Overexpression of Enzymatically Active Human Cytosolic and Mitochondrial Thioredoxin Reductase in HEK-293 Cells

Abstract

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxRoverexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1-or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.Thioredoxin reductases (TrxRs) 1 are members of the nucleotide-disulfide oxidoreductase family and are ubiquitously found in mammalian tissues. Two main isoforms of thioredoxin reductases exist in mammals: the classical cytosolic form (TrxR1, the TXNRD1 gene product) (1, 2), and the mitochondrial form (TrxR2) (3, 4). A third form, thioredoxin and glutathione reductase, is mainly expressed in testis (5). All mammalian TrxRs are homodimeric selenocysteine-containing enzymes (6) that share high sequence homology between each other and contain an NADPH binding domain and a FAD binding domain per subunit (7). TrxRs reduce and thereby activate thioredoxins (Trxs), which are small ubiquitous proteins with a conserved active site sequence (-Trp-Cys-Gly-ProCys-) that catalyze many redox reactions through the reversible oxidation of the active site dithiol to a disulfide. Trxs are involved in a variety of reactions, such as redox regulation of transcription factors (8, 9), reduction of hydroperoxides (4, 10), and modulation of cell growth (11) and apoptosis (12, 13). The selenocysteine residue (Sec) that is found at the C terminus in TrxRs (-Gly-Cys-Sec-Gly-COOH) is encoded by a UGA codon that is normally identified as a stop codon, but which can be transcribed as Sec in the presence of a selenocysteine insertion sequence (SECIS) element in the 3Ј-UTR of the corresponding mRNA (14). The C-terminal Cys-Sec motif is located on a presumably flexible arm of the enzyme that is kept reduced by the N-terminal redox active motif (-Cys-Val-Asn-Val-Gly...

The R-Ras interaction partner ORP3 regulates cell adhesion

“…Recent studies have pointed out that HEK293 cells express several factors that are usually present only in cells of neuronal origin (Shaw et al, 2002;Nalvarte et al, 2004). Therefore, the membrane protrusions formed in HEK293 cells may bear resemblance to polarized neurites (Shaw et al, 2002;Nalvarte et al, 2004;Govek et al, 2005). In this context it is noteworthy that a close homolog of ORP3, ORP6, was identified as one of the fifteen proteins that promote the differentiation of neurons from murine embryonic stem cells (Falk et al, 2007).…”

“…In neuronal cells, serum starvation has been shown to induce integrin-dependent neurite outgrowth, which is highly dependent on Ras-mediated PI3K, Cdc42 and Rac1 activation (Sarner et al, 2000). Recent studies have pointed out that HEK293 cells express several factors that are usually present only in cells of neuronal origin (Shaw et al, 2002;Nalvarte et al, 2004). Therefore, the membrane protrusions formed in HEK293 cells may bear resemblance to polarized neurites (Shaw et al, 2002;Nalvarte et al, 2004;Govek et al, 2005).…”

Redox Regulation in the Extracellular Environment

“…[195][196][197] In fact, a study by Mehta et al demonstrated that Sec incorporation efficiency in an in-vitro luciferase reporter system is only approximately 5-8% the levels of the wild-type luciferase. 198 …”

Mammalian Selenoprotein Thioredoxin-glutathione Reductase

“…These enzymes are key components of the thioredoxin system, which is one of major redox systems in cells. They control the redox state of thioredoxin but were also implicated more broadly in redox regulation, cell growth, and other functions (4,5). Two TRs, TR1 (also known as TrxR1 and Txnrd1) and TR3 (also known as TrxR2 and Txnrd2), have been characterized in detail and are components of cytosolic and mitochondrial thioredoxin systems, respectively.…”