A NOVEL REGULATORY ROLE of TRAPPC9 in L-PLASTIN-MEDIATED ACTIN RING FORMATION and OSTEOCLAST FUNCTION a Thesis Submitted to K

Home , Cathepsin D, Osteocalcin

A NOVEL REGULATORY ROLE OF TRAPPC9 IN L-PLASTIN-MEDIATED ACTIN

RING FORMATION AND OSTEOCLAST FUNCTION

A Thesis submitted

To Kent State University in partial

fulfillment of the requirements for the

degree of Master of Science

Nazar Jabbar Hussein

December 2016

Except for previously published materials Thesis written by

Nazar J. Hussein

B.Ed., University of Tikrit, Department of Biology, Samarra, Salah Addin, Iraq 2011

Approved by

Dr. Fayez Safadi ______, Chair, Master thesis Committee

Dr. Denise Inman ______, Members, Master thesis Committee

Dr. Michael Model ______,

Accepted by

Dr. Ernest J. Freeman

______, Director, School of Biomedical Sciences

Dr. James L. Blank ______, Dean, College of Arts and Sciences TABLE OF CONTENTS

TABE OF CONTE

LIST OF vi

LIST OF ABBREVIATION

I. ABSTRAC

II.

III. MATERIALS AND METHODS

IV. RESUL ..54

V. 77

VI. 82

VII. LITTERATE 85

LIST OF FIGURES

Figure 1.1: |Schematic Diagram of Bone anatomy of Osteon ...... 8

Figure 1.2 I A model of osteoclast differentiation induced by stimulatory factors ...... 14

Figure 1.3: | Bone remodeling ...... 20

Figure 1.4: | Structural diagram representi

Figure 1.5: | TRAPPC9 structur

Figure 1.6: I Role of TRAPPC9 in intra-Golgi

Figure 1.7: I Structural representation of osteoclast cytos

Figure 1.8: I Schematic diagram of podosome

Figure 1.9: | L-

Figure 2. 1: | PMX vectro

Figure 3.1: | TRAPPC9 expression increased during os

Figure 3. 2: | Cathepsin.K and NFATc1 expression increased during osteoclast differentiat

Figure 3. 3: | TRAPPC9 localization in p

Figure 3. 4: | TRAPPC9 co-localizes with Cathepsin.K during OC differentiation

Figure 3.5: | Figure 3.6: |

Figure 3.7: |

Figure 3.9: | LPL expression during osteocl

Figure 3.10: | Assesment of TRAPPC9 overexpression transfuction and transduction

Figure 3.11: | Assesment of TRAPPC9 overexpression transfuction and transduction

Figure 3.12: | Assesment of TRAPPC9 overexpression transfuction and transduction

Figure 3.13: | TRAPPC9 overexpression increases recruitment of LPL to

Figure 3.14: | TRAPPC9 overexpression is not associated with LPL mRNA

Figure 3.15: | TRAPPC9 overexpression enhanc

Figure 3.16: | Mechanism of TRAPPC9/LPL bining and meadatign the actin podosomes/actin rign formation i

LIST of TABLES

Table 1: List of approved treatment for osteoporosis and its side

Table 2: List of antibod

Table 3: Osteoclast prim

Table 4:

LIST OF ABBREVIATIONS

ALP Alkaline Phosphatase

BMM Bone marrow macrophage

OCN Osteocalcin

OPG Osteoprotegrin

OSX Osterix

RANKL Receptor Activator of Nuclear factor-Kappa B ligand

RUNX2 Runt-related Transcription Factor

TRAPPC9 Trafficking Protein Particle Complex 9

TRAPP Transport Particle Protein

LPL L-PLastin

BMMs bone marrow-derived macrophages

COPI coat Protein I

OSCAR Osteoclast-associated immunoglobulin-like receptor

NFATc1 Nuclear factor of activated T cells

GEF Guanine Exchange Factors

Ypt1p Yeast Protein Two 1 Protein

TRAP Tartrate-Resistant Acid Phosphatase

Dedication

I would like to dedicate this study to my country, family, and to everyone who supported me during these years.

To my father in his second death anniversary, I will be always as you wanted me to be, kind and successful. You always inspire me even though I cannot hear you or see you physically anymore. You will be always in my heart, mind, and prayers.

and support despite of the long distance between us.

To my brothers and sisters. Thank you for being there and thank you for all the advice and warm wishes.

To all my friends from all over the world. Thank you for all your support, kind words, and great wishes. You are all so precious to me.

To my advisor and mentor Dr. Fayez Safadi, without your wisdom, knowledge, and

positive attitude, patience, and encouragement. Thank you for all the valuable experience that you have provided me during my training in your lab.

To my lab members and colleagues: Asaad, thank you for all the delicious lunches that we had together, and the tremendous help and companionship as well. Greg, thank you for your willingness to help no matter what you were involved in, for your kindness and politeness. Tom, thank you for all your experience and precious notes. Fouad, thank you for all your kind words and your outgoing personality. Fatima, thank you for all the fine sweets and candies that you brought after each experiment. Kevin, thank you for all the help. Kim, thank you for being there where I needed help. Thank you all for being there when I needed someone to talk to and joke with. You are more than lab members; you are my best friends.

- -

- - ur hypothesis is that

TRAPPC9 plays a regulatory role in L-Plastin-mediated actin ring formation and osteoclast function and the overall objective of this research was to evaluate TRAPPC9/

LPL interaction as a possible target for modilities to enhance bone repair and regeneration.

CHAPTER I

INTRODUCTION

The skeletal system is a complex and dynamic organ that undergo changes needed to maintain bone and calcium homeostasis through a process called bone remodeling.

This process is crucial for repairing damaged bone and maintaining the homeostasis of minerals. This process is carried on through highly balanced function between local resorption of bone by the osteoclasts and bone formation by the osteoblasts. Indeed, an imbalance between these activities leads to and/or favors several diseases due either to an excess of bone resorption (e.g., osteoporosis, rheumatoid arthritis, and metastatic cancer) or bone deposition/formation (e.g., osteopetrosis and pycnodysostosis). The main forming cells are osteoblasts, which originate from the mesenchymal stem cell lineage. Osteoblasts produce a bone matrix that they later mineralized to form bone.

Usually matured osteoblasts get trapped in the bone matrix and become osteocytes during the process of bone formation. Osteoclasts are polynucleated cells that are derived from the differentiation of monocyte/macrophage precursors close to the bone surface in response to two stromal hematopoietic factors, the receptor activator of nuclear-factor- (RANKL) and colony-stimulating factor-1 (CSF1). It has been shown that TRAPPC9, a newly identified protein of the TRAPP II complex 45, binds to

-kB-inducing kinase (NIK), thus regulating both the canonical and alternate

NF- Trafficking protein particle complex 9 (TRAPPC9) is a protein subunit of the Transport Protein Particle II (TRAPPII) and it plays an important role in endoplasmic reticulum (ER) exiting to Golgi complex, intra-Golgi, and endosome-to-

Golgi transports in yeast cells 15, 74.

Osteoclasts become polarized and undergo significant morphologic changes during differentiation into mature cells, which leads to the complete remodeling of the actin cytoskeleton and organization of the podosome contacts into a dense circumferential band of actin (a podosome ring). This ring will result in bone erosion by forming a tight adhesive contact (the sealing zone). The degradation products (both inorganic and organic) are taken up by the osteoclasts and further processed. The fully mature osteoclast can detach from the bone and move away from the resorption lacuna to participate in several rounds of resorption. Collectively, the integrity of the cytoskeleton depends on tightly regulated bone remodeling and is carried out according to the physiological needs of the organism8, 48

Our hypothesis was the focus of the study (chapter III), we showed that TRAPPC9 binds to LPL thus, TRAPPC9 overexpression promotes LPL recruitment to podosome/actin ring region during osteoclastogensis. To prove this hypostasis, we first sought to measure TRAPPC9 expression during osteogenic differentiation of bone marrow-derived macrophages (BMMs). Then, we investigated the potential role of

TRAPPC9 in LPL-mediated actin ring formation and osteoclast function. Then we proposed a mechanism that could explain the Role of TRAPPC9 overexpression on osteoclast cytoskeleton organization by examining the physical binding between LPL and TRAPPC9. LPL was found to participate in early sealing zone formation of osteoclast. In addition, LPL has a role along with actin-binding protein cortactin, in the osteoclast sealing zone formation66. It has been shown that TRAPPC9 binds to several proteins such as TRAPPC10, TRAPPC2, and P150Glued a protein sub unit of Dynactin

113. In addition, TRAPPC9 was found to bind to COPI (coat Protein I). Therefore, we

were interested to examine the potential role of TRAPPC9 in LPL- mediated actin ring

formation and osteoclast function since our data approved the physical binding between

TRAPPC9 and LPL. We were interested to examine the effect of TRAPPC9

overexpression on LPL recruitment to podosomes region. Such a recruitment may

enhance the binding of LPL to F-actin thereby form stabilized podosomes.

Skeleton

The skeleton can be divided into two main structures: the appendicular skeleton, which is comprised of 139 bones, and the axial skeleton, which is comprised of 74 bones.

Bones are generally grouped into four general categories: long bones, short bones, flat bones, and irregular bones. The long bones category involves the clavicle, humeri, radii, ulnae, metacarpals, femurs, tibia, fibulae, metatarsals, and phalanges. The short bones category involves the carpal and tarsal bones, patellae, and sesamoid bones. The flat bones category consists of the skull, mandible, scapulae, sternum, and ribs. Finally, the category of irregular bones includes the vertebrae, sacrum, coccyx, and hyoid bone.

The skeleton function can be divided into three main roles. First, the skeleton system provides structural support, protects vital internal organs such as the heart, lungs, and kidneys. Second, the skeleton plays an essential structural role that provides highly maintained status of mineral homeostatic and acid-base balance. Third, the skeleton permits and eases the movement and the locomotion by providing levers for the muscles16.

The adult human skeleton is mainly made of two distinguishable forms. Cortical bone consists of about 80% of overall the system. The other 20% is made of trabecular bone.

Both cortical and trabecular bones are composed of osteons. The Harversian system is the name of osteons in cortical bone and it has a cylindrical shape form of nearly 400 mm long and 200 mm wide at their base and that results in an expanded branching network5. The Harversian system walls are formed of concentric lamellae and are aligned with the long axis of the bone. formed, which run perpendicular to the long axis of the bone. Both Harversian and s include blood vessels and nerves. Periosteum is the outer layer that covers the long bone and consists of a fibrous connective tissue layer that covers the outer cortical surface of bone except joints. Periosteum contains blood vessels, osteoblasts, osteoclasts, and nerve fibers16. While the endosteum is the connective tissue lining the inner surface of the bone cavity, it also contains blood vessels, osteoblast, and osteoclast16 ( Figure 1.1).

