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Wade Redick Wade Redick Dr. Bert Ely Biology 303 03 November 2009 The Genetics behind Temporary Excess Mucus in the Respiratory Pathways Mucus is a sugar-coated collection of proteins that is part of the body’s natural selfdefense. Mucus collects waste from air passages, which can then be released from the body in numerous ways. It is produced and secreted through differentiated epithelial cells that line certain pathways in the body. The average person produces approximately a liter of mucus per day (Hoffman). Mucus is essential to human life. Without it, almost every breath taken in would bring in pathogens that could infect the respiratory epithelial cells. Excess amounts of mucus are produced when an allergen is introduced into the system, usually into the respiratory tract. Normal epithelial tissue of the respiratory tract is lined with both Clara cells and goblet cells. Clara cells are normal, beneficial non-mucus producing cells. Goblet cells, on the other hand, are the cells that produce and secret mucus. They exist in various concentrations in many different types of epithelial linings. For instance, there is a much larger concentration of goblet cells in the intestinal tract than in respiratory tract (Chen et al. 2009). Another factor that affects goblet cells in addition to environmental allergens is the FOXA2 mRNA product. It is known that deletion or suppression of the FOXA2 mRNA transcript leads to an increased number of goblet cells (Chen et al. 2009). It is also hypothesized that the transcription factor SPDEF regulates many downstream mRNAs related to mucus production in goblet cells. The transcription factor SPDEF in healthy humans and mice is normally turned on in mucus-producing regions. Mucus is one of the first of the body’s defenses and certainly one of its most important; however, an excess of Wade Redick mucus in the body, especially in the respiratory tract, can create problems. For instance, cystic fibrosis is a genetic disorder that affects the mucus secretory glands especially in the respiratory tract, causing these glands to produce an excess amount of mucus. Cystic Fibrosis is the most common fatal disease in North America and Western Europe (Kresge). Because cystic fibrosis is a genetic disorder, the only way to cure this disease is through gene therapy. Up until now there has been no gene therapy for cystic fibrosis because scientists have not completely understood the mechanisms and genetics behind mucus secretion cells in the respiratory track. A process known as “hyperplasia” describes the previous thought about the mucusproduction at the cellular level (Chen et al. 2009). According to “hyperplasia”, scientists believed that the goblet cells were present at all times; however, when an allergen was introduced to the system, these cells would divide rapidly producing an excess amount of mucus. Also, until now little was known about the genetics behind these mucus producing cells and what caused abnormalities in these cells to produce excessive, harmful amounts of mucus. Without these crucial pieces of information, genetic diseases like cystic fibrosis that affect mucus-secreting cells cannot be cured; therefore, in the recent years there has been a large movement for research to make gene therapy readily available to prevent such diseases. A team of researchers at the Cincinnati’s Children Hospital Medical Center recently set out to change this lack of knowledge about excessive mucus-secretion in the body. Their research was focused into two main areas: the derivation of the goblet cells that produce the mucus in the respiratory epithelium and also the genes that code for these goblet cells. The team used mice in order to determine the origin of the goblet cells in the respiratory epithelium. They first labeled the Clara cells in the respiratory tract epithelium using Cre recombinase. Cre recombinase is an enzyme that can be used to turn specific genes on or off by Wade Redick recombining certain loci. In this experiment, it was used to recombine in a locus that permanently increased the expression of the lacZ reporter gene in the Clara cells in the respiratory tract (Chen et al. 2009). The lacZ reporter gene codes for β-galactosidase (β-gal), an enzyme that can be used to stain cells that produce this enzyme. This in essence “stains” the targeted cells because they have abnormally high amounts of β-gal since the lacZ reporter gene is on. Within five days of labeling the Clara cells, the labeled cells had shown extensive differentiation in the respiratory epithelium (Chen et al. 2009). At this point, no β-gal was detected in any other cells besides the Clara cells in the epithelial lining of the respiratory tract. Then, ovalbumin (OVA) was administered to the transgenic mice to create an allergic reaction and, therefore, produce excessive amounts of mucus. The results for this experiment stunned the entire team. The majority of the goblet cells now contained β-gal. This goblet cell differentiation also occurred without any signs of cell proliferation (Chen et al. 2009). In other words, few new cells were created when the ovalbumin was added. Instead, the goblet cells appeared to have originated from the Clara cells. This experiment provides strong evidence that the mucusproducing goblet cells in the respiratory epithelial originate from Clara cells. The team also believes that this process of Clara cells converting into goblet cells is “rapidly reversible” (Chen et al. 2009). This means that when the cause of the transformation was removed from the system, the goblet cells turned back into the original healthy Clara cells. In essence, the team was able to control this conversion by removing the allergen. This reversibility shocked the entire scientific community as this could open many doors for possible therapies. An independent study led by Dr. Takeyama observed the reversibility of this transformation into goblet cells as well; furthermore, they found two inhibitors that block the transformation of Clara cells into goblet cells. According to their findings, “the first of the inhibitors is able to impede the activity of Wade Redick epidermal growth factor receptor (EGFR). EGFR was persistently overactive in the ciliated airway cells in mice with the asthma-like condition. By blocking EGFR, the inhibitor prevented the buildup of ciliated cells [that produce excessive mucus]” (Takeyama et al. 1999). The second method interferes with major signaling