University of Southern California Dissertations and Theses

Peripheral myelin protein 22 promotes intestinal epithelial cell survival and barrier function maintenance

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Peripheral myelin protein 22 promotes intestinal epithelial cell survival and barrier function maintenance

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Content Peripheral myelin protein 22 promotes intestinal epithelial cell
survival and barrier function maintenance
by
Zihe Shi
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
[BIOCHEMISTRY AND MOLECULAR MEDICINE]
August 2024
Copyright 2024 Zihe Shi

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Acknowledgments
I would like to express my heartfelt gratitude to my mentor, Dr. Mark Frey. Dr. Frey is an easygoing, responsible, and caring mentor who has shown great patience and support since I joined the
lab. Despite my initial lack of laboratory experience, he has been accommodating. It has been an
honor to learn a variety of experimental techniques in our lab, including mammalian cell culture,
microscopy, and mouse handling.
I am also immensely thankful for the support and assistance of my fellow lab members, including
Jonathan Hsieh, Ashley Lehr, and Edie Bucar. I would like to extend special thanks to Jonathan
Hsieh for continuously teaching me various experimental skills, introducing new concepts, guiding
me through learning processes, and helping me refine posters and assignments. I truly appreciate
all the help I received during my time in the lab.
My gratitude also extends to my committee members, Dr. Xu Jian and Dr. Ching-ling Lien, for their
willingness to serve on my committee and for their invaluable feedback and suggestions on both my
project and thesis.
Last but not least, I am forever indebted to my parents, Chunmao Shi and Li Hu, for their
unwavering support and play significant roles in all aspects of my life. Thank you all for being my
constant support and encouragement.

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Table of Contents
Acknowledgments .......................................................................................................................................................................................... ii
List of Figures ................................................................................................................................................................................................... iv
Abstract .............................................................................................................................................................................................................. iv
List of Abbreviations .......................................................................................................................................................................................v
Chapter 1. Background and Introduction ............................................................................................................................................. 1
1.1 Background ........................................................................................................................................................................................... 1
1.2 ErbB3 deletion affects epithelial cells' barrier function and reduces Pmp22 expression ................................. 2
1.3 Pmp22 knockdown decreases barrier function and epithelial wound healing ...................................................... 4
1.4 Exploring the Impact of PMP22 on Epithelial Cell Physiology ....................................................................................... 5
Chapter 2 Materials and Methods ............................................................................................................................................................ 5
2.1 Cell Culture and Maintenance ....................................................................................................................................................... 5
2.2 Pmp22 Knockdown in Caco-2bbe Cells..................................................................................................................................... 5
2.3 Cell Counting Assay ........................................................................................................................................................................... 6
2.4 EdU Labeling Assay ........................................................................................................................................................................... 6
2.5 Adhesion Assay ................................................................................................................................................................................... 6
2.6 Apoptosis Assay .................................................................................................................................................................................. 6
2.7 Protein extraction .............................................................................................................................................................................. 7
2.8 Protein Assay........................................................................................................................................................................................ 7
2.9 Western Blotting ................................................................................................................................................................................. 7
Chapter 3 Pmp22 knockdown cells show reduced cell counts and increased apoptotic responses. ......................... 8
3.1 Decreased Cell Counts with Pmp22 Knockdown .................................................................................................................. 8
3.2 EdU Labeling Reveals No Change in Proliferation Rate After Pmp22 Knockdown. .............................................. 9
3.3 Adhesion Assay Shows Pmp22 Knockdown Does Not Affect Cell Adhesion. ......................................................... 10
3.4 Increased Apoptosis in Pmp22 Knockdown Cells Under Stress Conditions. ......................................................... 11
Chapter 4 Elevated p-AKT and BAX Expressions in PMP22 Knockdown Cells ................................................................. 12
4.1 Increased BAX expression and unchanged Bcl-2 levels in Pmp22 knockdown cells ......................................... 12
4.2 Increased p-AKT Expression in Pmp22 Knockdown Cells Following NRG-1β Treatment............................... 13
Chapter 5 Discussion and Future Directions .................................................................................................................................... 15
References ........................................................................................................................................................................................................ 17

