MICROENVIRONMENTAL FORCES REGULATE NOTCH SIGNALING THROUGH INTEGRINS by Michael Allen Detweiler A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology Boise State University December 2018 © 2018 Michael Allen Detweiler ALL RIGHTS RESERVED BOISE STATE UNIVERSITY GRADUATE COLLEGE DEFENSE COMMITTEE AND FINAL READING APPROVALS of the thesis submitted by Michael Allen Detweiler Thesis Title: Microenvironmental Forces Regulate Notch Signaling Through Integrins Date of Final Oral Examination: 25 October 2018 The following individuals read and discussed the thesis submitted by student Michael Allen Detweiler, and they evaluated his presentation and response to questions during the final oral examination. They found that the student passed the final oral examination. Allan Albig, Ph. Chair, Supervisory Committee Brad E.
Member, Supervisory Committee Troy Rohn, Ph. Member, Supervisory Committee The final reading approval of the thesis was granted by Allan Albig, Ph., Chair of the Supervisory Committee. The thesis was approved by the Graduate College. ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor, Dr.
Allan Albig, for being an incredible mentor and friend. With his guidance and moral support, I have been able to fully understand and integrate the ethics and investigative nature of science. I would also like to thank Dr. Brad Morrison and Dr.
Troy Rohn for being on my thesis committee and for their very helpful suggestions. Through my time at Boise State I have had the pleasure of working with and learning from my fellow colleagues, and for this I would like to thank the Albig lab members, Jacob Crow, Bryce Lafoya, Jordan Munroe, Shelby Havel, and Dr. I would also like to give thanks to the National Institute of Health for providing the funding to support this thesis. iv ABSTRACT The extracellular microenvironment contributes significantly to a cell’s function and behavior.
For instance, cell-cell interactions, cell-substrate interactions, and physical forces are all factors of the extracellular environment that can alter cellular behavior. Cells can receive these signals and forces through various membrane channels and receptors that transmit the signals from the extracellular to the intracellular space. Canonical Notch signaling is induced by ligand interactions with neighboring cells, but recent evidence has revealed that Notch signaling can occur through a variety of extracellular stimuli including hyperglycemia, hypoxia, multiple growth factors, fluid shear stress, and extracellular matrix (ECM) composition. Although Notch activation through ligand interactions with adjacent cells have been well established, non-canonical Notch signaling through the microenvironment is poorly understood.
Previous evidence suggests a novel activation of Notch signaling through an integrin pathway, proposing Notch as a microenvironmental sensor. Integrins are cell membrane receptors that are mainly recognized for cell-ECM attachment and induction of cellular signaling cascades but have also been shown to respond and transmit signaling through fluid shear stress and ECM stiffness. Since integrins have been shown to regulate Notch signaling and both exhibit a response to fluid shear stress, we hypothesized that Notch signaling responds to fluid shear stress through integrin activation. To test this, we compared Notch activation following exposure to fluid shear stress and Notch activation following shear stress after inhibiting integrin function.
Our data confirms that Notch activation is significantly v upregulated from fluid shear stress compared to a static control and inhibiting integrin function attenuates this response, suggesting integrins are required for Notch’s upregulation from shear stress. Because integrins also respond and transmit signals from varying ECM stiffness and Notch has been shown to be upregulated during conditions of fibrosis, we hypothesized that Notch signaling will be regulated by varying degrees of ECM stiffness through integrin activation. To investigate this hypothesis, we cultured cells on hydrogels with multiple levels of stiffness and measured Notch signaling. Our results indicate that like shear stress, Notch signaling is influenced by ECM stiffness.
Collectively our results indicate that Notch signaling is regulated through microenvironmental forces like fluid shear stress and ECM stiffness and is regulated through integrins. This furthers our understanding of the variations of Notch signaling in response to microenvironmental stimuli and the mechanisms involved. Notch has been implicated in a variety of diseases and our results improve our knowledge of Notch signaling in pathological conditions of the microenvironment including abnormal shear stress (e. atherosclerosis) and tissue stiffness (e.
vi TABLE OF CONTENTS ACKNOWLEDGEMENTS. v LIST OF TABLES .xi LIST OF FIGURES .xii LIST OF ABBREVIATIONS. xvi CHAPTER ONE: INTRODUCTION. 1 The Cellular Microenvironment.