Trabecular bone

Nerves

Blood vessels

Harversian canal

Periosteum Lamella

Osteocyte

Endosteum

Figure 1. 1: |Schematic Diagram of Bone anatomy of Osteon A: A cross section through the osteon shows the blood vessels (red and

blue), nerves (yellow), an B: A magnified

section of osteon shows the imbedded osteocyte (pink) and the inner lining

layer (Endosteum). Diagram adapted and modified from Servier Medical Art

Bone development

The skeletal system of all vertebrates consists mostly of cartilage and bone. The mesoderm is a germ layer where the skeletal system derived from. Mainly, the skeletal system is formed by chondrocytes and osteoblasts. To develop a healthy skeletal system, many cellular and molecular processes are involved including proliferation, migration, and differentiation of a variety of cell types, such as cartilage-forming chondrocytes, bone-forming osteoblast, and the bone and cartilage-resorbing osteo/chondroclast. The origin and source of the mesenchymal cells will be determined based on the distribution of the skeletal system. Three mesenchymal cell lineages are known to contribute to the formation of most skeletal tissues. The craniofacial bones originate from the neural crest cell lineage, the axial skeletons originate from the sclerotome compartment of the somites, and the appendicular skeleton is formed from the lateral plate mesoderm106. There are two essential processes that control bone ossification known as intramembranous and endochondral ossification.

Intramembranous ossification mostly results in the formation of the flat bone of the face and cranium. On the other hand, endochondral ossification is the process where most the skeletal system is formed following a cartilaginous template. Endochondral ossification begins with the mesenchymal condensations at the sites of future bone. The condensed mesenchymal cells undergo chondrogenic differentiation rather than osteogenesis, which gives rise to a cartilaginous template of the future bone 79, 80.

Bone Composition

T mainly consists of bone and it is mostly composed of a mineralized matrix. Throughout a bone tissues continually regenerate and undergo changes to , as well as biological, demands as a part of bone turnover. Bone as an organ generally contains cartilaginous joints, calcified cartilage in the growth plate, marrow space, and the cortical or compact bone (around 80%) and cancellous mineralized structures or trabecular or spongy bone (around 20%)19. In the skeletal system, the ratio of trabecular bone to compact bone is different from one location to another. The cortical (compact) bone is almost the solid phase of the bone and it consists of 10% porosity88 and can be found in the long bones (tibia and femur), short bones (wrist and ankle), and flat bones

(skull vault and irregular bones)88. The other form of the bone tissue is the trabecular

(spongy) bone, its porosity is around 50-90% and it is about 20 times lower than cortical bone in intensity compressive strength88. As a tissue, bone consists of mineralized and non-mineralized (osteoid) components of the cortical and cancellous regions of the long bone and flat bones (Figure 1.1, A). There are three types of cells that mostly regulate the bone homeostasis and maintenance: (1) osteoblast, the bone forming cells and when imbedded in the minerals they will become (2) osteocyte, and (3) osteoclast, the bone-resorbing cells. All three types of cells interact with each other and respond to one, as well as other cells through cell-to-cell contact or molecular signaling38. The main compositions of bones are mineralized extracellular matrix, collagen, water, non- collagenous proteins, and lipids. The bone extracellular matrix is made of the nano- crystalline, hydroxyapatite (HA) [Ca10(PO4)6(OH)2]38, which makes up 65 - 70% of bone composition by dry weight37.

Cellular Composition of Bone

Osteoblast

Differentiation

Osteoblasts (OB) are bone-forming cells derived from pluripotent mesenchymal stem cells. Differentiation of MSCs can lead into a variety of tissues, including, muscle, and fat11, 51. Osteoblast differentiation is regulated by several cytokines such as the transforming growth factor beta (TGF- , Wingless-type MMTV integration site family member (WNTs), bone morphogenic proteins (BMPs), and parathyroid hormone (PTH).

Osteoblast differentiation in vitro can be divided into multiple stages, including proliferation, extracellular matrix deposition, matrix maturation, and mineralization61, 93.

To measure osteoblast differentiation, the osteoblast expresses specific markers such as type I collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), and osteopontin (OPN). The marker for early differentiating osteoblast and their matrix is alkaline phosphatase, whereas osteocalcin is usually considered the marker for the later differentiation stage. ALP is generally considered an earlier marker of osteoblast differentiation whereas OC is usually measured at a late stage. Runt-related transcription factor 2 (Runx2) is another transcription factor that controls OB differentiation. Runx2 also regulates expression of various osteoblast-specific genes including Col1, ALP, OPN, osteonectin (ON), and OC40, 52, 60, 87. Bone formation and mineralization are first initiated by the deposition of an extracellular matrix rich in collagen type I by OB18, known as the osteoid, which is then mineralized through the

accumulation of calcium phosphate in the form of hydroxyapatite. This process

produces the hard but light-weight composite that is the major constituent of bone.

Osteoclast

Osteoclast cells are polynucleated cells (Figure 1.2) that differentiate from the

monocyte/macrophage precursor. Two major factors play a crucial role in osteoclast

differentiation from macrophage precursor, TNF-related cytokine; RANKL and the

polypeptide growth factor CSF-1 (colony-stimulating factor-1)57 107. Osteoclast

differentiation is inhibited by the osteoprotegrin (OPG) a decoy receptor for RANKL

produced by osteoblast cells56. The main function of osteoclast is to resorb bone.

Osteoclast expresses tartrate-resistant acid phosphatase (TRAP), calcitonin receptors95,

103, and cathepsin K. The osteoclast, which is the sole established resorptive cell,

degrades the skeletal matrix by forming a microenvironment between itself and the

bone surface. Hydrogen and chloride ions will be transported into this isolated space,

thereby creating an acidic milieu

process exposes the ing largely of type 1 collagen, which

is subsequently degraded by cathepsin K. The process of bone resorption requires a

bone micro-environment called lacunae composed of a sealing zone that is made of

densely packed podosomes that surround the apical membrane of the osteoclast6. The

actin ring isolates the resorptive microenvironment from the general extracellular space.

Actin rings are transient and present only when cell is attached to bone. Once the

osteoclast detaches from the bone surface to move to another site of skeletal degradation, the actin rings disappear, thereby, the organization of osteoclast cytoskeleton is an es 98.

Pre

osteoclast

Bone M-CSF M-CSF marrow + precursor RANKL

RANKL

Mature Multinuclear osteoclast osteoclast

Figure 1.2 I A model of osteoclast differentiation induced by stimulatory factors

Bone marrow precursors differentiate into mature osteoclast, under the stimulation of both RANKL and M-CSF, which cause cellular fusion of 10-20 individual cells.

Treatment of bone marrow precursor cells with M-CSF induced the expression of M-

CSFR, the receptor for M-CSF. M-CSF induces expression of RANK receptor on these cells, priming them for further differentiation in response to RANKL. Osteoclast Signaling pathway

RANKL/RANK/OPG

Precursors cells turn into mature osteoclast throughout osteoclastogenesis. Both the macrophage-stimulating factor (M- ligand (RANKL) are needed55, 57, 107. RANKL is a type II transmembrane protein abundantly expressed on the cell surface of BM stromal cells, osteoblasts (OB), and osteocytes71. The interaction between the RANKL and RANK receptor expressed on the surface of the cell surface is the key for the OC differentiation from precursor to mature

OC. Moreover, mice with both RANKL and RANK mutation have severely osteopetrosis and altered OC7, 58. On the other hand, osteoprotegerin (OPG) is also produced by OB.

OPG is the decoy receptor for the RANKL receptor, whereas overexpression of OPG in mice results in preventing OC formation and that results in osteopetrosis. Conversely,

OPG gene deletion results in osteoporosis90. TRAF-6 is another essential protein in OC differentiation. TRAF-6 gets activated once RNAKL binds to the RANK receptor and that will result in activation of all tree mitogen-activated protein kinase (MAPK) pathways, specifically extracellular signal-regulated kinase (ERK), Janus N-terminal kinase (JNK), and p38, as well as the phosphatidylinositol 3-kinase (PI-3K) and NF- transcription factors. TRAF-6 deletion in mice results in a severe osteopetrosis known by dysfunctional OC65.

Integrin Signaling

Integrins can be defined based on their mechanical function by attaching the cell cytoskeleton to the extracellular matrix (ECM), and biochemically, by sensing whether adhesion has occurred. Structurally, Integrins family exists in alpha and beta subtypes, which form transmembrane heterodimers. Integrins can transmit information both outside-in and inside-out by transducing biochemical signals into the cell and vice versa47. ntegrins are highly expressed in osteoclast42, 44. Integrins play a role in osteoclast adhesion to the RGD sequence of ECM proteins such as vitronectin, osteopontin, and bone sialoprotein33, 34, 41. integrin expression is increased in the precursor cells that are induced by RANKL during the osteoclast differentiation, proto- oncogene c-srcis mediating t by acting as a protein kinase and adopter protein that regulates lamellipodia formation and podosomes disassembly. Osteoclast bone resorption was shown to be partially inhibited by both anti- - rin in a RGD-dependent manner75.

Small GTPASes

The Rho family of GTPases plays an essential role throughout actin filaments assemble/dissemble process, thereby remodeling the cytoskeleton49. Extracellular matrix-cell interaction or adhesion mediates the Rho and Rac binding to GTP and their translocation to the cytoskeleton. In OC, Rho activation and expression of GTPase result in stimulation of actin ring formation, podosomes aggregation, osteoclast motility, and bone degradation. In contrast, alteration of Rho family expression results in inhibition of all these event12. Additionally, lamellipodia in osteoclast precursors can be formed by the Rac stimulation, thereby the frontal part of OC that it migrates will be

35.

NFATc1

Nuclear factor of activated T cells (NFATc1) is the calcineurin and calcium-regulated transcription factor NFATc1 and it is also activated through RANKL-RANK binding. OC differentiation can be induced during NFATc1 expression even in the absence of

RANKL96. Ca2+ signaling and c-Fos mediated signal through TRAF-6/NF- modulate NFATc1 expression96. NFATc1 is a known regulator of osteoclast specific genes, such as calcitonin receptor, TRAP96, Cathepsin K68, Osteoclast-associated immunoglobulin-like receptor (OSCAR)54, 22.

Bone remodeling

Bone remodeling is a process where the bone tissue is dynamically renewed during a person life. The importance of this process is to keep the homeostasis and to maintain a constant bone mass17, 109. Remodeling begins at birth and continues until death. Bone remodeling consists of four stages that happen continually and sequentially. The bone remodeling stages are: activation, resorption, reversal, and finally formation. The target site for remodeling can develop randomly, but also to areas that require repair10, 78.