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List of Figures
Figure 1.1 Assessing Intestinal Barrier Permeability in ErbB3-Deficient
Mice Using FITC-Dextran(by Jonathan Hsieh). .............................................................................................................2
Figure 1.2 RNA seq data shows Pmp22 expression decreased in
ErbB3-IEKO mice (by Jonathan Hsieh). ...........................................................................................................................3
Figure 1.3 Pmp22 knockdown decreases barrier function in
Caco2bbe monolayers (by Jonathan Hsieh). ..................................................................................................................4
Figure 2.1 Pmp22 Knockdown Reduces Cell Numbers Over Time ......................................................................8
Figure 2.2 EdU Incorporation was Unaffected by Pmp22 Knockdown .............................................................9
Figure 2.3 Adhesion Capacity Unaffected by Pmp22 Knockdown ....................................................................10
Figure 2.4 Pmp22 Knockdown Cells show exaggerated apoptosis after
TNF and CHX Treatment ......................................................................................................................................................11
Figure 3.1 Differential Expression of BAX and Stable Bcl-2 Levels in
Pmp22 Knockdown cells .....................................................................................................................................................13
Figure 3.2 Increased p-AKT Expression in shPmp22 Knockdown Cells .........................................................14

Abstract

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This study explores the role of Peripheral Myelin Protein 22 (PMP22) in the regulation of intestinal
epithelial cell functions. PMP22 is present at the tight junctions in intestinal epithelia, and
preliminary data from our lab show that loss of expression results in barrier leakage. Here, we
tested the effects of PMP22 knockdown on key cellular processes that contribute to barrier
function. We found that loss of PMP22 decreases cell survival and impairs wound
healing/migration, while not significantly altering cell proliferation or simple adhesion to the
culture substrate. Notably, PMP22 knockdown cells showed increased sensitivity to apoptosis,
particularly under stress conditions induced by tumor necrosis factor (TNF) and cycloheximide
(CHX). Western blot analysis further revealed an upregulation of phospho-AKT following treatment
with neuregulin-1β, suggesting compensatory activation of survival pathways. Additionally, an
increase in BAX expression without a corresponding change in Bcl-2 was observed, indicating a
shift towards pro-apoptotic signaling. These results suggest that PMP22 is involved in protective
mechanisms that maintain epithelial barrier integrity, which is crucial for preventing disorders
such as inflammatory bowel disease (IBD).
List of Abbreviations
ERBB3 - ErbB3 Receptor Tyrosine Kinase

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ErbB3-IEKO - ErbB3 Intestinal Epithelium-specific Knockout
DSS - Dextran Sulfate Sodium
FITC-dextran - Fluorescein Isothiocyanate-dextran
TEER - Trans-epithelial Electrical Resistance
Nrg-1β - Neuregulin-1 beta
PMP22 - Peripheral Myelin Protein 22
IBD - Inflammatory Bowel Disease
PBS - Phosphate Buffered Saline
RIPA - Radioimmunoprecipitation Assay Buffer
SDS - Sodium Dodecyl Sulfate
BSA - Bovine Serum Albumin
TNF - Tumor Necrosis Factor
CHX - Cycloheximide
TBST - Tris-buffered saline, 0.1% Tween 20
RT - Room Temperature
MES - 2-(N-morpholino) ethane sulfonic acid

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Chapter 1. Background and Introduction
1.1 Background
Inflammatory bowel disease (IBD), a set of chronic conditions including Crohn's disease and
ulcerative colitis, is characterized by a complex etiology involving an unregulated immune reaction
and recurring damage to the intestinal epithelial barrier. A regulated epithelial barrier is crucial for
enabling the absorption of nutrients while maintaining separation between the organism and the
gut contents. The barrier is actively sustained through processes like epithelial cell proliferation,
migration, and differentiation. All these processes can be disrupted in the development of IBD.
Recent studies have shown that the ERBB3 receptor tyrosine kinase is an important regulator of the
intestinal barrier. Our lab’s earlier research showed that mice with a targeted deletion of ErbB3 in
the intestinal epithelium (ErbB3-IEKO) display an increased level of inflammatory cytokines such as
IFN-ɣ and TNF and are more susceptible to DSS-induced colitis (Almohazey et al., 2017). These
results implicate ERBB3 as a key participant in maintaining the integrity of the intestinal epithelial
barrier, but the mechanisms are not well-understood.

