Expression of Capn6 During Osteoclast Differentiation in Wild-Type and HDAC4 Knockout Mice a THESIS SUBMITTED to the FACULTY OF

Home , Bone sialoprotein, CAPN6, CAPN8, Cathepsin K

Expression of Capn6 During Osteoclast Differentiation in Wild-type and

HDAC4 Knockout Mice

A THESIS

SUBMITTED TO THE FACULTY OF THE

UNIVERSITY OF MINNESOTA

Molly Kopf

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

MASTER OF DENTISTRY

Kim Mansky, PhD; Amy Tasca, DDS, PhD; and John Beyer, DDS, PhD

June 2019

Acknowledgements

First, I would like to thank my thesis committee chair Dr. Kim Mansky for her time and dedication to this project. It would not have been successful without her support and guidance. I would also like to thank my other committee members Dr. Amy Tasca and Dr. John Beyer for all their help and input for my master’s thesis. I greatly appreciate the time they committed to reviewing and shaping this thesis.

Additionally, I would like to express my gratitude to those individuals working in the Mansky lab at the University of Minnesota. They taught me several lab procedures, contributed to the study by providing samples, and created a fun yet productive environment for completing my research. These individuals include but are not limited to Dr. Eric Jensen, Bora Faulkner, Kristina Astleford,

Andrew Norton and Emily Campbell.

Furthermore, I’d like to thank the full-time and part-time faculty members of the orthodontic clinic for their commitment to my education over the last two years. I’m eager to use the wisdom and knowledge I gleaned from them in my future career. Lastly, I’d like to acknowledge my fellow orthodontic co-residents. I value their support and will always cherish the memorable experience of our orthodontic residency.

Dedication

I dedicate my master’s thesis to my husband Blake. Even though we were separated by hundreds of miles, you were there supporting me through it all.

Abstract

Introduction: The process of osteoclast differentiation and function consists of a network of complex signaling pathways with multiple negative and positive regulators. Previous studies suggest histone deacetylase (HDAC) proteins play a suppressive role in osteoclast differentiation; however, not much is known about the specific role of HDAC4. Expression of calpain 6 (Capn6) has been linked to increased organization of osteoclast microtubules for bone resorption.

In this study, we observe the expression of Capn6 in wild-type and HDAC4 knockout osteoclasts. Methods: qRT-PCR was preformed to assess Capn6 expression in wild-type and HDAC4 knockout mice over days 0 to 4 of osteoclast differentiation. Immunoblot analysis was used to assess CAPN6 levels in both groups. Results: Levels of Capn6 expression increased later in osteoclast differentiation in the wild-type osteoclasts, though the results were not significant.

There was a significant increase in Capn6 in osteoclasts from HDAC4 knockout mice after 3 days of RANKL stimulation. This was also significant when comparing HDAC4 knockout to wild-type osteoclasts. Conclusion: HDAC4 may be a negative regulator of osteoclast function, suppressing the expression of

Capn6. More studies are indicated to understand the interaction of HDAC4 and

Capn6 in the regulation of osteoclast activity.

iii

Table of Contents

List of Tables v

List of Figures vi

Introduction 1

Materials and Methods 12

Results 15

Discussion 21

Conclusions 24

References 25

List of Tables

Table 1. Capn6:Hprt expression in ten biologically independent wild-type 17 mice samples

Table 2. Capn6:Hprt expression in five biologically independent HDAC 4 19 knockout mice samples

List of Figures

Figure 1. Stimulation of osteoclast precursor by RANKL and M-CSF 3

Figure 2. The signaling pathways involved in the activation of NFATc1 5

Figure 3. Process of osteoclast resorption with HCl acid and 7

Cathepsin K

Figure 4. Expression of RNA sequencing genes in wild-type vs. knockout 16

mice on day 2 of osteoclast differentiation

Figure 5. A) Immunoblot analysis of Capn6 in wild-type mice B) Mean 18

Capn6:Hprt expression in wild-type mice over four days of

differentiation

Figure 6. Mean Capn6:Hprt expression over days of differentiation 19

between wild-type and HDAC4 knockout mice

Figure 7. Mean Capn6:Hprt expression over days of differentiation 20

between wild-type (black) and HDAC4 knockout mice (white)

