Proteomic Analysis of Membrane Bound and Associated Proteins of Human Gingival Fibroblasts and Periodontal Ligament Fibroblasts

PROTEOMIC ANALYSIS OF MEMBRANE BOUND AND ASSOCIATED PROTEINS OF HUMAN GINGIVAL FIBROBLASTS AND PERIODONTAL LIGAMENT FIBROBLASTS

A Thesis

Presented in partial fulfillment of the requirements for the degree Master of Science in the Graduate School of The Ohio State University

Holly Ann McKnight, D.D.S.

* * * * *

The Ohio State University 2012

Master’s Thesis Committee

Dr. Angelo J. Mariotti, Advisor Approved by

Dr. Thomas C. Hart Advisor Dr. Dimitris N. Tatakis Dentistry Graduate Program

Holly Ann McKnight, D.D.S.

2012

ABSTRACT

Background: The primary cell of the gingiva and periodontal ligament, the fibroblast, mainly functions to provide tissue homeostasis, integrity, communication, and immunity. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) exhibit several similarities, such as proliferation, migration, collagen synthesis, phagocytosis, expression of non-collagenous proteins, and outward appearance. However, many differences do exist between GF and PDLF and have been noted in culture, whether based on proliferative capacity and glycosaminoglycan content, chemotactic response, protein and collagen synthesis, or capacity to form mineralized tissue. Explanation for the differences in behavior and content of these cells may be evident in the proteome of the two cell types. The purpose of this study was to describe and compare the insoluble, membrane bound and associated proteins of GF and PDLF.

Methods: GF and PDLF were established from non-inflamed tissue of four systemically healthy subjects grown in cell culture consisting of MEM supplemented with 10% FBS. GF and PDLF were harvested in the 4th passage and membrane bound and associated proteins were isolated from fibroblasts. The protein samples were submitted for enzyme digestion and identification using liquid chromatography/ tandem mass spectrometry. Samples were separated on a capillary column using an UltiMate™ 3000 HPLC system from LC-Packings A Dionex Co (Sunnyvale, CA). Each sample was injected into the Precolumn Cartridge (Dionex, Sunnyvale, CA) and desalted with 50 mM acetic acid. Mobile phase A was 0.1% formic acid in water and 0.1% formic acid in acetonitrile was used as mobile phase B. Flow rate was set at 2µl/min. MS/MS data was acquired with a spray voltage of 2 KV at a capillary temperature of 175°C. A MS/MS scan generated product ion spectra to determine amino acid sequence. The mass accuracy of the precursor ions were set to 2.0 Da given that the data was acquired on an ion trap mass analyzer and the fragment mass accuracy was set to 0.5 Da. Considered modifications (variable) were methionine oxidation and carbamidomethyl cysteine. Scaffold (Proteome Software, Portland, Oregon, USA) was used to validate protein identifications derived from MS/MS sequencing results. Differences in proteins were considered statistically significant when means were at or greater than a 95% confidence interval.

Results: A total of five hundred nineteen proteins were identified from the eight samples analyzed via LC-MS/MS. Four hundred fifty proteins were common to both GF and PDLF. Forty

ii proteins were unique to GF and 29 were unique to PDLF. Of the proteins identified two hundred thirteen are known membrane bound or associated proteins. Twenty-eight proteins, identified from the 450 proteins common to both GF and PDLF, were detected in statistically significant greater quantities by either GF or PDLF. Five membrane proteins were detected in greater quantities by GF, while seven membrane proteins were detected in greater quantities by PDLF. Nineteen and ten membrane proteins were identified only in GF and PDLF respectively.

Conclusion: The current study is the first report in the literature to use proteomic analysis to identify and quantify the membrane bound and associated proteins of GF and PDLF. Furthermore it is also the first report to compare and contrast GF and PDLF membrane bound and associated proteins. The use of label free analysis allowed for the most inclusive description of the GF and PDLF proteomes for these 4 matched pairs. By exploring the functional characterization of the differences found between GF and PDLF proteomes we can speculate how the descriptive differences reported in this study relate to fibroblast function.

iii

DEDICATION

To my family, Curt, my husband, whose patience, understanding, and encouragement enables me to accomplish all my life goals, including this project. To Wayne and Carol, Ryan and Kala, and Jared whose love and support has been essential to my successes throughout life. To Tom and Evelyn who have taught me many lessons in professionalism and perseverance.

ACKNOWLEDGEMENTS

I would like to sincerely thank Dr. Angelo Mariotti, my advisor and mentor, for his guidance and expertise with this project to help make our vision a possibility.

Additional thanks to Deb Hooper, laboratory director, who was most encouraging during the most trying points of the project and has unparalleled stamina for research.

Thank you to the director, Dr Greg Ness, and residents in the Division of Oral and Maxillofacial Surgery who for the last three years helped to collect samples again and again.

My sincere gratitude to Drs. Tom Hart and Dimitris Tatakis who took time despite their demanding professional schedules to serve on my thesis committee.

Finally, I would like to express my gratitude to Dr. Kari Green-Church and Dr. Cindy James at The Ohio State University Mass Spectometry and Proteomics Facility for the time and expertise they spent on our project and then additional time teaching me the trade.

VITA

May 25, 1983……………………………………………………………………………..Born, Fremont, Nebraska United States of America

May 13, 2001………………………………………………………………………………High School Diploma Central Catholic High School West Point, Nebraska

May 14, 2005……………………………………………………………………………..Bachelor of Arts Creighton University Omaha, Nebraska

May 16, 2009……………………………………………………………………………..Doctor of Dental Surgery Creighton University Omaha, Nebraska

June 23, 2009- June 22, 2012…………………………………………………….Resident, Division of Periodontology The Ohio State University Columbus, Ohio

FIELDS OF STUDY

Major Field: Dentistry

TABLE OF CONTENTS

Page

ABSTRACT……………………………………………………………………………………………………………………….... ii

DEDICATION …………………………………………………………………………………………………………………….. iv

ACKNOWLEDGEMENTS……………………………………………………………………………………………………… v

VITA………………………………………………………………………………………………………………………………….. vi

LIST OF TABLES…………………………………………………………………………………………………………………. viii

LIST OF FIGURES……………………………………………………………………………………………………………….. ix

CHAPTERS:

1. INTRODUCTION……………………………………………………………………………………………………….. . 1

2. MATERIALS AND METHODS………………………………………………………………………………………. 22

3. RESULTS……………………………………………………………………………………………………………………. 30

4. DISCUSSION………………………………………………………………………………………………………………. 35

APPENDIX A: TABLES………………………………………………………………………………………………………… 41

APPENDIX B: FIGURES………………………………………………………………………………………………………. 67

REFERENCES……………………………………………………………………………………………………………………… 109

vii

LIST OF TABLES

TABLE Page

TABLE 1. Participant demographics……………………………………………………………………………….42

TABLE 2. Proteins identified by LC/MS-MS……………………………………………………………………43

TABLE 3. Membrane bound or associated proteins preferentially identified in GF……….. 59

TABLE 4. Membrane bound or associated proteins preferentially identified in PDLF….… 60

TABLE 5. Membrane bound or associated proteins only detected in GF………………………. 61

TABLE 6. Membrane bound or associated proteins only detected in PDLF……..……………. 62

TABLE 7. Membrane bound or associated proteins detected in greater quantities by GF reported by location and function………….……………………………………………………63

TABLE 8. Membrane bound or associated proteins detected in greater quantities by PDLF reported by location and function……………………………………………………………64

TABLE 9. Membrane bound or associated proteins detected only in GF in two or more strains reported by location and function…………………………………………………………65

TABLE 10. Membrane bound or associated proteins detected only in PDLF in two or more strains reported by location and function……………………………………………….66

viii

LIST OF FIGURES

FIGURE Page

FIGURE 1. Proteins identified by fibroblast type……………………………………………….. 68

FIGURE 2. Proteins identified by cellular component……………………………………….. 69

FIGURE 3. Protein quantification for Aminopeptidase N…………………………………… 70

FIGURE 4. Protein quantification for Microtubule associated protein 4…………... 71

FIGURE 5. Protein quantification for Annexin A11…………………………………………… 72

FIGURE 6. Protein quantification for Prohibitin-2……………………………………………..73

FIGURE 7. Protein quantification for NADH-ubiquinone oxudireductase 75 kDa subunit, mitochondrial……………………………………………………74

FIGURE 8. Protein quantification for Calcium/ calmodulin-dependent protein kinase type II subunit delta………………………………………………………..75

FIGURE 9. Protein quantification for Desmoplakin……………………………………….….76

FIGURE 10. Protein quantification for Membrane-associated progesterone receptor component 2…………………………………………………………….. 77

FIGURE 11. Protein quantification for Voltage-dependent calcium channel subunit alpha- 2/delta-1………………………………………………………….. 78

FIGURE 12. Protein quantification for 4F2 cell-surface antigen heavy chain…….. 79

FIGURE 13. Protein quantification for Nucleobindin-1…………………………………….. 80

FIGURE 14. Protein quantification for Ubiquitin carboxyl-terminal hydrolase isozyme L1………………………………………………………………………………. 81

FIGURE 15. Protein quantification for LETM1 and EF-hand domain-containing protein 1 mitochondrial………………………………………………………………….82

FIGURE 16. Protein quantification for Early endosome antigen 1………………………….83

FIGURE 17. Protein quantification for Macrophage-capping protein…………………….84

FIGURE 18. Protein quantification for 2-oxoglutarate dehydrogenase, mitochondrial……………………………………………………………………………….. 85