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1.2 ErbB3 deletion affects epithelial cells' barrier function and reduces
Pmp22 expression
Figure 1.1 Assessing Intestinal Barrier Permeability in ErbB3-Deficient Mice Using FITC-Dextran (by Jonathan
Hsieh). A) Serum fluorescence was measured four hours post-gavage to assess the tracer's translocation into the
bloodstream. B) Individual fluorescence values for both ErbB3 FF (control) and ErbB3 IEKO (knockout) mice. A marked
increase in serum fluorescence in the ErbB3 IEKO group suggests a compromised barrier. Statistical analysis revealed
significant differences (**p < 0.01).
In preliminary studies conducted by Jonathan Hsieh in the lab (Figure 1.1), FITC-dextran 4kDa, a
fluorescent tracer, was administered orally to mice to assess intestinal permeability. Blood samples
were collected four hours post-administration for analysis. Results showed ErbB3-IEKO mice
exhibited a significantly higher FITC signal in their bloodstream than wild-type littermates. This
outcome suggests that the intestinal barrier in ErbB3 deletion mice is more permeable, or “leaky,”
compared to normal mice. These findings lend further support to the crucial role of ErbB3 in
maintaining the integrity of the intestinal barrier.

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Figure 1.2 RNA seq data shows Pmp22 expression decreased in ErbB3-IEKO mice (by Jonathan Hsieh).
RNA sequencing analysis reveals significant changes in gene expression between wild-type (FF) and ErbB3 intestinal
epithelium knockout (KO) mice. Expression of the gene coding for peripheral Myelin Protein 22 (Pmp22) is notably
downregulated in KO mice, as highlighted by the heatmap and confirmed by a fold change of 0.290 and a q-value of 1.78E05.
Analysis of the ErbB3-deficient murine intestinal epithelium through RNA-sequencing by Jonathan
Hsieh (Figure 1.2) identified a marked reduction in the expression of Peripheral Myelin Protein 22
(Pmp22) RNA. While the PMP22 protein is integral to the myelin sheath in peripheral nerves and is
found in multiple tissues such as the gastrointestinal tract, its function in epithelial cells is not welldefined. However, PMP22's known interactions with tight junctions in other tissues and its role in
regulating myelin permeability (Guo et al., 2014) suggest it may be a mediator of ERBB3-related
epithelial barrier function.

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1.3 Pmp22 knockdown decreases barrier function and epithelial wound
healing
Figure 1.3 Pmp22 knockdown decreases barrier function in Caco2bbe monolayers (by Jonathan Hsieh).
Caco2bbe colonic epithelial cells stably transfected with shRNA against Pmp22 (shPmp22) or non-targeting shRNA (shNT)
were assessed for trans-epithelial electrical resistance (TEER). n = 3. *, p < 0.05.
In Jonathan Hsieh's exploration of PMP22’s role in maintaining epithelial barrier function (Figure
1.3), we assessed the impact of reduced PMP22 in Caco2bbe cell monolayers. Using trans-epithelial
electrical resistance (TEER), a measure of barrier tightness that gauges the ease of ion flow across
cell layers, we were able to quantify barrier integrity. The Caco2bbe cells, a well-established model
for studying barrier function, were modified to express lower levels of PMP22 (shPmp22) and
compared to cells with unaltered PMP22 expression (shNT). The result revealed that a decrease in
PMP22 led to reduced TEER, indicating a compromised barrier.
shPmp22

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1.4 Exploring the Impact of PMP22 on Epithelial Cell Physiology
The preliminary data described above suggest that the loss of PMP22 detrimentally impacts
epithelial barrier function and wound repair mechanisms. This could be through several
mechanisms. PMP22 depletion could result in diminished cell proliferation, elevated apoptotic
activity, altered cell migration/wound healing, or purely a change in tight junction structure. In this
study, the aim is to delineate how PMP22 influences proliferation, apoptosis, and wound healing in
order to understand its potential as a key regulator of epithelial homeostasis and tissue repair.
Chapter 2 Materials and Methods
2.1 Cell Culture and Maintenance
Caco-2bbe cells (ATCC #CRL-2102) were cultured in DMEM medium supplemented with 10% heatinactivated FBS, 1% penicillin-streptomycin, and insulin transferrin selenium supplement (BD
Biosciences). The cells were passaged using 0.05% trypsin-EDTA upon reaching 70-80%
confluency.
2.2 Pmp22 Knockdown in Caco-2bbe Cells
To create a PMP22-deficient model, Caco-2bbe cells were transfected with MISSION lentiviral
particles containing shRNA against Pmp22, supplied by Sigma. A control group with a non-specific
scrambled shRNA sequence was also developed. Following the transfection, the G418 antibiotic was
used to select successfully transfected cells, and the reduction in Pmp22 was verified through
quantitative real-time PCR.

