| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
EDITORIAL FOCUS
Department of Physiology and Biophysics, Chicago Medical School, Rosalindfranklin University of Medicine and Science, North Chicago, Illinois
EXPRESSION IN HETEROLOGOUS cell systems is a mainstay of modern biomedical research, where it is used to study the cell biology and function of both wild-type and mutant proteins. Although recombinant proteins are generally expressed in naive cells, it is becoming increasingly important to express proteins within a more native environment, particularly if the endogenous protein is hard to detect and the recombinant protein is tagged in some way to facilitate tracking. For example, many ion channels are expressed at very low copy numbers in cells, often making biochemical detection problematic, or there is poor availability of good antibodies against the native channel. In addition to basic research, protein expression systems are also being employed in drug development. For example, gene therapy, in which a cDNA is the therapeutic agent, attempts to establish protein expression of the wild-type counterpart of the endogenously expressed mutant protein. Cell-based high-throughput screening (HTS) assays using a heterologous expression system also are being increasingly employed not only in pharmaceutical laboratories but also within the academic environment to detect novel therapeutic agents. Many of these HTS assays are focused on developing therapeutic agents to treat genetic diseases caused by the misfolding of the aberrant protein (2).
One such disease is cystic fibrosis (CF), a genetic defect caused by mutations in a chloride channel called the cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of the codon for phenylalanine-508 (
F508) accounts for 
70% of all disease-causing alleles (12) and yields a protein that is unable to exit from the endoplasmic reticulum (ER) and traffic to the plasma membrane, where it functions as a chloride channel in the apical membranes of epithelia. Initially, there was much excitement surrounding the possibility that CF may be an excellent candidate for gene therapy. Although most assays have traditionally focused on detecting expressed CFTR protein either biochemically (Western blots, immunofluorescence) or functionally (patch clamp, short-circuit current), little attention has been directed to understanding how expression of CFTR cDNA is related to endogenous expression of the native CFTR gene. In the current article in focus (Ref. 11; see p. C756 in this issue), Beb
k et al. show that expression of CFTR from the endogenous gene is handled somewhat differently from they way in which exogenously expressed CFTR is processed. Conformational maturation of CFTR is very inefficient in heterologous expression systems (40% efficiency), yet for those cells expressing endogenous CFTR, folding and maturation of the endogenously expressed CFTR protein is very high (90% efficient) (9, 14, 15). The data presented by Bebk et al. show that there is a high degree of transcriptional and translational control over the expression of endogenous CFTR, control that is absent from heterologously expressed CFTR even when expressed in polarized epithelial cells.
For many biological systems, differences between responses are often highlighted under stress conditions. Under ER stress, several stress response elements are activated, including the unfolded protein response (UPR) and the ER-associated degradation pathway (ERAD). In many cases, ER stress leads to a decrease in the protein load experienced by the ER, at both a transcriptional and a posttranslational level (7, 8). Upon activation of the UPR in airway epithelial cells, the levels of genomic CFTR mRNA undergo a significant decrease, whereas the mRNA levels for exogenously expressed CFTR cDNA in the same cell are unaffected. Thus exogenous CFTR expression seems able to bypass the normal UPR limiting CFTR expression. Precisely why endogenous CFTR levels are affected by the UPR is not clear, since Bebk et al. report that the mRNA levels for another transmembrane protein, the transferrin receptor, are not altered. It is possible that since CFTR utilizes ATP for its channel gating, a reduction in CFTR levels in response to stress would lower the ATP utilization in cells. A parallel stress-related regulatory mechanism has been proposed by Hallows et al. (6), who proposed that under metabolic stress conditions, the AMP-dependent protein kinase (AMPK) inactivates CFTR, leading to reduced channel activity and, presumably, reduced ATP usage. Although the current study in focus uses pharmacological reagents to induce stress, it is important to note that oxidative stress (a more physiologically related stressor) also leads to a reduction in the levels of cellular CFTR (4, 5). In contrast to these observations, a previous report from Bebk et al. (3) argues that exposure of renal epithelial cells to 95% oxygen leads to an increase in CFTR protein and function. Although this latter case is clearly aphysiological, the role of oxidative species at levels likely seen by cells, and their subsequent impact on ER stress and CFTR expression, is clearly an area for investigation.