FIGURE 19. Protein quantification for A-kinase anchor protein 12………………………. 86

FIGURE 20. Protein quantification for Sideroflexin-3………………………………………….. .87

FIGURE 21. Protein quantification for Kinectin……………………………………………………. 88

FIGURE 22. Protein quantification for Synaptic vesicle membrane protein VAT-1 homolog…………………………………………………………………………………….…. 89

FIGURE 23. Protein quantification for Glucosylceramidase……………………………….…. 90

FIGURE 24. Protein quantification for Syntaxin-12…………………………………………….... 91

FIGURE 25. Protein quantification for Ras-related protein Ral-A……………………….…. 92

FIGURE 26. Protein quantification for Epidermal growth factor receptor…………….. 93

FIGURE 27. Protein quantification for Filamin-B………………………………………………….. 94

FIGURE 28. Protein quantification for Microsomal glutathione S-transferase 3…… 95

FIGURE 29. Protein quantification for Apoptosis-inducing factor 1, mitochondrial..96

FIGURE 30. Protein quantification for Epsin-1……………………………………………………… 97

FIGURE 31. Protein quantification for NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 7………………………………………………………. 98

FIGURE 32. Protein quantification for Clathrin light chain A…………………………………99

FIGURE 33. Protein quantification for Chondroitin sulfate proteoglycan 4……………100

FIGURE 34. Protein quantification for Integrin alpha-8………………………………………….101

FIGURE 35. Protein quantification for Lactadherin………………………………………………..102

FIGURE 36. Protein quantification for Ezrin…………………………………………………………..103

FIGURE 37. Protein quantification for Junction plakoglobin………………………………….104

FIGURE 38. Protein quantification for Matrix metalloproteinase- 14…………………….105

FIGURE 39. Protein quantification for Matrix-remodeling-associated protein 7…….106

FIGURE 40. Protein quantification for Cadherin-2…………………………………………………107

FIGURE 41. Protein quantification for Desmocollin-1……………………………………………108

CHAPTER 1: INTRODUCTION

The Gingiva and the Periodontium

Teeth are supported and maintained by a physiologically dynamic periodontium. The periodontium is comprised of gingiva, periodontal ligament, alveolar bone, and cementum.

Gingiva, the soft tissue that covers the surface of teeth and underlying bone, consists of epithelium on the surface and its underlying connective tissue. The predominant cell type of healthy gingival connective tissue is the fibroblast, approximately 5% of the overall composition of healthy connective tissue1. Approximately, sixty to sixty-five percent of the connective tissue of the gingiva is collagen, 10 to 15% is vascular tissue including, blood and lymph vessels and nerves, 15 to 20% is ground substance, while 3% is other cell types, such as leukocytes. 2

Although additional studies3 have concluded that the cellular component of connective tissue may comprise as much as 49%, gingival fibroblasts consistently account for the greatest numeric and volumetric amount of cells. The ground substance, also known as extracellular matrix, which surrounds the cellular components of connective tissue and collagens, consists of glycoproteins and proteoglycans.

The main function of the gingival connective tissue is to give tone and substance to the free gingival margin and contour and support to the attached gingiva. 2 In addition it protects the root surface and alveolar bone from insults in the oral cavity. 4 The connective tissues of the gingiva heal remarkably well. Most often repairing gingival tissue after surgical insult results in reconstruction of the tissues without evidence of scarring as opposed to wound healing of

dermal skin which often scars.5 Although gingival connective tissue has good reparative qualities, it is not as great as the reparative capacity of the periodontal ligament.4 Incidentally, collagen bundles of the gingival connective tissue are contiguous with the periodontal ligament.2

The interdependent arrangement of gingival and periodontal ligament fibers characterizes health in the periodontium and shields the periodontal ligament from pathologic insult.

The periodontal ligament (pdl) is responsible for retaining the tooth inside the bony housing of the jaw. The 0.15-0.40 mm space2 between the cementum of the tooth and the bone of the jaw is filled with specialized connective tissue that is the periodontal ligament. The pdl is comprised of bundles of dense fibrous connective tissue. It is responsible for nutrition, metabolism, and sensory perception of the ligament space as well as parts of the gingiva. As with gingiva, fibroblasts are by far the most numerous cell type of the periodontal ligament.1

The primary cell of the gingiva and periodontal ligament, the fibroblast, mainly functions to provide tissue homeostasis. Fibroblasts produce structural collagens, as well as elastins, glycoprotiens, proteoglycans, and glycosaminoglycans 6 which compose the extracellular structure of both gingiva and periodontal ligament. Fibroblasts also facilitate the breakdown of tissue. They secrete the inactive precursor collagenases that when activated degrade the extracellular matrix. 7 Fibroblasts are able to phagocytose cross-linked collagen to maintain homeostasis.

In vivo, fibroblasts tend to be isolated from each other; dispersed and attached amongst surrounding collagens and glycoproteins.4 Fibroblasts grown in vitro have more intimate contact

with each other, however observation of such a distinction in the behavior of fibroblasts may be entirely mute based on the general heterogeneity of fibroblast populations, a topic that will be addressed later in this report. Current understanding of gingival fibroblast (GF) morphology in health is that they are sparse and flattened in appearance indicative of low metabolic turnover.

When under injury from incision or insult from bacterial pathogens, GF migrate to the site at increased density, divide and produce new matrix at increased metabolic turnover.

Collagens account for the bulk of matrix proteins in periodontal tissue. 8 Type I collagen is the dominant collagen species throughout gingival connective tissue. 9, 10 Collagen fibers are arranged into either dense large bundles or loose thin fibers. The dense bundles are predominately comprised of Type I collagen, while the fine reticular collagen network contains both type I and type III collagen. 11 Type IV collagen is predominant at the basement membrane interface of epithelium to underlying connective tissue. Type-IV collagen also surrounds blood vessels found within connective tissue. Types V and VI collagens have also been identified diffusely in gingival connective tissue 9, usually associated with bundles of type I collagen.

Proteoglycans are also ubiquitous to the extracellular matrix of connective tissue.

Specific antibody and DNA probes have been used to identify several proteoglycans associated with gingival tissues, including decorin, biglycan, syndecan, and versican. 12-15 At their core proteoglycans are proteins that are heavily glycosylated and form bonds with glycosaminoglycans. Integrins are cell surface receptors of fibroblasts that interact with the collagens and proteoglycans of the extracellular matrix.16

Sixty, thirty and ten percent of the glycosaminoglycans of connective tissue are dermatan sulfate, chondroitin sulfate and equal proportions of hyaluronic acid and heparin sulfate, respectively.17 The glycoprotein fibronectin is distributed throughout gingival connective tissue and found concentrated at collagen fibers.18 Gingiva also contains varying amounts of the glycoproteins osteonectin, vitronectin, elastin, and tenascin. 4

The organization of the pdl is rich with extracellular matrix (ECM) and has low cell density. In contrast, the intercellular space in gingival epithelium, for example, is small due to the dense formation of cell layers; therefore only small amounts of ECM are present.19

Periodontal ligament fibroblasts (PDLF) undergo extensive turnover and are principally located around the blood vessels of the pdl and endosteal spaces of the alveolar bone.20, 21 Primary cultures of PDLF deposit heavy amounts of collagen type I and fibronectin. 19 PDLF respond to mechanical forces by significantly increasing the production of collagen type-1 and fibronectin.22

Cells found in the pdl proliferate and differentiate into cells capable of synthesizing bone, cementum and the ECM of the pdl.23 Some PDLF express varying amounts α-smooth muscle actin, osteopontin, and alkaline phosphotase24-26 which may be helpful in explaining the homeostasis of the pdl, as well as osteogenesis by regulating proteins secreted by PDLF.

Alkaline phosphatase, in particular, is expressed by pdl cells forming mineralized tissue. Most cells of the pdl have osteogenic potential, but may not express the full repertoire of differentiation-associated proteins implicated in osteogenesis.27 None the less, it is interesting to note that studies of pdl cells have been able to demonstrate osteoblast differentiation.

Function of Fibroblasts

Once thought to be static cells of connective tissues, fibroblasts have reemerged as dynamic cells responsible for tissue’s integrity, communication, and immunity. Fibroblast attachment to ECM maintains cell shape and function in addition to tissue’s integrity. They essentially control the balance of the synthesis and degradation of ECM.28 As discussed previously, fibroblasts remodel ECM by synthesizing connective tissue components, predominately collagens. 29 Phagocytosis of collagen by fibroblast allows for tissue to turnover and remodel. This remodeling process allows for changes to occur in tissue shape or structure.30

When the regulatory mechanism of fibroblasts becomes perturbed for one reason or another a net gain of connective tissue results in overgrowth while a loss of connective tissue leads to tissue destruction. An example of this type of tissue imbalance occurs in gingival enlargement seen in patients undergoing cyclosporine therapy.31

Fibroblasts communicate with each other and with cells of other tissue origins. GF interact with overlying epithelial cells to determine specific epithelial cell differentiation and characterization. 32 They also migrate to the site of injury during wound healing and repair or regenerate tissue at the site. By rearranging cellular microstructures including myosin, vimentin, and actin filaments, fibroblasts elongate and thus migrate by chemotaxis to inflammatory sites.33, 34

Fibroblasts participate in host immunity and inflammatory events of periodontal disease. In terms of host immunity, antigen studies have shown that the GF may themselves be

antigen presenting cells.35, 36 Furthermore, GF interact with neutrophils and lymphocytes during periodontal disease initiation and progression. Receptors for proinflammatory cytokines, IL-1β,

TNF-α, and IL-6 are present on the surface of fibroblasts. These cytokines target fibroblasts to produce collagenolytic enzymes, specifically metalloproteinases (MMP) that lead to tissue destruction. At the same time, cytokines like IL-6 can stimulate fibroblast proliferation and structural protein production in therapeutic concentrations. 37

Fibroblasts exhibit a range of morphological and functional characteristics.