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2.3 Cell Counting Assay
To begin to evaluate cellular proliferation and survival, we performed a cell-counting assay. Cells
were plated at a density of 1 × 10^5 per well in 4-well chamber slides, with treatments including
both the addition and omission of neuregulin (Nrg)-1β, which activates ErbB3 signaling. After 1, 3,
and 6 days of culture, the cells were fixed with 4% formaldehyde, stained with Hoechst 33342, and
counted using a fluorescent microscope.
2.4 EdU Labeling Assay
To specifically investigate cell proliferation, an EdU labeling assay was performed. After a 6-day
culture period, cells were exposed to EdU for 2 hours before fixation. Cells actively incorporating
EdU into DNA (a surrogate for S phase of cell division) were labeled using the Click-iT EdU Alexa
Fluor 488 Imaging Kit (Thermo Fisher Scientific, Catalog #C10337). The percentage of cells
incorporating EdU was then determined through fluorescence microscopy.
2.5 Adhesion Assay
In the adhesion assay, equal numbers of control and Pmp22 knockdown cells were seeded. After 4
h, nonadherent cells were washed away with PBS and the remaining cells were fixed with
formaldehyde and stained with Hoechst 33342. The adherence of cells to the substrate was then
observed and counted under a fluorescence microscope.
2.6 Apoptosis Assay
Knockdown and wild-type cells were seeded in 96-well plates at a density of 1 × 10^5 per well.
After 48 h cultures were exposed to vehicle, TNF, and CHX, either alone or together, for 5 hours.

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After treatment, apoptosis was evaluated using a caspase-3/7 green ReadyProbesTM reagent
(Thermo Fisher Scientific, Catalog #R37111) detection assay.
2.7 Protein extraction
Protein Extraction Cells were washed with phosphate-buffered saline (PBS) and lysed on ice using
RIPA buffer for 10 minutes. The lysates were then centrifuged at 10,000 rpm for 10 minutes at 4°C.
The supernatant was transferred to a new tube, with a small aliquot (approximately one-fifth)
reserved for protein quantification. The remaining lysate was mixed with an appropriate amount of
4X sample buffer, heated for 5 minutes at 95°C, vortexed, and briefly spun down. Samples were
then stored at -80°C until further analysis.
2.8 Protein Assay
Protein concentrations were determined using a standard curve prepared with known
concentrations of bovine serum albumin (BSA). Standards included a blank, 2.5 µL, 5 µL, and 10 µL
of BSA (2 mg/mL). After adding reagents A, S, and B, the reaction was incubated for 15 minutes
before measuring absorbance using a protein assay machine.
2.9 Western Blotting
Proteins were resolved on Bolt™ Bis-Tris Plus Mini Protein Gels (4-12%, 1.0 mm, WedgeWell™
format, Thermo Fisher Scientific, Catalog #NW04122BOX) using 1X Bolt MES SDS running buffer.
Protein (30 µg) was loaded per well, and 5 µL of Pageruler Plus Prestained Protein Ladder was
added to the first lane. Electrophoresis was conducted at 110V. Proteins were transferred to
nitrocellulose membranes using the iBlot Gel Transfer Device. Membranes were blocked with 5%
milk for 1 hour at room temperature, incubated with primary antibodies at 1:1000 dilution

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overnight at 4°C, followed by several washes with TBST, and incubated with fluorescently labeled
secondary antibodies suitable for detection at 700 nm and 800 nm. After further washes, the
membranes were scanned using the Odyssey M imaging system and quantified using the Empiria
Studio software.
Chapter 3 Pmp22 knockdown cells show reduced cell counts and
increased apoptotic responses.
3.1 Decreased Cell Counts with Pmp22 Knockdown
The cell counting assay was performed to assess the impact of Pmp22 knockdown on Caco-2bbe cell
density over time. Over periods of 1, 3, and 6 days, cells with reduced Pmp22 levels exhibited a
notable decrease in cell count compared to control groups, even in the presence of the survival
factor neuregulin-1β (Nrg-1β). This suggests that Pmp22 either plays a crucial role in maintaining
cell viability or driving proliferation (Figure 2.1).
Figure 2.1 Pmp22 Knockdown Reduces Cell Numbers Over Time
Caco-2bbe cells with or without Pmp22 knockdown were plated at equal densities and the remaining cells were counted

