Extracellular Matrix Protein CCN1 (Cyr61) Negatively Regulates Endothelial Cell Adhesion ...

151 Pages·2015·4.85 MB·English

Extracellular Matrix Protein CCN1 (Cyr61) Negatively Regulates
Endothelial Cell Adhesion Molecules

by
Katherine Ringo

Submitted in Partial Fulfillment of the
Requirements for the Degree
Doctor of Philosophy

Supervised by Professors David A. Dean and Jennifer L. Young

Program in Toxicology
School of Medicine and Dentistry
University of Rochester
Rochester, NY

2015
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Biographical Sketch

Katherine Ringo was born in Rochester, NY in 1984. She attended Stanford University,
and graduated with a Bachelor of Science degree in Biological Sciences in 2008. After
college, she worked as a technician in the laboratory of Jennifer L. Young. She began
doctoral studies in Toxicology at the University of Rochester in September 2009. She
pursued her research on the role of CCN1 in pulmonary inflammation under the direction
of Jennifer L. Young and David A. Dean, and received a Masters of Science in Toxicology
in 2011.

The following publication was the result of work conducted during doctoral study:
Ringo, Katherine, Rosemary Norman, and Jennifer L. Young (2015) “Extracellular Matrix
Protein CCN1 (Cyr61) Negatively Regulates Endothelial Cell Adhesion Molecules.” In
Preparation.
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Acknowledgements
There are many people I would like to thank for their support and help as I worked on this
dissertation.
Past and present members of the Dean and Young labs, I am so lucky to have you all as
co-workers, so that I am able to say that my lab has been my second family. My fellow
graduate students, Melissa, Mootaz, Marta, Ben, Khatera, Fredrik, Christiaan, and Haiqing
have been great friends and great sympathizers in times of epic experimental/life fails.
The indomitable Xin has been a fantastic teacher to me in the lab. In the dadlab, Mike,
Holly, Lynn, Becky, Matt, and Pratik have all made the lab a wonderful place. Rosemary
has been tremendously helpful with experiments and, of course, organizing.
I would like to thank my mentor, Jen, whose ceaseless support, encouragement,
enthusiasm have been instrumental in getting me through graduate school. I have given
her enough frustration for a career’s worth of grad students, but she has not only helped
me develop as scientist, but also to grow significantly as a person. I know how fortunate I
was to land in her lab as a technician six years ago! Her advice and example will guide
me through the rest of my life.
I would like to also thank my co-mentor, David, for all of his guidance and patience.
Discussions with David have honed my critical thinking—I can count on him to find the
weak point in my logic. His off-the-cuff scrap paper diagrams have turned many previously
confusing concepts crystal clear.
Finally, I would like to thank my family for their support, particularly my mom. She believed
in me, even during times when I didn’t, and has buoyed me through uncountable hurdles.
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Abstract
The extracellular matrix (ECM) is now recognized as an important cellular modulator,
providing essential contextual cues for appropriate cellular behavior. CCN1 (Cysteine-rich
61 or Cyr61) is an ECM-associated protein that binds to and signals through integrins and
heparan sulfate proteoglycans, and has been shown to mediate a number of cellular
responses including proliferation, differentiation, adhesion, migration, and apoptosis.
Gene array studies have identified CCN1 as one of the most highly upregulated genes in
a number of inflammatory lung disorders, including chronic obstructive pulmonary disease,
hyperoxia-induced lung injury, ventilator-induced lung injury, and asthma. The role of
CCN1 in lung inflammation, however, has only begun to be explored. The pathology of
acute lung injury is driven by excessive inflammation, revolving around excessive
neutrophil infiltration. Neutrophil infiltration is mediated by cell adhesion molecules, such
as intercellular adhesion molecule 1 (ICAM1) expressed on vascular endothelial cells.
Inflammatory cytokines such as tumor necrosis factor (TNF) play a central role in lung
inflammation and induce expression of endothelial cell adhesion molecules. Our lab has
previously shown that CCN1 can modulate TNF signaling in fibroblasts. Based on this, I
hypothesized that CCN1 modulates expression of cell adhesion molecules in endothelial
cells. In this thesis, I demonstrate that CCN1 negatively regulates the cell adhesion
molecules ICAM1 and vascular cell adhesion molecule 1 (VCAM1) in response to multiple
inflammatory stimuli. Further, I use inhibitor studies to identify the binding site on CCN1
that carries this functionality and demonstrate that integrins, but not heparan sulfate
proteoglycans, are required for this regulation. Finally, I demonstrate that CCN1-mediated
regulation of ICAM1 occurs at the mRNA level, is independent of canonical NF-κB
signaling, and involves Rac1 signaling. This novel role of CCN1 contributes to our
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understanding of the physiology of lung inflammation and provides a potential therapeutic
target for treatment of acute lung injury.
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Contributors and Funding Sources