Phenotypically distinct and functionally different subpopulations of fibroblasts are found in the adult periodontium despite the physical appearance of the cells being identical under light and electron microscopes.2 Cultured fibroblasts are highly diverse populations of cells with a great degree of heterogeneity.38-40 Evidence from fibroblast cultures of tissue explants indicates that subtypes of fibroblasts differ in function including growth rate and collagen synthesis; additionally they respond differently to signaling molecules such as estrogen, transforming

41, 42 growth factor-β, interferon-γ, prostaglandin E2, and other substances. Studies also support the notion that fibroblasts vary in cytoskeletal protein expression, surface markers, and even physical size.43, 44 Inflammatory mediators play a role in the increased presence of select subtypes of fibroblasts particularly those phenotypes present in inflammation and fibrosis.8

Fibroblast subpopulations not only exist but play an active role in the immune system, synthesizing and responding to cytokines and antigen presentation.45 The heterogeneity of

fibroblasts is seen from site to site (ie. between GF and PDLF), but also within the same anatomical site, (ie. between subpopulations of GF or PDLF).

GF population subtypes produce varying amounts of molecular components and proteins according to their respective origin4. GF of the papillary portion are characterized by production of migration stimulating factor (MSF), an isoform of fibronectin; smaller morphology, and greater proliferative potential than deeper reticular connective tissue fibroblasts.46 MSF is of particular interest because it has been found to have a parallel distribution pattern to the glycoprotein tenascin which has only been found in fetal connective tissue, tumor-associated stroma, and healing wounds in the adult.47, 48 Other subpopulations of fibroblast are capable of forming fibrosis of the connective tissue when a subtype responsible for structural protein production dominates. Alternatively subtypes responsible for an increase in collagenase production may be responsible for connective tissue breakdown seen in periodontal disease states.49 An explanation for these phenomena has led some GF to be subdivided by their receptors for Cyclosporine A, C1q (a component of complement 1) or estrogen.42, 45

PDLF are composed of subpopulations that can be categorized on their function in collagen production, morphology, and glycogen pools.45 Subpopulations of PDLF have been shown to express varying amounts of osteopontin, alkaline phosphatase and α-smooth muscle actin based on their apical-cervical location within the pdl space.27 The expression of α-smooth muscle actin in fibroblasts develops features of myofibroblasts; therefore PDLF have the potential to acquire a myofibroblastic phenotype. 50

GF and PDLF exhibit several similarities, such as proliferation, migration, collagen synthesis, phagocytosis, expression of non-collagenous proteins, and outward appearance.27

However, many differences do exist between GF and PDLF and have been noted in culture whether based on proliferative capacity and glycosaminoglycan content6, chemotactic response51, protein and collagen synthesis52, or capacity to form mineralized tissue53.

PDLF are derived from the inner layer of the dental follicle and are ectomesenchymal in origin.54 Derivation of GF is not completely understood, but it is thought these cells develop from either from the dental follicle or embryonic ridge mucosa and are mesenchymal cells.29

A prominent feature of PDLF is cytoplasmic glycogen pools and abundant bands of contractile type microfilaments. GF by contrast do not contain prominent contractile microfilaments.55

GF undergo more rapid proliferation rates than PDLF.6 In vitro wound healing demonstrates that GF proliferate and migrate significantly faster than PDLF.56 This observable event has led to the established principle in guided tissue regeneration that without GF exclusion, connective tissue and epithelial attachment will predominate over periodontal regeneration.

GF and PDLF have been found to respond differently to Porphyromonas gingivalis, a prominent pathogen of periodontal disease. Both GF and PDLF in the presence of P. gingivalis exhibit increased inflammatory expression of IL-1β, IL-6, IL-8, TNF-α, and MCP-1. However the expression of osteoprotegerin was reduced in GF while PDLF were not affected.57

PDLF produce more collagen and protein in vitro compared to GF.52 Collagen turnover is slower in the gingiva than the periodontal ligament58. An example of this phenomenon is the response of connective tissue and pdl collagens to orthodontic rotational forces which may contribute to post-orthodontic relapse.

Microarray analyses of GF and PDLF reveal differences in gene expression in GF and

PDLF. 59 Pdl stem cells are capable of developing into adipocytes, osteoblast-like and cementoblast-like cells and demonstrate the capacity to produce cementum and periodontal ligament tissues.60 Alkaline phosphatase has been detected in the cellular extracts of PDLF, not

GF. 61 GF are unable to regenerate pdl demonstrating that the fibroblast types are distinct and unable to transform into the other cell type.52 In addition, GF are better able to inhibit osteoclast formation than PDLF, possibly through the production of osteoprotegerin.62

Both PDLF and GF have been found to respond to several growth factors, however the responses are tissue type specific. Platelet derived growth factor-BB (PDGF-BB) is mitogenic to both GF and PDLF. GF are activated in the presence of growth factors leading to increased proliferation. PDGF-BB also affects the synthesis of collagens by PDLF 8; furthermore PDLF have a significantly greater proliferative response to PDGF-BB than GF.63 In response to enamel matrix derivative protein, PDLF attach and spread along root surfaces more readily and express an increase in alkaline phosphatase compared to GF.64 Cell signaling by growth factors is just one avenue of research ongoing regarding fibroblast function.

Avenues of Research for GF and PDLF

Observations of the physical and functional heterogeneity of GF and PDLF has triggered ongoing research to define the biologic basis for these differences including investigations of fibroblast genetics, proteomics, cell signaling pathways, and secretory phenotypes. Studies have attempted to explain variations in GF and PDLF function on the basis of differential expression of specific genes.59, 65 DNA microanalysis and reverse transciptase- polymerase chain reaction (RT-

PCR) studies have been used to investigate differential gene expression. In an in vitro study, Han and Amar (2002), through cell expansion cultures and DNA microarray analysis, found one hundred sixty-three genes to be differentially expressed between GF and PDLF. Genes encoding transmembrane proteins and cytoskeleton-related proteins tend to be up-regulated in PDLF, while cell-cycle regulation protein and metabolism-related protein genes are up-regulated in

GF.59

RT-PCR uses commercially available gene arrays to profile gene expression of multiple genes simultaneously.65 Use of RT-PCR with fibroblasts attempts to differentiate the cell types based on gene transcription. Lallier, Spencer and Fowler (2005) used RT-PCR to identify potential transcriptional markers that could be used to identify different periodontal cell populations (PDLF, GF, and osteoblasts) and lineages.66 RT-PCR revealed all three cell types expressed scripts for several ECM proteins including Types I, III, and V collagen, vimentin, fibronectin, rheumatoid arthritis-related antigen 47, and osteonectin. The proteoglycan lumican may be useful in identifying osteoblasts from fibroblasts, since it was not expressed in

osteroblast RT-PCR. Osteonectin is expressed in greater amounts in osteoblasts than GF and

PDLF. Fibromodulin is selectively expressed in PDLF. 66

Proteome analysis may also be useful in gaining a deeper understanding of the molecular properties of fibroblasts. Briefly, proteomics is the study of protein expression, a developing science that will be described in more detail in the following sections of this report.

Proteomic analysis in the study of GF and PDLF is limited. Only one publication of a descriptive study is available at this time. The technique used for the study revealed 900 protein spots, however the authors were only able to consistently and confidently identify an eighth of these proteins present.67 PDLF proteins identified by proteomic analysis in this study were associated with cell scaffolding, cellular motility, membrane trafficking, chaperone proteins, stress proteins, folding proteins, metabolic enzymes, detoxification proteins, biodegradative proteins, translation and transduction proteins, cell cycle regulation proteins, and extracellular proteins.67

Since information on GF proteome and PDLF proteome is limited perhaps other tissue fibroblasts may be exemplary of informatics provided by such analysis. Proteomic analysis of dermal fibroblasts has been used to determine collagen and collagen-related structures of skin.68 Proteomics can provide information on fibroblast response to tissue conditions.

Proteomic study of dermal fibroblasts with induced hypoxia revealed that reduced oxygen did not significantly alter proliferation or protein content of fibroblasts, however it did alter the quantitative expression of 56 proteins.69

Fibroblasts play an active role in the cell signaling of tissues, both as signalers by release of their cell products and as receptors of endogenous and exogenous molecules (e.g., cytokines, hormones, and growth factors), which has become of vast interest in cell biology research.

One aspect of interest in pdl cell signaling is the fibroblast’s ability to self-regulate to maintain homeostasis. Since the pdl exhibits a steady-state of homeostasis in health, the cell populations of the pdl must also exhibit a steady-state of renewal, expansion, and cell death. The population of fibroblasts of the periodontium contains apoptotic fibroblasts comparable in number with those that are proliferative to remain homeostatic.70 The cell signaling involved in homeostasis is also critical to the regulation of the ECM and quite frankly vice versa. ECM molecules, such as collagen, fibronectin, and tenascin on tooth root surfaces have been found to be chemotactic for PDLF and promote cell proliferation, migration, and attachment.51

Studies have also been able to draw some fascinating conclusions on the fibroblast response to cytokines and polypeptide growth factors. As discussed previously, polypeptide growth factors such as platelet-derived growth factor-ββ are both chemotactic and mitogenic to

PDLF.71 While proinflammatory cytokines IL-1β, IL-6 and TNF-α play important roles in mediating inflammatory responses of both GF and PLDF in periodontal disease. 72-74 Cytokine IL-

1β, IL-6, and TNF-α receptors have even recently been suggested as therapeutic molecules to regulate the inflammatory response of GF in periodontal disease.37

Secretory phenotype has long been discussed in cell biology to describe the physical route by which cell products are expelled from the cell. The classic pathway is endoplasmic

reticulum and golgi body dependent.75 Proteins transcribed at the ribosome are packaged, delivered and mature as they progress from endoplasmic reticulum to golgi bodies.