Once the activation stage begins, recruitments of mononuclear monocyte-macrophage osteoclast precursors from circulation and activation occur82, and the endosteum that contains lining cells of the bone surface is lifted off bone surface and multiple mononuclear cells fuse to form multinucleated preosteoclast. To create bone resorption pits between the bone surface and the ventral space of osteoclasts, the multinucleated osteoclasts form a sealing zone that can provide a tight attachment to the bone surface.

Resorption of bone occurs when the osteoclast starts secreting ions through H+-

ATPase proton pumps and chloride channels in their cell membranes into the resorption pit to lower the pH, which helps mobilize bone mineral89. Other lytic enzymes are secreted throughout the bone resorption stage such as: tartrate-resistant acid phosphatase, cathepsin K, matrix metalloproteinase 9, and gelatinase from cell cytoplasmic lysosomes23. At the end of resorption, multinucleated osteoclast undergoes apoptosis and the resorption site is populated with mononuclear cells29, 81. The reversal stage is the transition stage between resorption and bone formation. Further investigation is needed to understand this stage. Initially, after the osteoclast-bone resorption stage, undissolved demineralized collagen matrix aggregates and covers the Howship lacunae31. Monocytic phagocytes were first thought to play a role in cleaning the Howship lacunae from the collagen remnants on the bone surface101. Finally, the bone formation stage in human ranges between 4 to 6 months to be completed. During this process, mesenchymal stem cells or early osteoblast progenitors are returned to the resorption lacunae. It is known that osteoblasts lead the bone formation process by secreting bone formation molecules such as collagen type I; non-collagenous proteins, which include proteoglycans; glycosylated proteins such as tissue non-specific alkaline phosphatase; small integrin-binding ligand proteins; Gla-containing proteins; and lipids, which compose the remaining organic material3. Bone remodeling process is summarized in (Figure 1.3).

Pre-osteoclast

Figure 1.3: | Bone remodeling stages

Bone is a dynamic organ; thus, it resorbs and replaces continually through a sequential remodeling process. To keep bone density and skeletal strength within 10% of the skeleton being replaced annually, bone remolding must occur. Diagram from Servier

Medical Art.

Metabolic bone diseases

Introduction

Metabolic bone disease is a widely defined term. It includes diseases that cause abnormal bone formation such as osteoporosis, which affects 54 million Americans by

2020 (The National Osteoporosis Foundation). Rickets is another bone disease that is caused by a vitamin D deficiency, and although not common in the U.S, the Rochester

Epidemiology Project identified 768 cases in Minnesota100. In addition, osteogenesis immperfecta is a known bone disease; however, the exact number of people suffering from osteogenesis immperfecta is unknown. It is estimated that a minimum of 20,000, and possibly as many as 50,000 people, suffer from osteogenesis immperfecta in the

USA (Osteogenesis Imperfecta Foundation), 1,250 adults were affected in the U.S by a rare disease called marble bone disease (osteopetrosis) according to the National

Organization of Rare Disorders. https://rarediseases.org.

Osteoporosis

As we live in a very dynamic and rapid environment, our health and bodies have been affected drastically. Living for a long time can cause age related problems such as osteoporosis. Osteoporosis can be defined as a skeletal disorder in which bone integrity and strength are altered, predisposing an individual to an increased risk of fracture.

Osteoporosis-related fractures most commonly occur in the hip, wrist, or spine. Many researchers have documented the effects of Osteoporosis on both men and women of all races, but particularly older white and Asian women who are past menopause are at a higher risk. More than 9 million osteoporotic fractures occur worldwide each year, including 1.6 million hip fractures that are caused by osteoporosis20. In the United

States and other countries, osteoporosis is considered the most widespread metabolic bone disease. It has been estimated that almost 2 million osteoporosis-related fractures are characterized as follow: nearly 547,000 vertebral fractures, 297,000 hips fractures,

397,000 wrist fractures, 135,000 pelvic fractures, and 675,000 fractures in other bones have occurred in the United State alone in 2000. Those numbers are expected to increase to 3 million by 20259. In a healthy bone organ, the coupling of bone resorption by osteoclasts and bone formation by osteoblasts is what maintains the balance between the two processes, keeping the bone organ strong. Osteoporosis alters the coupling of the two interactions resulting in osteoclast activity that far outweighs that of the osteoblast. This process is enhanced in women after menopause as mentioned earlier, whereby the loss in estrogen is associated with an increase in osteoclast activity. Estrogen plays a crucial regulatory role in bone formation by protecting bone form resorption. Estrogen has an impact on the osteoclast by suppressing its formation and stimulating osteoclast apoptosis46, 53. The regulatory role of estrogen in the osteoclast is essential. Estrogen can inhibit the RANKL production is produced by bone marrow stromal/osteoblast precursor cells, T cells, and B cells28. In addition, Estrogen mediates the osteoclast differentiation, and thereby mediates bone resorption by the regulation of RNAKL/OPG. Such an alteration of the RANKL/OPG ratio in menopausal patients leads to increased osteoclast development and activity97.

Osteoporosis treatment

Most osteoporosis treatments target osteoclast function, thereby decreasing bone resorption83. Below, there are few treatment modalities for osteoporosis.

Calcitonin

Calcitonin is one of the antiresorptive agents used for osteoporosis treatment. Calcitonin can decrease the osteoclast calcitonin receptor activity. Salmon calcitonin preparation

(SCT) is the most widely used calcitonin formula and it exists as a nasal spray. The efficacy of the nasal spray was estimated during a clinical trial, where a daily use of 200

IU of SCT showed a 20% decrease in osteoporosis biomarkers, with a small effect on

BMD in the spine13, 14, 69.

Calcium and vitamin D

Calcium and vitamin D have long been recognized as important and required nutrients for bone health and maintenance. The continuation of calcium and vitamin D regiments in a patient with bone loss is critical for optimal care. Unfortunately, 90% of women may not be getting enough calcium and over 50% of women treated for bone loss have inadequate vitamin D levels43. Calcium and vitamin D combination therapy has been accepted as the baseline treatment for osteoporosis. These treatments have been studied where adequate calcium and vitamin D supplementation had been achieved43.

Estrogen receptor

Raloxifene is known as a selective estrogen receptor modulator and its treatment mechanism is to block the effect of estrogen. Researchers have reported that in postmenopausal women who used Raloxifene for 3 years, those women showed a reduction in the incidence of vertebral fractures in about 30%30. Based on a study that was conducted by the Health Initiative, a group of healthy women aged 50-79 showed a 34% risk reduction for hip and vertebral fractures and a 24% reduction in incidence of osteoporotic fractures. On the other hand, other studies have reported that the long- term use of estrogen replacement can be linked to several side-effects that associated with breast cancer84, 92.

Strontium ranelate

This treatment can work on both bone remodeling processes. The treatment stimulates bone formation and inhibits bone resorption, and it is available in European countries.

To evaluate the clinical effects of strontium ranelate, clinical trials were conducted. Data form those clinical trials showed that treatment with 2 g/day of strontium ranelate resulted in increases BMD at all sites, a reduction of 37% in vertebral fractures, and a reduction 14% in non-vertebral fractures over 3 years77.

Bisphosphonates

Many bisphosphonates have been approved by the FDA, and bisphosphonates are currently a very common as a treatment for osteoporosis. Alendronate was the first one approved by the FDA and it is taken a once daily. Other bisphosphonates have been approved by the FDA such as clodronate, risedronate, ibandronate, and pamidronate.

Bisphosphonates clinical effects start from their ability to bind hydroxyapatite crystals, and thus high affinity to bone. Bisphosphonates are released from the bone matrix by active osteoclasts once the exposure to acid and enzymes occurs25, 105. It has been shown that the bisphosphonates treatment increased the mineral bone density (BMD) of the hip by 3%-6% and the spine by 5% -8%. In women, on the other hand, a decrease in nonvertebral fractures by 25%-40%, including hip fractures by 40%-60%, was observed with alendronate and risendronate treatment105. Clinical reports documented that the use of bisphosphonates for a long time can be a reason for several side effects seen with osteonecrosis of the jaw. Several patients with cancer who were treated with zoledronic acid unexpectedly developed osteonecrosis of the jaw27.

Inhibitors

Inhibitors are a potential treatment for osteoporosis that are currently being studied. The inhibitors mechanism is , such as cathepsin K, and

D. Cathepsin K is the main enzyme that is secreted by the osteoclast to degrade bone, elastin, collagen, and gelatin. Many inhibitors still under study are relactib, odanacatib, and the cathepsin K inhibitor. The orally administered drug available is sarcatininb and it is a competition of Src kinase. Saracatininb has shown in vitro a reduction of bone resorption39.

Drug name Expected side Drug action Reference

effect

Calcitonin Rhinitis, nasal lowers blood calcium levels P.F. Hirsch et (Fortical, Miacalcin) irritation, by suppressing osteoclast al. J dizziness, nasal activity in the bones and Musculoskel dryness increasing the amount of Neuron calcium excreted in the urine Interact. 2001 Estrogen Thromboembolic Increases B. Lawrence events; osteoprotegerin (OPG) and Riggs. Journal cerebrovascular decreases M-CSF and of Clinical accident, stroke, RANK. Investigation. and breast 2000 cancer (when combined with progestin); gynecologic problems (endometrial bleeding); breast abnormalities (pain, tenderness, and fibrocytosis) Monoclonal Dermatitis, rash, Monoclonal antibody against Henry G et al. antibody mild bone/muscle RANK ligand. JCEM. 2010 Denosumab pain, Urinary (prolia) Tract Infections (UTIs) Romosozumab Dermatitis, rash, increases bone formation Michael R et (AMG) mild bone/muscle by binding to sclerostin, an al. New pain, Urinary osteocyte-derived inhibitor England Tract Infections of osteoblast activity. Journal of (UTIs) Medicine. 2014 Selective estrogen VTE, arthralgia, The biological actions of Gizzo, receptor leg cramps, flu raloxifene are largely Salvatore et modulator syndrome, mediated through binding to al. Obstetrical (SERMs) peripheral estrogen receptors. This & Raloxifene edema, hot binding results in activation Gynecological (Evista) flashes of estrogenic pathways in Survey.2013 some tissues (agonism) and blockade of estrogenic pathways in others (antagonism) Bisphosphonates Lower-limb An antagonist to estradiol David J. Tamoxifen lymphedema (E2) in estrogen receptor Bentrem et al. (Nolvadex, Istubal, Blood clots, deep David J. Valodex, Genox) vein thrombosis Bentrem et al. (DVT) Endocrinology. 2000 Aendronate Mild upper GI Affinity for bone minerals Russell et al. (Fosamax) events, and inhibition effects on Osteoporos esophageal osteoclasts (induction of Int. 2008 ulcerations, osteoclast apoptosis) perforations, and bleeding events Etidronate Mild upper GI Affinity for bone minerals Russell et al. (Didronel) events, and inhibition effects on Osteoporos esophageal osteoclasts (induction of Int. 2008 ulcerations, osteoclast apoptosis) perforations, and bleeding events Ibandronate Esophageal Affinity for bone minerals Russell et al. (Boniva, Bonviva) ulcerations, and inhibition effects on Osteoporos perforations, and osteoclasts (induction of Int. 2008 bleeding events osteoclast apoptosis) Pamidronate Mild upper GI Affinity for bone minerals Russell et al. (Aredia) events, and inhibition effects on Osteoporos esophageal osteoclasts (induction of Int. 2008 ulcerations, osteoclast apoptosis) perforations, and bleeding events Residronate Esophageal Affinity for bone minerals Russell et al. (Actonel) ulcerations, and inhibition effects on Osteoporos perforations, and osteoclasts (induction of Int. 2008 bleeding events osteoclast apoptosis)