This work was supported by a dissertation committee consisting of Professors Jacob N.
Finkelstein and Arshad Rahman of the Department of Pediatrics and Professor Minsoo
Kim of the Department of Microbiology and Immunology. Technical assistance was
provided by Rosemary Norman of the Department of Pediatrics. All experiments and data
analysis were completed by the student independently. Graduate study was supported by
a Provost Fellowship from the University of Rochester, a Toxicology Training Grant from
the NIEHS, the American Heart Association, and grants from the NHLBI.
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Table of Contents

Chapter 1: Introduction
CCN1……………………………………………………………………………… ……. 1
ARDS/ALI………………………………………………………………………… ……. 5
Inflammation……………………………………………………………………... ……. 8
Endothelial Cells………………………………………………………………….…… 12
ICAM1……………………………..…………………………………………………… 13
Summary………………………………………………………………………………. 16
Chapter 2: Characterization of CCN1 Regulation of Endothelial Cell
Adhesion Molecules
Introduction……………………………………………………………………………..17
Materials and Methods
Cell Culture…………………………………………………………………… 21
Plasmids and electroporation……………………………………………….. 21
Matrix-bound CCN1………………………………………………………….. 23
Immunoblotting……………………………………………………………….. 23
Polymerase Chain Reaction…………………………………………….…... 25
Antibodies and reagents…………………………………………………….. 26
Expression and purification of CCN1……………………………………….. 27
Statistical analysis……………………………………………………………. 28
Results
CCN1 downregulates TNF-induced ICAM1 and VCAM1 in
endothelial cells…………………………………………………………. … .. 29
Inhibitory Effect of CCN1 on ICAM1 is not specifically dependent on
TNF stimulation…………………………………………………………. …... 35
Soluble and matrix-bound forms of CCN1 have the same inhibitory
activity on endothelial ICAM1………………………………………….. …... 40
Discussion………………………………………………………………………………42
Chapter 3: Identification of CCN1 Binding Sites and Endothelial Cell
Receptors Required for Regulation of Intercellular Adhesion
Molecule 1
Introduction……………………………………………………………………………. 49
Integrins……………………………………………………………………….. 49
Heparan sulfate proteoglycans……………………………………………... 53
Low-density lipoprotein receptor-related proteins…………………..…….. 55
CCN1 binds distinct sets of receptors to regulate different cell
behaviors ...……………………………………………………………… …... 55
Materials and Methods
Cell Culture …………………………………………………………………… 58
Adhesive matrix proteins ..………………………………………………….. 58
Immunoblotting ..…………………………………………………………….. 59
Antibodies and reagents ...………………………………………………….. 60
Statistical analysis ...…………………………………………..…………….. 61
Results
CCN1 regulation of endothelial ICAM1 is integrin-dependent…………… 63
Heparan sulfate proteoglycans are not required for CCN1
regulation of ICAM1…………………………………………………….. …... 65
The V2 binding site on CCN1 is required for regulation of ICAM1………. 67
Integrin function blocking antibodies failed to identify specific
integrin(s) required for CCN1-mediated regulation of ICAM1……….. ….. 67
Vitronectin does not regulate endothelial ICAM1 in the manner of
CCN1…………………………………………………………………….. …... 70
Discussion…………………………………………………………………..…………. 74
Chapter 4: Mechanism and Signaling Pathway of CCN1 Regulation of
Endothelial ICAM1
Introduction…………………………………………………………………..………... 79
Materials and Methods
Antibodies and reagents……………………………….……………………. 83
Fluorescent immunocytochemistry…………………………………………. 83
Quantitative PCR ……………………………………………………………. 84
Concentration of conditioned media………………………………………... 84
Enzyme-Linked Immunosorbent Assay (ELISA)………………………….. 85
Results
CCN1 regulation of endothelial ICAM1 is independent of canonical
NF-κB signaling ………………………………………………………… …... 86
CCN1 regulates ICAM1 message in endothelial cells………………......... 90
Shedding does not mediate CCN1 negative regulation of ICAM1………. 92
Rac1 mediates CCN1 regulation of endothelial ICAM1 ………..….……. 94
Discussion……………………………………………………………………….…….. 98
Chapter 5: Conclusion
Regulation of ICAM1 independently of canonical NF-κB signaling……..….. …. 102
Proposed Model: CCN1 acts as an integrin antagonist to suppress Rac1
activity…………………………………………................................................. …. 106
Other potential effects of CCN1 on Endothelial Cells…………………………….. 110
CCN1 as a therapeutic……………………………………………………………… 112
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List of Tables

Chapter 2
2.1 List of primers……………………………………………………………………….… 26

Chapter 3
3.1 CCN1 binding site inhibitory peptides………………………………………….…… 62