the PARISTM kit (Ambion) according to the manufacturer’s instructions. Quantitative Real Time RT-PCR—1 ␮g of total RNA extracted from cell lysates was converted to cDNA using a high capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s instructions. SiRNA Knockdown—The cells were post-seeded at ϳ1 ϫ 105 cells/6 wells and either mock transfected or transfected with siPORT NeoFx transfection reagent and negative control #1 siRNA or Silencer௡ predesigned siRNAs for SP1 and MTF-1 at a final concentration of 30 nM using the reverse transfection method according to the manufacturer’s instructions.

RESULTS

MNK Overexpressed Fibroblasts

DISCUSSION

Tau is a microtubule associated protein that forms the major