Table 1: List of approved treatment for osteoporosis and its side effect

Table modified from Amir et al. Ann Intern Med. 2008

TRAPP Complex

Protein transport and trafficking in cells is a biological mechanism by which proteins are transported to the appropriate destinations in the cell or outside of it. Carrier proteins are used to transport proteins across cellular membranes such as the plasma membrane, endoplasmic reticulum, and nuclear envelope. Proteins are also trafficked between membrane-bound organelles inside membrane vesicles. Vesicular trafficking is at the basis of cellular life, it governs cell communication via secretion and uptake of signaling molecules, enzymes, and adhesion molecules21. Intracellular vesicles have to go through many stages to become mature and functional vesicles. The very early forming stage of vesicles starts with budding from the donor compartments (e.g. the trans-Golgi network) and they are subsequently transported to a different destination compartment (e.g. the plasma membrane), where membrane fusion occurs21. It has been indicated that trafficked vesicles have a crucial role in regulating osteoclasts and osteoblasts, and thus affect bone remodeling112. Recently, many studies explained the molecular mechanisms that are linked to the trafficking process and reported the most frequent impact factors that bind and regulate trafficked vesicles. These factors include proteins that coat the vesicle, such as GTPases of the Rab family, fusogenic SNAP

(Soluble NSF Attachment Protein) REceptor (SNARE) proteins, and tethering factors that coordinate or participate in membrane tethering (TRAPP Complex)94. The Rab protein family is a major player in binding and trafficking protein vesicles. Rab proteins

transport vesicles as a general mechanism for regulating traffic between organelles2. It has been stated that Rab GTPases need to be switched to the active form by some Guanine Exchange Factors (GEF) such as the TRAPPII complex. TRAPPII functions in the membrane tethering of coated vesicles during intra-Golgi and early endosome to late Golgi traffic50. Another study reported that TRAPPII is an exchange factor for yeast protein two 1 protein (Ypt1p), and TRAPPII was also reported to be an exchange factor for Ypt32p. In yeast, Ypt1p and Ypt32p are homologous with human Rab family proteins32. The TRAPPII complex has multiple subunits such as TRAPP1,

TRAPP2,TRAPP3, and TRAPP9 (Figure 1.4).

Yeast TRAPPII

Mammalian TRAPPII

Figure 1.4: | Structural diagram representing TRAPPII Complexes TRAPPII complex consists of multiple core subunits in both the yeast and mammal. It appears that both complexes have common and specific subunits.

TRAPPC9

Structurally, TRAPPC9 has two distinctive domains. The first domain is known as

ASPM, SPD-2, Hydin (ASH) on the C-terminal of TRAPPC9 protein, and the second one is multiple tetratricopeptide repeats(TPR), -solenoid bearing that stretches and repeats86. (Figure 1.5). Trafficking protein particle complex 9 (TRAPPC9) is a protein subunit of the Transport Protein Particle II (TRAPPII). TRAPPC9 is implicated in endoplasmic to Golgi trafficking pathway and intra Golgi trafficking in yeast cells 74 (Figure 1.6). TRAPPC9 is a highly-conserved protein without any explored functional domains110. The TRAPPC9 sequence has similarity up to 92% in mice, 87% in chicken, and 85% in zebrafish. In addition, TRAPPC9 is encoded by the gene located on the 8q24.3 locus and contains 23 exons85. Recent studies showed that TRAPPC9 is expressed in mouse neurons and deep gray matter72. It is expressed in the small intestine and colon of the mouse, too. Another study showed that TRAPPC9 is expressed in low levels in the brain and heart 110. It has been documented that

TRAPPC9 binds to several proteins such as TRAPPC10, TRAPPC2, and P150Glued 113.

In addition, TRAPPC9 was found to bind to COPI (coat Protein I). TRAPPC9 overexpression might compete away p150Glued from the Sec23/Sec24 binding complex and uncoupling COPI form p150Glued thus, disrupt the architecture of microtubules113.

The role of TRAPPC9 in the skeletal system has not yet been studied in detail.

However, our lab is studying TRAPPC9 in bone cells. T. Mbimba et al. from our lab showed that TRAPPC9 has a possible regulatory role in osteoblast/osteoclast differentiation and function through NF-kB regulation. Recently, data showed that

TRAPPC9 interacts with NIK and IKK2, and thereby regulates NF-kappa-B signaling indirectly45. Recent studies have linked mutations in TRAPPC9 to a mental retardation disease increasingly found in populations of Israeli-Arabic descendent 67, 72.

Figure 1.5: | TRAPPC9 structural form

A: 2D structural representation of TRAPPC9 (PDB (http://www.pdb.org). B: TRAPPC9 domains show the ASH and TPR regions.

Figure 1.6: I Role of TRAPPC9 in intra-Golgi vesicles trafficking

COPII- coated vesicles are mediating the vesicle trafficking from ER to Golgi, which interacts with the TRAPPI complex. TRAPPII also mediates the vesicle tethering to Golgi and intra-Golgi. TRAPPC9 is a specific subunit of the TRAPPII complex. TRAPPC9 is mostly interacting with Golgi proteins.

Osteoclast cytoskeleton

The main role of osteoclast is to resorb bone and degrade the extra cellular matrix. The skeletal matrix will be degraded after forming a microenvironment between the osteoclast itself and the bone surface. In order to form such a separated microenvironment, polarization of the osteoclast cytoskeleton is required for interaction between the bone matrix/cell interface and integrin to mediate the physical interaction with the extracellular matrix. expressed by the -binding heterodimer is

v 3, which recognizes the amino acid motif arginine-glycine aspartic acid (RGD).

Another remarkable structure in the osteoclast cytoskeleton is the sealing zone (actin ring). The sealing zone isolates the resorptive microenvironment from the general extracellular environment and it is formed only during cell/bone matrix adhesion.

However, the sealing zone will be disappeared after the osteoclast detaches from the bone surface to access a new site of skeletal degradation98. In addition, the osteoclast is a migratory cell and its cytoplasm is flexible, dynamic, and can be reshaped and modified into distinct ordered structures that meet specific physiological demands such as actin aggregates known as podosomes. Podosomes are generally redundant and associated with easily detectable actin8. Bone resorption is associated with another cytoskeletal structure of the osteoclast known as the ruffled border. Ruffled borders are morphologically distinctive and they made up mainly of complicated folds of plasma membrane. Moreover, ruffled borders exist only in the osteoclast during the resorptive process98 (Figure 1.7).

A Basolateral membrane

Nucleus

Ruffled border B

Podosomes belt

Actin ring

Figure 1.7: I Structural representation of osteoclast cytoskeleton (resorptive state)

A: A lateral view of mature OCI represents the resorptive state, osteoclast nuclei are pulled up. B: A cross section of mature OC shows the actin rings are bounded by belts of podosomes (over bone). Attachment to the bone matrix is facilitated by integrin receptors. The osteoclast releases hydrogen ions through ruffled borders into the resorptive cavity.

Osteoclast cytoskeleton structures

Podosomes/ actin ring

Podosomes are circular adhesions that are formed by several types of cells such as macrophages, dendritic cells, and osteoclasts. Structurally, podosomes consist of a dense actin core surrounded by a ring of integrins and various cytoskeletal adaptor proteins such as talin, vinculin, gelsolin, cortactin, and L-Plastin 70. Podosomes organization is like large clusters and it is associated within the actin network. It has been shown that the actin network organization is antiparallel arranged, which nicely correlates with the proposed stabilizing and the proposed protrusive function of the core64. Recent studies that used fluorescent recovery after photobleaching (FRAP) reported that the actin polymerization continues into the podosomes core in addition to the accumulation of involved proteins in actin polymerization24. During bone resorption, the assembly of podosomes was increased and regulated by cytosolic Ca2+ and PKC.

It is clear that such a tight adhesive contact between the cell and its substrate leads to a very low-pH environment below the osteoclasts into which lysosomal proteases are secreted99. It has been proposed that podosomes transform into the actin ring. The actin ring structure is similar to the podosome, one which consists of condensed actin bands.

Additionally, actin ring colocalizes with vinculin, talin43, and proline-rich tyrosine kinase-

2 (PYK2). It has been documented that the formation of the actin ring might participate in the reorganization of actin and associated proteins from individual podosomes into the continuous ring. However, the precise components remain to be defined8, 26

(Figure1.8).

F-Actin

Actin L-PLastin Gelsolin Actin + Cortactin proteins cloud Vinculin Integrins

Cell membran ECM

Figure 1.8: I Schematic diagram of podosome in mature osteoclast

A: Podosome unit consists of F-actin cone (podosome core) with the actin polymerization activator surrounded by a cloud of adaptor proteins. (Top view) B: Different regulatory proteins form

L-Plastin (LPL)

Plastins are actin-bundling proteins essintial for actin regulation in eukaryotes. Plastins exist in three isoforms: L-, I-, and T-plastin. LPL was initially found in transformed human fibroblasts, although later it was found that normal expression of LPL is restricted to cells of the hematopoietic lineage62. LPL, also called lymphocyte cytosolic protein 1 (LCP1), has been described as one of the 15 most abundant proteins in human monocytes and T cells73. Structurally, LPL has three N-terminals, two N- terminal EF-hands that are homologous to calmodulin-calcium-binding domains, followed by two actin-binding domains (ABDs), and a third N-terminal that is the site of serine phophorylation. ABDs are where LPL binds to F-actin and they have two domins known as calponin-homology domains. Because of this orgnization, LPL is classified as a protein member of -actinin family59 (Figure 1.9). It has been reported that LPL activity can be regulated through both calcium binding and serine 5 residue phosphorylation. A study showed that rapid actin assembely was noticed in membrane ruffles and microspkes during the posphorylation of LPL, thereby enhancing LPL-F-actin binding73.