Introduction

Bone is often thought of as the strong, rigid supporting structure of the bodies of various organisms. It is necessary for the vitality of those organisms allowing for mobility, protection of significant organs, production of blood cells, and storage of minerals like calcium. Bone tissue is made up of a collagenous matrix, inorganic elements, and cells. Even though bone is strong and rigid, it is not static. Throughout a lifetime, bone is constantly being reshaped, remodeled, and repaired. This continual dynamic process of resorption (bone degradation) and apposition (bone formation) is completed at such a rate that in one year, approximately 10% of the bone content is replaced in a human adult.1

Bone remodeling cycle

The process of resorption and apposition is facilitated by specific cells called osteoclasts and osteoblasts, respectively. Through various signaling pathways, bone remolding occurs first by osteoclasts recruitment to the site of repair. Resorption creates Howship’s lacunae on the bony surface. Once these lacunae get to a depth of about 50µm, certain signals recruit mesenchymal cells to differentiate into osteoblasts at the site and start bone apposition.2 A disruption in the process of removing and adding bone can cause harmful effects in humans.2,3 For example, an increase in osteoclastic activity will lead to an imbalance where more bone will be resorbed. This will weaken the bone and cause bone disorders such as osteoporosis, rheumatoid arthritis, periodontal disease, multiple myeloma, and certain metastatic cancers.1–5 A decrease in 1 resorption processes by osteoclasts leads to an increase in the amount of bone and cause diseases such as osteopetrosis.5,6

Osteoclast differentiation

Since osteoclasts play a key role in most bone disorders, studies have been conducted to learn more about their origin and differentiation process.4,5

Osteoclasts are multinuclear giant cells derived from hematopoietic precursors of the monocyte and macrophage lineage that surround the bone.3 Original studies looked at failed osteoclast recruitment leading to osteopetrosis to determine transcription factors involved in differentiation. One of the first transcription factors that plays a role in osteoclastogenesis is PU.1.4,5,7 This is a positive regulator of transcription involved in the generation of the common progenitors for both osteoclasts and macrophages; therefore, a deletion of PU.1 will cause a lack of both of those cell types, and knockout mice exhibit an osteopetrotic phenotype.1,7 In addition, many genes required for osteoclast and macrophage differentiation have PU.1 binding sites in their promoters.7

Studies have shown that there are two main factors that stimulate osteoclastogensis in vitro: macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL)1–3. RANKL is a membrane-bound cytokine derived from the tumor necrosis factor (TNF) family.1–

3 Osteoprotegerin (OPG), another TNF-derived receptor, was used to discover the importance of RANKL in osteoclast differentiation.4 OPG was simultaneously discovered by two groups: Amgen and Snow Brand Milk Group.6 It is a secreted 2 protein and was found to be a decoy receptor competing with RANK to bind

RANKL, and thus inhibiting osteoclast differentiation and inducing osteopetrosis in vitro and in vivo. 2,5,8 Both RANK and RANKL are found in other organs of the body such as skeletal muscle, thymus, liver, colon, adrenal glands, lungs, brain and kidneys.6 The OPG/RANK/RANKL regulation system is important in the regulation of resorption by negatively and positively controlling the activation of

RANK on osteoclasts.

Figure 12: Stimulation of osteoclast precursor by RANKL and M-CSF

PU.1 stimulates expression of colony-stimulating factor 1 receptor

(CSF1R) on macrophage progenitors. This is a receptor for CSF1, also known as

M-CSF which causes proliferation of osteoclast precursor cells as well as upregulates RANK. Again, M-CSF knockout mice have a lack of proliferating osteoclast cells, leading to an increase in bone density.7 M-CSF also activates microphthalmia-associated transcription factor (MITF), which plays a key role in

3 later stages of osteoclastogensis. MITF regulates the expression of anti-apoptotic protein Bcl-2 and promotes the survival of osteoclasts and macrophages.1,5,7

RANKL leads to osteoclast differentiation through its complex signaling pathway9. Both osteoblasts and osteocytes have been shown to be the source of

RANKL that binds to RANK in osteoclasts.10,11 In vivo, it was demonstrated that a direct contact with osteoblasts or osteocytes is not needed for RANKL to bind to their receptors on the osteoblast progenitor cells.3,12 Additionally, it has been discovered that vesicular RANK secreted by osteoclasts exhibits a reverse signaling pathway that in turn upregulates osteoblast differentiation.12 This shows the unique coupling of bone formation and bone apposition. Therefore, therapies that inhibit osteoclast differentiation will also decrease the amount of bone formation due to this coupling phenomenon.

