From the Department of Surgery, University of Colorado Health Sciences Center, Denver.
It is the regulation of gene expression that determines phenotype and cellular response. Several families of proteins control gene expression in cells and influence the pathogenesis of multiple organ failure, the acute phase response, atherosclerosis, and graft-vs-host disease. Understanding the basics of the regulation of gene transcription will allow the knowledgeable surgeon to target gene expression as a therapeutic modality in multiple diseases. We examine nuclear factor kappa B as an example of a transcription factor that is involved in multiple surgical diseases and has pharmacological inhibitors available to knowledgeable surgeons.
It is hard to believe but Pope John Paul II's and Bill Clinton's genes are 99% identical. It is the expression of their genes that is the dominant determinant of phenotype. Anyone who still thinks genes count must live with this fact. In developing cells, the timing and duration of gene expression is everything. Even in mature cells, however, gene expression is a dynamic and plastic process. Of course, in each patient all cells carry the same genes. Again, it is the temporal expression of these identical genes that drives some cells to become pancreas while others create the tip of your nose.
The conversion of genetic information into functional protein is a 2-step process, transcription and translation. Messenger RNA (mRNA) synthesis, determined by the nucleotide sequence encoded within DNA, is termed transcription. Surprisingly, cells are profligate in their mRNA synthesis and 80% of mRNA is degraded before it ever leaves the cell nucleus.1Translation is the synthesis of a final protein product based on the nucleotide sequence within the surviving mRNA. Thus, the architectural plans (or genetic code) establish the potential construct of the building. The transcription and translation of these plans determines not only what the building looks like but also how it responds to a hurricane. Thus, the regulation of transcription and the cell's control of translation are really the determinants of cellular behavior.
In recent years it has become increasingly clear that a patient's cellular response to shock or sepsis may be as important in determining outcome as the initial ischemic or bacterial insult. A cell's response options are encoded into its DNA. Current consensus concludes that the actual response to shock or sepsis, however, is determined by regulation of gene transcription.1,2 The purpose of this review is to examine families of proteins that regulate gene transcription in response to surgical stress. We propose that an understanding of transcriptional regulation will lay the foundation for future therapeutic manipulation of gene expression by surgeons. This review is directed toward the surgeon/scientist rather than the molecular biologist, with the conviction that the clinician is essential to the application of molecular techniques.
DNA, the instruction manual, contains all the information necessary to construct all types of cells. The DNA double helix is traditionally compared with a stepladder in which the rungs are either 2 matching purines—adenosine (A) or thymidine (T)—or 2 matching pyrimidines—guanine (G) or cytosine (C). The sides of the ladder are series of deoxyribose sugars linked by robust phosphodiester bonds. The rungs of the ladder, connecting adenosine to thymidine and guanine to cytosine are relatively weak hydrogen bonds. By breaking these weak hydrogen bonds, a single DNA strand is peeled free to serve as a template for mRNA synthesis.
Again, the entire DNA instruction manual is contained in each cell. This fact presents the following 2 opposite but perplexing information conundrums.
Problem A: "How can we build a pope (or a president) with only 30000 separate genetic instructions?" Physicists will never understand the answer. They believe that atomic particles and molecules should have a limitless repertoire of velocities and positions. Just ask Heisenberg. However, once you string together a series of amino acids to form a protein, the secondary and tertiary structure is predetermined. And only several quaternary configurations are physically possible. In that structure is function, the ultimate utility of each cellular protein prescribed by the primary DNA-encoded amino acid sequence. Einstein and Rutherford, however, got religion because they appreciated that organic vitality cannot be reduced to physical-chemical principles. All unifying attempts to reduce biology (not to mention physiology and psychology) to physics have failed.3
Problem B: "If all cells contain a complete DNA instruction manual, why don't you grow a nose on the side of your pancreas?" The answer reinforces the conclusion that genes don't count—it is the expression of genetic information (not the genes themselves) that distinguishes Madonna from Mother Teresa. Terminally differentiated cells have whole chapters of the DNA instruction manual glued inaccessibly together into nucleosomes. Nucleosomes are globs of chromosomal DNA that physically hide the DNA, making whole sections of genetic information inaccessible to RNA polymerase (Figure 1).
The cellular architectural and behavioral instruction manual is encoded in the chromosomal double DNA helix. Terminally differentiated cells, however, have "locked up" irrelevant information for that cell into globs of DNA (nucleosomes) that are inaccessible to RNA polymerase. When a cell wants to make a statement, it peels off a segment of single-stranded DNA that is a comfortable fit for RNA polymerase. The inclination to peel off seductive messenger RNA (mRNA) is controlled by enhancer regions (like the glucocorticoid response element [GRE]) that can either stimulate or inhibit "upstream" promoter element enthusiasm. Lipopolysaccharide (LPS) activates nuclear factor kappa B (NF-κB)—a transcriptional regulatory protein consisting of p50 and p65 components—that translocates to the nucleus encouraging tumor necrosis factor (TNF) gene transcription by binding to its promoter elements. Conversely, steroids activate the cytosolic steroid receptor that binds the GRE and not only discourages TNF gene transcription directly but also prevents the NF-κB–TNF promoter element complex from stimulating transcription. IκBα indicates inhibitory kappa Bα.
To "fine tune" this physical (nucleosomal) control of enzyme-protein production (the retina knows it will never want to produce sperm), chromosomes are equipped with upstream accelerators/rheostats/brakes termed promoters and enhancers (Figure 1). These gene-control elements permit constituitive synthesis of myosin heavy chain (no one can ever have enough heart) or inducible synthesis of tumor necrosis factor (TNF) or inducible nitric oxide synthase (iNOS) in response to a septic challenge.