However, LPL may also be regulated through direct binding by other proteins such as cortactin, vimentin , and calmodulin. Functunally, LPL was shown to play an essential role for normal T- and B-Cell motility by moving into and out of chemokine-receptor- associated lipid rafts following chemokine signaling along with coronin 1A63. In addition,

LPL was found to participate in early sealing ring formation of osteoclast. A recent study demonstrated that LPL has a role along with the actin-binding protein cortactin, in the osteoclast sealing ring formation66.

EF EF CH CH CH CH

Headpiece ABD1 ABD2

Actin filament

LPL molecules

Figure 1.9: | L-Plastin structural form.

A: 2D structural representation of L-Plastin (PDB (http://www.pdb.org)).

B: N terminal of LPL has two (EF) domains known as EF hand loops, which

participate calcium regulation of LPL; the C-terminal has two tandem ABDs, each

of which consists of two calponin-homology (CH) domains.

C: LPL folds into a compact structure that bundles actin filaments.

Specific Aims The purpose of this thesis is (1) to characterize the role of TRAPPC9 in osteoclast function using bone marrow-derived macrophages (BMMs) and Identify TRAPPC9 binding proteins partners that might play a role in osteoclast cytoskeleton reorganization and function. (2) Determine the effects of modulating TRAPPC9 and/or LPL using genetic approaches on OC trafficking, function and get better understanding how is that might affect the cytoskeletal structures/re-organization in OC function mediated by

TRAPPC9/LPL interaction.

Our hypothesis is that TRAPPC9 plays a regulatory role in L-Plastin-mediated actin ring formation and osteoclast function. The overall objective of this research was to evaluate

TRAPPC9/ LPL interaction as a possible target for modilities to enhance bone repair and regeneration.

Aim 1: To identify TRAPPC9 binding proteins partners that might play a role in

osteoclast cytoskeleton reorganization and function. To do so, we used mass

spectrometery technuiqe usuing mature OC.

Aim 2: To Determine the effects of modulating TRAPPC9 and/or LPL using

retrovirus to overexpress TRAPPC9 on OC trafficking, function and examine how

is that might alter the cytoskeletal structures/re-organization in OC function

mediated by TRAPPC9/LP interaction during osteoclastogenesis.

Chapter II

Materials and Methods

2.1. Reagents and antibodies

Recombinant M-CSF, RANKL were purchased from R&D Systems (MN, USA).

Minimum Essential Medium (MEM) Alpha Medium and Dulbecco's Modified Eagle's

Medium were purchesd form Mediatech, Inc (VA, USA). pGFP-C-shLenti expressing

TRAPPC9 was purchased from Origene (MD, USA) along with empty vectors. Lentiviral packaging vectors; pMDLg/pRRE and pRSV-Rev were purchased from the plasmid depository Addgene (MA,USA) along with the envelop plasmid pMD2. G. Polyclonal antibody against TRAPPC9 construct was purchased from Proteintech (IL, USA).

Polyclonal antibody against L-Plastin, NFATc1 were purchased from from Cell Signaling

Technology (MA, USA) (table 2). Polyclonal antibody against Cathepsin-K was purchased form Bioss Inc (MA,USA). All the secondary antibodies were purchased from

Cell Signaling Technology (MA, USA). All fluorescence secondary antibodies were purchased from Thermo-Scientific (NY, USA), Santa Cruz Biotechnology, Inc (TX,USA), and Jackson ImmunoResearch Laboratories, Inc (PA, USA). BCA Protein Assay Kit was purchased from Thermo-Scientific (NY, USA). 10% formalin was purchased from Fisher

Scientific (NY, USA). RIPA lysis buffer was purchased from EMD Millipore (MA, USA).

PBS solution was purchased from Bio Basic (NY, USA).

Table 2: List of antibodies were used

Anti body Company name

TRAPPC9 Proteintech (IL, USA)

L-Plastin Cell Signaling Technology (MA, USA)

Cathepsin.K Bioss Inc (MA,USA).

NFATc1 Cell Signaling Technology (MA, USA)

actin Santa Cruz Biotechnology, Inc (TX,USA)

GAPDH Cell Signaling Technology (MA, USA)

2.2. Cell culture

Male Black 6 (C57BL/6) mice between 6 8 weeks of age were used to collecte bone marrow form their femurs and tibias as following, Mice were sacrificesd by using the

CO2 asphyxia methed as descriped in the protocol.femurs and tibias of the mice were collecte after removing all the muscles and soft tissues. Bone marrow cells were flushed using 21 gage needle attached to a 10 cc syringe containing total culture media

-streptomycin. After the flushing step,

of M-CSF (R&D) for 3 days in 95% air and 5% CO2 incubator at 37°C. Attached cells were considered to be bone marrow-derived macrophages (BMMs) and were further used for osteoclasts (OCs) differentiation. 2.3. Retroviral production and transfection

Retroviral infectious particles expressing the mouse cDNA or the GFP protein were produced by co-transfection the retrovector (PMX) (Figure.2.1) in Plat-E cells using X-

TREMEGENE 9 DNA transfection technique (MO, USA). 72 hours post-transfection the viral media was collected and mixed with with dilution ration (1:1).

BMMs were infected with infectious retrovirus particles expressing GFP, and TRAPPC9

efficiency of transfection was evaluated under a fluorescence microscope. The media was changed the next day and differentiation was induced 48 hours post transfection by adding -CSF and 40 of RANKL.

Figure 2. 1: | PMX vectro map

PMX vector and Plat-E cells were genurosly provided by Dr. Yousef Abu-Amer lab,

Department of Orthopaedics, Washington University.

2.4. Immunoblotting analysis

The total protein was extracted form cell by using 1X RIPA buffer (Millipore) mixed with proteinase inhibitor cocktail (Thermo). Lystae were kept on ice for 30 mins and then centrifuged at high speed for 30 mins. The supernantent transofrmed to another 1.5m mcirocentrifuge tube and the pellet was discard. BCA kit (Thermo) was used to measure the protein concentration according to manufacture recommendation, and 20- total protein were resolved by electrophoresis on a 12% SDS-polyacrilamide gel (Bio-

Rad) under reducing condition and then transferred on PVDF membrane (Bio-Rad). The membrane was blocked for one hour in 5% BSA in TBST buffer in 4Co and immediatelly probed with primary antibody for total NFATc1, L-Plastin (Cellsignaling); TRAPPC9

(Proteintech, IL, USA); Cathepsin-K (Bioss in, MA, USA) followed by appropriate HRP- conjugated secondary antibodies (Cellsignaling, MA, USA). Proteins expression was evaluated by chemiluminescence using luminata forte westernblot HRP substrate

(Millipore, MA, USA) and imaged with Syngen Pxi imaging system. Relative protein expression was evaluated using ImageJ ver. 1.50i.

2.5. Mass Spectrometry

To investigate the interaction between TRAPPC9 complex and other proteins partenrs,

BMMs cells were grown in total culture media supplemented with M-

concentration was calculated by following BCA protocol (Bio-Rad, CA , USA). 100µg of protein was combined with 10µg of TRAPPC9 antibody (Proteintech Group, Inc. IL,

USA) and then the reaction was incubated overnight at 4ºC with mixing. 25µL (0.25mg) of Pierce Protein A/G Magnetic Beads (Thermo Scientific Fisher Inc., MA, USA) were placed into a 1.5mL microcentrifuge tube to be washed by washing buffer. The

TRAPPC9 antigen/antibody mixture was added to a 1.5mL microcentrifuge tube containing pre-washed magnetic beads and was incubated at room temperature for 1 hour with mixing. The beads were collected with a magnetic stand and then the flow- through was removed. The beads were washed twice with washing buffer and then were mixed with 100µL of SDS-PAGE reducing sample buffer for 10 minutes then the mix were resolved by electrophoresis on a 12% SDS-polyacrilamide gel (Bio-Rad) to perform the mass spectrometry as following, Five most abundant bands including one at the molecular weight of ~130 kD (aimed at TRAPPC9, MW 128,233.2 D) were excised from the SDS-gel and cut to little pieces. The gel pieces were washed and destained with 50% ethanol containing 5% acetic acid. To alkylate cysteine residue, the gels were reduced with dithiothretol and then alkylated using iodoacetamide. The gel pieces were incubated in 40 µL of sequencing grade trypsin (20 ng/µL) for overnight digestion. The peptides are extracted twice using 50% acetonitrile with 5% formic acid. Extracted peptide mixtures were dried in SpeedVac. Peptides were dissolved in 40 µL of loading buffer (2% acetonitrile with 0.1% formic acid). Only 4 µL of sample was injected onto the analytical column. The Q Exactive plus is a high-resolution mass spectrometer and is coupled to a Dionex UltiMate 3000 nano-flow LC system. The analytical column is

Dionex Acclaim PepMap RSLC 75 µm × 15 cm (P/N: 164534) with a trapping Column

(C18 PepMap 100, 5 µm). The instrument was operated in data-dependent mode with a mass range of 400-1600. The MS survey scan with a resolution of 70,000 was followed by twelve MS/MS the ESI spray voltage was kept at +2.0 kV.

2.6. Immunoprecipitation

To investigate TRAPPC9-L-Plastin interaction, BMMs were grown in total culture media supplemented with M- osteoclasts were lysed and protein concentration was calculated by following BCA protocol (Bio-Rad, CA , USA). 100µg of protein was combined with 10µg of TRAPPC9 antibody (Proteintech Group, Inc. IL, USA) and then the reaction was incubated overnight at 4ºC with mixing. 25µL (0.25mg) of Pierce Protein A/G Magnetic Beads

(Thermo Scientific Fisher Inc., MA, USA) were placed into a 1.5mL microcentrifuge tube to be washed by washing buffer. The TRAPPC9 antigen/antibody mixture was added to a 1.5mL microcentrifuge tube containing pre-washed magnetic beads and was incubated at room temperature for 1 hour with mixing. The beads were collected with a magnetic stand and then the flow-through was removed. The beads were washed twice with washing buffer and then were mixed with 100µL of SDS-PAGE reducing sample buffer for 10 minutes. Immunoprecipitates were resolved by electrophoresis on a 12%

SDS-polyacrilamide gel (Bio-Rad) and then transferred on PVDF membrane (Bio-Rad).

The membrane was blotted for one hour in 5% BSA in TBST buffer and subsequently probed with primary antibody of L-Plastin and then followed by appropriate HRP- conjugated secondary antibodies. Proteins expression was evaluated by chemiluminescence using luminata forte westernblot HRP substrate (Millipore) and imaged with Syngen Pxi imaging system. Relative protein expression was evaluated using Licor ImagJ ver. 1.50i.

