Luận án: Điều hòa vận chuyển chủ động ở ống thận người bởi MT-3: Vai trò của miền C

University of North Dakota UND Scholarly Commons Theses and Dissertations Theses, Dissertations, and Senior Projects January 2015 Regulation Of Vectorial Active Transport In Human Proximal Tubule Cells By MT-3: The Role Of The C-Terminal Domain On E-And N- Cadherin Expression And The Confirmation Of Protein-Protein Interactions Andrea Marie Nore Follow this and additional works at: https://commons.edu/theses Recommended Citation Nore, Andrea Marie, "Regulation Of Vectorial Active Transport In Human Proximal Tubule Cells By MT-3: The Role Of The C- Terminal Domain On E-And N-Cadherin Expression And The Confirmation Of Protein-Protein Interactions" (2015). Theses and Dissertations.edu/theses/1938 This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact zeineb.

REGULATION OF VECTORIAL ACTIVE TRANSPORT IN HUMAN PROXIMAL TUBULE CELLS BY MT-3: THE ROLE OF THE C-TERMINAL DOMAIN ON E-AND N-CADHERIN EXPRESSION AND THE CONFIRMATION OF PROTEIN-PROTEIN INTERACTIONS by Andrea Marie Nore Bachelor of Science, Minnesota State University Moorhead, 2010 A Dissertation Submitted to the Graduate Faculty of the University of North Dakota School of Medicine and Health Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy Grand Forks, North Dakota December 2015 Copyright 2015 Andrea Nore ii This dissertation, submitted by Andrea Nore in partial fulfillment of the requirements for the Degree of Doctor of Philosophy from the University of North Dakota, has been read by the Faculty Advisory Committee under whom the work has been done and is hereby approved. Garrett __________________________________________ Seema Somji __________________________________________ Donald A. Dunlevy This dissertation is being submitted by the appointed advisory committee as having met all of the requirement of the School of Graduate Studies at the University of North Dakota and is hereby approved. __________________________________ Wayne Swisher Dean of the School of Graduate Studies _________________________________ Date iii PERMISSION Title REGULATION OF VECTORIAL ACTIVE TRANSPORT IN HUMAN PROXIMAL TUBULE CELLS BY MT-3: THE ROLE OF THE C- TERMINAL DOMAIN ON E-AND N-CADHERIN EXPRESSION AND THE CONFIRMATION OF PROTEIN-PROTEIN INTERACTIONS Department Biochemistry and Molecular Biology Degree Doctor of Philosophy In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection.

I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work or, in his absence, by the Chairperson of the department or the dean of the School of Graduate Studies. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation. Andrea Nore November 5, 2015 iv TABLE OF CONTENTS LIST OF FIGURES.

vii LIST OF TABLES. 1 Hypothesis and Rationale. 3 Epithelial Cell Adhesion and Polarity. 6 Epithelial-Mesenchymal Transition.

8 Cadmium is Nephrotoxic. 18 Metallothionein-3 Protein-Protein Interactions. 22 Laser-Capture Microdissection. 26 RNA isolation and qRT-PCR.

34 Expression of E- and N-Cadherin in Human Kidney Proximal Tubules. 34 Expression of E- and N-Cadherin in HK-2 Cells as a Function of Growth Medium Composition. 38 Comparison of E- and N-Cadherin Expression in HK-2 and HPT Cell Cultures. 39 Alterations in Tight Monolayer Development.

44 Association of the Unique C-Terminal Domain of MT-3 With MT-3 Induced MET in HK-2 Cells. 46 Effect of Forced E-cadherin Expression on HK-2 Vectorial Active Transport, N-Cadherin Expression, and Cell Morphology. 52 Zn7MT-3 mediated pulldowns in HK-2 lysates. 55 V5-mediated immunoprecipitations in MT-3 expressing HK-2 lysates.

60 HK-2 Cells have Features Associated with the Initial Stages of EMT. 60 HK-2 Cells Undergo MET Mediated by MT-3. 65 MT-3 Interacts with Proteins that Promote an Epithelial Phenotype. 82 vi LIST OF FIGURES Figure Page III-1.