Once RANK and RANKL bind, RANK has the ability to bind TRAF6 in osteoclasts, which has a major role in signal transduction for osteoclast differentiation.1,2,13 TRAF6 activates NF-κB and mitogen-activated kinases

(MAPKs) pathway which led to activations of NFATc1, one of the most critical factors in osteoclastogenesis.1,14 TRAF6 stimulates calcinuerin which dephosphorylates NFATc2.15 Dephosphorylated NFATc2 can then translocate to the nucleus of osteoclasts and bind to c-Fos and c-Jun proteins, both of which have an increased expression due to the activity of TRAF6.15,16These transcription factors then activate the NFATc1 promotor which then activates genes for osteoclast differentiation.16 NFATc1 also exhibits an autoamplification function where it can activate its own promoter causing an even greater 4 expression of genes involved in osteoclastogensis.14 Like many transcription factors involved in osteoclast differentiation, mice with loss of expression of

NFATc1 or c-Fos exhibit osteopetrosis.14,16

Figure 21: The signaling pathways involved in the activation of NFATc1

Bone resorption

The exact mechanism by which osteoclasts are recruited to the site of remodeling bone is unknown. Once the osteoclasts encounter the bony surface, they become polarized and the nuclei accumulate away from the site of the resorption. Due to the transportation of acidic cytoplasmic vesicles, they also form a unique ruffled resorptive border next to the surface of the bone. An actin ring is formed around the ruffled surface creating a sealing zone.2

Osteoclasts attach to the bone using αvβ3 integrins that recognize a specific amino acid sequence of Arg-Gly-Asp (RGD). This sequence is found in several bone matrix proteins including osteopontin and bone sialoprotein.2,17

αvβ3 integrins are absent in bone marrow macrophage (BMM) precursors that

5 have not yet committed to becoming osteoclasts. αvβ3 knockout mice have osteoclasts that do not develop a ruffled border or actin ring and the mice have an increase in bone density.18 The activation of c-Src kinase and its association with αvβ3 integrins is also necessary for the formation of the sealing zone and ruffled border, which are absent in mice where c-Src is not expressed.18

Activated c-Src can phosphorylate and activate Syk, a tyrosine kinase, which aids in the arrangement of the cytoskeleton.17

The organization of the osteoclast cytoskeleton and the formation of the ruffled border next to bone is crucial in the resorptive process of osteoclasts. The area under the ruffled border that is sealed in by the actin ring is highly acidic.19

H+ ATPase are arranged in vesicles and transported to the resorptive surface of the osteoclasts, forming the ruffled borders. The H+ ATPases pump H+ into the extracellular space to create the acidic environment.20 Since osteoclasts use Cl-

- - /HCO3 exchangers to regulate their own neutral intracellular pH, the Cl ions are deposited into the resorptive space containing H+ ions creating HCl acid that then degrades the hydroxyapatite present at the bone surface.21 In order to break down the collagen type I, an acidic protease, cathepsin K, is released from the ruffled borders of the osteoclasts.22,23 In mice that lack cathepsin K, the resorbing bone demonstrated naked collagen fibrils without attached hydroxyapatite, indicating that acid environment itself can remove the bone minerals but can’t completely resorb the bony matrix.22

Figure 32: Process of osteoclast resorption with HCl acid and Cathepsin K

Regulation of osteoclast by HDACs

Acetylation of proteins is a major regulating mechanism found in eukaryotic cells. The addition of an acetyl group on lysines significantly changes proteins properties. This action is reversible and controlled by both acetyltransferases and deacetlyases.24–28 Histone acetyltransferases (HATs) and deacetylases (HDACs) were among the first to be studied and found to be linked to transcription activation and suppression respectively.25–28 Since histone proteins are responsible for the organizing and packaging of our DNA into chromatin, a change in histone properties by the addition or removal of an acetyl group can expose or hide segments of the DNA.28,29 Even though they were first found to regulate histones, HATs and HDACs have been found to add and remove acetyl groups on non-histone proteins as well.24