When Darwin was born, however, we only needed to live to the age of procreation. Now, the American Association or Retired Persons is a big lobby. An extravagant posttraumatic cytokine response, while innocuous to a roaming streetwise d'Artagnan from Detroit, can prove lethal to Jessie Helms. Again, multiple organ failure is partially due to the initial bacterial or ischemic hit, but in large part it is a result of the inducible systemic inflammatory conflagration perpetrated on the unsuspecting cell by evolutionarily helpful, but potentially uncivilized, proinflammatory genes. Molecular biologists deal with these guys in the library. Surgeons confront these marauders in hand-to-hand combat daily in our surgical intensive care units. It is incumbent on the clinically active, surgical cognoscenti to, as so aptly stated by former heavyweight boxing champion Joe Frazier, "know your enemy."
Families of transcription factors modulate all aspects of gene expression. Nuclear factor kappa B (NF-κB) will serve as an example of a regulatory protein that appears central to many surgically relevant clinical events (Figure 2).
Nuclear factor kappa B (NF-κB) is a cytosolic protein that, when activated by extracellular inflammatory signals, translocates to the nucleus where it docks on DNA "upstream" of several proinflammatory cytokine genes like tumor necrosis factor or interleukin-1. Thus, sepsis (or oxidants and lipopolysaccharide) begets more inflammation (via tumor necrosis factor and interleukin-1). In this fashion, NF-κB is central to transforming the initial traumatic insult into raging multiple organ failure. ARDS indicates acute respiratory distress syndrome.
NF-κB was first identified in 1986 as an inducible regulator of the expression of the κ light chain immunoglobulin gene in B cells.4 Subsequently, functional NF-κB binding sites have been identified in the promoter regions of many genes. Multiple stimuli activate NF-κB–induced gene transcription.5 As such, NF-κB plays a pivotal role in balancing the healthy immune response with pathologic inflammatory states. Understanding the mechanism of NF-κB activation during the normal inflammatory response may offer novel therapeutic strategies against exaggerated proinflammatory conditions.
Multiple stimuli, including proinflammatory cytokines, lipopolysaccharide (LPS), and reactive oxygen intermediates, activate NF-κB (Figure 2). These stimuli act through distinct signaling pathways that ultimately phosphorylate inhibitory kappa B (IκB). Phosphorylation identifies IκB for degradation by proteases.6 Once IκB is degraded, NF-κB is free to translocate into the nucleus and initiate gene transcription. Evidence favors convergence of these separate signaling pathways at IκB kinase7 (Figure 1).
Teleologically, a little proinflammatory gene expression must be good. Indeed, genetic knock-out of NF-κB in mice is associated with liver degeneration and lethal developmental abnormalities.8 Similarly, mice lacking the p50 component of NF-κB are susceptible to infection and exhibit multifocal immune defects.9 The cell appears to recognize the autodestructive inflammatory potential of unbridled NF-κB activation, and cells have evolved. Several tight autoregulatory networks act directly on NF-κB . The IκB-α gene contains a functional NF-κB promoter site. IκB production is stimulated by NF-κB .10 Thus, as soon as any NF-κB is produced, a negative feedback loop begins to shut itself down. The newly made IκB appears to suppress NF-κB activity by binding it both in the nucleus and in the cytosol.11 IκB also blocks DNA binding of NF-κB . Indeed, IκB -deficient animals exhibit high levels of activated NF-κB following stimulation.12
While IκB participates in a negative feedback loop, proinflammatory products of genes regulated by NF-κB may conversely amplify the inflammatory signal. Both TNF-α and interleukin-1 (IL-1) are NF-κB gene products, and in turn activate NF-κB. This type of positive feedback loop has the potential to extend healthy local inflammation into the pathogenic systemic inflammatory response syndrome.
Multiple agents have been described that inhibit activation of NF-κB and the subsequent production of proinflammatory cytokines.13 Glucocorticoids are potent anti-inflammatory agents that exert much of their effect through inhibition of the synthesis of proinflammatory mediators. The classic mechanism of glucocorticoid action is through activation of glucocorticoid receptors in the cytoplasm, which then move into the nucleus and activate transcription of steroid-responsive genes. However, glucocorticoids decrease production of many inflammatory mediators that have no identifiable steroid-responsive element. Evidence is accumulating that glucocorticoids exert most of their anti-inflammatory action through inhibition of NF-κB. Glucocorticoids appear to inhibit NF-κB through 2 mechanisms. First, the activated glucocorticoid receptor binds to NF-κB and prevents it from binding to DNA and activating gene transcription.14 Second, glucocorticoids induce the production of IκB which acts to suppress NF-κB activation.15,16
Aspirin is probably the oldest synthetic drug in the pharmacy today. While aspirin inhibits cyclooxygenase enzymes, aspirin and sodium salicylate also inhibit activation of NF-κB by preventing the phosphorylation or degradation (or both) of IκB.17
Other clinically accessible inhibitors of NF-κB include prostaglandin E2, tacrolimus, cyclosporin, antioxidants like vitamin E, and nitric oxide.13 In each instance, surgeons are the dominant purveyors of these agents.
The clinical importance of gene regulation is evident. The surgical scientist is uniquely positioned not only to perform a bowel anastomosis but also to manipulate gene expression at the time of surgery.
Corresponding author: Brian D. Shames, MD, Department of Surgery, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Box C-320, Denver, CO 80262.
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