Immunohistochemical staining of E-cadherin and N- Cadherin in the human kidney. Expression of E-and N-cadherin in microdissected proximal tubules. Influence of growth medium on E-and N-cadherin expression in HK-2 cells. Comparison of E-cadherin expression in HPT and HK-2 cell cultures.

Comparison of N-cadherin expression in HPT and HK-2 cell cultures. The pattern of E- and N-cadherin expression is unaltered when cells are grown on transwell inserts. Transepithelial resistance comparison of HPT and HK-2 cell cultures. Connexin 32 expression in HK-2, HPT, and HK-2 cells expressing MT-3.

Mutated metallothionein constructs. Effect of the altered domains of MT-3 on the formation of domes in stably transfected HK-2 cells. Effect of the altered domains of MT-3 on the expression of E-and N-cadherin in stably transfected HK-3 cells. Effect of the altered domains of MT-3 on the expression of connexin 32 in stably transfected HK-3 cells.

The effect of forced overexpression of E-cadherin on the expression of N-cadherin and on dome formation in HK-2 cells. Schematic representation of the methodology used to pulldown MT-3 interacting proteins. MT-3 interacts with β-actin, Tropomyosin 3, enolase-1, and aldolase A. Zinc supplementation 6 hours post transient transfection of V5-tagged MT-3 into HK-2 cells increases MT-3 protein expression.

MT-3 interacts with several proteins in cultured proximal tubule cells. Co-immunoprecipitation of MT-3 protein complexes in HK-2 cells transfected with V5 tagged-MT-3. Immunostaining of MT-3 in Skin Pigment Lesions. Immunostaining of MT-3 in Nevi and Melanoma Cells.

Expression of MT-3 in primary normal human keratinocytes (NHEK), Immortalized Human Keratinocytes (HaCaT), and normal human melanocytes (HEMa-LP). 94 viii LIST OF TABLES Table Page I-1. MT-3 interacting proteins. Primary antibodies used for western blotting.

Transepithelial resistance and dome formation in HK-2 cells overexpressing MT-3 with altered protein domains. Immunostaining of MT-3 in normal skin, SCC, BCC, Nevi, and Melanoma. 89 ix ACKNOWLEDGMENTS First and foremost I would like to thank Dr. Don Sens, Dr.

Van Doze, Dr. Jim Porter and Dr. Joe Provost for recognizing my potential when I was an undergraduate researcher. Their combined support and mentorship has greatly impacted my scientific success and I am forever grateful.

I would like to recognize the many individuals who made this body of work a possibility. Xudong Zhou, Dr. Yun Zhen, and Dr. Mary Ann Sens procured the tissue and aided in all of immunohistochemistry related data.

I would also like to thank Dr. Chandra Bathula for designing the mutant metallothionein constructs, identifying putative protein interactions with the assistance of Dr. John Shabb, and Wallace Muhonen, and finally for performing the microdissection of proximal tubules from paraffin-embedded blocks of tissue. I would like to thank Dr.

Dunlevy for her assistance with microscopy, expertise in cell biology, and mentorship. Brent Voels performed much of the cloning and purification of the 6.2/V5 vectors prior to kindly giving them to me for linearization and transfection. Seema Somji and Dr. Scott Garrett have been fantastic mentors as well, always available to assist with any struggles I encountered and their independent strengths have made me a well-rounded researcher.

Finally, I would like to thank my family and friends that have supported me as I achieved my goals. My husband (and best friend) John, who has been with me from the beginning of graduate school, is my rock. The many great friends who started and ended this journey with me, in particular Elizabeth Sandquist, Katie Collette, and Dani Rastedt. x These girls hold a special place in my heart, and I will cherish our many “knit nights,” happy hours, potlucks, seminars, practice sessions, comprehensive test review, and cakes.

