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Transcript
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
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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
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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
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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
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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
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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&ampcpsextcurrchannel=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.