Procollagenases of fibroblasts are exported from the cell to the ECM by the classic pathway.

Uncoventional secretory pathways export proteins or products by direct translocation across plasma membranes, as with cytokine IL-1β or by intracellular transport intermediates like acetyl

CoA.

Functionally different fibroblast subpopulations have been demonstrated in both the gingiva and periodontal ligament based on secretory phenotype. Subpopulations of PDLF have been found to express alkaline phosphatase, but this expression varies within the pdl as a function of distance to cementum or bone.26 Within GF specific subpopulations are thought to be responsible for gingival repair and proliferation.45 Proteome studies may show the most promise in explaining protein expression largely responsible for both cell signaling pathways and the secretory phenotypes of fibroblasts.

Cell Function of Membrane Bound and Associated Proteins

The functional complexity of fibroblasts is dependent on the orchestra of cellular components. As characteristic of active synthesizing cells, fibroblasts contain a great amount of rough endoplasmic reticulum, golgi bodies and mitochondria.2 Their cytoplasm is abundant with mitochondria, vacuoles, and vesicles.4 Intracellular microfilaments are few in non-motile fibroblasts, however are prominent when cell migration occurs.4 The conductor of all these

cellular inhabitants is the nucleus, most specifically the nuclear envelope and its associated proteins that coordinate cell function by being the gatekeeper of the cell. The cell membranes that compartmentalize organelles from cytoplasm are rich with insoluble proteins affixed to a phospholipid bilayer.

The structural and functional organization of the nucleus is intimately linked to gene expression of the cell. 76 The nuclear envelope that encases the nucleus is comprised of the outer membrane (ONM), the inner membrane INM), nuclear pore complexes, and the nuclear lamina.77 Prior to the development of proteomic analysis the nuclear envelope, often referred to incorrectly as the nuclear membrane—which refers only to the OMN and IMN complex-- had been least characterized78. Nonetheless, the nuclear envelope was heavily targeted by pharmacotherapuetics often without specific explanation for the modes of action. The outer nuclear membrane is contiguous with endoplasmic reticulum and interacts with the cytoplasm of the cell, while the inner nuclear membrane intimately interacts with the nucleoplasm of the nucleus. Nuclear pore complexes may be adherent to the ONM, INM or may be transmembraneous. The nuclear lamina is a proteinacous structure composed of lamins, a class of intermediate filament proteins, sandwiched between the nuclear membrane and peripheral chromatin of the nucleus.

Since the advent of proteomic analysis, the protein database has more than 950 identified human nuclear proteins.79 Emerin, MAN-1, LBR, and SUN-1 are just a few of INM proteins that have been associated with lamins responsible for, amongst other nuclear envelope

functions, the assembly of chromatin. 80 Mutations in lamins and associated proteins cause approximately twenty different diseases including but not limited to lung, gastrointestinal, skin, genitourinary cancers, herpes simplex virus, and human cytomegalovirus. 80-82 Diseases caused by mutations in the integral proteins of the INM include Reynolds syndrome, atypical progeria syndrome, and Emery-Dreifuss muscular dystrophy to name a few.81

Mitochondria by virtue of being dual membrane organelles also contain numerous membrane-bound insoluble proteins that are critical to cell function. Mitochondria are known to be the energy powerhouse of the cell, but have other important roles in cell processes including cell signaling, calcium homeostasis, cell cycle regulation, apoptosis, free radical production and thermogenesis.83

Mitochodrial dysfunction alters and decreases ATP production in the cell which leads to oxidative stress and induces apoptosis. During routine energy production, via ATP production in the cell, oxygen molecules are largely reduced to water molecules, however 1-2% of the oxygen molecules are reduced incompletely and become free radicals in the form of superoxide anions or hydrogen peroxides.83 Oxidative damage induces cellular apoptosis. Alterations to mitochondrial membrane proteins can sway an increase in oxidative free radicals. Disturbances in mitochondrial calcium, ATP or oxygen metabolism play a significant role in disease pathogenesis, especially in oxygen dependent tissues such as neural or brain tissues.84

Mitochondrial dysfunction and oxidative damage has been linked to Parkinson’s disease,

Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and multiple sclerosis amongst a number of inherited mitochoriapathies.85-87

Intermediate filaments (IFs) are the major constituents of cellular cytoskeleton of tissues including epithelium, periodontal connective tissues and bone. 19 Structurally, IFs are a tripartite structure with a highly consistent central α-helical rod domain with variable N- head and C-tail termini.88 Individual filaments bundle and branch to form cellular cytoskeleton. IF proteins are subclassified into seven groups: five cytoplasmic, one nuclear (lamins, discussed previously), and one subcortical.88 They play an important role in the biological function of the cell including contractility, migration, structural integrity, and proliferation. 89 Mechanical forces which lead to the deformation of the IF of cytoskeleton especially near the boundary of the cell nucleus have been implicated in the induction of tooth movement in orthodontic therapy.90

Vimentin, desmin, glial fibrillary acidic protein and peripherin are a nominal few of many intermediate filament proteins of interest in cell function. 91, 92 Vimentin, an intermediate filament protein of mesenchymal cells, is profuse in subgingival connective tissue. Vimentin can be detected in cell culture by use of the vimentin specific antibody. Vimentin present in PDLF suggests strong proliferation, synthesis and extracellular deposition of collagen Type I and fibronectin. 19 Abnormalities of IF proteins cause epidermolysis bullosa and epidermolytic hyperkeratosis, in addition to the laminopathies.91

Endoplasmic reticulum and golgi bodies are membrane bound organelles responsible for synthesizing, folding, and trafficking proteins. 93 Golgi bodies are organized into several stacks

of about seven flattened cisternae.94 Golgi receive newly synthesized and folded proteins from the endoplasmic reticulum which mature by transversing through golgi stacks. Defective proteins are recycled, while properly assembled proteins are delivered to their appropriate destination or undergo further post-translation modifications.94 This process ensures protein assembly quality control which involves recognition and cleavage of unique sequences in protein prodomains to form mature proteins.95

Golgi-associated proteins and proteases play key roles in tumor formation and viral and bacterial pathogen propagation.95 Alkaline phosphatase, a protein found in golgi bodies, is inhibited by protease cleavage by furin, a golgi body protein associated with the misprocessing of coagulation factors in von Willebrand disease and growth factors like bone morphogenic protein-4 (BMP-4).96 Defining the proteome of fibroblasts has the potential to allow for new understanding of protein expression as well as alterations associated with disease states such as periodontitis.

Proteomics

Proteomics is the study of the “proteome”, a term coined in the late 1990’s used to describe the entire protein complement of the genome.79, 97, 98 Protein structures have long been of interest in the discovery of cell function. High quality analytical techniques and methods are critical in facilitating protein identification and linking the protein to a function.99

The disparity between the genetic code of the cell and the actual protein content is being

examined and filled by the field of proteomics. Proteomic analysis is able to account for all proteins present in a cell; those translated by a cell’s genetic code as well as proteins that have been modified by post-translational changes or alternative splicing.97, 98

The human genome consists of 30,000 to 50,000 genes that yield more than 100,000 proteins.100 Analyzing DNA sequences can yield information regarding proteins possibly encoded, but may not be congruent to actual protein expression. DNA sequence information cannot provide information on post-translation alterations or explain domains that are physiologically relevant and translated verses those that are spliced out or skipped in translation.98 The application of proteomic analysis does allow for discovering proteins that are expressed and therefore are critical to cell function.

As alluded to the previous section of this report, proteins are great in number, highly variable and complex. Therefore proteomic studies rely on sophisticated and evolving technologies to provide specific informatics on human cellular proteins. The basis for proteomic studies involves two basic analysis systems: 2-D gel electrophoresis or mass spectrometry. This discussion includes the essential components to each process, however many additions and variations to the processes can be made which has been described in previous work.101

2-D gel electrophoresis separates protein mixtures by charge and molecular weight into high resolution, reproducible patterns in two dimensions oriented at 90 degrees to each other.

This allows proteins to spread over a larger surface area, increasing resolution102. The first dimension, isoelectric focusing (IEF), separates proteins by charge. The second dimension,

sodium dodecyl sulphate-polyarcrylamide gel electrophoresis (SDS-PAGE) separates proteins by size or molecular weight. From the pattern of spots displayed, quantitative and qualitative differences in proteins can be assessed. For identification of complex proteins or a mixture of proteins, the spots can be transferred from gel to membranes and identified by isotopic labeling, antibody coagulation, or mass spectrometry.98

2-D gel electrophoresis is advantageous as a technique since the equipment is relatively inexpensive and resolution can allow up to 11,200 proteins to be identified from a mixture of protein material.98 The disadvantages of 2-D gel electrophoresis include issues with reproducibility, difficulty in performing the procedure, failure in detection when more than 1000 spots are present, and containment of multiple proteins in a detected spot.98 Improved technologies and modifications in pre-treatment protocols have improved 2-D gel electrophoresis’s usefulness. Application of 2-D gel electrophoresis provides for simple comparison of diseased and normal cell lysates.103

Mass spectrometry (MS) has become the analysis system of choice for many aspects of proteomics. MS provides information on peptide (digested protein) mass and different structural modifications, such as gylcosylation and phosphorylation.98 MS can also generate amino acid sequence information on proteins that have not been described previously.104 MS can be performed on proteins that have been generated into a gas phase, a technique called matrix assisted laser desorption ionization (MALDI), or by liquid electrospray ionization (ESI).