HDACs fall into two families: class I (HDAC1, 2, 3, and 8) and class II

(HDAC4, 5, 6, 7,9, and 10).24,28–30 Class I HDACs are primarily contained within a cell’s nucleus while class II HDACs can shuttle between the nucleus and the cytoplasm.30 These HDAC proteins have a role in osteoclast differentiation and bone resorption. HDACs 1 and 2 from the class I family have been found to suppress osteoclast formation.30 When suppressed, HDAC3 inhibits osteoclast differentiation, whereas HDAC 7 has the opposite affect when suppressed.31

Overexpression of HDAC5 has been shown to be a negative regulator of osteoclastogenesis.28 HDAC6 and HDAC9 suppress osteoclast activity.28,30

Additionally, in osteoclasts, HDAC inhibitors for class II HDACs have been suggested to suppress bone resorption.32 Currently, there are no studies exploring the effect of HDAC4 on osteoclast differentiation and function.

Calpain 6 (Capn6)

Proteases are also important modulators of cellular function. A unique family of cysteine proteases called calpains are involved in many basic cellular functions. The classic cytoplasmic calpain-1 and calpain-2 are structurally unique in that have a cysteine-proteinase domain and a Ca2+-binding domain.33 Since these enzymes are regulated by Ca2+ concentrations, they are thought to be active in several intracellular signaling pathways that involve Ca2+, such as signal transduction, apoptosis, cell cycle progression, proliferation, differentiation, membrane fusion, and platelet activation.33,34 Most of these mammalian calpains

8 are so widely expressed that their physiologic function has been difficult to determine.34,35

The human genome sequencing project discovered 15 different calpains.36

These calpains have been divided into classical calpains (Capn1-3, 8, 9, 11-14) that have the same domains as the classic calpain-1 and calpain-2: calpain-like protease (CysPc), C2-domain-like (C2L), and penta-EF-hand (PEF). The non- classical calpains (Capn5-7, 10, 15, 16) have the CysPc domain with either the

C2L or PEF domains, or neither. 36,37 Additionally, calpains have can be divided into categories based on their location within the body. Most calpains are expressed throughout the body (Capn1, 2, 5, 7, 10, 13-16) while Capn3 is in skeletal muscle, Capn8/9 are present in the gastrointestinal system, and Capn11 is primarily found in testis.37

Unlike the classic calpains, calpain-6 does not have protease activity because it lacks an active-site catalytic cysteine residue. Originally, Capn6 mRNA was found to be more abundantly expressed in the placenta in mice, rats and humans; however, further studies show that Capn6 is also expressed early in development in the kidney and lungs in mice.35,38 Additionally, Capn6 is noted in early skeletal muscle development, but is significantly down-regulated shortly postpartum.38 However, little is known about the function of calpain-6.

In a study conducted by Tonami Kazuo et al., Capn6 deficient mice showed advanced muscular development compared to controls. The authors also revealed that in mice injected with cardiotoxin, a snake venom that targets muscle fibers, the mice without Capn6 showed more nuclei in each myofibril and 9 a larger cross-sectional area of the healing muscles. These results indicated that

Capn6 may have a suppressive role in skeletal muscle growth and healing.37

Capn6 and Bone

Capn6 also has a role in bone remodeling. After determining that Capn6 was upregulated during osteoclastogenesis, Jung Min Hong et al. conducted a study to assess the role of Capn6 in bone biology. Their results indicate that

Capn6 functions to stabilize the microtubules of osteoclasts through acetylation.

These microtubules are then attached to the actin ring and radiate toward the center of the cell. A decrease in Capn6 causes less microtubule organization and decreases the amount of β3-integrin, an important key in cytoskeleton regulation.

They conclude that Capn6 plays a role in the stability of osteoclasts cytoskeleton and an inhibition of Capn6 would halt the resorptive function of osteoclasts.39

Aims

Our study aims to better understand the relationship between osteoclast differentiation regulators and expression of genes necessary for normal osteoclast function. To determine the mechanism of HDAC4 regulation on osteoclastogensis, the Mansky lab at the University of Minnesota preformed RNA sequencing of HDAC4 null (4cKO) osteoclast and wild-type (4WT) osteoclasts to observe changes in gene expression between the two. From this analysis,

Capn6, Dok2, Fbln2 and Serpine1 were significantly different between the wild- type and HDAC4 null osteoclasts, and all four genes appeared to play a role in 10 bone resorption. Using qRT-PCR shown in the results section, we determined that Capn6 had the most significant change HDAC4 null osteoclasts compared to wild-type. In this study we examine the differences of Capn6 expression in

HDAC4 negative and wild-type osteoclasts over day 0 to 4 of osteoclast differentiation.