2.7. TRAP staining and activity

Infected and non-infected (control) BMMs were plated in 96-well plates at 600k cells/cm2 density for differentiation and staining. OC differentiation was induced by treatment of BMMs -CSF (R&D) and 40 culture media containing 10% FBS, 1% penicillin and streptomycin. Then, the wells were fixed after 7 days in culture, and OCs were visualized by histochemical staining for

TRAP staining and activity was measured to correlate with stain. In brief, the cells were fixed in 10% formalin for 20 minutes at room temperature then washed twice with Di- water. Then, cells were permeabilized with methanol/Acetone (1:1) solution for 3 minutes at room temperature and allowed to dry for 3 minutes. After, cells were incubated with was prepared of mixing (1mg/mL of pNpp in TRAP buffer (12 mg/ml Sodium Tartrate in 120 mM Sodium Acetate buffer, pH

5.2)) for 1 hour at 37°C. After an hour, 10 transferred to

96 well-plate contains plate-reader at 405nm. For staining, the remaining solution was removed and the cells were washed with Di-

-Dimethyl Formamide in TRAP buffer) at 37°C for 30 minutes. OCs images were captured using Envos microscope equipped with a fix camera.

2.8 Osteo assay surface and Von Kossa staining

Infected and non-infected (control) BMMs were plated on Corning 96-stripwell at 600k cells/cm2 density for resorption area measurement and Von Kossa staining to differentiate between the resorbed area versus non resorbed area. OC differentiation was induced by treatment of BMMs -CSF (R&D) and 40

(R&D) in total culture media containing 10% FBS, 1% penicillin and streptomycin. Next, the media was aspirated from and 10% bleach solution was added for 5 minutes. The wells were washed twice with distilled water and stained with Von Kossa minerals stain as following, wells were stained with fresh 5% AgNO3 and were put under UV light for an hour. After, wells were washed 3 times with dH2O to make sure that the all the silver nitrate were removed then the stain were developed with fresh 5% Na2CO3 in 10% formalin up to 5 minutes for minerals and matrix staining in bone osteo assay. Next, wells were washed again with dH2O 3 times. Finally, wells were fixed with 5% Na2S2O3 for 2 minutes and then washed with dH2O 3 times to be prepared for relative area resorption quantification by Nikon microscope.

2.8. Immunofluorescence staining

BMMs were grown on 4-Well Chamber Slides 1.7 cm2 Growth Surface 2.5mL (Corning,

NY,USA) and differentiated to OCs as described above. Immediately, OCs were rinsed in cold PB then fixed in 4% paraformaldehyde for 20 minutes. The cells were then washed twice for 2 minutes each in cold PBS. After washing, the cells were blocked in

5% BSA in TBST buffer for one hour at room tempretuare and permeabilized in a solution containing 10% BSA and 0.1 Triton X1oo in PBS for 1 hour at room temperature. The cells were washed 3 times in cold PBS and incubated with primary antibodies overnight at 4°C. The next day the cells were washed 3 times in cold PBS for

5 minutes each and with gentle shaking and incubated with secondary antibody for 1 hour at room temperature then washed 3 times for 5 minutes each in cold PBS. The process was repeated for detection of the second protein in the case of co-localization, then cells were incubated with DAPI-containing mounting medium (Abcam).

Fluorescence was detected using a NIKON microscope and Olympus confocal microscopes.

2.9. Real time PCR analysis

A total RNA was extracted using miniRNeasy kit (QIAGEN), and cDNA was generated

recommendations. By using the following osteoclast gene specific primers Real-time

-PCR was performed using an ABI prism 7500 sequence Detection system with SYBRGreen PCR Master Mix

(Applied Biosystems). Primers amplifications were performed in triplicate and normalized against a GAPDH gene using the following parameters: 40 cycles of 15 sec denaturation at 95°C and 1 min amplification at 60°C. The relative gene expression was evaluated using the comparative cycle threshold (CT) method.

Table 3: Osteoclast primers

TRAPPC9 Forward; GCT GTC ACC TTG GAG AAC AT Reverse; GCT CAA GAA GTC GCC ATA CA Cathepsin K Forward; -CTT AGT CTT CCG CTC ACA GTAG- Reverse; -ACT TGA ACA CCC ACA TCC TG- L-Plastin Forward; -GCT CTT GGC CTT GTT CAG AC- Reverse; -CAG TGT TGC AGT CTC CCT GA- NFATC1 Forward; -CTC GAA AGA CAG CAC TGGAGC AT-3 Reverse; -CGG CTG CCT TCC GTC TCAT AG- GAPDH -CGA CTT CAA CAG CAA ATA CCA CTC TTC C- -TGG GTG GTC CAG TTC TTA CTC CTT-

2.10. Data analysis

The Prism 6 software version 6.01 (GraphPad, CA, USA) was used to analyze statistical differences between individual groups. All individual experiments were independently repeated for a minimum of three times with similar results. Comparison of multiple groups was analyzed by, a one-way analysis of variance (1-way ANOVA) followed by a

-test was performed to compare two groups. Differences were considered statistically significant with a P value less than 0.05. Group means or means ± standard error of the mean (±SEM) was graphed.

Chpter III

Results

3.1. TRAPPC9 expression during osteoclast differentiation

To better understanding of the role of TRAPPC9 in osteoclast (OC), we first examined

TRAPPC9 expression during OC differentiation. TRAPPC9 expression was analyzed in

RANKL-induced OC differentiation of Bone Marrow-derived Macrophages (BMMs).

using immunofluorescence staining, BMMS culture was terminated at time points and probed with anti-TRAPPC9 antibody. Confocal microscopy images of mature OC showed (Figure 3.3) TRAPPC9 expression and accumulation on differentiated of multinucleated OC cells. Our data showed that the expression of TRAPPC9 is increased during the cell differentiation as shown by staining intensity. Using the confocal imaging we were able to localize TRAPPC9 expression in the cytoplasm and the perinuclear around the cell periphery. In addition, our data showed that during OC differentiation, TRAPPC9 co-localizes with Cathepsin K around the cell periphery (Figure 3.4)

D0 D3 D5 D7 Myeloid Precursor BMM Osteoclast Precursor Mature Osteoclast

M-CSF M-CSF M-CSF + + RANKL RANKL

Figure 3.1: | TRAPPC9 expression increased during osteoclast differentiation

BMMs cells were cultured in the presence of M-CSF (20 ng/ml) for 3 days then exposed to RANKL (40ng/ml) for 4 days one dose each two day. RNA were extracted at different time points. To determine gene expression, total RNA was converted to cDNA and subjected to qRT-PCR using primers specific for TRAPPC9. Experiment was repeated 3 time with similar results. Data presented in all graphs represent Mean ± SE (*p<0.05) compared to day 0.

Figure 3. 2: | Cathepsin.K and NFATc1 expression increased during osteoclast differentiation

BMMs cells were isolated form 6-8 weeks old C57BL6 mice then cultured in the presence of M-CSF (20 ng/ml) for 3 days then exposed to RANKL (40ng/ml) in the presence of M-CSF (20ng/ml) for 4 days. Total RNA was extracted at different time points. To determine gene expression, RNA was isolated and converted to cDNA then subjected to qRT-PCR using primers specific for Cathepsin.K (A) and primers for

NFATc1 (B). Representitive data are shown n=2. Data presented in all graphs represent

Mean + SE (*p<0.05) compared to day 0. DAPI TRAPPC9

Mature OC

Figure 3. 3: | TRAPPC9 localization in pre and mature OC

BMMs cells were seeded onto chamber slide at 600k cell/cm2 and cultured in

differentiation media containing 20ng/ml M-CSF and 40ng/ml RANKL. Cultures were

terminated at day 7 and followed by immunofluorescence using anti-TRAPPC9 (red)

antibody to detect TRAPPC9 and DAPI for the nucleus. Top panel shows the pre

mature OC which TRAPPC9 localizes around the nucleus and ER. Bottom panel shows

the mature OC where TRAPPC9 localizes around the cell periphery. Experiment was

repeated 2 times with similar results.

Cathepsin.K TRAPPC9 Merged

Figure 3. 4: | TRAPPC9 co-localizes with Cathepsin.K during OC differentiation

BMMs cells were seeded onto chamber slide at seed densitiy of 600k cell/cm2 and cultured in differentiation media containing 20ng/ml M-CSF and 40ng/ml RANKL.

Cultures were fixed at day 7 and followed by incubation of anti-Cathepsin.K antibody

(green) and anti-TRAPPC9 (red) antibody to detect TRAPPC9. Merged image represents the colocalization around the cell periphery. Experiment was repeated 3 times with similar results.

3.2. TRAPPC9 binds to L-Plastin and other protein parteners

We invistigated

- (Figure 3.5). Our data showed that

TRAPPC9 binds to more than a hundered different protein partenrs (table 3), LPL ,

Vinculin, Gelsolin, and other proteins were found in our massspecterometry data that bind to TRAPPC9. It has been shown that LPL with other structural proteins play an essential structrul role in podosme/ acting rign intitation and formation in OC8. To confirm the interaction/binding between TRAPPC9 and LPL, BMMs were plated on a 6 well plate in the presence of 20 ng/ml of M-CSF and 40ng/ml of RANKL. The cell lysate was collected and immunoprecipitated with anti TRAPPC9 antibody and the immunoprecipitates were resolved by electrophoresis on a 12% SDS-polyacrilamide gel. The membrane subsequently was probed with primary antibody against LPL

(Figure 3.6). Immunoprecipitation (IP) analysis showed that TRAPPC9 was co- precipitated with LPL. We next examined the co-localization between TRAPPC and

LPL by using the immunofluorescence staining technique. Our data showed that

TRAPPC9 and LPL co-locolized with each other in the cytoplasim and around the cell periphery (Figure 3.7).

Figure 3.5: |

the TRAPPC9 antigen/antibody mixture was mixed with 100µL of SDS-PAGE reducing sample buffer for 10 minutes then the mix were resolved by electrophoresis on a 12% SDS-polyacrilamide gel to perform the mass spectrometry.The gel pieces were incubated in 40 µL of sequencing grade trypsin (20 ng/µL) for overnight digestion. Extracted peptide mixtures were dried in SpeedVac and then peptides were dissolved in 40 µL of loading buffer and only 4 µL of sample was injected onto the analytical column.

Table 3:

I.P Lysate

M.W.70KD

Figure 3.6: | -

To approve the interaction between TRAPPC9/LPL in mature OC, Immunoprecipitation technique was performed as follow. BMMs were isolated from 6-8 weeks old C57BL6 mice and plated in a 6 well plate. Cells were stimulated with20 g/ml of M-CSF and

40 g/ml of RANKL for 7 days. After 7 days, cell lysate then immunoprecipitated with anti

TRAPPC9, IgG (negative control), and normal protein lysate (positive control) then were analyzed by immunoblotting using anti-LPL antibody.