3 Downstream Notch Signaling. 5 Canonical vs Non-Canonical Activation. 6 Notch Signaling and Disease. 8 Integrin Downstream Signaling.
12 Notch and Shear Stress. 18 CHAPTER TWO: INITIAL SHEAR STRESS-INDUCED NOTCH SIGNALING IS REGULATED THROUGH AND INTEGRIN PATHWAY. 24 Notch Signaling is induced by Shear Stress. 24 Notch Activation from Shear Stress Is Regulated by Integrins.
27 Notch Activation from Shear Stress Is Regulated Via Integrin Downstream Pathways Src and Enos. 37 mRNA Expression Analysis:. 38 Western Blot Analysis:. 38 CHAPTER THREE: MATRIX STIFFNESS REGULATES NOTCH ACTIVATION.
42 viii Notch Activation Changes with Varying Degrees of Matrix Stiffness in Endothelial Cells. 63 Notch: A multi-functional integrating system of microenvironmental signals. 64 The Cellular Microenvironment:. 66 Notch as an Integrator of Cellular Microenvironments:.
69 ECM-Notch Interactions:. 70 Direct ECM-Notch Interactions That Control Notch Signaling. 70 Indirect ECM-Notch Interactions that Control Notch Signaling (Transcriptional Mechanisms). 76 Direct ECM Notch Interactions That Control Notch Signaling (Crosstalk Mechanisms).
78 Notch crosstalk with other signaling networks. Integrins, TGF-, WNT, and VEGF:. 80 Crosstalk between Notch and Integrins:. 80 ix Notch and TGF-:.
84 Notch and WNT:. 86 Notch and VEGF:. 88 Other Microenvironment Conditions That Control Notch (Shear stress, hypoxia, and hyperglycemia). 90 Notch and Shear Stress:.
91 Notch and Hypoxia. 93 Notch and Hyperglycemia:. 108 x LIST OF TABLES Table A.1: Basic and proposed mechanisms by which Notch responds to various microenvironmental signals. 98 xi LIST OF FIGURES Figure 1.1: Domains of the Notch receptor.
The extracellular domain (NECD) of Notch contains 36 epidermal growth factor (EGF)-like repeats, followed by a transmembrane portion containing Lin-12-Notch repeats. The intracellular domain (NICD) consists of RAM, Ankyrin repeats, transactivation domain (TAD) and proline, glutamine, serine, and threonine (PEST)-rich domains and is responsible for transcriptional control.2: Blood vessels consist of 3 main layers. The tunica intima is composed of endothelial cells surrounded by a basement membrane and regulates vessel permeability and integrity. The tunica media has smooth muscle cells and matrix proteins and is mainly responsible for contraction/dilation of the vessel, and the tunica adventitia consists of mostly ECM proteins and associates the vessels with the surrounding tissue and provides elasticity.
Illustration showing the proposed method of mechanotransduction. As shear stress forces are implemented to the apical surface of cells, the force applies tension to the cytoskeleton where it will then transmit to the focal adhesions and junctions of the cell. Illustration of experimental hydrogels. A glass coverslip is used to attach polyacrylamide hydrogel.
The hydrogel is then crosslinked to an ECM protein using crosslinkers like L-DOPA or Sulfo- sanpah. The cells are then cultured on the surface attaching to the ECM protein with focal adhesions.1: Notch signaling is induced via shear stress. HMEC1s exposed to ~12dyn/cm^2 of orbital shear stress. A) Notch intracellular domain (N1ICD) and full length Notch1 whole cell lysates detected by Western blot and normalized to Vinculin control.
b) Downstream Notch genes Hey1, Hes1, and Hey2 expression levels detected by qPCR, Klf2 used as a positive shear stress control. Values are mean +/- SEM; * = p < 0. c) Downstream Notch genes Hey1 and Hes1 expression levels detected by qPCR following treatment with Doxycycline in N1ICD transgenic HMEC1s to overexpress Notch signaling.2: Shear Stress-induced Notch is regulated through integrins. HMEC1s exposed to ~12 dyn/cm^2 of shear stress following integrin inhibition.
a) xii Notch downstream genes Hey1 and Hes1 mRNA expression detected by qPCR following treatment with RGD peptides (5 g/ml) and 90 minutes of orbital shear stress. Klf2 used as a positive shear stress control. b) Notch downstream genes Hey1 and Hes1 mRNA expression detected by qPCR following treatment with MBCD (10 mM) and 90 minutes of orbital shear stress. Klf2 used as a positive shear stress control.