Materials and Methods

Osteoclast differentiation

Cultures of murine osteoclast differentiation was performed as previously described.28 Bone marrow cells were flushed from the femora and tibiae of 4WT or 4cKO 4-week-old male mice. Harvested cells were incubated in culture dishes overnight in phenol red-free alpha-MEM (Gibco) containing 5% fetal bone serum

(Atlanta Biologicals), 25 units/mL penicillin/streptomycin (Invitrogen), 400 mM L-

Glutamine (Invitrogen) in the presence of 1% CMG 14-12 culture supernatant containing M-CSF. Then, non-adherent cells were counted, plated in tissue culture plates, and cultured for 2 days with 1% CMG culture supernatant containing M-

CSF. After 2 days of culture, the cells are considered to be bone marrow-derived macrophages (BMMs). The BMMs were cultured for additional 4-5 days in phenol red-free alpha-MEM containing 5% fetal bone serum, 25 units/mL penicillin/streptomycin, 1% CMG 14-12 culture supernatant containing M-CSF and

10 ng/mL RANKL (R&D Systems) to obtain osteoclasts.

cDNA and qRT-PCR

cDNA samples used in this study were obtained from osteoclasts of 4cKO and 4WT mice provided Dr. Kim Mansky at the University of Minnesota. These samples were taken at day 0, 1, 2, 3, and 4 of osteoclast differentiation.

Quantitative real-time PCR (qRT-PCR) was performed using CFX Connect Real-

Time PCR system (Bio-Rad). The 20µl reactions contained 1µl cDNA, 10µl iTaq 12

Universal Syber Green Supermix, 8.8µl distilled water, 0.1µl Capn6 forward primer (5’-AGG GCA GAT TGG GGA ACA AG-3’) and 0.1µl Capn6 reverse primer (5’-CAC CAC CTC GGT CCA TTC TC-3’). The PCR conditions were as follows: 95°C for 3 minutes followed by 40 cycles of 95°C for 15 seconds, 58°C for 30 seconds and 72°C for 30 seconds. This was followed by melt curve analysis: 95°C for 5 seconds, 65°C for 5 seconds, and then 65°C to 95° with

0.5°C increase for 5 seconds. Capn6 genes were normalized to Hprt and the frequencies were equally amplified.

Immunoblot analysis

Protein cell lysates were extracted from primary osteoclasts using a modified

RIPA buffer (50 mM Tris pH 7.4, 150mM NaCl, 1% IGEPAL, 0.25% sodium deoxycholate and 1mM EDTA) with HALT Protease & Phosphatase Inhibitor

Cocktail (Thermo Scientific). After clearing lysates through centrifugation, proteins were resolved by SDS-PAGE then transferred to PVDF membrane

(Millipore). The proteins were then blocked and blotted in 1:1000 dilution of

CAPN6 primary antibody (Abcam) overnight at 4°C. They were then incubated with horseradish peroxidase conjugated secondary antibody at a 1:10,000 dilution of secondary antibody (rabbit) for 1 hour at room temperature. A western bolting detection kit (Western Bright Quantum, Advansta) was used to detect antibody binding.

Statistical Analysis

Unpaired t-test or one-way ANOVA analysis were used to compare the data using Graph-Pad Prism version 7. All results are expressed as a mean ± standard deviation.

Results

RNA Sequencing

To determine the mechanism(s) by which HDAC4 regulates osteoclast differentiation, the Mansky lab preformed RNA sequencing from wild-type and

HDAC4 null osteoclasts. RNA sequencing gives an unbiased analysis of gene changes occurring in HDAC4 null osteoclasts. From the genes identified by RNA sequencing there was a group of 4 genes, Capn6, Dok2, Fbln2 and Serpine1, that appeared to play a role in resorption and were significantly changed between

4WT and 4cKO osteoclasts. To confirm which of these genes were significantly changed between wild-type and HDAC4 null osteoclasts, we performed RT- qPCR and measured expression from cells that had been stimulated with 2 days of RANKL. As shown in Figure 4, Capn6 was the only gene whose expression trended towards a significant increase compared to wild-type. Because Capn6 was the only gene with a change in the HDAC4 null osteoclasts, we wanted to further characterize Capn6 expression during osteoclast differentiation.