DAPI LPL

TRAPPC9 Merged

Figure 3.7: | TRAPPC9 co-localizes with LPL in mature OC

BMMs cells were seeded onto chamber slide at 600k cell/cm2 and cultured in differentiation media containing 20ng/ml M-CSF and 40ng/ml RANKL. Cells were fixed at day 7 and followed by immunofluorescent staining using anti-TRAPPC9 (red) antibody to detect TRAPPC9, anti-LPL (green) antibody, and DAPI (blue) for the nucleus. The merged image (yellow) shows the co-localization between TRAPPC9 and

LPL in mature OC. Experiment was repeated 2 times with similar results.

3.3. L-Plastin expression during osteoclast differentiation

Although It was prviousley shown that normal expression of LPL is restricted to cells of the hematopoietic lineage62, we sought to determine the expression of LPL during OC differntioaiton. LPL expression was determined in RANKL-induced OC differentiation of

Hematopoietic Cells (BMMs). We invistigated the LPL gene expression during OC differintaion by extractin RNA at different time point. LPL gene expression analysis was obtianed as following, total RNA and cell lysate were collected from HSCc cultured in

OC differentiation media. Result showed that RANKL-induced OC differentiation increased LPL expression during OC differentiation as shown by qRT-PCR (Figure 3.

8.A) and western blot analysis (Figure 3.8.B). These data suggested that LPL expression might play an essential role during osteoclast differentiation. Next, the expression of LPL was also confirmed by using immunofluorescence staining, BMMs culture was terminated at differentiation and probed with anti-LPL antibody. We examined the localization of LPL in mature OC. Confocal microscopy images of mature

OC showed LPL expression and accumulation on differentiated multinucleated OC cells

(Figure 3.9). Our data showed that the expression of LPL is increased during the cell differentiation as shown by staining intensity. Using the confocal imaging, we were able to localize LPL expression in the cytoplasm and the perinuclear around the cell periphery.

B LPL

M.W 70 KD

GAPDH

M.W 37 KD

Pre.OC OC Mature OC

Figure 3.8: | LPL expression increased during osteoclast differentiation

Total RNA was converted to cDNA and subjected to qRT-PCR using primers specific for

LPL (A). Furthermore 30 lot and probed with anti-LPL and GAPDH (loading control) antibodies (B). Data presented in all graphs represent Mean ± SE (*p<0.05) compared to day 0.

DAPI LPL

Mature OC

Figure 3.9: | LPL expression during osteoclast differentiation

BMMs cells were seeded onto chamber slide at 600k cell/cm2 and cultured in differentiation media containing 20ng/ml M-CSF and 40ng/ml RANKL. Cultures were fixed at day 5, 7 and followed by immunofluorescence using anti-LPL (green) antibody to detect LPL and DAPI (blue) for the nucleus. Experiment was repeated 2 times with similar results.

3.4. TRAPPC9 overexpression recruites LPL to intitate podosome/ actin ring formation

It has been previously shown that TRAPPC9 interacts with TRAPPC10 subunit of the complex TRAPPII which is believed to function as guanine exchange factor GEF for

Ypt/Rab GTPase114 by activating Rab1. In additon, Zong et al113 demonstrated that

TRAPPC9 binds and interact with p150 (Glued) with the same carboxyl terminal domain of p150 (Glued) that binds Sec23 and Sec24. We invistagated the effect of TRAPPC9 modulation on LPL recrutiemnt and thereby podosome/actin ring formation. To determine the effect of TRAPPC9 on LPL, TRAPPC9 was overexpressed by using retroviral particles expressing cDNA clone. Plat-E cells were transfected with (PMX) vector expressing either TRAPPC9 cDNA clone or GFP. 72 hours post-transfection, The efficiency of transfection was evaluated under a fluorescence microscope (Figure

3.10.A). Next, BMMs were transduced with infectious particles expressing GFP,

TRAPPC9 cDNA overnight. The efficiency of transduction was assesed under a fluorescence microscope (Figure 3.10.B). To confirm the transduction method further,

Transduced HCSc were lysed and total RNA and protein were extracted. RNA was converted to cDNA and TRAPPC9 gene expression was measured (Figure 3.11. A) then followed by western blot analysis (Figure 3.11. B). In addition, the transduction of

BMMs were assesed by visualizing the mature OC under the light microscope (Figure

3.12.A) or stained with TRAP for positive osteoclast (Figure 3.12.B). Our immunoflourecnece data showed that TRAPPC9 noraml expression is not asscoiated with LPL recrutiment (Figure 3.13A). On the other hand, TRAPPC9 overexpression recruites LPL to initiate the formation of podosomes/actin ring and thus, more actin aggregates were formed (Figure 3.13.B).

Transfected Plat-E cells. (cDNA)

Transduced

OC.

(TRAPPC9)

Figure 3.10: | Assesment of TRAPPC9 overexpression transfuction and transduction technique

Retroviral infectious particles expressing the mouseTRAPPC9 cDNA and the GFP protein were produced by co-transfection the retro vector (PMX) with packaging vector plasmids (pMDLg/pRRE, pRSV-Rev) and envelop vector plasmid (pMD2.G) Plat-E cells using X-tremeGENE 9 DNA Transfection Reagent. 72 hours post-transfection the transfection was evaluated under the fluorescence microscope (A). BMMs were transduced with infectious retrovirus particles expressing GFP and TRAPPC9 cDNA overnight mixed with to efficiency of transfection was evaluated under a fluorescence microscope. Experiment was repeated 4 times with similar results.

Figure 3.11: | Assesment of TRAPPC9 overexpression transfuction and transduction technique

TRAPPC9 overexpression method was evaluated by both qPCR and western blot techniques as following, BMMs cells were isolated form 6-8 weeks old C57BL6 mice and divided into two groups, non transduced group (control) and transduced group.

Both were cultured in the presence of M-CSF (20 ng/ml) for 3 days then exposed to

RANKL (40ng/ml) in the presence of M-CSF (20ng/ml) for 4 days. RNA was extracted at day 7. To determine gene expression, total RNA was converted to cDNA and subjected to qRT- lysate was subjected to western blot and probed with anti-LPL and (loading control) antibodies (B). Experiment was repeated 2 times with similar results.

Normal OC. Transduced OC. A

Figure 3.12: | Assesment of TRAPPC9 overexpression transfuction and transduction technique

Infected and non-infected (control) BMMs were plated in 96-well plates at 600k cells/cm2 density for differentiation and staining. OC differentiation was induced by treatment of BMMs - culture media containing 10% FBS, 1% penicillin and streptomycin. Then, the wells were fixed after 7 days in culture, and OCs were visualized first by the light microscope

(A), and then visualized by histochemical staining for TRAP staining (B). Experiment was repeated 2 times with similar results.

LPL DAPI LPL DAPI B A

Actin Merged Actin Merged

Figure 3.13: | TRAPPC9 overexpression increases recruitment of LPL to podosomes BMMs were plated into two chamber slides with density 600k cells per chamber and

divided into Infected and non-infected (control) groups. OC differentiation was induced

by treatment of BMMs - in total culture media

containing 10% FBS, 1% penicillin and streptomycin. Then, the chambers were fixed

after 7 days in culture followed by immunofluorescence using anti-LPL (green) antibody

to detect LPL and DAPI (blue) for the nucleus and probed with Phalloidin (red) to detect

Actin filaments. Merged image showed normal reorganization of LPL and Actin in non

transduced group (A). However, Merged image of TRAPPC9 overexpression group (B)

showed highly aggregation and formation of actin/LPL aggregates (arrowheads)

associated with TRAPPC9 overexpression. Experiment was repeated 2 times with

similar results. 3.5. TRAPPC9 overexpression is not involved in LPL mRNA expression induction in mature OC

We wanted to determine whether TRAPPC9 oveexpression is involved in LPL inducing signalling pathways thereby modulating the LPL mRNA expression. Our qPCR data showed that TRAPPC9 is dispensable for LPL gene expression thus, TRAPPC9 is only modulating the physical recruitments of LPL but not the signaling induction (Figure 3.14) to that end, RNA was converted to cDNA then TRAPPC9 gene and LPL gene expression were mesured. Data showed that there was no siginificant increase in LPL gene expression (transduced group) as compared with (non transduced group).

Figure 3.14: | TRAPPC9 overexpression is not associated with LPL mRNA expression

BMMs were infected with infectious retrovirus particles expressing GFP (Control),

TRAPPC9 cDNA overnight in total culture media containin . The media was changed the next day and differentiation was induced 48 hours post

- After 7 days, cells were laysed and total RNA using the High Capacity Reverse Transcription Kit. By using LPL, PCR was

. Primers amplifications were performed in triplicate and normalized against a GAPDH.The relative gene expression was evaluated using the comparative cycle threshold (CT) method. Data presented in all graphs represent Mean

± SE (*p<0.05) compared to control. Experiment was repeated 2 times with similar results 3.6. TRAPPC9 overexpression enhances osteoclast resprption function in vitro

We determined the role of TRAPPC9 on osteoclast function by modulating TRAPPC9 expression. As previously mentioned, osteoclasts main function is to resorb bone.

Therefore, to assess osteoclast function, osteoclasts were plated onto a 96 well Corning

Osteo Assay plate (mimics living bone material) and stained with Von Kossa minerals stain. Visualization of resorption pits from mature OCs were demonstrated by using the microscope and images showed that TRAPPC9 overexpression group has more pits formed than the control group ( Figure 3.15.A). Next, we quantified the realtive resorption area and our data showed that TRAPPC9 overexpression enhances OC function by increasing the realtive resorption area as compared with control group were the TRAPPC9 gene expression was normal (Figure 3.15.B).

cDNA A

Control

Figure 3.15: | TRAPPC9 overexpression enhances OC function

Relative resorption area was measured after staining with Von Koss to visualize the resorbed area versus non resorbed one in both group (TRAPPC9 overexpression and control group). BMMs were cultured onto Corning Osteo Assay Surface made of an inorganic crystalline calcium phosphate coating that mimics native bone. BMMs differentiation was induced by M-CSF (20 ng/ml) and RANKL (40 ng/ml) for 7 days.

After, mature OCs were bleached and wells were stained with Von Kossa and visualized under the microscope (A). Relative resorption area was measured using

Nikon microscope software (B), difference between experimental and control groups statistically significant Experiment was repeated 2 times with similar results.