An advancement in MS technology, liquid chromatography tandem MS (LC-MS/MS), allows for

complicated mixtures of proteins in varied concentration within a sample to be separated even with similar molecular weights by differences in hydrophobicity.98

MS is advantageous in proteomics for analyzing samples of thousands of proteins rapidly with less laboratory processing error.105 Also, small amounts of protein are required for

MS analysis which makes identification of less abundant proteins possible.98 LC-MS/MS has been used for exploring differential protein populations, to identify disease markers, to discover new drugs, and study cellular functions.

In dentistry proteome research has focused on oral flora and pathogens, salivary secretions, and protein expression of hard tissues. Proteomic biomarkers in caries pathogen profiles have been found to be useful in predicting caries in a pediatric population.106 In endodontic lesions proteomics has been used to demonstrate the complexity of microbiota involved in the pathogenesis of the necrotic pulp.107 Proteomic analysis of dental hard tissues has been used to describe the protein expression of dentin to define the organic matrix of dentin and characterized odontoblasts. 108

In review of published studies regarding proteomes of oral soft tissues, only three articles pertain to the human gingiva or periodontium. The first proteomic study in periodontal literature describes protein expression of PDLF.67 In an analysis of neutrophils of patients with aggressive periodontitis, an increase Caldesmon combined with neutrophil dysfunction may have predictive value in determining aggressive disease.109 For example, Galectin 3 has been found to be upregulated in GF in patients with Cyclosporin A-induced gingival overgrowth.110

To our knowledge no previous work has attempted to describe and compare the proteome of gingival fibroblasts with periodontal ligament fibroblasts. The purpose of this investigation is therefore to describe and compare the insoluble proteins of GF and PDLF.

The working hypothesis for this study is GF membrane bound and associated protein content varies in composition from PDLF membrane bound and associated proteins.

The specific aims of our study are:

1. Isolate fibroblasts (GF and PDLF) in matched pairs from individuals

2. Isolate membrane bound and associated proteins

3. Perform proteomic analysis to determine membrane bound and associated

proteins of PDLF and GF

4. Compare PDLF and GF membrane bound and associated proteins

CHAPTER 2: MATERIALS AND METHODS

Collection of Samples

Fibroblasts were established from human gingiva and human periodontal ligament harvested from individuals treatment planned for extraction of impacted third molars at the

Division of Oral and Maxillofacial Surgery at The Ohio State University College of Dentistry.

Teeth included for the harvest of periodontal ligament fibroblast had three-fourths or more root formation and were completely or partially bony impacted with radiographic evidence of a dental follicle present and lack of communication with the oral cavity. Potential participants had a diagnosis of gingival health, which was confirmed by absence of gingival inflammation or infection. Interested subjects who met inclusion criteria provided signature of informed consent approved by the Institutional Review Board of The Ohio State University. Inclusion criteria for this study were: healthy patients without a history of systemic disease, 16 years of age or older, a patient of record of The Ohio State University College of Dentistry and a previously signed treatment plan for extraction of third molar(s). Twenty subjects consented to participate in the study aged 16 to 36 years of age.

Culture of Gingival Fibroblasts

Human GF were harvested from the gingiva of the overlying flap of the impacted third molars. All gingival samples appeared clinically healthy (firm, nonedematous, and coral pink) at biopsy. At the time of sampling, tissue explants were immediately placed in 10 ml of biopsy

media comprised of Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum, 350 μg/ml L-glutamine, 100 μg/ml penicillin and 100 μg/ml streptomycin.

Using aseptic technique under a laminar flow hood, the gingival samples were segmented in pieces approximately 0.5-1.0mm in diameter using a sterile #10 blade. Tissue segments were pipetted along with a small of amount of biopsy media into 25ml flasks.

Fibroblasts were propagated in 25 cm2 flasks in the same biopsy media at 37°C in a humidified incubator with 5% CO2 and 95% air atmosphere. Media were exchanged in the flasks every 48 hours.

At approximately 90% confluence, growth media were removed, cells were washed with phosphate buffered saline(PBS) pH 7.4, treated with 0.25% trypsin/EDTA, and incubated for five to seven minutes to remove fibroblasts from the base of the flask. The fibroblasts were then transferred to 75ml flask and further propagated or split between three 75 ml flask to facilitate cell line passage. In the 4th passage fibroblasts were transferred to sequentially larger 250 ml flasks. Experiments were performed on cells in the 4th passage to have sufficient cells for processing and minimize changes that may occur at increased passage of cells.

Culture of Periodontal Ligament Fibroblasts

Human PDLF were harvested from the middle third of the root of impacted third molars.

All teeth samples were third molars from complete or partial bony impacted sites. At the time of sampling, explants were immediately placed in 10 ml of biopsy media.

Using aseptic technique under a laminar flow hood, the tooth roots were segmented into thirds by scoring the root surface. Periodontal ligament was harvested from the middle third of the root surfaces into pieces approximately 0.25-0.5mm in diameter using a sterile #10 blade. Segments were pipetted along with a small of amount of biopsy media into 25ml flasks.

Fibroblasts were grown and propagated in 25 cm2 flasks in the same biopsy media at 37°C in a humidified incubator with 5% CO2 and 95% air atmosphere. Media were exchanged in the flasks every 48 hours.

At approximately 90% confluence, growth media were removed, cells were washed with phosphate buffered saline, treated with 0.25% trypsin/EDTA, incubated for five to seven minutes to remove fibroblasts from the base of the flask. The fibroblasts were then transferred to 75ml flask and further propagated or split between three 75 ml flask to facilitate cell line passage. In the 4th passage fibroblasts were transferred to sequentially larger 250 ml flasks.

Cells for the analysis were all harvested in the 4th passage.

Isolation and solubilization of membrane bound and associated proteins

Prior to confluence, cells from the 4th passage were washed with phosphate buffered saline (PBS) pH 7.4, treated with 0.25% trypsin/EDTA, and incubated for five to seven minutes to harvest fibroblasts from the flask. The fibroblasts were pelleted from solution by centrifugation at 3000 rpm for 5 minutes at which time the trypsin/EDTA solution was removed. The membrane bound and associated proteins were isolated from fibroblasts according to the

modified protocol of Chevallet, M.et al (1998)111 by The Ohio State University Mass

Spectrometry and Proteomics Facility/Protein Expression and Purification Facility112.

Briefly, cell pellets were suspended in 40mM Tris-hydrocloride (pH 8.0) with protease inhibitor at a concentration of 200μL/ 1 mL A600 pellet size and transferred to 1.5mL microcentrifuge tube for cell fraction. Cells were gently sonicated on ice with medium power for

10 seconds and rested on ice for 20 seconds. Alternating sonication and rest cycles were repeated three times. The cell lysate was centrifuged for 15 minutes at 4°C at 10,000 rpm to pellet insoluble cellular components. The supernatant of soluble proteins was removed and transferred to a 1.5 ml microtube and stored at -80°C.

The pellet of insoluble protein was resuspended in 200μL of lysis buffer consisting of

40mM Tris-HCl (pH 8.0), 7M urea, 2M thiourea, 0.25% w/vASB-14, and 0.25% of 100% NP-40.

Samples were incubated at room temperature (21°C) for 30 minutes. Subsequently samples were vortexed for 1 minute. Pellet lysate was centrifuged for 30 minutes at room temperature at 13,200 rpm. The supernatant containing the membrane proteins with some lipid, nucleic acid, and sugar components was diluted to 500μL with distilled water (dH2O). Twenty percent fresh trichloracetic acid (TCA) was added to suspension and incubated on ice for 1 hour to remove contaminants, such as Tris-HCL or urea from previous step. Samples were centrifuged for 15 minutes to pellet and the supernatant was carefully removed from pellet and stored in a microtube at -80°C. Pellets were centrifuged again for 15 minutes at 13200 rpm to remove the remaining TCA.

Without disturbing the pellet, 10μL of supersaturated Tris base was added to the pellet and allowed to rest for 5 minutes. 1 mL of iced acetone was added to the pellet and vortexed.

The protein suspension was centrifuged for 15 minutes at 4°C at 13200 rpm. Acetone was removed and the dry protein pellet was allowed to air dry. The protein pellet was resuspended in 2μL/200μL Invirtosol protein solubilizer (Invitrogen) and 2M urea and submitted for enzyme digestion and identification at the Mass Spectrometry and Proteomics Facility/Protein

Expression and Purification Facility at The Ohio State University.

Peptide separation, protein identification, and quantitation

The protein samples were submitted for enzyme digestion and identification at the

Mass Spectrometry and Proteomics facility for label-free proteomic quantitation methodology.

Briefly, protein was reduced by adding 10 µL of DTT (5 mg/mL solution prepared in 100 mM ammonium bicarbonate) and the reaction proceeded on at 60 °C for 30 minutes. To block reduced cysteine residues, 10 µL of Iodoacetimide solution (15 mg/mL in 100 mM ammonium bicarbonate) was added to the sample and allowed to incubate at room temperature for 15 minutes in the dark. Trypsin was prepared in 50 mM ammonium bicarbonate and added to the protein solution with an enzyme to substrate ratio of 1:25 (w/v). The sample was incubated for

2 hour at 37 °C before quenched by acidification.