pathways that are responsible for the transformation into goblet cells in the respiratory tract. Conclusively, both studies do not support the previous theory of “hyperplasia.” Rather, they support a new theory dubbed “metaplasia,” where Clara cells convert into goblet cells when an allergen is introduced into the system. The second objective of the Cincinnati team was to determine a way to genetically combat excessive mucus. As mentioned earlier, the transcription factor SPDEF is believed to have a relationship to the goblet cells in the epithelial linings throughout the body, but little evidence has been shown to support this up until now. The team at Cincinnati Children’s Hospital Medical Center saw this as their opportunity to genetically inhibit excessive mucus production. They used Laser Capture Microdissection (LCM), which is the use of a laser to remove cells to sample the epithelial tissue (Chen et al. 2009). Doxycycline, an antibiotic that can also be used genetically to turn genes on/off, was used to induce the SPDEF transcription factor in the respiratory tract which induced goblet cell production. The team obtained epithelial cells using LCM prior to administering the doxycycline and 3 days after adminstration. The team used microarrays to show that the SPDEF transcription factor influenced the expression of 306 unique mRNAs (Fig. 1). Many of the genes that were affected are involved with mucin production, which is the major component of mucus. For instance, mucin 16 (Muc16) was heavily induced by the SPDEF transcription factor; on the other hand, SPDEF also inhibited certain genes that coded for Clara cell differentiation products. For instance, FOXA2, which leads to prevention of the proliferation of goblet, was blocked by SPDEF. These experiments Wade Redick provide evidence for the notion that the transcription factor SPDEF regulates genes that code for mucus components and also inhibits genes that code for anti-mucus agents. Figue 1: SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production. Left bar indicates gene expression prior to administration of antibiotic where right bar indicates 3 days after administration. The research team at Cincinnati’s Children Hospital Medical Center took it one step further by experimenting with the idea that SPDEF is essential to mucus production. Two groups of mice were genetically engineered: mice with SPDEF turned on and those with SPDEF turned off. Then, ovalbumin was introduced into the lungs of the two groups of mice. The overall anatomy of the lungs did not change (Chen et al. 2009). The mice with SPDEF off showed no morphological signs of goblet cells. They also did not show any expression of the previously mentioned genes that coded for mucus products. On the other hand, in the mice with SPDEF turned on large numbers of goblets cells were observed using a microscope. Also, the genes that were responsible for mucus production, such as Muc16, were expressed at higher levels when SPDEF was turned on. These results provide strong evidence for the idea that the transcription Wade Redick factor SPDEF is essential to goblet cell differentiation in the respiratory tract. In other words, SPDEF regulates genes that regulate the production of mucus. The most interesting aspect to take away from the studies is the reversibility of the differentiation of Clara cells into goblet cells. If the genetic defect is removed, the goblet cells that produce excessive mucus and cause problems for the body will turn back into to healthy Clara cells. This reversibility provides a possible pathway for treatments in the future. With this knowledge about the transcription process behind mucus production, cures and preventions for diseases, such as cystic fibrosis, are more likely. Even the common cold, which currently has no cure, possible could be treated more effectively; however, it may be a while before this therapy becomes a viable option. Dr. Whitesett of the Cincinnati’s Children Hospital team believes it will be a couple years before this knowledge and technology can start to be applied to therapeutic uses (Chen et al. 2009). However, recent progress in a study led by Dr. Nadel of the University of California- San Francisco may bring a cure sooner than scientists believe. Like the team at Cincinnati’s Children Hospital, Dr. Nadel and his colleagues studied the induction of goblet cells through introduction of an allergen into mice pulmonary system. They hypothesized that when an allergen is introduced, a chemical signal is sent to the genes that the team at Cincinnati’s Children Hospital discovered that code for mucin producing proteins. This chemical messenger appeared to be produced by tyrosine kinase, an enzyme that produces signals in many internal cellular communications (Tyner et al. 2006). Dr. Nadel believes that they can use specific antibiotics to block the tyrosine kinase and, therefore, prevent the relay of the signal to the mucus-related genes. Although it may appear gene therapy and cures for mucus diseases like cystic fibrosis are far away, scientists, such as Dr. Nadel and Dr. Holtzman, believe that they are on the brink of a breakthrough. Wade Redick Works Cited Cincinnati Children's Hospital Medical Center. "Scientists Identify Gene For Short-circuiting Excess Mucus In Lung Disease, Common Colds." ScienceDaily 15 September 2009. 5 November 2009 <http://www.sciencedaily.com /releases/2009/09/090914172332.htm>. Hoffman, Douglas. "Mucus Flakes from Nose." Your Total Health. NBC, 2009. Web. 6 Nov. 2009. <http://yourtotalhealth.ivillage.com/mucus-flakes-fromnose.html&cpsextcurrchannel=1>. Kresge, Nicole. "Scientists discover basic defect in cystic fibrosis airway glands." EurekAlert! 17 Mar. 2006: n. pag. Web. 5 Nov. 2009. <http://www.eurekalert.org/pub_releases/200603/asfb-sdb031706.php>. Chen, Korfhagen, Xu, Kitzmiller, Wert, Maeda, Gregorieff, Clevers, and Whitsett. "SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production." Journal of Clinical Investigation 119.10 (2009): 2914-24. PDF file. Takeyama, Kiyoshi, et al. "Epidermal growth factor system regulates mucin production in airways." Proceedings of the National Academy of Sciences 96.6 (1999): 3081-3086. Proceedings of the National Academy of Sciences. Web. 22 Nov. 2009. <http://www.pnas.org/content/96/6/3081.full.pdf+html>. Tyner JW, et al. “Blocking airway mucus cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferention signals.” Journal of Clinical Investigation 116(2) (2006). Web. 22 Nov. 2009.