Podosmes/Actin ring TRAPPC9

Golgi

ECM

TRAPPC9

LPL

Figure 3.16: | Mechanism of TRAPPC9/LPL binding and meadating the actin podosomes/actin ring formation in osteoclas

Proposed mechanism of TRAPPC9/LPL binding in osteoclast actin ring formation. We proposed that TRAPC9 promotes LPL recruitment and bring it to close proximity of actin filaments of podosmoes/actin ring resulting in highly stabilized cytoskeleton of OC during osteoclasteogenisis. TRAPPC9/ LPL binding may be unbinded and recycled to

Golgi again. Chapter IV

Discussion

In this study, we sought to understand the role of TRAPPC9/L-Plastin (LPL) during RANKL-induced osteoclast (OC) differentiation and function of Bone Marrow

Macrophage (BMM) cells. We examined the overall hypothesis that TRAPPC9/LPL interaction may play a regulatory role in actin ring formation and thus, osteoclast differentiation and bone resorption, expecting that overexpressing TRAPPC9 would result in increased of LPL recruitment to podosomes and eventually actin ring formation.

Such a recruitment may stabilize the actin ring structure thus, increasing bone resorption.

First, we evaluated the expression of TRAPPC9 during OC differentiation and how is that may affect the OC function. TRAPPC9 expression was vastly studied. In mouse, TRAPPC9 expression was localized in neurons of the cerebral cortex, hippocampus, and deep gray matter.72 Another study showed that TRAPPC9 is expressed in tissues of mouse colon and small intestine by the Conventional RT-PCR analysis110. Wen et al110 analyzed human tissues and showed high TRAPPC9 expression levels in muscle and kidney, and low expression in brain, heart, and placenta. TRAPPC9 was also found to be very weakly expressed in immune organs and cells such as thymus, spleen, and peripheral blood leukocytes. In addition, TRAPPC9 expression was also identified in osseous cells and tissues such as murine calvarias cell line MC3T3-E1. To assess the expression of TRAPPC9 during OC differentiation by measuring TRAPPC9 gene expression, we showed that TRAPPC9 gene expression is increased during OC differentiation resulting in an increased gene expression of NFATc1. During RANKL-induced OC differentiation, TRAPPC9 enhanced the phosphorylation and activation of NF-kB which resulted in the activation of both c-Fos and NFATc1 thus, NFATc1 has been implicated as a master regulator of OC differentiation4. The importance of NF-kB activation in OC differentiation has been vastly studied in both cells and animal models. NF-kB activation contributes to the RANKL- induced OC differentiation via downstream activation of NFATc14. Next, we examined the role of TRAPPC9 in OC function. Given that TRAPPC9 binds to p150 glued a subunit of dynein activator complex113, it is also possible that the role of TRAPPC9 in osteoclast differentiation is an essential component of the dynein motor complex, organization of microtubule, intracellular vesicular trafficking and dynein motor activity are all critical for the activation and function of osteoclasts.1, 36, 76, 104, 108. In mature OC, secretory pathways regulate osteoclastic bone resorption therefore, proteins have newly synthesized must be transported from the endo plasmic reticulum (ER) to the Golgi complex by the secretory pathway. To ensure the directionality and fidelity of transported proteins process, such a process has to be completed and regulated by intracellular membrane traffic complexes. Cathepsin. K which is the principal lysosomal acidic hydrolase degrading the organic matrix of bone, is secreted into the resorption lacuna in resorbing osteoclasts 111, indicating that lysosome secretion is a major pathway of intracellular protein traffic thus, osteoclastic bone resorption. To that end, it appears that Cathepsin. K gene expression is increased along with the increasing of TRAPPC9 gene expression during OC differentiation. We showed also that TRAPPC9 co-localizes with Cathepsin.K on the cell periphery suggesting an essential role of TRAPPC9 associated with intracellular protein traffic thereby, OC bone resorption.

Second, we sought to examine what other proteins partners might bind to

TRAPPC9 and play a role in osteoclast cytoskeleton reorganization and function. In this part of our study, we focused on podosomes/actin ring formation and which proteins might be involved in this process. The importance of OC cytoskeleton comes from the ability to manipulate the accessory proteins that involved in assemble and disassemble of actin filaments therefore, podosomes/ actin ring formation. It has been shown that during bone resorption, the assembly of podosomes was increased and regulated by cytosolic Ca2+ and PKC. It is clear that such a tight adhesive contact between the cell and its substrate leads to a very low-pH environment below the osteoclasts into which lysosomal proteases are secreted99. It has been proposed that podosomes transform into the sealing zone (actin ring) thus, the actin ring structure is similar to the podosome, one which consists of condensed actin bands. Additionally, actin ring colocalizes with vinculin, talin43, LPL, and proline-rich tyrosine kinase-2 (PYK2). Interestingly, our mass spectrometry data showed that TRAPPC9 binds to very various proteins that involved in many cellular structures and molecular mechanisms such as Vinculin, Gelsolin,

TRAPPC10, Cathepsin.D, and LPL. To that end, we were interested to investigate and test our hypothesis that such an interaction between TRAPPC9 and LPL might play a regulatory role of podosome/actin ring formation in mature OCs. To that extend, LPL is highly expressed in hematopoietic lineage and it is required for T- and B-cell motility and activation.

Recently, LPL was implicated as a critical factor in the assembly of precursor sealing zones at the early phase of sealing ring formation. Additionally, previous studies strongly suggested that LPL plays a major role in bone remodeling since LPL-/- mice are osteopetrotic. OC bone-resorbing capacity in these mice is significantly impaired, while OB function remains unaltered. Despite progress in the field, many gaps in knowledge are still unsolved relative to the biology of sealing ring formation in OCs. We sought to better understanding the potential regulatory role of TRAPPC9 in LPL by manipulating the expression of TRAPPC9 using genetic approaches. We found that

TRAPPC9 overexpression resulted in an increased recruitment of LPL to podosomes/actin ring structures. Our data suggested that TRAPPC9 physically binds to

LPL and bring it to close proximity of actin filaments of podosmoes/actin ring resulting in highly stabilized cytoskeleton of OC. Given that the actin ring was highly stabilized, our data from osteoassay and Von Kossa stain showed that the relative resorption area of overexpressed TRAPPC9 group was higher than control group and that supports the idea where our hypothesis was built on. In fact, the osteoclast cytoskeleton is unique in that it forms an actin ring which isolates the resorptive microenvironment from the general extracellular space and therefore, in order for OCs to attach and detach the bone surface and resorb bone, the actin ring structure must be highly stabilized and organized. It would be interesting to investigate whether TRAPPC9 induces the LPL gene expression or not by looking at the entire signaling pathways that might be involved in LPL gene expression. We found that TRAPPC9 has no regulatory role in

LPL gene expression induction even when TRAPPC9 was overexpressed suggesting that the mainly role of TRAPPC9 is to physically bind to LPL and transport it to podosome/actin ring.

Taken together, the findings in this study show for the first time the role of

TRAPPC9 in LPL-mediated actin ring formation and osteoclast function. In addition, we can propose TRAPPC9/LPL binding as a potential target for treatment of bone metabolic disorders such osteoporosis.

Chapter V

Conclusion and future studies

The main goal of this project was to identify the role of TRAPPC9 in bone resorption (osteoclast differentiation and function). First identified in yeast, Trafficking

Protein Particle Complex subunit 9 (TRAPPC9) is a subunit of TRAPPII was first identified in yeast and it is a tethering complex that acts in intra-Golgi and endosome-

Golgi transport. However, it has been shown recently that TRAPPC9 physically binds and interacts with both NIK and IKK2 thus regulates NF-

Mutations in TRAPPC9 have been associated with the congenital Non-syndromic

Autosomal-Recessive Intellectual Disability (NS-ARID) which is mostly attributed to a defect in NF- -ARID patients showed the presence of mild to severe musculoskeletal defects.

Western blot and qPCR analysis of bone marrow-derived macrophages (BMMs) extracted from 8-weeks-old C57BL6 mouse showed expression of TRAPPC9 in both bone cells, furthermore immunofluorescence staining of osteoclasts confirms the expression of TRAPPC9 in various bone cells including osteoblast, osteoclast, bone lining cells and cells in the marrow cavity. TRAPPC9 was expressed differentially in

C57BL6 long bone at different ages. In both osteoblast and osteoclast. TRAPPC9 expression during osteoclastogenesis was measured and shown a significant increase during their differentiation.

To answer the question how TRAPPC9 expression might regulate OC differentiation and function, we used gain of function experiment in vitro using retroviral particles expressing TRAPPC9 cDNA clone.

These data document that 1) In osteoclast TRAPPC9 positively regulates NFATc1 and cathepsin K expression, thus osteoclast differentiation and function; 2) TRAPPC9 binds to various protein partners play a role in cellular structures and molecular mechanisms of osteoclastogenesis such as LPL. Therefore, TRAPPC9/LPL binding mediates the podosome/actin ring formation by bringing LPL to close proximity of actin filaments thus, resulting in highly stabilized cytoskeleton of OC. TRAPPC9 overexpression promotes the recruitment of LPL to podsome/actin ring region. Such a stabilization will result in a firm attachment of OCs over osteo assay surface wells thereby, increases the relative resorption area.

We conclude from the above results that TRAPPC9 expression is important for osteoclast differentiation, cytoskeleton reorganization, and function. TRAPPC9 mutation is associated with mild to severe NS-ARID a disease that affect mostly patient of Arabic-

Israeli descent. NS-ARID patients have mild to severe skeletal phenotypes such as polydactyly, cranial-facial defect, osteopetrosis, and microcephaly due to increased bone deposition. These data show the importance of TRAPPC9 in the skeletal system.

While these results provide support for a mechanistic/ structural role of TRAPPC9 in bone resorption, many questions remain to be answered. Osteoclast excretes enzymes important for catalytic degradation of bone matrix during bone resorption by the osteoclast, expression of such catalytic enzymes is important for osteoclast function therefore, a defect in this pathway should affect osteoclast differentiation and function. We like also to further understand the role of TRAPPC9 in earlier verses late differentiation of OC since TRAPPC9 expression shows an increase in early stage of differentiation of all bone cells. As mentioned before, TRAPPC9 is a trafficking protein that binds LPL and other proteins associated with cytoskeleton. Cytoskeleton rearrangement is a critical component of both osteoblast and osteoclast differentiation and function. It is important to understand the role of TRAPPC9 expression in cytoskeleton rearrangement and its subsequent effect on osteoclast function. For that reason, we like to do

- -

therapeutic products to deal with diseases associated with bone loss and fracture repair are highly demanded. In that regard, TRAPPC9 might play a role as a possible therapeutic target due to its impact on osteoclast differentiation, cytoskeleton organization and function. One can reduce bone resorption during inflammation or age related bone loss by disrupting the

TRAPPC9/LPL interaction.

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