Capillary-liquid chromatography-nanospray tandem mass spectrometry (Capillary-

LC/MS/MS) of global protein identification was performed on a Thermo Finnigan LTQ orbitrap

mass spectrometer equipped with a microspray source (Michrom Bioresources Inc, Auburn, CA) operated in positive ion mode. Samples were separated on a capillary column (0.2X150mm

Magic C18AQ 3µ 200A, Michrom Bioresources Inc, Auburn, CA) using an UltiMate™ 3000 HPLC system from LC-Packings A Dionex Co (Sunnyvale, CA). Each sample was injected into the

Precolumn Cartridge (Dionex, Sunnyvale, CA) and desalted with 50 mM acetic acid for 10 minutes. The injector port was then switched to inject and the peptides were eluted off of the trap onto the column. Mobile phase A was 0.1% formic acid in water and 0.1% formic acid in acetonitrile was used as mobile phase B. Flow rate was set at 2µl/min. Typically, mobile phase B was increased from 2% to 50% in 90-250 min, depending on the complexity of the sample, to separate the peptides. Mobile B was then increased from 50%-90% in 5 min and then kept at

90% for another 5 min before being brought back quickly to 2% in 1 min. The column was equilibrated at 2% of mobile phase B (or 98% A) for 30 min before the next sample injection.

MS/MS data was acquired with a spray voltage of 2 KV at a capillary temperature of 175 °C.

The scan sequence of the mass spectrometer was based on the data dependant

TopTen™ method: the analysis was programmed for a full scan recorded between 300 – 2000 Da and a MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive scans of the ten most abundant peaks in the spectrum. The resolution of full scan was set at 30000 to achieve high mass accuracy MS determination. The CID fragmentation energy was set to 35%. Dynamic exclusion is enabled with a repeat count of 30 s, exclusion

duration of 350 s and a low mass width of 0.50 and high mass width of 1.50 Da. Multiple

MS/MS detection of the same peptide was excluded after detecting it three times.

Sequence information from the MS/MS data was processed by converting the .raw files into a merged file (.mgf) using an in-house program, RAW2MZXML_n_MGF_batch (merge.pl, a

Perl script). The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.3.2 (Boston, MA) and the database searched against the full SwissProt database version 2010_10 (521,016 sequences; 183,900,292 residues) or NCBI database version 2001005

(11,961,441 sequences; 4,083,117,772 residues). The mass accuracy of the precursor ions were set to 2.0 Da given that the data was acquired on an ion trap mass analyzer and the fragment mass accuracy was set to 0.5 Da. Considered modifications (variable) were methionine oxidation and carbamidomethyl cysteine. Scaffold (Proteome Software, Portland, Oregon,

USA)113 was used to validate protein identifications derived from MS/MS sequencing results.

Scaffold verifies peptide identifications assigned by SEQUEST and Mascot using the X!Tandem database searching program (1). Scaffold then probabilistically validates these peptide identifications using PeptideProphet (3) and derives corresponding protein probabilities using

ProteinProphet (2).

Statistical analysis

The mean, standard deviation, confidence interval, paired T-test and Fisher extact test for all proteins were determined using the data sets of the four matched GF and PDLF

membrane pellets using bioinformatic software and statistical program analysis by Scaffold

(Proteome Software, Portland, Oregon, USA). Algorithms for calculating protein probabilities from peptide probabilities were carried out by Scaffold (Proteome Software, Portland, Oregon,

USA).

CHAPTER 3: RESULTS

Four matched pairs of GF and PDLF strains were successfully propagated, underwent insoluble protein isolation, and proteomic analysis. The four source individuals (Table 1) ranged in age from 16.4 to 25.4 years old with a mean age of 20.1 years. Three source individuals were female, one male. All participants were systemically healthy with no medication use reported with exception of one female who reported taking oral contraceptives. All participants were never smokers. Intraoral examinations determined all participants were free of any signs or symptoms of gingival inflammation, periodontal disease or oral infections.

A total of five hundred nineteen proteins were identified from the eight samples analyzed via LC-MS/MS (Table 2). Four hundred fifty proteins were common to both GF and

PDLF. Forty proteins were detected only in GF and 29 were detected only in PDLF (Figure 1). Of the proteins identified two hundred thirteen are known membrane bound or associated proteins (Figure2, Table 2).

Twenty-eight proteins, identified from the 450 proteins common to both GF and PDLF, were detected in statistically significant greater quantities by either GF or PDLF. Fifteen proteins were detected in significantly greater quantities by GF using Paired- T test or Fischer extact test.

These proteins include Aminopeptidase N, A-kinase anchor protein 2, Annexin A6, Vimentin,

Annexin A11, UPF0556 protein C19orf10, Prohibitin-2, NADH-ubiquinone oxidoreductase 75kDa mitochondrial, Protein AHNAK2, Annexin A4, Acetyl-CoA acetyltransferase mitochondrial,

microtubule-associated protein 4, SH3 domain-binding glutamic acid rich like protein 3,

Trifunctional enzyme subunit beta mitochondrial, and Profilin-1.

Five membrane or associated proteins were detected in greater quantities by GF compared to PDLF (Table 3). These proteins include Aminopeptidase N (Figure 3), Microtubule associated protein 4 (Figure 4), Annexin A11 (Figure 5), Prohibitin-2 (Figure 6), and NADH- ubiquinone oxidoredcutase 75kDa subunit (Figure 7).

Thirteen proteins were detected in significantly greater quantities by PDLF compared to

GF. These proteins include Alpha-2-HS-glycoprotein, Calcium/calmodulin-dependent protein kinase type II subunit delta, Desmoplakin, Serum albumin, Membrane-associated progesterone receptor component 2, Basement membrane-specific heparin sulfate proteoglycan core protein,

EGF-like repeat and discoidin protein i-like domain-containing protein 3, Filaggrin, Voltage- dependent calcium channel subunit alpha-2/delta-1, 4F2 cell-surface antigen heavy chain,

Nucleobindin-1, Four and a half LIMdomains protein 2, and Ubiquitin carboxyl-terminal hydrolase isozyme L1.

Seven of the proteins identified preferentially in PDLF compared to GF are membrane bound or associated proteins (Table 4). These proteins include Calcium/calmodulin-dependent protein kinase type II subunit delta (Figure 8), Desmoplakin (Figure 9), Membrane-associated progesterone receptor component 2 (Figure 10), Voltage-dependent calcium channel subunit alpha-2/delta-1 (Figure 11), 4F2 cell-surface antigen heavy chain (Figure 12), Nucleobindin-1

(Figure 13), and Ubiquitin carboxyl-terminal hydrolase isozyme L1 (Figure 14).

Forty of the 519 proteins identified by proteomic analysis were only detected in GF.

These proteins were Protein AHNAK2, Cathepsin K, Sulfide quinone oxidoreductase mitochondrial, SH3 domain-binding glutamic acid-rich-like protein 3, Microtubule-associated protein 4,LETM1 and EF-hand domain-containing protein 1 mitochondrial,Early endosome antigen 1,Succinyl-CoA ligase [GDP-forming] subunit alpha mitochondrial,Macrophage-capping protein,Fibulin-2, 2-oxoglutarate dehydrogenase mitochondrial, A-kinase anchor protein 12,

Cathepsin Z, Keratin, type II cuticular Hb3, L-lactate dehydrogenase B chain, 60S ribosomal protein L13,Sideroflexin-3, NADH-ubiquinone oxidoreductase 75 kDa subunit mitochondrial,

Succinyl-CoA 3-ketoacid-coenzyme A transferase 1 mitochondrial, Kinectin, Synaptic vesicle membrane protein VAT-1 homolog, Protein S100-A4, F-actin-capping protein subunit beta,

Uveal autoantigen with coiled-coil domains and ankyrin repeats, Glucosylceramidase, Keratin, type I cuticular Ha1, Syntaxin-12, Transcription intermediary factor 1-beta, Ras-related protein

Ral-A, Beta-hexosaminidase subunit beta, Phosphatidylethanolamine-binding protein 1,

Cathepsin L1, Epidermal growth factor receptor, Filamin-B, Microsomal glutathione S- transferase 3, Apoptosis-inducing factor 1 mitochondrial, Cysteine-rich protein 2, Epsin-1,

Isocitrate dehydrogenase [NADP] mitochondrial, and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7.

Of the forty proteins only detected in GF, nineteen were membrane bound or associated proteins (Table 5). These proteins include Microtubule-associated protein 4 (Figure

4), LETM1 and EF-hand domain-containing protein 1, mitochondrial (Figure 15), Early endosome

antigen 1 (Figure 16), Macrophage-capping protein (Figure 17), 2-oxoglutarate dehydrogenase, mitochondrial (Figure 18), A-Kinase anchor protein 12 (Figure 19), Sideroflexin-3 (Figure 20),

NADH-ubiquinone oxidoreductase 75kDa subunit,mitochondrial (Figure 7), Kinectin (Figure 21),

Synaptic vesicle membrane protein VAT-1 homolog (Figure 22), Glucosylceramidase (Figure 23),

Syntaxin-12 (Figure 24), Ras-related protein Ral-A (Figure 25), Epidermal growth factor receptor

(Figure 26), Filamin-B (Figure 27), Microsomal glutathione S-transferase 3 (Figure28), Apoptosis- inducing factor 1, mitochondrial (Figure 29), Epsin-1 (Figure 30), and NADH dehydrogenase

(ubiquione) 1 alpha subcomplex subunit 7 (Figure 31).