Interestingly, Bebk et al. have shown that overexpression of exogenous CFTR in airway cells does not activate ER stress. This is particularly important for gene therapy applications, since activation of the UPR by overexpression of improperly folded proteins can lead to cell damage and apoptosis (13), as seen in amyloid-
-induced neural degeneration (10) and pancreatic -cell destruction in diabetes mellitus (1). The observation that expression of exogenous CFTR is unaffected by the UPR may be a double-edged sword for CFTR gene therapy. On one hand, mRNA levels for exogenous CFTR (and presumably levels of mature CFTR protein and hence channel function) will not be affected by ER stress; however, as noted above, CFTR channels at the cell surface may be subject to AMPK-dependent regulation. The potential downside for gene therapy is that exogenous CFTR expression will not be subject to normal cellular regulation. Whether the latter issue is a cause for concern in gene therapy awaits a more thorough investigation of the reasons why endogenous CFTR expression is apparently so tightly regulated.
 |
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
 TOP REFERENCES |
|---|
-cells and diabetes mellitus. Exp Biol Med (Maywood) 228: 1213–1217, 2003.2. Aridor M, Hannan LA. Traffic jam: a compendium of human diseases that affect intracellular trafficking processes. Traffic 1: 836–851, 2000.[CrossRef][Web of Science][Medline]
3. Bebk Z, Tousson T, Schwiebert LM, Venglarik CJ. Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers. Am J Physiol Cell Physiol 280: C135–C145, 2001.
4. Bebk Z, Varga K, Hicks JK, Venglarik CJ, Kovacs T, Chen L, Hardiman KM, Collawn JF, Sorscher EJ, Matalon S. Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl– secretion in airway epithelia. J Biol Chem 277: 43041–43049, 2002.
5. Cantin AM, Bilodeau G, Ouellet C, Liao J, Hanrahan JW. Oxidant stress suppresses CFTR expression. Am J Physiol Cell Physiol 290: C262–C270, 2006.
6. Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK. Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. J Clin Invest 105: 1711–1721, 2000.[Web of Science][Medline]
7. Iwawaki T, Hosoda A, Okuda T, Kamigori Y, Nomura-Furuwatari C, Kimata Y, Tsura A, Kohno K. Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress. Nat Cell Biol 3: 158–164, 2001.[CrossRef][Web of Science][Medline]
8. Kimata Y, Ishiwata-Kimata Y, Yamada S, Kohno K. Yeast unfolded protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface proteins. Genes Cells 11: 59–69, 2006.
9. Lukacs GL, Mohamed A, Kartner N, Chang XB, Riordan JR, Grinstein S. Conformational maturation of CFTR but not its mutant counterpart (F508) occurs in the endoplasmic reticulum and requires ATP. EMBO J 13: 6076–6086, 1994.[Web of Science][Medline]
10. Pereira C, Ferreiro E, Cardoso SM, de Oliveira CR. Cell degeneration induced by amyloid- peptides: implications for Alzheimer’s disease. J Mol Neurosci 23: 97–104, 2004.[CrossRef][Web of Science][Medline]
11. Rab A, Bartoszewski R, Jurkuvenaite A, Wakefield J, Collawn JF, Bebk Z. Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression. Am J Physiol Cell Physiol 292: C756–C766, 2007.
12. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FS, Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073, 1989.
13. Rutkowski DT, Kaufman RJ. A trip to the ER: coping with stress. Trends Cell Biol 14: 20–28, 2004.[CrossRef][Web of Science][Medline]
14. Varga K, Jurkuvenaite A, Wakefield J, Hong JS, Guimbellot JS, Venglarik CJ, Niraj A, Mazur M, Sorscher EJ, Collawn JF, Bebok Z. Efficient intracellular processing of the endogenous cystic fibrosis transmembrane conductance regulator in epithelial cell lines. J Biol Chem 279: 22578–22584, 2004.
15. Ward CL, Kopito RR. Intracellular turnover of cystic fibrosis transmembrane conductance regulator. J Biol Chem 269: 25710–25718, 1994.
Related articles in Am J Physiol Cell Physiol:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