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after 1, 3, and 6 days, both with and without NRG-1β (n=4). (A) Representative images showing cell density at day 6. (B)
Quantification over time. **, p < 0.01, ns denotes no significant difference.
3.2 EdU Labeling Reveals No Change in Proliferation Rate After Pmp22
Knockdown.
The EdU labeling assay was used to determine if Pmp22 depletion affects the proliferation of
intestinal epithelial cells. By marking newly formed DNA, this assay identifies cells in the process of
dividing. Additional DAPI staining allowed for a count of total cells. The analysis showed no
significant difference in the proportion of EdU-positive cells between the control and the Pmp22
knockdown groups, regardless of NRG presence or absence. This indicates that the decrease in cell
numbers seen in Pmp22 knockdown cultures may not be due to changes in proliferation rates
(Figure 2.2).
Figure 2.2 EdU Incorporation was Unaffected by Pmp22 Knockdown.
(A) Representative images showing proliferation signal in control Caco2bbe cells. EdU (green) marks actively cycling cells
with DAPI (blue) as a counterstain for all cells. (B) Control and Pmp22 knockdown cells +/- NRG-1β were counted (n=3).
No significant differences were detected.

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3.3 Adhesion Assay Shows Pmp22 Knockdown Does Not Affect Cell
Adhesion.
To rule out the possibility of cell count results (Figure 2.1) reflecting decreased adhesion to the
culture plate, control and knockdown cells were seeded in equal numbers for 4 hours. After this
period, non-adherent cells were washed away, and adherent cells were fixed for counting.
Observations made using a fluorescence microscope showed no significant differences between
control and Pmp22 knockdown groups (Figure 2.3). These results suggest that Pmp22 knockdown
does not affect the cells’ adhesion capabilities to the extracellular matrix.
Figure 2.3 Adhesion Capacity Unaffected by Pmp22 Knockdown.
Equal numbers of shNT control and shPmp22 cells were seeded on plates. After 4 hours, plates were washed, fixed, and
the remaining cells counted.

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3.4 Increased Apoptosis in Pmp22 Knockdown Cells Under Stress
Conditions.
The apoptosis assay demonstrated a notable increase in cell death in shPmp22 knockdown cells
relative to shNT when treated with both tumor necrosis factor (TNF) and cycloheximide (CHX)
(Figure 2.4). This increased vulnerability suggests that Pmp22 may help protect cells under stress, a
function that could be vital for preserving epithelial barrier integrity during inflammatory
conditions.
Figure 2.4 Pmp22 Knockdown Cells show exaggerated apoptosis after TNF and CHX Treatment.
Caco2bbe cells expressing shPmp22 or shNT were challenged with TNF (100 ng/ml), CHX (1 ug/ml), or both. Apoptosis
rates were quantified by incubation with a fluorescent caspase substrate (n=3), quantified in (A) with example images in
(B). ** for p < 0.01, *** for p < 0.001

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Chapter 4 Elevated p-AKT and BAX Expressions in PMP22 Knockdown
Cells
4.1 Increased BAX expression and unchanged Bcl-2 levels in Pmp22
knockdown cells
The Western blot analysis examined the expression levels of BAX and Bcl-2 in response to Pmp22
knockdown in Caco2bbe cells. The BAX/Bcl-2 ratio is a significant indicator of a cell's susceptibility
to apoptosis, affecting intrinsic apoptotic pathways. An increase in this ratio suggests a higher
apoptotic potential, due to BAX's pro-apoptotic and Bcl-2's anti-apoptotic roles (Oltvai et al., 1993;
Gross et al., 1998). The results show a significant increase in BAX expression in the Pmp22
knockdown lines (A2, A3, 3D) compared to the control (shNT), while Bcl-2 levels remained
consistent across all samples (Figure 3.1). This change indicates a potential shift towards a proapoptotic state in the Pmp22-deficient cells, which could lead to increased cell death under stress
conditions.