Values are mean +/- SEM; * = p < 0. c) N1ICD fragment whole cell lysate detected via western blot and folded to Vinculin following treatment with RGD peptide and MBCD and exposed to orbital shear stress for 45 and 90 minutes.3: Shear stress-induced Notch activation is regulated by integrin downstream pathways, Src & eNOS. HMEC1s exposed to ~12 dyn/cm^2 of shear stress following treatment with AZM and DPI. a) mRNA expression of Hey1 and Hes1 detected by qPCR when AZM (10 m) is folded to vehicle control following shear stress.
b) mRNA expression of Hey1, Hes1, and Hey2 detected by qPCR when DPI (100 M) treatment is folded to static control following shear stress. Values are mean +/- SEM; * = p < 0. c) Western blot analysis of NICD with AZM, DPI, and vehicle control.4: Shear stress induces Notch activation through Integrin-Src-eNOS pathway. Illustration representing the suggested regulation of Notch activation following shear stress via an integrin-Src-eNOS pathway.1: Notch downstream genes are regulated through ECM stiffness.
a) Downstream Notch genes Hey1 and Hes1 were analyzed via qPCR following culturing on polyacrylamide hydrogels with stiffnesses of 10, 20, and 40 kPa. B) Downstream Notch gene Hes5 was analyzed via luciferase assay after being cultured on various stiffness of polyacrylamide hydrogels at 10, 20, & 40 kPa. Values are mean +/- SEM; * = p < .1: Canonical Notch signaling and Notch conservation between human and Monosiga brevicollis. Throughout Figure 1, conservation of Notch proteins or domains between human and M.
brevicollis is indicated by green (positive), yellow (unknown), or red (negative) shading according to references (King, Westbrook et al. 2008) and (Gazave, Lapebie et al. (A) Conservation of mammalian Notch receptor domains in M. Mammalian (human) Notch receptors contain 36 EGF-like repeats and three LNR or NRR (Lin-12 Notch Repeats or Negative Regulatory Region) repeats in the extracellular domain.
The intracellular portion of human Notch contains seven ankyrin domains and a PEST sequence at the C-terminal. For simplicity, the intracellular RAM (RBPj Association Module) domain, two NLS (Nuclear Localization sequence) domains, and TAD (Transactivation Domain) are not shown in this figure. xiii Please refer to references (Kopan and Ilagan 2009, Kopan 2010) for complete details. Three separate proteins (N1, N2, and N3) in M.
brevicollis contain six Ankyrin domains, two LNR domains, and 36 EGF- like repeats respectively (King, Westbrook et al. (B) Model of canonical Notch activation mechanism. Notch receptors are modified in the secretory pathway (ER/golgi) by Furin cleavage (S1 cleavage) and glycosylation of EGF-like domains by O-fucosyltransferase (O-fut), Rumi/Poglut1 (Protein O-Glycosyltransferase 1), and fringe family glycosyltransferases. The Furin cleavage products remain non-covalently associated in the membrane where a pulling force initiated by Notch ligand endocytosis in sending cells enables further cleavage by -secretase (S2 cleavage, NEXT fragment) at the LNR domain, and -secretase (S3 cleavage, NICD fragment) in the membrane of receiving cells.
Several regulatory proteins including Numb, Notchless, and Deltex control Notch availability at the membrane. After -secretase cleavage, the NICD fragment translocates to the nucleus where it displaces the transcriptional co-repressor SMRT from CSL/RBP-jk. NICD participates in a transcriptional complex with CSL, MAML, and p300 to drive transcription of Notch targets such as Hes and Hey genes. NICD steady- state levels are controlled by nuclear export, ubiquitination (Ub) by Sel10, and subsequent degradation in the proteasome.2: Summary of ECM control of Notch signaling.
Canonical activation of Notch receptors by Notch ligands can be manipulated in three ways by cellular interactions with ECM.) Direct interactions between Notch receptors or ligands and various ECM molecules can either inhibit or promote activation of Notch signaling.) Indirect interactions between ECM and Notch are characterized by ECM mediated increased or decreased expression of Notch ligands on sending cells or Notch receptors on receiving cells.