Figure 4: Expression of RNA sequencing genes in wild-type vs. knockout

mice on day 2 of osteoclast differentiation.

Capn6 Expression in Wild-type

Since the pattern of expression during osteoclast differentiation of Capn6 had not been previously reported, I measured Capn6 expression at day 0 (M-

CSF stimulation only) and days 1 through 4 of M-CSF and RANKL stimulation.

The mean expression of Capn6 in the wild-type mice (Table 1; Figure 5B) had a trend to increase from day 0 up to day 3 of osteoclast differentiation and then slightly decreased on day 4 (D0 = 0.05506 ± 0.043, D1 = 0.2207 ± 0.2, D2 =

1.282 ± 1.282, D3 = 1.53 ± 0.866, D4 = 1.243 ± 0.537). These values are not statistically significant between the days of differentiation. Immunoblot analysis shows a similar trend with an increase in protein from day 0 to day 2 with a slight

16 decrease from day 3 to day 4 (Figure 5A). This expression pattern suggest that

Capn6 gene expression is activated by RANKL stimulation.

Day 0 Day 1 Day 2 Day 3 Day 4 1 0.0003947 4.1831067 0.4881633 5.8895397 0.6430588 2 0.0003542 8.520622 0.5650046 1.3991284 1.2119916 3 1.3244512 0.1328072 3.3413981 2.4917447 4.9508801 4 ------0.1608324 5.573903 2.7320885 9.5985062 5 0.0245907 0.4346618 1.899889 2.0220817 0.6445749 6 0.0031402 0.9158682 0.5796387 0.0462268 0.9204921 7 ------0.0367271 1.362286 2.0386185 1.6819611 8 0.0855213 0.1908068 0.5827853 0.5307552 1.4033493 9 0.0010649 0.0021822 0.6270385 2.6246164 7.6433318 10 0.004869 0.0167547 0.5518585 0.8636681 0.6089835

Table 1: Capn6:Hprt expression in 10 biologically independent wild-type mice

samples. Dashes indicate samples with no results

Figure 5: A) Immunoblot analysis of Capn6 in wild-type mice. B) Mean

Capn6:Hprt expression in wild-type mice over four days of differentiation.

Capn6 Expression in HDAC4 Knockout Mice

Next, we performed the same analysis with osteoclasts from the HDAC4 knockout mice. The mean Capn6 expression in the HDAC4 knockout mice (Table

2; Figure 6) increased from day 0 to day 3 with a decrease at day 4 of osteoclast differentiation (D0 = 0.03984 ± 0.041, D1 = 0.04252 ± 0.03, D2 = 1.172 ± 0.383,

D3 = 6.118 ± 3.032, D4 = 4.254 ± 0.93). ANOVA analysis revealed a statistically significant increase in Capn6 expression between day 0 and day 3 (p = 0.0028), day 0 and day 4 (p =0.0304), day 1 and day 3 (p = 0.0028), day 1 and day 4 (p =

0.0305), and day 2 and day 3 (p = 0.0116). As with the wild-type osteoclasts,

Capn6 expression appears to be stimulated with RANKL stimulation in HDAC4 null osteoclasts, but has greater expression at later days of osteoclast differentiation (compare days 3-4 of 4WT and 4cKO)

Day 0 Day 1 Day 2 Day 3 Day 4 1 0.0257348 0.0098109 1.1336976 3.6042235 1.0734253 2 0.0061078 0.0023691 0.4083642 0.6025746 0.5408956 3 0.00386 0.0098003 0.7492106 3.9726561 5.3219426 4 0.0315707 0.0689341 1.4936812 9.5870577 3.6093849 5 0.0840753 0.0488342 1.2746045 4.7937234 3.831786

Table 2: Capn6:Hprt expression in 5 biologically independent HDAC 4 knockout

mice samples

Figure 6: Mean Capn6:Hprt expression in HDAC4 knockout mice over four days

of differentiation

Capn6 Expression in Wild-type vs. HDAC4 Knockout mice

Comparing Capn6 expression in both wild-type and HDAC4 knockout osteoclasts, we determined that Capn6 expression was similar from day 0 to 2 in both wild-type and HDAC4 knockout mice (Figure 7). There was a significant increase in Capn6 expression in HDAC4 knockout mice when compared to wild- type mice on day 3 (p= 0.0022). There was also a greater decrease in Capn6 expression in the wild-type mice on day 4 compared to the HDAC4 knockout mice; however, the difference was not significant.