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Figure 3.1 Differential Expression of BAX and Stable Bcl-2 Levels in Pmp22 Knockdown cells.
(A) Representative Western blot showing BAX, Bcl-2, and Actin protein expression levels. Control cells (shNT) and three
Pmp22 knockdown cell lines (A2, A3, 3D) are included. BAX shows varied expression across the samples, while Bcl-2
expression is faint and exhibits no discernible differences between control and knockdown cells. Actin is a loading control
to ensure equal protein loading across the lanes. (B) Quantification of BAX expression normalized to Actin shows
significantly increased BAX levels in shPmp22 cells compared to shNT (n=2). * for p < 0.05
4.2 Increased p-AKT Expression in Pmp22 Knockdown Cells Following
NRG-1β Treatment
p-AKT, a phosphorylated form of AKT kinase, plays a critical role in cellular processes such as
survival, growth, and proliferation, primarily mediated through signal transduction pathways that
prevent apoptosis and promote cell viability (Manning and Toker, 2017; Hers et al., 2011). In this

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context, we aimed to explore the modulation of p-AKT in response to genetic alterations in PMP22,
particularly under conditions influenced by NRG-1β, a known activator of the AKT signaling
pathway.
Before analyzing p-AKT, we treated cells with NRG-1β for 15 minutes. Without this activation step,
p-AKT levels showed minimal differences between shPmp22 knockdown and control cells, being
barely detectable. Following NRG-1β treatment, there was a noticeable enhancement in p-AKT
expression in shPmp22 cells, especially in the A2 cell line, compared to controls (Figure 3.2). This
indicates that the absence of PMP22 may alter the cell's response to AKT pathway activation.
To ensure that the observed changes in p-AKT levels were not due to variations in the total AKT
protein availability, we also assessed the expression of total AKT across all cell lines. The analysis
confirmed that all cell lines expressed normal levels of total AKT, thus excluding the possibility that
the differences in p-AKT were due to inherently low AKT protein levels in certain lines.

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Figure 3.2 Increased p-AKT Expression in shPmp22 Knockdown Cells
(A) Representative Western blot displaying levels of p-AKT, after cells have been treated with NRG-1β for 15 mins before
protein extraction, and Actin proteins. Samples include control cells (shNT) and three Pmp22 knockdown lines (A2, A3,
3D), with Actin as the loading control. (B) Quantitative analysis of p-AKT expression normalized to Actin shows significant
increases in p-AKT levels in the shPmp22 cells, especially in A2, compared to shNT. * for p < 0.05, ** for p < 0.01, *** for p
< 0.001.
Chapter 5 Discussion and Future Directions
This study underscores the significant role of Pmp22 in intestinal epithelial cells, particularly its
ability to protect against cell death under stressful conditions—a key consideration in diseases such
as inflammatory bowel disease (IBD) where barrier function is compromised. The results show that
while Pmp22 knockdown leads to increased apoptosis. In contrast, it does not impact cell
proliferation or basic adhesion, indicating that the effects on apoptosis are selective.

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Further analysis examined the expression of p-AKT and the BAX/Bcl-2 ratio in Pmp22 knockdown
cells. Notably, we observed an increase in p-AKT expression following NRG-1β treatment, which
might suggest that the cells are trying to compensate for the loss of PMP22. Essentially, the cells
might be boosting their survival signals, through the AKT pathway, to make up for the instability
caused by lower PMP22 levels. This could potentially be a way for the cells to maintain their health
and function despite the disruption. Moreover, the increase in BAX expression without a
corresponding change in Bcl-2 levels highlights a shift towards pro-apoptotic signaling. This pattern
mirrors findings in human small intestinal adenocarcinoma, where increased apoptosis and BAX
expression, alongside stable Bcl-2 levels, have been reported (Gao and Wang, 2009), supporting the
relevance of these markers in intestinal pathology.
To further investigate PMP22's role in epithelial integrity and inflammation, developing an in vivo
mouse model could be highly beneficial. One practical approach is to generate Pmp22 knockout
mice. We could also perform rescue experiments, which involve reintroducing PMP22 back into the
knockout mice, potentially through targeted gene therapy using viral vectors. We can assess if
restoring PMP22 reverses the symptoms associated with its absence by comparing specific
biomarkers, such as inflammation markers or other indicators of gastrointestinal health.
Such experiments would extend our understanding of PMP22's roles, as outlined by Naef and Suter
(1998), who discuss PMP22's involvement in the stability of myelin and axonal maintenance,
suggesting its broader regulatory roles could also influence intestinal epithelial cell functions.