Twenty-nine proteins of the 519 proteins identified by proteomic analysis were only detected in PDLF. These proteins were Filaggrin, Synaptopodin-2, Sequestosome-1, Clathrin light chain A, Chondroitin sulfate proteoglycan 4, Integrin alpha-8, DAZ-associated protein 1,

GTP-binding protein SAR1a, Alpha-2-macroglobulin, Lactadherin, Ferritin heavy chain,

Phosphoenolpyruvate carboxykinase [GTP] mitochondrial, Ezrin, Eukaryotic translation initiation factor 6, Keratin, type I cytoskeletal 17, Junction plakoglobin, T-complex protein 1 subunit delta,

Heterogeneous nuclear ribonucleoprotein F, Tubulin beta-3 chain, Matrix metalloproteinase-14,

Matrix-remodeling-associated protein 7, Cadherin-2, Desmocollin-1, Nidogen-2, 60S ribosomal protein L38, Serine/arginine-rich splicing factor 2, Fibrillin-2, G-rich sequence factor 1, and

Ubiquitin-associated protein 2-like.

Ten of the 29 proteins only identified in PDLF were membrane bound or associated proteins (Table 6). These proteins include Clathrin light chain A (Figure 32), Chondroitin sulfate

proteoglycan 4 (Figure 33), Integrin alpha-8 (Figure 34), Lactadherin (Figure 35), Ezrin (Figure

36), Junction plakoglobin (Figure 37), Matrix metalloproteinase-14 (Figure 38), Matrix- remodeling- associated protein 7 (Figure 39), Cadherin-2 (Figure 40), and Desmocollin-1 (Figure

41).

CHAPTER 4: DISCUSSION

The current study is the first report in the literature to use proteomic analysis to identify and quantify the membrane bound and associated proteins of GF and PDLF. Furthermore it is also the first report to compare and contrast GF and PDLF membrane bound and associated proteins. Fibroblasts (GF and PDLF) were isolated in matched pairs from individuals to propagate four pairs of GF and PDLF strains, membrane bound and associated proteins solubilized for protein isolation and lysates of the GF and PDLF analyzed by use of liquid chromatography/ tandem mass spectrometry (LC-MS/MS).

The use of label free analysis allowed for the most inclusive description of the GF and

PDLF proteomes. Merits of label free analysis that led to use of this type of analysis in the current study include its ease in approach over isotope labeling since isotope labeling would have been expensive and cumbersome for the number of proteins expected and then identified by our study. Also label free analysis allows for identification of proteins not previously described or labeled for with isotopes. Certainly the added advantage over 2D gel technique is the ability to identify and quantify proteins in the same process.

However, all current techniques used in proteomic analysis have shortcomings that should also be recognized as potential limitations of study. Disadvantages of LC-MS/MS used in this study include difficulties with accuracy and reproducibility of proteins identified, especially in proteins low abundance in complex samples. Some difficulty with reproducibility is due to the

ionization of peptides in mass spectrometry which is dependent on rate and size of the molecules during ionization that can lead to misinterpretation by mass spectrometry.

Review of the available protein databases (Mascot and SwissProt) identified 519 known proteins. Two hundred thirteen of the proteins identified are known membrane bound and associated proteins. This is the most comprehensive and richest report in the literature of GF and PDLF proteomes. Previous characterizations of GF and PDLF were mostly observational or derived from gene probes instead of direct proteomic analysis as used in the current study.

Only one study in the literature has previously attempted to use proteomic analysis for exploration of the periodontal proteome. Reichenberg et al.67 described the protein components of PDLF in 2005. In the study, three periodontal ligament fibroblast cultures were established and analyzed using a 2-dimensional gel electrophoresis and mass spectrometry technique. In total they identified 117 gel spots to correspond to 74 different proteins. The limitations of the 2-gel electrophoresis are apparent from their results. Many proteins have such similar molecular weights and isoelectric points that separation of proteins from complex samples seen in cell lysates result in clouded, and often indistinguishable, protein spots. To avoid the perils of this type of result by 2-DE, the current study used LC/MS-MS that digests proteins into peptides allowing for better solubilization that enhances the sheer quantity of proteins identified. In addition, the use of matched pairs in the current study of GF and PDLF strains allowed for meaningful analysis of the proteins identified in common and unique to GF and PDLF beyond a descriptive list of proteins.

Despite the differences in technique the proteomic analysis completed by Reichenberg et al (2005) by use of whole cell lysis and 2-D gel electrophoresis and the analysis in this study,

71% of proteins identified by Reichenberg et al were also identified in the current study. Some of the common identified proteins accounted for are both membrane bound and non- membrane bound proteins this is since despite enriching our samples for membrane bound proteins many non-membrane bound proteins cling to these proteins by nature. Additionally perhaps not all membrane bound proteins have been previously described as such. However, the current study was able to identify hundreds of more proteins than the previously described.

This descriptive proteomic study is the initial step in expanding the knowledge and determining the importance of GF and PDLF protein similarities and differences which may be indicative of functional similarities and differences. By exploring the functional characterization of the differences found between GF and PDLF proteomes we can speculate how the descriptive differences reported in this study relate to fibroblast function.

A brief explanation of membrane or associated proteins that were synthesized in greater quantities by GF compared to PDLF is listed in Table 7. The proteins found in greater quantity in GF suggest important roles in cell metabolism, growth, immunity and energy regulation. Recent literature has taken noted interest in a particular cell membrane protein detected in greater quantities by GF, Aminopeptidase N.

Aminopeptidase N (AMPN) is a zinc-containing metalloprotease that removes amino acids sequentially from the N-terminals of peptides and proteins114 . AMPN is widely distributed

in the body of mammals, and can be expressed on the surface of various types of cells and be involved in angiogenesis115. AMPN was shown to be involved in the degradation of extracellular matrix in tumor invasion and may serve as a target receptor for drug delivery into tumors116.

A brief explanation of membrane or associated proteins that were synthesized in greater quantities by PDLF compared to GF is listed in Table 8. In PDLF, the membrane proteins synthesized in greater quantities are associated with the homeostasis of calcium, for calcium dependent cell processes, and attachment to the extracellular membrane. Two of these proteins, calcium/calmodulin-dependent protein kinase type II subunit delta and desmoplakin, are widely considered to function as adhesion molecules. This assumption is based in part on their homology to the cadherin family of calcium-dependent adhesion molecules117.

A brief explanation of membrane or associated proteins that were unique to GF and were detected in two or more of the fibroblast strains are listed in Table 9. These five proteins are associated with multiple membranes in the cell. In general they play important roles in shape and function of the mitochondria and energy production.

A brief explanation of membrane or associated proteins that were unique to PDLF and were detected in two or more of the fibroblast strains are listed in Table 10. These two proteins are important for the assembly and organization of the cytoskeleton, thereby influencing the function of cellular processes118.

The diversity of the functional characterization of membrane bound and associated proteins speaks to the diversity of GF and PDLF cellular components and biological processes.

However the differences seen in the results of the current study are likely reflective of true differences in nature since the propagation of GF and PDLF in vitro has been found to be a valid study model previously. PDL cells have been found to express similar phenotype in vitro and in vivo. A study comparing in vivo to in vitro concluded that the in vitro models used for assessment of PDLF cell differentiation appears to be appropriate and independent of the cell sampling method, whether collected from the extraction socket or root surface.27

However a potential limitation of using cultured cells strains is that repeated passaging of cells tends to reduce cell heterogeneity in vitro. Conventional culture methods cannot easily assay for the phenotype of non-proliferating or slow proliferating subtypes. 119 An alternative to overcome this issue is to study clonal populations, which has its own limitations in efficieny, efficacy, and sample size limitations. In addition, different cell types may exist in the cells harvested from the pdl that are all fibroblast-like cells but may be truly different cell types or lineages; for example, cementoblast, osteoblast precursors, and fibroblasts. However, well- defined cell lineage markers are currently unavailable.29

A final limitation of the current study is that by design this study is meant to be descriptive of the similarities and difference in GF and PDLF proteomes. In contrast to functional proteomic studies we are unable to extrapolate if differences in the results are interrelated or interdependent on each other. In reality many cell functions are interdependent on other cellular processes, but this significance cannot be reported by descriptive proteomic analysis.

A complication that occurred in the propagation of matched pair strains within the current study was loss of cell cultures do to environmental contaminations and fibroblast recovery from fresh tissue cultures. In total consent and tissue sample were collected from twenty individuals for the analysis of four matched pairs. This type of cell culture failure has been reported previously. Studies of fresh tissue cultures of periodontal ligament cells and gingival fibroblast have reported success in culturing pdl cells from biopsies at 76%, while success rates for gingival fibroblasts reported at 48%.120 Despite this complication our study was able to richly describe and compare GF and PDLF proteomes.

In conclusion, the current study was able to identify 519 GF and PDLF proteins, 213 of which are known membrane bound and associated proteins. Five membrane proteins were identified in greater quantities by GF, while seven membrane proteins were identified in greater quantities by PDLF. Nineteen and ten membrane proteins were detected only in GF and PDLF respectively. Future avenues for research should consider use of techniques for functional proteomic analysis to determine the interplay and functional significance of the current findings.