Figure 7: Mean Capn6:Hprt expression over days of differentiation between wild-

type (black) and HDAC4 knockout mice (white)

Discussion

Even though the expression of Capn6 did not have significant differences between the days of osteoclast differentiation in the wild-type mice, the trend I observed would suggest that Capn6 increases later in osteoclast differentiation.

This is reinforced by the immunoblot analysis, which shows larger amounts of

CAPN6 in days 2, 3 and 4. This trend is expected since CAPN6 is an important component in stabilizing microtubules during osteoclast function.39 Organization of osteoclast cytoskeleton during differentiation would be essential to allow for bone resorption. A larger sample size would be beneficial to decrease the amount of error and determine significance.

In the HDAC4 knockout mice, there was a significantly higher expression of Capn6 at day 3 of differentiation. Expression of Capn6 then decreased on day

4, though the difference was not significant. This, again, suggests an increase in acetylated microtubules in HDAC4 knockout osteoclasts. Increased expression at this specific point in differentiation may be noted because organization of the osteoclast’s cytoskeleton occurs around that time, allowing osteoclasts to resorb bone. More studies that look at cytoskeletal organization of osteoclasts during differentiation with Capn6 expression is necessary to determine timing.

When comparing Capn6 expression between the wild-type and HDAC4 knockout mice, there was significantly more Capn6 expressed on day 3 of differentiation in the knockout mice. Since suppression of HDAC4 increases

Capn6, which has an important role in osteoclast function, this suggests that

HDAC4 may have a suppressive role in osteoclast activity. This may at first seem 21 contrary to the observed phenotype of HDAC4 null osteoclasts which are unable to resorb bone; however, it may be the elevation of Capn6 expression does not allow for the correct pattern of acetylated tubulin during bone resorption.

Currently the Mansky lab is analyzing the expression of acetylated tubulin in the

HDAC4 null osteoclasts to determine if acetylated tubulin expression is altered.

The suppressive function of HDAC4 is similar to other HDAC proteins like

HDAC1, 2, and 6.28,30,31 Our study only extensively analyzed gene expression of

Capn6. Additional studies that address differences in osteoclast structure and function in wild-type and HDAC4 knockout mice would be beneficial. Further studies that look at other genes involved in osteoclast differentiation in mice with suppressed HDAC4 would help to determine HDAC4’s role in that process.

In orthodontics, the mechanism of bone resorption and apposition is vital to level and align teeth. The disruption of this mechanism through bisphosphonate use in osteoporotic individuals slows down orthodontic movements and can cause an increase in root resorption, lack of root parallelism following orthodontics, and an increased risk of osteonecrosis of the jaw.40 With the long half-life of bisphosphonates, patients who have taken these medications can be at risk for these side effects years after they have ceased taking the medication.41 In recent years, there has been an increase in adult patients pursuing orthodontic treatment and proper management of patients who have taken bisphosphonates is essential.

Our research may be useful in discovering different medications to suppress osteoclast activity that have shorter half-lives and don’t bind as strongly 22 to bone. Promotion of HDAC4 or similar HDAC proteins could inhibit osteoclastogenesis, thus slowing or halting bone resorption. Additionally, suppressing Capn6 could destabilize osteoclast’s microstructure and prevent them from functioning at a normal capacity. Additional studies are needed to determine the viability of these methods as osteoporosis therapies.

Conclusions

Capn6 expression increases in wild-type mice later in osteoclast differentiation, though the increase was not significant. Capn6 expression significantly increased on day 3 of osteoclast differentiation in HDAC4 knockout mice. HDAC4 may be a negative regulator of osteoclastogenesis. Additional studies are indicated to confirm if HDAC4 directly regulates Capn6 expression.

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