I couldn’t have done it without any of you. xi ABSTRACT The proximal tubule of the kidney is particularly susceptible to toxicant-induced damage and cell cultures of human proximal tubule cells are widely utilized to study the role of epithelial-mesenchymal transition (EMT) in renal disease. Cadmium is a toxic metal ion that is known to produce renal tubular necrosis and accumulate in the proximal tubule. This metal binds to a family of cysteine rich metal binding proteins known as metallothioneins (MT) that are found in abundance in the kidney.

Previous studies from our laboratory have shown that the third isoform of metallothionein (MT-3) is expressed in the epithelial cells of the human kidney, including those of the proximal tubule. An immortalized proximal tubule cell line does not express MT-3 and does not demonstrate vectorial active transport. Transfection of the MT-3 gene into the HK-2 cells restores vectorial active transport as evidenced by dome formation. This suggests that MT-3 is involved in mesenchymal to epithelial transition (MET), the reverse of EMT, and promotes and epithelial phenotype.

The goals of the present study were to examine the role of growth media composition on classic EMT responses, quantitatively evaluate the expression levels of E- and N-cadherin, define the functional epitope of MT-3 that mediates MET in HK-2 cells, and identify proteins that interact with MT-3 to promote epithelial features in the proximal tubule. It was shown that both E- and N-cadherin mRNA and protein are expressed in the human renal proximal tubule. Based on the pattern of cadherin expression, vectorial active transport, and transepithelial resistance, it seems that the HK-2 cell line has already undergone many of the early features associated xii with EMT. Our data indicates the unique, six amino acid C-terminal sequence of MT-3 is required to induce MET in HK-2 cells.

A combination of co-immunoprecipitation and western blotting indicate that MT-3 interacts with myosin-IIa, β-actin, enolase-1, tropomyosin-3, and aldolase-a in vitro. Together, the data suggests the HK-2 cell line can be an effective model to study later stages in the conversion of the renal epithelial cell to a mesenchymal cell and when transfected with MT-3 it may be an effective model to study the process of MET. MT-3 protein-protein interactions provide insight into the potential mechanism by which MT-3 promotes cytoskeletal organization in non-diseased epithelial proximal tubule cells and offers the opportunity to investigate these interactions under pathological conditions. xiii CHAPTER I INTRODUCTION Significance The incidence of chronic kidney disease (CKD) is steadily rising and has reached epidemic proportions in the western and industrialized world.

Chronic kidney disease is associated with albuminuria, decreased creatinine clearance, altered glomerular morphology, and tubular degeneration (Eddy & Neilson, 2006). Epidemiological evidence and animal models have demonstrated chronic cadmium exposure as a significant contributory factor for developing CKD (Gobe & Crane, 2010; Klaassen, Liu, & Diwan, 2009; Walter C Prozialeck & Edwards, 2012; Thévenod, 2003) Clinicopathological studies have shown tubulo-interstitial fibrosis to be the hallmark of CKD progression (Bohle, Müller, Wehrmann, Mackensen-Haen, & Xiao, 1996; Eddy & Neilson, 2006; Fine, Ong, & Norman, 1993; Zeisberg & Neilson, 2010). This suggests that halting the progression of CKD could be achieved by stopping the progression of or even by inducing remission of fibrosis. Renal fibrosis is defined as the scarring of the tubulo-interstitial space after kidney damage of any type.

It appears to be initiated at random in small areas that are preceded by interstitial inflammation, then expands to become diffuse if drivers of fibrosis persist (Prunotto et al. Accumulation and proliferation of activated fibroblasts (myofibroblasts) in these small areas are linked to the risk of progression of fibrosis (Hinz et al. The exact source 1 of renal myofibroblasts remains to be definitely defined. Several hypotheses exist in the literature including: migration of circulating fibrocytes to the site of the lesion, differentiation of local fibroblasts or pericytes, direct transformation of resident endothelial cells by endothelial-mesenchymal transition (endoMT), or transformation of resident epithelial cells through epithelial mesenchymal transition (EMT).

Studies in experimental models have shown that it is the pericytes that respond to chronic injury and profibrotic signals through proliferation and differentiation into myofibroblasts (S.