APPENDIX A: TABLES

Participant Age Sex Systemic Medications Smoking Tissue Health Number Conditions Status (Inflammation/Infection)

094 25.4 M None None Never (-/-)

089 21.2 F None Orthotricyclen Never (-/-)

084 17.5 F None None Never (-/-)

082 16.4 F None None Never (-/-)

Table 1. Participant demographics

Table 2. Proteins identified (bold face indicative of membrane bound protein)43

Table 2 Continued. Proteins identified (bold face indicative of membrane44 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane45 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane46 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane47 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane48 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane bound protein) 49

Table 2 Continued. Proteins identified (bold face indicative of membrane50 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane51 bound protein)

Table 2 Continued. Proteins identified (bold face indicative ofof membranemembrane52 boundbound protein)protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane53 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane54 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane55 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane56 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane57 bound protein)

Table 2 Continued. Proteins identified (bold face indicative of membrane bound protein)

Table 3. Membrane bound or associated proteins preferentially detected by GF

Table 4. Membrane bound or associated proteins preferentially detected by PDLF

Protein Microtubule-associated protein 4 LETM1 and EF-hand domain-containing protein 1, mitochondrial Early endosome antigen 1 Macrophage-capping protein 2-oxoglutarate dehydrogenase, mitochondrial A-Kinase anchor protein 12 Sideroflexin-3 NADH-ubiquinone oxidoreductase 75kDa subunit,mitochondrial Kinectin Synaptic vesicle membrane protein VAT-1 homolog Glucosylceramidase Syntaxin-12 Ras-related protein Ral-A Epidermal growth factor receptor Filamin-B Microsomal glutathione S-transferase 3 Apoptosis-inducing factor 1, mitochondrial Epsin-1 NADH dehydrogenase (ubiquione) 1 alpha subcomplex subunit 7

Table 5. Membrane bound or associated proteins only detected by GF

Protein Clathrin light chain A Chondroitin sulfate proteoglycan 4 Integrin alpha-8 Lactadherin Ezrin Junction plakoglobin Matrix metalloproteinase-14 Matrix-remodeling- associated protein 7 Cadherin-2 Desmocollin-1

Table 6. Membrane bound or associated proteins only detected by PDLF

Protein Name Protein Location and Function Aminopeptidase N Cell membrane (single pass). Involved in the metabolism of regulatory peptides and has been found to cleave antigen peptides bound to major histocompatibility complex class II molecules therefore playing roles in cell metabolism, regulation, and immunity. Microtubule associated Plasma membrane, cytoplasm, cytoskeleton. MAP4 is protein 4 responsible for the connection of cytoskeletal microtubules to the cellular plasma membrane.

Annexin A11 Nuclear envelope, nucleoplasm (cell cycle spedific) Involved in cellular growth and signaling cell replication. Required for midbody formation and completion of the terminal phase of cytokinesis. Prohibitin-2 Mitochondrion Inner membrane, nuclear membrane. It acts as a mediator of transcriptional repression by nuclear hormone receptors via recruitment of histone deacetylases. In mitochondria it is probably involved in regulating mitochondrial respiration activity and aging. NADH-ubiquinone Mitochondrion inner membrane. It is a core subunit of the oxidoreductase 75kDa mitochondrial membrane respiratory chain NADH subunit dehydrogenase (Complex I) that is believed to belong to the minimal assembly required for cell catalysis.

Table 7. Membrane bound or associated proteins synthesized in greater quantities by GF reported by location and function

Protein Name Protein Location and Function

Calcium/calmodulin- Peripheral membrane protein. One of four isoform chains dependent protein kinase type (alpha, beta, gamma, and delta) that form homo- or II subunit delta heteromultimeric enzymes that are calcium dependent and are responsible for the phosphorylation necessary to carry out many cell processes. Desmoplakin Plasma membrane protein. Involved in the organization of the desmosomal cadherin-plakoglobin complexes into discrete plasma membrane domains and in the anchoring of intermediate filaments to desmosomes Membrane-associated Cell membrane protein (single pass). Steroid receptor progesterone receptor protein; steroid receptor proteins are often used as target component 2 receptors for medication administration.

Voltage-dependent calcium Cell membrane protein. Regulates calcium current density channel subunit alpha-2/delta- and activation or inactivation kinetics of the calcium 1 channel

4F2 cell-surface antigen heavy Cell membrane protein (single pass). Required for the chain function of light chain amino-acid transporters. Involved in the sodium-independent, high-affinity transport of large neutral amino acids such as phenylalanine, tyrosine, leucine, arginine and tryptophan. Nucleobindin-1 Golgi network membrane protein, peripheral membrane protein. A major calcium-binding protein of the Golgi. It is expected that NUCB1 plays a role in calcium homeostasis. Ubiquitin carboxyl-terminal Endoplasmic reticulum membrane. Involved in the hydrolase isozyme L1 processing of ubiquitin precursors. Ubiquitin proteins direct protein recycling within the cell. This enzyme is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin.

Table 8. Membrane bound or associated proteins synthesized in greater quantities by PDLF reported by location and function

Protein Name Protein Location and Function Microtubule-associated Plasma membrane, cytoplasm, cytoskeleton. MAP4 is protein 4 responsible for the connection of cytoskeletal microtubules to the cellular plasma membrane. LETM1 and EF-hand Mitochondrion inner membrane. Maintenance of domain-containing protein mitochondrial tubular network shape and cistae organization. 1, mitochondrial Assembly of the supercomplexes of the respiratory chain of mitochondria. Early endosome antigen 1 Peripheral membrane. Binds phospholipid vesicles containing phosphatidylinositol 3-phosphate and participates in endosomal trafficking Macrophage-capping Nuclear membrane. Calcium-sensitive protein critical to nuclear protein actin filament function. May play a role in regulating cytoplasmic and/or nuclear structures through potential interactions with actin by binding DNA and macrophage function 2-oxoglutarate Mitochdrion membrane. Catalyzes the overall conversion of 2- dehydrogenase, oxoglutarate to succinyl-CoA and CO2; therefore it is critical to mitochondrial cellular glycolysis A-Kinase anchor protein 12 Plasma membrane. Anchoring protein that mediates the subcellular compartmentation of protein kinase A and protein kinase C. Antibodies against this protein are seen in the disease state of myasthenia gravis Kinectin Endoplasmic reticulum membrane, plasma membrane. Involved in kinesin-driven vesicle motility. It accumulates in integrin-based adhesion complexes upon integrin aggregation by fibronectin Synaptic vesicle membrane Cell membrane (integral protein). Possesses ATPase activity protein VAT-1 homolog and plays a part in calcium-regulated keratinocyte activation in epidermal repair mechanisms. However VAT1 has been found to have no effect on cell proliferation Ras-related protein Ral-A cell membrane (surface protein). Multifuntional GTPase involved in a variety of cellular processes including gene expression, cell migration, cell proliferation, oncogenic transformation and membrane trafficking Table 9. Membrane bound or associated proteins unique to GF in two or more strains reported by location and function

Protein Name Protein Location and Function

Clathrin light chain A Cytoplasmic vesicle membrane, plasma membrane. Critical to vesicle assembly and endocytosis

Junction plakoglobin Cell membrane. Membrane-associated plaques that are architectural elements to influence the arrangement and function of both the cytoskeleton and cells within the tissue. The presence of plakoglobin in both the desmosomes and in the intermediate junctions suggests that it plays a central role in the structure and function of submembranous plaques

Table 10. Membrane bound or associated proteins unique to PDLF in two or more strains reported by location and function

APPENDIX B: FIGURES

Figure 1. Proteins identified by fibroblast type

Figure 2. Proteins identified by cellular component69

Figure 3. Protein quantification for Aminopeptidase N

Figure 4. Protein quantification for Microtubule associated protein 4

Figure 5. Protein quantification for Annexin A11

Figure 6. Protein quantification for Prohibitin-2

Figure 7. Protein quantification for NADH-ubiquinone oxudireductase 75 kDa subunit, mitochondrial

Figure 8. Protein quantification for Calcium/ calmodulin-dependent protein kinase type II subunit delta

Figure 9. Protein quantification for Desmoplakin

Figure 10. Protein quantification for Membrane-associated progesterone receptor component 2

Figure 11. Protein quantification for Voltage-dependent calcium channel subunit alpha- 2/delta-1

Figure 12. Protein quantification for 4F2 cell-surface antigen heavy chain

Figure 13. Protein quantification for Nucleobindin-1

Figure 14. Protein quantification for Ubiquitin carboxyl-terminal hydrolase isozyme L1

Figure 15. Protein quantification for LETM1 and EF-hand domain-containing protein 1 mitochondrial

Figure 16. Protein quantification for Early endosome antigen 1

Figure 17. Protein quantification for Macrophage-capping protein

Figure 18. Protein quantification for 2-oxoglutarate dehydrogenase, mitochondrial

Figure 19. Protein quantification for A-kinase anchor protein 12

Figure 20. Protein quantification for Sideroflexin-3

Figure 21. Protein quantification for Kinectin

Figure 22. Protein quantification for Synaptic vesicle membrane protein VAT-1 homolog

Figure 23. Protein quantification for Glucosylceramidase

Figure 24. Protein quantification for Syntaxin-12

Figure 25. Protein quantification for Ras-related protein Ral-A

Figure 26. Protein quantification for Epidermal growth factor receptor

Figure 27. Protein quantification for Filamin--B

Figure 28. Protein quantification for Microsomal glutathione S-transferase 3

Figure 29. Protein quantification for Apoptosis-inducing factor 1, mitochondrial

Figure 30. Protein quantification for Epsin-1

Figure 31. Protein quantification for NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 7

Figure 32. Protein quantification for Clathrin light chain A

Figure 33. Protein quantification for Chondroitin sulfate proteoglycan 4

100

Figure 34. Protein quantification for Integrin alpha-8

101

Figure 35. Protein quantification for Lactadherin

102

Figure 36. Protein quantification for Ezrin

103

Figure 37. Protein quantification for Junction plakoglobin

104

Figure 38. Protein quantification for Matrix metalloproteinase- 14

105

Figure 39. Protein quantification for Matrix-remodeling-associated protein 7

106

Figure 40. Protein quantification for Cadherin-2

107

Figure 41. Protein quantification for Desmocollin-1

108

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