Page | 17
References
Almohazey, D., Lo, Y. H., Vossler, C. V., Simmons, A. J., Hsieh, J. J., Bucar, E. B., Schumacher, M. A.,
Hamilton, K. E., Lau, K. S., Shroyer, N. F., & Frey, M. R. (2017). The ErbB3 receptor tyrosine kinase
negatively regulates Paneth cells by PI3K-dependent suppression of Atoh1. Cell death and
differentiation, 24(5), 855–865. https://doi.org/10.1038/cdd.2017.27
Frey, M. R., Dise, R. S., Edelblum, K. L., & Polk, D. B. (2006). p38 kinase regulates epidermal growth
factor receptor downregulation and cellular migration. The EMBO journal, 25(24), 5683–5692.
https://doi.org/10.1038/sj.emboj.7601457
Gao, C., & Wang, A. Y. (2009). Significance of increased apoptosis and Bax expression in human
small intestinal adenocarcinoma. The journal of histochemistry and cytochemistry : official journal of
the Histochemistry Society, 57(12), 1139–1148. https://doi.org/10.1369/jhc.2009.954446
Gross, A., Jockel, J., Wei, M. C., & Korsmeyer, S. J. (1998). Enforced dimerization of BAX results in its
translocation, mitochondrial dysfunction and apoptosis. The EMBO journal, 17(14), 3878–3885.
https://doi.org/10.1093/emboj/17.14.3878
Guo, J., Wang, L., Zhang, Y., Wu, J., Arpag, S., Hu, B., Imhof, B. A., Tian, X., Carter, B. D., Suter, U., & Li, J.
(2014). Abnormal junctions and permeability of myelin in PMP22-deficient nerves. Annals of
neurology, 75(2), 255–265. https://doi.org/10.1002/ana.24086

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Hers, I., Vincent, E. E., & Tavaré, J. M. (2011). Akt signalling in health and disease. Cellular
signalling, 23(10), 1515–1527. https://doi.org/10.1016/j.cellsig.2011.05.004
Manning, B. D., & Toker, A. (2017). AKT/PKB Signaling: Navigating the Network. Cell, 169(3), 381–
405. https://doi.org/10.1016/j.cell.2017.04.001
Naef, R., & Suter, U. (1998). Many facets of the peripheral myelin protein PMP22 in myelination and
disease. Microscopy research and technique, 41(5), 359–371. https://doi.org/10.1002/(SICI)1097-
0029(19980601)41:5<359::AID-JEMT3>3.0.CO;2-L
Oltvai, Z. N., Milliman, C. L., & Korsmeyer, S. J. (1993). Bcl-2 heterodimerizes in vivo with a
conserved homolog, Bax, that accelerates programmed cell death. Cell, 74(4), 609–619.
https://doi.org/10.1016/0092-8674(93)90509-o

Abstract (if available)

Abstract This study explores the role of Peripheral Myelin Protein 22 (PMP22) in the regulation of intestinal epithelial cell functions. Through a series of assays, we discovered that PMP22 knockdown leads to decreased cell survival and impaired migration, while not significantly altering cell proliferation or simple adhesion to the culture substrate. Notably, PMP22 knockdown cells showed increased sensitivity to apoptosis, particularly under stress conditions induced by tumor necrosis factor (TNF) and cycloheximide (CHX). These results suggest that PMP22 is involved in protective mechanisms that maintain epithelial barrier integrity, which is crucial for preventing disorders such as inflammatory bowel disease (IBD). Future studies will delve into the signaling pathways impacted by PMP22, with a focus on the PI3K pathway, known for its role in cell survival, and the p53 tumor suppressor pathway. Additionally, we aim to investigate more complex cell-to-cell adhesion interactions to further understand the effects of PMP22 knockdown on epithelial barrier properties.

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Creator Shi, Zihe (author)

Core Title Peripheral myelin protein 22 promotes intestinal epithelial cell survival and barrier function maintenance

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2024-08

Publication Date 07/26/2024

Defense Date 06/27/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag intestinal epithelial cell,PMP22

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Frey, Mark (committee chair), Lien, Chingling (committee member), Xu, Jian (committee member)

Creator Email ziheshi@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113998G12

Unique identifier UC113998G12

Identifier etd-ShiZihe-13275.pdf (filename)

Legacy Identifier etd-ShiZihe-13275

Document Type Thesis

Format theses (aat)

Rights Shi, Zihe

Internet Media Type application/pdf

Type texts

Source 20240730-usctheses-batch-1187 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu