Effect of Fluoride on Parathyroid Hormone Secretion Chaitanya

Effect of Fluoride on Parathyroid Hormone Secretion

Chaitanya Prakash Puranik

A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Dentistry (Oral Biology)

Chapel Hill

2013

Approved by:

Eric T. Everett

Timothy Wright

Mitsuo Yamauchi

Bryan Roth

Wolfgang Bergmeier

Chaitanya Prakash Puranik

ABSTRACT

Chaitanya Prakash Puranik: Effect of Fluoride on Parathyroid Hormone Secretion

(Under the direction of Eric Everett)

Systemic fluoride induces osteoclastogenesis in C3H/HeJ (C3H) mice with increased serum iPTH, sRANKL, and TRAP5b along with reduced levels of OPG; whereas, in C57BL/6J (B6) mice fluoride has anabolic actions (Yan et al 2007). In humans pseudo-hyperparathyroidism is sometimes seen with skeletal fluorosis.

Early events of fluoride’s actions on the parathyroid gland are not well understood.

Objective: This study investigated fluoride’s effects on iPTH secretion and its underlying mechanism. Methods: In vitro: Thryo-parathyroid complexes (TPC) from

5-6 week old C3H (n=18) and B6 (n=18) male mice were dissected and dispersed.

TPC were cultured in medium pre-optimized for calcium, phosphorus, and magnesium levels equivalent to mouse serum levels. TPC were treated with 0, 250 or 500µM [F-] for 24hrs and secreted iPTH levels determined by ELISA. HEK293T- cloned cells (containing luciferase reporter) were transfected with human calcium sensing receptor (CASR) or orphan GPCRs followed by 0 or 1mM fluoride treatment

(16hrs) to determine β-arrestin recruitment. In vivo: 5-6 week old C3H (n=78) and

B6 (n=78) male mice were gavaged with distilled or fluoride water (1µg [F-]/g-body weight). Serum iPTH, calcium, phosphorus, magnesium, and fluoride were

iii determined at 0, 3, 6, 12, or 24hrs. Results: A dose-dependent decrease in secreted iPTH was seen in vitro for C3H ( p<0.005) and B6 ( p<0.005) at 500µM [F -] exposure. Single gavage led to an increase in serum fluoride peaking at 0.5hr. iPTH also peaked at 0.5hr in both C3H ( p=0.002) and B6 ( p=0.01) followed by a decrease in C3H at 24hrs ( p=0.002) returning to baseline at 48hrs. At 12hrs, there was an increase in iPTH in B6 ( p<0.001) which returned to baseline at 24hrs. Fluoride treatment did not alter β-arrestin recruitment by CASR. GPR111, GPR142 and

GPRC5D demonstrated increased β-arrestin recruitment. Conclusion: Fluoride modulates iPTH secretion in vitro and in vivo . However, Fluoride’s action on the parathyroid gland is not mediated through CASR. While fluoride’s effects, in vitro , were equivalent between the two mouse strains, early strain-dependent effect on iPTH secretion was observed in vivo . Difference in fluoride-mediated gene expression in C3H and B6 suggests an underlying difference in physiologic handling of fluoride by the two strains.

ACKNOWLEDGEMENTS

This dissertation study was carried out at the School of Dentistry, University of North Carolina at Chapel Hill and is a result of the guidance, persistence and collaboration of my mentors and colleagues. First, I would like to express my gratitude to my mentor Prof. Eric T. Everett for giving me the opportunity to work in his lab and develop my research skills under his guidance. His enthusiasm, discipline, and commitment towards science motivated me to undertake quality research. His mentorship was paramount in providing a well-rounded experience consistent with my career goals. Dr. Everett’s critical analysis approach taught me how to be an efficient researcher, teacher and administrator. It was his vision that set goals for me and it was his patience which kept me on track to achieve those goals. I would like to extend my sincere gratitude towards my other committee members. Prof. Bryan Roth provided me with an opportunity to learn and perform experiments in his lab. His critical but positive methodology helped me learn new techniques and employ them towards my research. Prof. Mitsuo Yamauchi taught me to critically analyze data and make careful conclusions without over interpreting the data. Dr. Wolfgang Bergmeier provided me useful understandings to improve my research strategies and enhance the quality of my scientific experiments. Prof.

Timothy Wright provided me a deep insight in scientific research, clinical problems, and genetic approaches.

I am grateful to my co-workers and friends Dr. Yan Dong, Holly Cheeseman,

Kathleen Ryan, Dr. Melanie Alazzam, and Dr. Shijia Hu with whom I shared my research space and time. I would also like to thank Heather Zimmerman, Lauren

Katz, Eleni Boukas, Elise McGuire and Staci Love who helped me in my research and made my stay at UNC enjoyable.

I wish to thank Drs. Sylvia Frazier-Bowers, Ching Chang Ko, Patrick Flood, and

Asma Khan for their constant help and support. I am grateful for the support and motivation by Dr. Ceib Philips, Oral Biology program director. I am also thankful to the Oral Biology program manager, Ms. Cindy Blake, for patiently guiding me throughout my training.

I am grateful to Drs. Angeles E. Martinez-Mier, Patrick Giguere, Hemant Kelkar and John Preisser for their technical support. Finally, I am thankful to the Oral

Biology staff and students who stood beside me through the highs and lows of my graduate career. I extend my gratitude to my master’s thesis advisors Drs. Robert

Baier and Anne Meyer for their constant support and encouragement. I would like to thank my family for their trust and support towards my educational career. I am grateful to all those who stood by me and I promise to make them proud with my sincere efforts and successful results.

TABLE OF CONTENTS

TABLE OF CONTENTS ...... vii

LIST OF FIGURES AND TABLES ...... xii

LIST OF ABBREVIATIONS AND SYMBOLS ...... xiii

CHAPTER 1: REVIEW OF LITERATURE ...... 1

1.1 Fluoride ...... 1

1.1.1 Introduction ...... 1

1.1.2 Metabolism ...... 2

1.1.2.1 Absorption ...... 3

1.1.2.2 Distribution ...... 4

1.1.2.2.1 Distribution in the soft tissues ...... 5

1.1.2.2.2 Distribution in body fluids ...... 6

1.1.2.2.3 Distribution in mineralized tissues ...... 6

1.1.2.3 Renal clearance ...... 7

1.1.2.4 Fecal clearance ...... 9

1.1.2.5 Factors modifying metabolism ...... 9

vii

1.1.2.5.1 Acid-base equilibrium ...... 9

1.1.2.5.2 Renal impairments ...... 9

1.1.2.5.3 Altitude ...... 10

1.1.2.5.4 Physical activity ...... 10

1.1.2.5.5 Circadian rhythm ...... 11

1.1.2.5.6 Nutrition ...... 12

1.1.2.5.7 Genetic influences ...... 13

1.1.3 Toxicity ...... 15

1.1.4 Fluorides in dentistry ...... 16

1.1.4.1 Fluoride’s action on tooth enamel ...... 17

1.1.4.2 Enamel fluorosis ...... 18

1.1.5 Interactions on bone...... 19

1.1.5.1 Bone remodeling, osteoblasts and osteoclasts ...... 19

1.1.5.2 Characteristics of fluoride’s actions and hypotheses ...... 21

1.1.5.2.1 Tyrosine phosphate hypothesis ...... 22

1.1.5.2.2 G protein hypothesis ...... 24

1.1.5.5 Skeletal fluorosis ...... 25

1.2 Parathyroid hormone (PTH) ...... 31

1.2.1 Mineral homeostasis and bone-intestine-parathyroid-kidney (BPK) axis

...... 31

viii

1.2.2 PTH and PTHLH ...... 33

1.2.2.1 Synthesis and secretion of PTH ...... 34

1.2.2.2 Regulation of PTH secretion ...... 35

1.2.2.2.1 Extracellular calcium-mediated regulation ...... 35

1.2.2.2.2 1,25(OH)2D3-mediated regulation ...... 37

1.2.2.2.3 Phosphorous- FGF23-KLOTHO-mediated regulation ...... 37

1.2.2.3 Metabolism of PTH ...... 38

1.2.2.4 PTH- and PTHLH-PTH1R ...... 39

1.3 Calcium Sensing Regulator (CASR) ...... 40

1.3.1 Isolation of Calcium Sensing Receptor ...... 40

1.3.2 CASR gene ...... 41

1.3.3 Functions of CASR in tissues ...... 42

1.3.3.1 Parathyroid gland ...... 42

1.3.3.2 Parafollicular cells ...... 42

1.3.3.3 Kidney ...... 43

1.3.3.4 Intestine ...... 43

1.3.3.5 Bone ...... 44

1.3.4 CASR signaling ...... 44

1.3.5 CASR agonists ...... 45

CHAPTER 2: SPECIFIC AIMS ...... 47

CHAPTER 3: FLUORIDE MODULATES PARATHYROID HORMONE

SECRETION IN VITRO AND IN VIVO ...... 51

3.1 Abstract ...... 52

3.2 Introduction ...... 54

3.3 Materials and Methods ...... 56

3.3.1 Animals ...... 56

3.3.2 Parathyroid gland organ culture ...... 56

3.3.3 Gavage and sample collections ...... 57

3.3.4 ELISA and biochemistry assays ...... 57

3.3.5 RNA extractions and cDNA preparation ...... 58

3.3.6 Statistical analysis ...... 59

3.4 Results ...... 60

3.4.1 Responsiveness of TPC organ culture model ...... 60

3.4.2 Effect of fluoride on TPCs ...... 61

3.4.3 Serum fluoride levels after gavage ...... 61

3.4.4 Serum iPTH after single fluoride dose via oro-gastric gavage ...... 62

3.4.5 Serum total calcium, phosphorus, and magnesium levels ...... 64

3.4.6 qPCR: Expression of genes involved in BPK feedback loop ...... 66

3.5 Discussion ...... 68

CHAPTER 4: FLUORIDE DOES NOT TRIGGER BETA-ARRESTIN

RECRUITMENT (NON-CANONICAL SIGNALING) BY CASR ...... 72

4.1 Abstract ...... 73

4.2 Introduction ...... 75

4.3 Material and Methods ...... 77

4.3.1 CASR and orphan GPCRs constructs ...... 77

4.3.2 Cells and transfection ...... 77

4.3.3 β-arrestin recruitment assay ...... 78

4.3.4 Statistical analysis ...... 78

4.4 Results ...... 80

4.4.1 Effect of fluoride on human CASR ...... 80

4.4.2 Effect of calcimimetic (R568) on human CASR ...... 80

4.4.4 Effect of fluoride on R568-mediated CASR response ...... 81

4.4.5 Effect of R568 on CASR is specific ...... 82

4.4.5 Effect of R568 extends to CASR related GPCRs ...... 83

Figure 4.5: Effect of R568 on GPRC6A ...... 84

4.4.6 Effect of fluoride on 126 orphan GPCRs ...... 84

4.5 Discussion ...... 87

Bibliography ...... 92

LIST OF FIGURES AND TABLES

Figure 1.1: Fluoride action hypotheses ...... 24

Figure 1.2: Ground water fluoride concentration around the world...... 27

Figure 1.3: Clinical and radiographic features of skeletal fluorosis ...... 29

Figure 1.4: Bone-Parathyroid-Kidney (BPK) axis ...... 33

Figure 1.5: CASR signaling cascade ...... 45

Figure 3.1: iPTH levels in media supernatant ...... 60

Figure 3.2: Serum fluoride levels ...... 62

Figure 3.3: Serum iPTH levels 0-24 hrs ...... 63

Figure 3.4: Serum iPTH levels 48-96hrs ...... 64

Figure 3.5: Serum calcium, phosphorus, and magnesium levels...... 65

Figure 3.6: Expression of genes involved in BPK feedback loop ...... 67

Figure 4.1: Effect of fluoride on CASR ...... 80

Figure 4.2: Effect of calcimimetic (R568) on CASR ...... 81

Figure 4.3: Effect of fluoride on R568-mediated CASR response ...... 82

Figure 4.4: Effect of R568 on CASR is specific ...... 83

Table 4.1: Percentage RLU for screened orphan GPCRs ...... 85

Table 4.2: Orphan GPCRs activated by fluoride ...... 86

Figure 4.5: Schematic of the β-arrestin recruitment assay ...... 88

xii

LIST OF ABBREVIATIONS AND SYMBOLS

[F -] fluoride ion

1,25(OH)2D3 1, 25 dihydroxy vitamin D3

AC adenylyl cyclase

ALP alkaline phosphatase

B6 C57BL/6J mouse strain

BIPK bone-intestine-parathyroid-kidney

BMM bone marrow macrophages

BPK bone-parathyroid-kidney

C3H C3H/HeJ mouse strain

Ca 2+ ionic calcium cAMP cyclic adenosine monophosphate

CASR calcium sensing receptor cDNA complimentary deoxyribonucleic acid

CLCN5 chloride channel 5 c-Src tyrosine protein kinase

CT calcitonin

DC-STAMP dendritic cell-specific transmembrane protein

ELISA enzyme-linked immunosorbant assay

ER endoplasmic reticulum

ERK extracellular signal-regulated kinase

FAP fluorapatite

xiii

FGF1Rc fibroblast growth factor receptor 1c

FGF23 fibroblast growth factor 23

FMBD fluorotoxic metabolic bone disease

Gd 2+ ionic gadolinium

GI tract gastro-intestinal tract

GPCR G-protein coupled receptor

Grb2 growth factor receptor-bound protein 2

GTP guanosine triphosphate

HAP hydroxyapatite

HF hydrofluoric acid

HTL HEK293T-cloned cells containing integrated luciferase reporter

IP3 inositol triphosphate iPTH intact parathyroid hormone

MAPK mitogen-activated protein kinase

MC3T3-E1 osteoblastic cell line form mouse calvaria

M-CSF macrophage colony-stimulating factor

Mg 2+ ionic magnesium

MMP9 matrix metalloproteinase 9 mRNA messenger ribonucleic acid

NFATc-1 nuclear factor of activated T-cells, cytoplasmic 1

NSHPT neonatal severe hyperparathyroidism

OPG osteoprotegerin (current symbol TNFRSF11B, tumor necrosis

factor receptor superfamily, member 11b)

xiv

PDZK1 PDZ containing domain 1

PKC protein kinase C

PLA2 phospholipase A2

PLC phospholipase C

PLD phospholipase D

PPI polyphosphoinositide

PTH parathyroid hormone

PTH1R parathyroid receptor 1

PTH2R parathyroid receptor 2 qPCR quantitative real time polymerase chain reaction

Raf serine/threonine protein kinase

Ras family of small GTPase

SF skeletal fluorosis

SLC34A1 solute carrier family 34, member 1

SLC9A3R1 solute carrier family 9 member 3 regulator 1

SOS son of sevenless

Sr 2+ ionic strontium sRANKL soluble receptor-activator of NF-κB ligand (current name

TNFSF11, tumor necrosis factor (ligand) superfamily, member

11)

TPC thyro-parathyroid complex

TRAP5b tartrate-resistant acid phosphatase 5b

VDR vitamin D receptor

WHO World Health Organization

αKLOTHO alpha klotho

xvi

CHAPTER 1: REVIEW OF LITERATURE

1.1 Fluoride

1.1.1 Introduction

Fluorine is the 13 th most abundant element found in the earth’s crust (Fordyce et al., 2007). Due to the small radius of the fluorine atom, its effective surface charge is the highest among all elements. As a consequence, fluorine is the most electronegative element and seldom occurs in nature in an elemental form. It is found frequently as inorganic fluoride that is widely distributed (Ekstrand, 1978).

Fluorine exists in nature in compound forms such as: fluorite (CaF 2), fluorapatite

(Ca 5(PO 4)3F), and cryolite (Na 2AlF 6). Higher concentrations of fluoride are found in dust, air, soil, rocks and surface ground water in the areas with recent or past geologic and pyroclastic activities or geologic uplifts (Everett, 2011). Presence of fluoride in surface and ground water is a major source of exposure to humans.

Fluoride is also present in many processed food and drinks we consume. Soft drinks and fruit juices account for 75% of the fluoride intake in the United States. Fluorides as polytetrafluoroethyleneare (PTFE) are widely used in industrial processes for non-stick coatings. Fluoride has been used as an enzyme inhibitor in the past by biochemists in millimolar (mM) concentrations. Sodium fluoride is used as a general inhibitor of serine proteases and phosphatases. Fluoride can demonstrate two biologic actions (biphasic) based on its concentrations: mitogenic (inducing mitosis) at low concentrations and toxic at higher concentrations.

1.1.2 Metabolism

Ingested fluoride is primarily absorbed from the stomach and intestine. Fluoride ion can complex with calcium ion to form fluorite (CaF 2) (Kanbur et al., 2009). In the body, fluoride can combine with various elements to form insoluble complexes which can accumulate in the extracellular compartments (Mittal and Flora, 2006). Fluoride absorption (stomach and intestine), distribution (hard and soft tissue) and excretion

(kidney) are pH dependent process. Hydrofluoric acid (HF) is a weak acid having an acid dissociation constant (pK a) of 3.4. Therefore at 3.4 pH, 50% of fluoride exists as un-dissociated form (HF) while the remaining 50% is in the dissociated or ionic form

[F -]. When pH decreases from 3.4, the concentration of HF increases and when pH increases above 3.4, the concentration of [F -] increases (Whitford, 1996). HF has a high coefficient of permeability as compared to fluoride and hence HF can penetrate lipid bilayer more easily (Gutknecht and Walter, 1981). HF movement occurs in response to a pH gradient between body compartments (HF moves from the acidic compartment to the alkaline compartment along the pH gradient).

Plasma fluoride concentrations rise rapidly after absorption of fluoride (as HF) through the stomach (Whitford, 1996). Fluoride that is not absorbed from the stomach is absorbed from the intestine. However, unlike gastric absorption, intestinal absorption of fluoride is independent of pH (Nopakun et al., 1989). Fluoride which is not absorbed from the stomach or intestine is excreted in the feces

(Whitford, 1996).

Plasma fluoride concentrations reach peak after ingestion within 20-60 minutes

(Whitford, 1996). The fluoride is then deposited in the newly mineralized tissues or is excreted through urine. In adults, approximately half of the absorbed fluoride is deposited in the mineralized tissues (bone, enamel, and dentin). Mineralized tissue holds approximately 99% of body’s fluoride (Whitford, 1994). However, fluoride is not irreversibly bound to bone. Fluoride can be released back into the plasma during bone remodeling (Whitford, 2008). A small fraction of fluoride absorbed is found in soft tissues (Whitford, 1984). Most of the absorbed fluoride, not taken up by mineralized tissues, is excreted in urine. Very small fraction (<1%) of absorbed fluoride is excreted in sweat and feces. The plasma fluoride concentrations return to the baseline after 3-6 hours (Whitford, 1996).

1.1.2.1 Absorption

More than 80% of the ingested fluoride is absorbed from gastro-intestinal (GI) tract. (Whitford, 1996). Presence of high amounts of bi- and tri-valent cations

(calcium, magnesium, and aluminum) which form insoluble complexes with fluoride reduces net fluoride absorption. (Whitford, 1996). Although fluoride absorption from the stomach occurs rapidly, the rate of absorption is determined by gastric acidity

(Messer and Ophaug, 1993; Whitford and Pashley, 1984) and velocity of gastric emptying (Messer and Ophaug, 1991; Nopakun et al., 1989).

Fluoride absorption and gastric pH are inversely related. When the acidity of the stomach decreases (antacid administration) then the absorption of the fluoride decreases (Whitford and Pashley, 1984). When ionic fluoride enters the acidic

3 gastric lumen, it is converted into HF which is an uncharged molecule that can readily cross the gastric mucosa. Higher the acidity of the gastric content, faster will be the fluoride absorption from the stomach.

Additionally, gastric emptying interferes with gastric fluoride absorption. Animal studies have shown that delayed gastric emptying might result in slower and smaller increases in plasma fluoride levels (Messer and Ophaug, 1991; Nopakun et al.,

1989). More than half of the fluoride which escapes gastric absorption is absorbed in the proximal part of small intestine (Nopakun et al., 1989). The small intestine has a huge capacity for fluoride absorption and fluoride is rapidly absorbed following emptying from the stomach. Ionic fluoride crosses the leaky epithelia through tight junctions between the paracellular channels (Nopakun et al., 1989). Fluoride absorption from the small intestine compensates for the low gastric absorption due to high pH to ensure that overall fluoride absorption is relatively unaffected by gastric pH (Messer and Ophaug, 1993; Whitford and Pashley, 1984).

1.1.2.2 Distribution

Plasma is the central compartment for fluoride distribution. Fluoride achieves steady-state distribution in the soft tissues where less than 1% of absorbed fluoride is present. More than half of absorbed fluoride is taken up by the calcified tissues, where fluoride is reversibly bound, and can be released back into the plasma in case of demand. (Whitford, 1994).

Fluoride occurs in plasma in ionic and non-ionic forms. In the blood, ionic fluoride is not equally distributed between plasma and blood cells. Concentration of fluoride

4 in the plasma is twice as high as that found in the blood cells. The biological function of non-ionic fluoride has not been established yet. However, its concentration is usually higher than that of ionic fluoride. The non-ionic and ionic fluoride together constitute the total plasma fluoride (Whitford, 1996). Ionic fluoride concentrations in the plasma are dynamic; it depends on the amount of fluoride intake, deposition/removal (in/from soft and hard tissues), and urinary excretion (Whitford,

1996). Therefore, plasma fluoride levels have been used as ‘contemporary biomarkers’ of fluoride exposure.

1.1.2.2.1 Distribution in the soft tissues

Fluoride in plasma is rapidly distributed to various tissues of the body. The rate of blood flow to the different tissues determines the distribution kinetics of fluoride

(Whitford, 1996). When considering fluoride distribution the in soft tissues, it is important to note that fluoride, as HF, accumulates in the alkaline compartment in response to a pH gradient. The intracellular compartment is usually more acidic than extracellular and therefore, fluoride concentrations are typically lower in the intracellular compartment compared to those present in the plasma or extracellular compartment. In cases of acute fluoride poisoning, wherein toxic levels of fluoride are ingested, alkalization of the body fluids to promote a net efflux of intracellular fluoride is a recommended treatment.

1.1.2.2.2 Distribution in body fluids

Fluoride concentrations in body fluids are dynamic and different from those found in plasma. Cerebrospinal fluid and milk have fluoride concentrations 50% lower than that of plasma. Gingival crevicular fluid and ductal saliva (submandibular and parotid) fluoride levels are slightly higher and lower, respectively, than those in plasma. Reported ratio of fluoride concentrations in ductal saliva to plasma fluoride concentrations are around 0.9 and 0.8 for submandibular and parotid secretions, respectively (Whitford, 1994). Ductal saliva has also been employed as a

‘contemporary biomarker’ of fluoride exposure (Olympio et al., 2007; Wilson and

Bawden, 1991; Zero et al., 1992). Concentrations of fluoride in the whole saliva are more variable and higher than those seen in ductal saliva. Exogenous fluoride contamination of whole saliva renders it unsuitable for estimation of plasma fluoride concentrations as a ‘contemporary biomarker’. (Whitford et al., 1999).

1.1.2.2.3 Distribution in mineralized tissues

Approximately 99% of the fluoride is found in the mineralized tissues of the body such as; bone, enamel, and dentin (Whitford, 1994). Fluoride concentration in bone is not uniform. The concentrations of fluoride in long bone are higher in the periosteal and endosteal regions. As compared to compact bone, cancellous bone has higher fluoride concentrations due to its greater surface area in contact with the surrounding extracellular fluid. Bone fluoride concentrations tend to increase with age due to continuous fluoride uptake throughout the life (Richards et al., 1994).

It is estimated that approximately 36% of the absorbed fluoride becomes associated with the bone, while the remainder is excreted. In children, the degree of retention is much higher (55%) (Villa et al., 2010) due to the rich blood supply and large surface area of the growing bones (Whitford, 1994).

Fluoride concentrations in the dentin are similar to bone fluoride concentrations and increase with age. Dentin fluoride levels are higher close to the pulp and reduce progressively towards the dentino-enamel junction (Rao et al., 1995). Enamel fluoride concentrations are usually lower than the levels found in dentin. No correlation was found between the concentrations of fluoride in dentin and enamel

(Vieira et al., 2004; Vieira et al., 2006). Concentration of fluoride in the dental enamel decreases with age, especially in areas where dental biofilm accumulates.

The fluoride concentrations in the dental enamel reflect the level of fluoride exposure during amelogenesis (process of enamel formation) (Rao et al., 1995). A significant correlation between the severity of dental fluorosis and fluoride concentrations has been found only for the dentin. (Vieira et al., 2004; Vieira et al., 2006).

1.1.2.3 Renal clearance

The kidneys play a major role in fluoride removal from the body. Under normal conditions, approximately 60% of fluoride absorbed each day by healthy adults is excreted in urine. In children about 45% of the absorbed fluoride is excreted through urine (Villa et al., 2010). More fluoride is retained in children as compared to the adults may be due to the growing skeleton in the children and comparatively slow

7 bone remodeling in adults. Fluoride in the plasma and urine reflects a physiologic balance between fluoride intake and removal.

The concentrations of fluoride in the glomerular filtrate and plasma are comparable. After entering the renal tubules, a variable amount of the ion is reabsorbed and returned to the systemic circulation (Whitford, 1996). Tubular reabsorption of fluoride and glomerular filtration rate are the principal determinants of the urinary fluoride excretion. Compared to the renal clearance of other halogens

(~1-2 mL/min), fluoride has high renal clearance (35mL/min). However, individual variations in the renal clearance of fluoride are common (Whitford, 1994) and are attributed to the three major factors; differences in the glomerular filtrations rates

(Spak et al., 1985), pH of urine (Jarnberg et al., 1983) and urinary flow rate

(Ekstrand et al., 1982).

The mechanism of renal tubular reabsorption of fluoride is also pH-dependent and occurs by diffusion as HF (Whitford et al., 1976). Hence, when the pH of the tubular fluid is relatively high, the proportion of fluoride as HF is lower while there is a higher proportion of fluoride as [F-]. Only a small amount of HF crosses the epithelium of the renal tubule to be reabsorbed and a high amount of fluoride is excreted in urine. Conversely, when the pH of the tubular fluid is lower, high amounts of HF cross the tubular epithelium into the interstitial fluid where the pH is relatively neutral which promote the dissociation of HF. Dissociated fluoride then diffuses into peritubular capillaries and is returned to the systemic circulation.

1.1.2.4 Fecal clearance

Fecal fluoride represents less than 10% of the ingested fluoride. In other words, more than 90% of ingested fluoride is usually absorbed (Ekstrand et al., 1994).

When diets containing high amounts of calcium are consumed, unabsorbed calcium forms complexes with fluoride in the stomach. The complexes of calcium and fluoride do not easily diffuse through the GI lining and thereby reduces the total amount of fluoride absorbed. (Whitford, 1994).

1.1.2.5 Factors modifying metabolism

1.1.2.5.1 Acid-base equilibrium

Acid-base equilibrium plays an important role on the metabolism and tissue concentrations of fluoride. Factors that alter the acid-base equilibrium include diet composition (vegetarian diet increases urinary pH), altitude, drugs, metabolic and respiratory disorders. Acute respiratory acid-base disorders affect renal excretion of fluoride in the same manner as the metabolic disorders (Whitford, 1996).

1.1.2.5.2 Renal impairments

A direct relationship between renal impairment and enamel defects (such as enamel hypoplasia) was observed in humans (Al-Nowaiser et al., 2003; Farge et al.,

2006; Koch et al., 1999). In a study comparing children with renal disease to healthy children, the frequency of dental fluorosis was not significantly different between the two groups. However, children with renal disease demonstrated more severe dental fluorosis than children without renal disease (Ibarra-Santana et al., 2007).

1.1.2.5.3 Altitude

Rats raised in hypobaric chambers to stimulate high altitudes demonstrated increased enamel disturbances, regardless of the levels of ingested fluoride

(Whitford, 1996). Alterations in acid-base balance, caused by hypobaric hypoxia due to high altitude of residence were responsible for decreased urinary fluoride clearance. This decrease in urinary fluoride clearance resulted in greater retention of fluoride (Whitford, 1997). A significantly higher prevalence of fluorosis has been observed in Tanzanian communities at a high altitude (1,463 m), in contrast with a low altitude area (100m), with similar food habits and low levels of fluoride in the drinking water (Yoder et al., 1998). The severity of enamel disturbances at the high altitude area was not consistent with the fluoride concentration in drinking water.

Such a finding suggested that altitude, along with other factors, may contribute to the severity of enamel disturbances. Studies conducted in other high altitude locations around the world confirmed that physiological changes associated with residence at high altitude are able to exacerbate the effects of fluoride on mineralized tissues

(Akosu and Zoakah, 2008; Martinez-Mier et al., 2004; Pontigo-Loyola et al., 2008).

1.1.2.5.4 Physical activity

Prolonged physical activity reduces the pH gradient across cell membranes, especially skeletal muscle cells, which promote the diffusion of fluoride from the extracellular to the intracellular fluid. Physical activity-mediated catecholamines secretion can lead to renal vasoconstriction leading to decreased renal fluoride clearance. Depending on the balance of several factors, exercise could be

10 associated with either decrease or increased circulating fluoride levels (Whitford,

1996). Although physical activity may alter the pattern of fluoride excretion, the impact on the development of dental fluorosis seems negligible.

1.1.2.5.5 Circadian rhythm

Daily variations are observed in levels of calcium and phosphorus which are involved in bone remodeling (the process involving a balance between deposition and resorption of bone) under the influence by mineral homeostatic hormones.

Fluoride, similar to calcium and phosphorous, is primarily stored in the skeleton and therefore, it was hypothesized that plasma fluoride levels would exhibit circadian rhythm too (Talmage et al., 1975). Such rhythmicity was verified in dogs, with a mean peak fluoride concentration around 9 AM, followed by a decrease around 9

PM (Whitford, 1996).

Whitford et al demonstrated a rhythmicity of fluoride concentrations in human plasma, with mean peak (0.55 mol/L) at 11 AM and the lowest concentrations occurring between 5 and 8 PM (Whitford et al., 2008). Plasma fluoride concentrations were positively correlated with urinary fluoride excretion rates and with parathyroid hormone (PTH) levels, suggesting that both the renal system and mineral homeostatic hormone (PTH) might be involved in the rhythmicity of plasma fluoride.

1.1.2.5.6 Nutrition

A correlation between malnutrition and prevalence of dental fluorosis prevalence has been suggested in the past; however, the evidence for such a relationship is controversial. In a study with Tanzanian children, Yoder et al suggested a direct relationship between malnutrition and dental fluorosis (Yoder et al., 1998) whereas another study demonstrated that dental fluorosis is independent of nutritional status

(Buzalaf and Whitford, 2011). Nutritional status is commonly assessed by the height- for-age and weight-for-age indices as per the recommendations by the World Health

Organization (WHO). It is hypothesized that a malnourished child may absorb fluoride more quickly due to the inexistence of ions to form complexes in an empty stomach. On the other hand, a malnourished child may have low fluoride deposition due to slower bone growth.

The prevalence of dental fluorosis and severity of dental defects were compared between the children fed vegetarian or non-vegetarian diet (Awadia et al., 1999). In the vegetarian group, the enamel fluorosis severity and prevalence were 21% and

67% whereas; in non-vegetarian group it was 35% and 95% respectively.

Additionally, the calculated risk for developing enamel fluorosis was 7 time higher in non-vegetarian diet fed children as compared to the vegetarian diet fed children.

Tamarind extracts have been used to promote urinary fluoride excretion by increasing urinary pH in school children (Khandare et al., 2004). Tamarind paste contains 8.4-12.4% tartaric acid, which is not metabolized in the body and eventually is excreted through urine. Tartaric acid reduces the pH of urine which forces fluoride

12 to remain in a dissociated form as [F -] and hence fluoride cannot be reabsorbed (as

HF) in the kidneys. This ultimately leads to fluoride wasting.

High dietary concentrations of certain cations, especially calcium, can reduce the extent of fluoride absorption (Vieira et al., 2004; Vieira et al., 2006). A study reviewed by Buzalaf et al , conducted in the province of Jaingxi, China, where the prevalence of dental fluorosis was reported to be above 50%, the consumption of milk was a critical factor influencing the incidence of dental fluorosis in children. The incidence of dental fluorosis among the children consuming milk was lower (7.2%) as compared to the children not consuming milk (37.5%). (Buzalaf and Whitford,

2011). In India, where approximately 62 million people have dental fluorosis, studies have been conducted to identify increased risk of dental fluorosis due to dietary components, other than fluoride (Bhargavi et al., 2004). Low calcium concentrations in the drinking water and prevalence of dental fluorosis are inversely related.

Therefore, calcium supplementation in areas with endemic fluorosis will minimize the absorption of fluoride through water and will avoid excessive incorporation of fluoride in the mineralized tissues (Khandare et al., 2005).

1.1.2.5.7 Genetic influences

A study conducted in three distinct areas of Tanzania, which differed in water fluoride concentrations and altitude, demonstrated that at two out of three areas with severe fluorosis, several children had very little evidence of enamel disturbances

(Yoder et al., 1998). Urinary fluoride values and fluoride values in the food were

13 similar between those two groups. Differences between two homogeneous tribes raised the possibility of genetic influence on fluorosis prevalence.

Genetic predisposition to dental fluorosis was demonstrated in 12 strains of mice.

Other confounding variables such as age, sex, food and housing were controlled.

(Everett et al., 2002). The 12 inbred strains of mice showed variable susceptibilities to dental fluorosis. The A/J strain demonstrated highly susceptibility to dental fluorosis; whereas, 129P3/J mouse strain demonstrated minimal dental fluorosis.

The onset of dental fluorosis in the A/J strain was rapid with highest severity amongst the tested strains. Two strains; A/J and 129P3/J, demonstrated variable responses on the bone after fluoride exposure (Mousny et al., 2006). It was hypothesized that the different susceptibility to dental fluorosis between these two 2 strains was due to differences in fluoride metabolism (the resistant strain would excrete more fluoride which in turn would lead to decreased susceptibility to dental fluorosis). A metabolic study to test this hypothesis revealed an interesting finding.

The resistant strain (129P3/J) excreted a significantly lower amount of fluoride in urine than the susceptible strain (A/J) resulting in higher plasma and bone fluoride concentrations. The fluoride, however, did not affect the amelogenesis in the

129P3/J strain (Carvalho et al., 2009). In mouse, the quantitative trait loci associated with dental fluorosis were detected on chromosomes 2 and 11 (Everett et al., 2009).

Histological examination of maturing enamel showed that fluoride treatment resulted in accumulation of amelogenins in the maturing enamel of A/J mice, but not of

129P3/J mice. All these studies indicate that the mechanism of fluorosis can only be

14 partly explained with genetics. The differences in fluorosis susceptibility may involve physiological, biochemical, and molecular differences in theses mouse strains.

1.1.3 Toxicity

Systemic toxicity of fluoride can occur following fluoride exposure through dermal or parenteral route (Augenstein et al., 1991; Mullins et al., 1998; Yolken et al., 1976).

Toxicity of fluoride is due to electrolyte disturbances such as: hypocalcemia, hypomagnesemia, and hyperkalemia (Greco et al., 1988; Kao et al., 1999; Stremski et al., 1992). In 2001, the Toxic Exposure Surveillance System of American

Association of Poison control Centers reported about 1200 HF exposure incidents

(Litovitz et al., 2002). Severity of the exposure depends mostly on the concentration of the HF and the total surface area of body affected or exposed. Often, the HF exposure leads to mild to moderate dermal or ocular injuries, but exposure to very high doses of HF leads to life-threatening complications (Kao et al., 1999). Systemic exposure of high amounts of fluoride as HF leads to chelation of plasma calcium which leads to severe hypocalcemia which can be fatal (Mullett et al., 1987).

Fluoride readily forms complexes with calcium and magnesium ions; hence antacids containing calcium or magnesium cations are routinely administered to avoid toxic effects of fluoride. Although oral antacids containing calcium and magnesium salts are effective for gastric decontamination after systemic HF exposure, they do not alter survival time after toxic dose of HF (Heard and Delgado, 2003).

1.1.4 Fluorides in dentistry

The fluoride’s effect on prevention of dental caries was observed in the early

1800s. Fluoride is naturally present in the surface drinking water in variable amount.

Governmental agencies around the world have started programs to control the amount of fluoride in the drinking water depending on the average temperature and altitude of the area. Although controversial, the effectiveness of such programs have been established through the observations that water fluoridation programs led to a decline in the prevalence of dental caries. Billions of individuals in approximately 40 countries are exposed to fluoride from adjusted fluoridated water supplies (Browne et al., 2005; Pizzo et al., 2007). In the US, according to the Center for Disease

Control 2008 water fluoridation statistics, approximately two hundred million people receives fluoridated water. In other words, F reaches more than 70% of the US population through public water supply (Sampaio and Levy, 2011). Since the initial findings of the effectiveness of fluoride to prevent on caries, intensive research has been conducted to determine the benefits, safety, and the cost-effectiveness of fluoride delivery modalities other than water. Fluoridation of milk, salt, dietary supplements was undertaken as an alternative to augment fluoride supplementation to children and reduce caries incidence (Sampaio and Levy, 2011). Fluoride containing solutions, gels, foams, varnishes, rinses, and dentifrices can be applied topically to render teeth resistant to caries attack (Pessan et al., 2011).

1.1.4.1 Fluoride’s action on tooth enamel

Dental enamel is primarily composed of minerals of calcium and phosphate commonly termed as hydroxyapatite (HAP) (McCann, 1953). The HAP crystals form a closely packed lattice structure. Fluoride can replace hydroxyl (OH), carbonate

2- 3- (CO 3 ) and phosphate (PO 4 ) ions in HAP lattice to form fluorapatite (FAP). FAP renders enamel hard and resistant to decay and acid attack.

- - Ca 10 (PO 4)6(OH) 2 + 2F Ca 10 (PO 4)6F2 +2OH

(Hydroxyapatite) (Fluorapatite)

Calcium present on the outer surface of the enamel complexes with fluoride to form fluorite (CaF 2). Fluorite is resistant to acid dissolution and hence renders the outer surface of enamel resistant to demineralization after bacterial acid attack.

Fluorite also acts as a source for fluoride ions. Reversibly bound fluoride to the calcium can be released in saliva to raise salivary fluoride concentration. Fluoride

2- also diffuses to the underlying enamel layers where it replaces the OH, CO 3 or

3- PO 4 ions and forms FAP.

The rate of formation of FAP is dependent on the pH of saliva and concentration of fluoride in the saliva (McCann, 1953). Apart from the role of fluoride in reducing demineralization by formation of FAP, fluoride also has an antimicrobial effect (Marquis, 1995; Yoshihara et al., 2001). Fluoride slows down plaque metabolism by inhibiting glycolysis. Reduced metabolism decreases reproduction in bacteria which reduces the bacterial count and acid production. To summarize:

• Fluoride forms FAP which is acid-resistant and renders the dental enamel

resistant to demineralization.

• Fluoride promotes remineralization of demineralized tooth enamel by

precipitation of fluorapatite from saliva

• Fluoride inhibits bacterial growth in the plaque and prevents acid attack on

the teeth

1.1.4.2 Enamel fluorosis

Enamel fluorosis is defined by porous and discolored enamel with increased protein content and decreased mineral content (Robinson et al., 2004; Wright et al.,

1996). Matsuo et al demonstrated that fluoride exposure of ameloblasts leads to G protein-mediated disturbance in intracellular transport (Matsuo et al., 1996).

Fluoride treatment of the ameloblasts altered the composition and rate of synthesis of enamel proteins (Lyaruu et al., 1986). Bronckers et al demonstrated that fluoride treatment did not alter amelogenin synthesis by the ameloblasts however it altered the secretion of amelogenin (Bronckers et al., 1984). Demonstration of increased number of large vacuoles in the cytoplasm of the secretory ameloblasts after fluoride treatment provides further evidence of fluoride-mediated decrease in the vesicular fusion and secretion of the proteins (Mornstad and Hammarstrom, 1978). Similarly in rat ameloblasts, fluoride treatment led to an increased accumulation of secretory vesicles suggestive of derangement in protein trafficking due to fluoride (Matsuo et al., 1996). Guanosine triphosphate binding proteins are important for vesicular transport (Hall, 1987). On the golgi bodies the secretory proteins vesicles receives their coat protein complex. This process is regulated by the guanosine triphosphate binding proteins (Finazzi et al., 1994). In ameloblasts too, the guanosine

18 triphosphate binding proteins (such as heterotrimentric G proteins) regulates vesicle formation in the golgi complex (Leyte et al., 1992).

1.1.5 Interactions on bone

1.1.5.1 Bone remodeling, osteoblasts and osteoclasts

Bone remodeling is a process that involves a balance between osteoclast and osteoblast activity. Osteoblasts increase bone mass and osteoclasts resorb bone.

This fine balance between the osteoclasts and osteoblasts activity is essential to maintain bone homeostasis. Fluoride actions on bone are influenced by the concentration of fluoride. Physiological concentrations of fluoride promote osteoblast proliferation and increased matric production. Fluoride concentrations higher than physiological concentrations suppresses proliferation by inducing apoptosis of differentiated osteoblast (Allolio and Lehmann, 1999). The effects of fluoride on the osteoclasts are contradictory. Rodent bone marrow osteoclast precursor cells demonstrated increased tartrate-resistant acid phosphatase activity after fluoride treatment suggestive of increased fluoride-mediated osteoclast number and activity

(Debinski and Nowicka, 2004; Yan et al., 2007). Contrary to this finding, another study demonstrated a fluoride-mediated reduction in osteoclast number and bone resorption (Okuda et al., 1990). The underlying molecular mechanism for fluoride’s action on osteoclasts is not clearly understood.

Multinucleated osteoclasts are derived from hematopoietic progenitor cells.

Osteoclasts are terminally differentiated cells and are responsible for bone resorption. Osteoclasts are generated following mechanical response and the

19 number of differentiated osteoclast are very few long the bone surfaces. (Tanaka et al., 2006; Yavropoulou and Yovos, 2008). Osteoclasts can be induced from bone marrow macrophages (BMM) using two cytokines: receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)

(Boyle et al., 2003). Osteoclast precursors fuse together to form multinucleated, terminally differentiated osteoclast. NFATc1 upregulates DC-STAMP to stimulate osteoclast precursor fusion (Kim et al., 2008) which triggers formation of functional osteoclast. Functional, multinucleated osteoclasts attaches to the bone and forms a tight seal underneath which hydrogen ions are pumped to create an acidic environment for bone resorption (Song et al., 2009). Acidic environment help osteoclasts to degrade the inorganic bone matrix. Osteoclasts also secrete matrix metalloproteinase (MMP9) which is involved in the degradation of organic matrix

(Kim et al., 2008; Song et al., 2009).

Fluoride effect was studied on intracellular second messengers (calcium concentrations) in osteoblast-like cell line (Mertz, 1981). Fluoride increased intracellular calcium transiently independent of external calcium and the magnitude is dependent on culture dentistry (Kawase et al., 1988). Fluoride also increased cytosolic free calcium concentrations in hepatocytes (Blackmore and Exton, 1986), platelets (Poll et al., 1986) and neutrophils (Strnad and Wong, 1985), and thereby induced differentiated functions. It is interesting to know that, PTH and 1,25 hydroxyvitamin D3 (1,25 (OH)2D3) are also known to induce intracellular calcium mobilization and alter ALP activity (Boland et al., 1986; Yamaguchi et al., 1987).

Farley et al reported that micromolar concentration of sodium fluoride enhances

DNA synthesis and ALP activity in osteoblasts (Farley et al., 1983). Fluoride also demonstrated an increase in patients with osteoporosis (Riggs et al., 1980).

Fluoride action induced cell proliferation in bone cells at a concentration of 10

M. This concentration is comparable to the serum fluoride concentration in humans

(Farley et al., 1983). Mitogenic effects of fluoride on bone cells from other species including human was confirmed by various researches (Burgener et al., 1995; Hall,

1987; Kassem et al., 1993; Khokher and Dandona, 1990; Modrowski et al., 1992;

Reed et al., 1993; Wergedal et al., 1988).

There is now compelling evidence that fluoride at mitogenic (µM) concenrations stimulates mature osteoblast activities such as alkaline phosphatase expression

(Khokher and Dandona, 1990; Wergedal et al., 1988), collagen synthesis (Kassem et al., 1993; Modrowski et al., 1992) and osteocalcin production (Kassem et al.,

1994) in monolayer cultures. Fluoride at mitogenic concentrations also stimulated calcium uptake (Farley et al., 1983; Zerwekh et al., 1990) and sodium-dependent phosphate transport (Selz et al., 1991) in osteoblasts. Therefore, it is widely accepted that at low doses, fluoride stimulates osteoblast proliferation leading to increased bone formation.

1.1.5.2 Characteristics of fluoride’s actions and hypotheses

Mitogenic action of fluoride on the cells is seen at very low doses (µM). In the osteoblasts, the mitogenic actions of fluoride are reproducibly seen in an in vitro environment (Farley et al., 1983; Wergedal et al., 1988). The mitogenic actions of fluoride on the bone cells are typically evident in present of growth factors (Farley et

21 al., 1988; Reed et al., 1993). Phosphate concentration in the media used for in vitro experiments often influence the fluoride sensitivity of the cells to induce mitogenic changes (Farley et al., 1988). Fluoride acts on various cell types, however the effects of fluoride on the osteoblast precursor cells are more pronounced (Bellows et al., 1990; Farley et al., 1990). Fluoride’s mitogenic actions on the osteoblasts are commonly mediated through increased activation of various signaling proteins, including mitogen-activated protein kinase (MAPK) (Lau et al., 1989; Thomas et al.,

1996; Wu et al., 1997)

Fluoride is interestingly known to skew the balance between bone formation and resorption towards the bone formation. This overall shift results in increased bone deposition resulting in overall increase in bone mass. Several laboratories around the world are trying to uncover the mechanistic aspect of fluoride’s action on cell types involved in biomineralization especially osteoblast cells. The understanding the exact mechanism of fluoride’s action on bone formative cell, osteoblast, will further our understanding of the pathophysiology of degenerative bone diseases and thereby help development of efficient therapeutic strategies. Based on some literature findings, researchers have proposed two possible mechanism of fluoride’s action on bone cells. These two hypotheses are presented below.

1.1.5.2.1 Tyrosine phosphate hypothesis

High acid phosphatase activity on phosphotyrosine was noted in osteoblast-like cells after fluoride treatment. The acid phosphatase activity on phosphoserine and phosphothreonine after fluoride was relatively unaffected (Lau et al., 1989; Lau and

Baylink, 1998). These findings suggest that the effects of fluoride are mediated through the tyrosine phosphate pathway. Proteins harvested from chicken calvarial cells, which were exposed to fluoride, demonstrated increased amount of phosphorylated plasma proteins (Lau et al., 1989). We know that the effects of most growth factors are mediated through tyrosine kinase receptors present on the cell surface. Fluoride sensitizes the cells to readily respond to mitogenic actions of growth factors (Lau et al., 1987). These observations led to the formulation of this first hypothesis for the mechanism of fluoride’s action.

Cells derived from human osteosarcoma demonstrated an increase in amount of tyrosyl phosphorylated proteins in response to low doses of fluoride (~50-200 µM).

Increasing the fluoride concentration further increased the amount of phosphorylated proteins such as ERK 1/2 (Thomas et al., 1996). Similar response to fluoride was seen in the cells isolated from human mandibular bone chips (Thomas et al.,

1996). The phosphorylation events were observed after a relatively long time which might be suggestive of a fluoride-mediated inhibition of phosphotyrosyl phosphatases. Although various literature findings support this hypothesis further studies are required to conclusively prove this hypothesis.

Figure 1.1: Fluoride action hypotheses

Fluoride inhibits the tyrosine phosphatase leading to increased phosphorylation of signaling proteins such as ERK 1/2 triggering proliferation (A) (Lau et al., 1989; Thomas et al., 1996). Fluoride along with other multivalent ions stimulates GDP to convert into GTP which is required for the activation of G proteins which in turn activates and phosphorylated signaling proteins including ERK 1/2 which are required for cell proliferation (B) (Caverzasio et al., 1996; Caverzasio et al., 1997).

1.1.5.2.2 G protein hypothesis

In myocytes, fluoride as fluoroalumino complexes can activate ERK2 kinase, an enzyme involved in cell differentiation and proliferation (Anderson et al., 1991).

Fluoride, in presence of multivalent ions such as aluminum, induced cell proliferation in osteoblast live cells, MC3T3-E1 (Caverzasio et al., 1996).

Heterotrimeric G proteins (Gi and G q), triggers cell proliferation is widely accepted. However, the mechanism underlying this triggering of cell proliferation is not clear (Post and Brown, 1996; van Biesen et al., 1996). The involvement of Ras pathways leading to ERK phosphorylation by G protein has been studied in the past

(Post and Brown, 1996) along with the involvement of the Grb2 and Sos (Post and

Brown, 1996; van Biesen et al., 1996). Similar to the first hypothesis, this second hypothesis needs additional experimental evidence is required to understand the fluoride’s mode of action (van Biesen et al., 1996).

1.1.5.5 Skeletal fluorosis

Fluoride is predominantly stored in the mineralized tissues of the body such as enamel, dentin, and bones. In the bone fluoride demonstrate a biphasic actions

(Collier, 1980). Fluoride, at low doses, provides hardness to the bone and renders enamel less susceptible to acid dissolution (Parnell et al., 2009). However, excess systemic exposure to fluoride can lead to skeletal and dental fluorosis. Skeletal fluorosis (SF) is a bone condition characterized by spectrum of bone changes ranging from osteoporosis to osteosclerosis. SF is clinically characterized by stiffness and rigidity of bone, restricted joint movements, bending deformities of spine and rotational deformities of long bones and calcification of ligaments and tendons (Ismail and Hasson, 2008). Fluoride reduces mineral solubility in the bone thereby affecting calcium metabolism in the body (Messer et al., 1973).

Bone histomorphometric studies demonstrated that fluoride exposure lead to increased bone mass. Additionally, the number of functional osteoblasts was higher

25 after fluoride exposure. Fluoride-mediated proliferation of bone forming cells is indicative of anabolic effect of fluoride on the bone. It was previously speculated that fluoride renders bone hard and resistant to resorption. The increase in bone mass after fluoride exposure is attributed to excessive bone formation rather than decreased bone resorption (Briancon and Meunier, 1981; Riggs et al., 1980).

Although fluoride exposure increased bone mass and bone mineral density, it did not reduce the incidence of fractures undermining the effectiveness of fluoride treatment

(Kleerekoper et al., 1991; Pak et al., 1994; Riggs et al., 1990; Riggs et al., 1994).

Thus effectiveness of fluoride as a therapeutic agent can be enhanced if the benefit- to-risk profile of fluoride is improved. This improvement in benefit-to-risk profile can be achieved by understanding the mechanism of fluoride’s action.

SF has been extensively studied in fluoride endemic areas (Fig. 1.2) of the world.

Most of the SF related data comes from Brazil, China and India where levels of fluoride in drinking water are higher than optimal, 1.5 parts per million (ppm). SF features can be broadly categorized as: generalized osteosclerosis and fluorotoxic metabolic bone disease (FMBD). Both these clinical pictures are based on the amount of dietary intake of calcium. Children demonstrating low intake of calcium, show FMBD with significant serum biochemical abnormalities. Radiographs of patients, living in an area of China where fluoride in drinking water was >5ppm, were studied for SF. Radiographs of the patients were screened for features such as, osteosclerosis (increased bone density with cortical thickening), osteoporosis

(decreased bone density, thinned cortices and decreased number of trabaculae

26 without bone deformity), osteomalacia (osteoporosis with bone deformity), intermittent growth lines and diaphyseal widening and soft tissue calcification.

Figure 1.2: Ground water fluoride concentration around the world.

Reprinted with permission from Amini M, Mueller K, Abbaspour KC, Rosenberg T, Afyuni M, Møller KN, Sarr M, Johnson CA., Statistical modeling of global geogenic fluoride contamination in groundwaters., Environ Sci Technol . 2008 May 15;42(10):3662-8. Copyright 2008 American Chemical society.

It was observed that approximately half of the SF patients demonstrated osteosclerosis and rest demonstrated osteoporosis or osteomalacia (Wang et al.,

1994). Also, intermittent growth lines, diaphyseal widening and calcification of tendons and ligaments were seen as additional overlapping features. It was concluded that radiographic features of SF have a wide spectrum of radiographic characteristics. In another study undertaken in fluoride endemic parts of India with

>5ppm fluoride concentration in drinking water, variable radiographic features were reported in children affected with skeletal fluorosis.

Clinical findings of SF (Fig. 1.3) includes stiffness and rigidity of spine and joints, flexional deformities of bones, rotational deformities of legs, neurological complications, arthralgia, pseudo-hyperparathyroidism and restricted movements

(Teotia et al., 1998). Teotia et al also found that pseudo-hyperparathyroidism was a common finding in patients with SF (Teotia and Teotia, 1973). Another study undertaken in Turkish population indicated that PTH was elevated in SF patients along with a decrease in serum calcium (Koroglu et al., 2011). Harinarayan et al reported that in FMBD patients demonstrated serum phosphorus and 1,25(OH)2D3 values comparable to the control group. Serum alkaline phosphatase (ALP) and

PTH mid-molecule (PTH-MM) levels were higher (Harinarayan et al., 2006).

Additionally, serum calcium, creatinine clearance and phosphorus excretion index were decreased. It is interesting to note that although, the patients in this study demonstrated similar clinical picture, they were exposed to variable amount of fluoride through drinking water, ranging from 0.23-14ppm (Harinarayan et al., 2006).

Collectively, the findings can be summarized as:

• SF demonstrates variable radiographic and clinical picture

• Persistent elevation of PTH in fluoride endemic areas

• Pseudo-hyperparathyroidism is a common finding in fluoride endemic area

• Patients demonstrating similar clinical and radiographic picture were exposed

to variable fluoride levels

Figure 1.3: Clinical and radiographic features of skeletal fluorosis Reprinted with permission from : 1. Christie DP., The spectrum of radiographic bone changes in children with fluorosis. , Radiology. 1980 Jul;136(1):85 -90. Copyright 1980 Radiological Society of North America. 2. Whyte MP, Totty WG, Lim VT, Whitford GM., Skeletal fluorosis from instant t ea., J Bone Miner Res. 2008 May;23(5):759-69. Copyright 2008 American Society of Bone and Mineral Research.

1.1.5.6 Fluoride and parathyroid h ormone (PTH)

Yan et al in 2007 (Yan et al., 2007 ; Yan et al., 2011) studied the effect of fluoride on bone homeostasis in two strains of mice: C3H/HeJ (C3H) and C57BL/6J (B6).

These mice are extensively studied for their bone and bone cell properties. C3H has high bone mass, peak bone density, serum alkaline phosphatase activity and lower bone resorption rate as compared to B6 (Dimai et al., 1998; Judex et al., 2004 ;

Linkhart et al., 1999; Turner et al., 2000 ; Turner et al., 2001). Young C3H and B6 fe male mice were exposed to fluoride for 3 weeks. Bone fluoride content, serum

29 osteoclast biomarkers, in situ osteoclast numbers, osteoclast potential and hematopoietic colony forming cell assay, micro-computed tomography (µCT) analysis and biomechanical properties of bone were studied. After 3 weeks of fluoride exposure, C3H and B6 mice demonstrated variable effects. B6 mice began with higher bone fluoride and accumulated more fluoride in bone as compared to

C3H. Anabolic actions of fluoride were favored in B6, including increase in serum

ALP activity, increase in mineralized bone volume and trabacular thickness.

However, in C3H, fluoride favored osteoclastogenesis demonstrated by an increase in bone marrow osteoclast potential and increase in osteoclast number along with an increase in serum osteoclast biomarkers including intact PTH (iPTH), sRANKL,

TRAP5b and decrease in OPG. Similar results were found when the same strains of mice were exposed to long term fluoride exposure (Yan et al., 2011). Interesting finding of both the studies was elevated iPTH which was consistent with findings in fluoride endemic areas (Harinarayan et al., 2006; Teotia et al., 1998; Teotia and

Teotia, 1973).

1.2 Parathyroid hormone (PTH)

1.2.1 Mineral homeostasis and bone-intestine-parathyroid-kidney (BPK) axis

Calcium is required in the body for proper maintenance of neuromuscular function and hard tissue mineralization. The levels of the calcium in the body are tightly regulated. Parathyroid gland Chief cells are sensitive to the changes in the level of ionized calcium in the body which is maintained at 1.2-1.3mM. Even a slight

30 change in the ionized calcium levels triggers secretion of PTH by the Chief cells.

PTH actions are required to restore the normocalcemic state in the body. PTH normalizes plasma ionic calcium levels by three defined actions:

1. PTH mediates production of osteoclast in the bone which resorbs bone and

release calcium in the blood

2. PTH acts on kidney to induce calcium reabsorption

3. PTH has an indirect action on intestine. In the kidneys PTH signals to

increase 1 αhydroxylase production which promotes calcium absorption in the

intestine.

FGF23 and 1,25(OH)2D3 are involved in feedback regulation to check levels of

PTH. The interaction of these three hormones is required for the calcium homeostasis. The cross-talk between these three tissues to achieve calcium homeostasis has been traditionally referred as bone-parathyroid-kidney (BPK) axis.

However, the indirect involvement of intestine in the absorption of calcium is crucial for successful calcium regulation. Hence we now refer this control mechanism for calcium homeostasis as the BPK as bone-intestine-parathyroid-kidney (BIPK) axis

(Fig. 1.4)

Blood calcium levels are constantly monitored by a membrane bound G protein- coupled receptor, calcium sensing regulator (CASR), present on the parathyroid gland chief cells, bone and kidneys. Chief cells of parathyroid gland produce and store PTH in vesicles for quick release in case of demand. PTH acts on kidney through its receptor PTH1R to modulate production of 1α hydroxylase which produce

1,25(OH)2D3 to help calcium absorption in the intestine. Decreased 1,25(OH)2D3

31 production is sensed by vitamin D receptor (VDR) on parathyroid gland which signals production of PTH.

Action of PTH on kidneys leads to calcium reabsorption and phosphorus excretion through Solute carrier family 34 member 1 (SLC34A1), Solute carrier family 9 member 3 regulator 1 (SLC9A3R1), PDZ domain containing 1 (PDZK1), and chloride channel 5 (CLCN5). PTH production is also influenced by FGF23, a hormone produced by bone osteocytes and is responsible for phosphate regulation.

FGF23 acts on parathyroid gland through its receptor couple, αKLOTHO and

FGF1Rc, to suppress PTH production. FGF23 also acts on kidneys via its receptor couple to induce phosphorus excretion. Intestine plays a vital role in maintaining mineral absorption and hence mineral homeostasis is influenced by interaction of bone, parathyroid, intestine and kidneys. Therefore, it will be appropriate to call BPK axis as bone-intestine-parathyroid-kidney (BIPK) axis.

Figure 1.4: Bone-Parathyroid-Kidney (BPK) axis (Adapted from Donate-Correa et al., 2012; Landry et al., 2011; Razzaque, 2009; Strom and Juppner, 2008)

1.2.2 PTH and PTHLH

Full length PTH polypeptide has 84 amino acid with 9.4kDa molecular mass.

PTH is secreted by the Chief cells of the parathyroid gland. Developmentally, parathyroid tissues can get entrapped in the thymus in which case the thymic tissue can be a source of PTH (Gunther et al., 2000). Evolutionarily, two primitive forms of

PTH are seen in fishes which even lack an identifiable parathyroid gland tissue.

(Gensure et al., 2004; Hogan et al., 2005). In spite of the sequence similarity, the functional potency of these primitive forms of PTH is lower than human PTH.

Therefore, similar to human PTH, these primitive forms of PTH might be involved in the development of skeletal and neural systems in these fishes (Guerreiro et al.,

2007).

The sequence of parathyroid hormone like hormone (PTHLH) in the amino terminal region (1-34) is homologous to PTH. PTHLH is required for endochondral bone formation and also mediates hypercalcemia in malignancy (Broadus et al.,

1988; Strewler et al., 1987; Suva et al., 1987). PTHLH is necessary for development of teeth and mammary glands due to its involvement in epithelial-mesenchymal interactions (Gensure et al., 2005). Due to the sequence similarity between PTH and

PTHLH, both these may have a common ancestral origin (Abbink and Flik, 2007).

1.2.2.1 Synthesis and secretion of PTH

In humans, the PTH gene is present on the short arm of chromosome 11

(11p15.3-p15.1). A 25 amino acid presequence and a 6 amino acid prosequence is present in the precursor PTH molecule along with an 84 amino acid full length mature PTH sequence (Usdin et al., 2008). The presequence is required for the shuttling of the precursor PTH polypeptide across the endoplasmic reticulum (ER).

The prosequence function is not clearly defined yet; however, the involvement prosequence in precursor PTH polypeptide folding has been postulated. (Wiren et al., 1989). After the movement of precursor PTH polypeptide through ER and subsequent folding events, mature PTH is packed into secretory vesicles. The mature PTH is then secreted in response to hypocalcaemia after vesicle fusion and exocytosis. PTH in the vesicle can have an alternative fate. In case of

34 hypercalcemia, calcium-sensitive proteases cleave PTH inside the secretory vesicles leading to the production of small PTH fragments which are devoid of classical PTH response. (Habener et al., 1975). PTH can be removed from the circulation after secretion through its cleavage in the kidneys and the liver (D'Amour,

2006). Peripheral clearance of PTH by cleavage is required for abolishing the prolonged activity of PTH in the body. Peripheral clearance ensures that the PTH response is transient. The amino- and carboxyl-terminal fragments, generated after cleavage, may have distinct biologic roles (Murray et al., 2005).

1.2.2.2 Regulation of PTH secretion

1.2.2.2.1 Extracellular calcium-mediated regulation

The primary function of the parathyroid gland Chief cells is to secrete PTH, a calcitropic hormone. Calcium sensing receptors (CASR) present on the Chief cells of parathyroid gland senses ionized calcium in the blood. Based on the blood ionized calcium levels, Chief cells modify PTH secretion. Increased ionized calcium levels lead to decreased PTH secretion, decreased ionized calcium levels increases PTH secretion. The response of the parathyroid gland chief cells to blood ionized calcium level is steep sigmoidal. Minor fluctuations in the blood ionized calcium levels can trigger appreciable changes in PTH secretion by the chief cells (Potts, 2005).

Blood ionized calcium levels influences PTH secretion by the parathyroid Chief cells. Decrease in the blood ionized calcium levels increases parathyroid chief cell number (Proliferation) and thereby increase the expression of PTH . Increase in the levels of ionized calcium in the blood can reduce PTH secretion by two underlying

35 mechanisms. When there is an increase in the blood ionized calcium levels the proteases in the PTH vesicles are activated which then cleaves the PTH and generates functionally inactive small size PTH fragments. Similarly, when there is an increase in the blood ionized calcium levels it leads to the inhibition of PTH vesicle fusion with the parathyroid Chief cell membrane leading to a decrease in PTH secretion. (Potts, 2005). The sensitivity of parathyroid Chief cells is achieved by a unique calcium sensing receptors, CASRs, present in the cell membrane. CASR have high affinity for ionized calcium. CASR dimer can bind to ionized calcium (at mM concentration) which leads to the receptor (CASR) activation (Ward et al.,

1998).

The essential role of the CASR in humans was witnessed in loss-of-function mutations in the CASR gene (Brown and MacLeod, 2001). Heterozygous mutations of CASR results in familial hypocalciuric hypercalcemia (FHH), which clinically demonstrate increased circulatory levels of PTH along with hypercalcemia. FHH patients also demonstrate reduced number of CASR on the parathyroid Chief cells.

Hence, due to absence of functional CASRs on the parathyroid Chief cells, calcium supplementation does not reduce PTH levels in the individuals with FHH.

Homozygous mutations of CASR lead to neonatal severe primary hyperparathyroidism (NSHPT) which is characterized by abnormally high PTH levels and severe hypercalcemia (Grant et al., 2012). Heterozygous and homozygous mutation of Casr in mice demonstrates comparable clinical picture as seen in heterozygous and homozygous CASR mutations in humans respectively. (Liu et al.,

2011).

Alternative form of CASR is expressed in the bone, cartilage, kidneys, intestine and thyroid gland parafollicular cells. (Brown and MacLeod, 2001). Increase in blood ionized calcium leads to activation of CASR in the parathyroid Chief cells which inhibits PTH release. In the kidneys, activation of CASR leads to inhibition of calcium reabsorption. Thus blood ionized calcium can individually regulate functions of these two tissues to achieve normal blood ionized calcium level. (Grant et al., 2012).

1.2.2.2.2 1,25(OH)2D3-mediated regulation

From the BIPK axis it is clear that 1,25(OH)2D3 negatively regulates PTH levels.

Hence deficiency of 1,25(OH)2D3 can lead to PTH overproduction (Metzger et al.,

2013). 1,25(OH)2D3 binds to the vitamin D receptor (VDR) present of the parathyroid Chief cells. VDR is associated with a transcriptional repressor of PTH

(Ritter and Brown, 2011; Steingrimsdottir et al., 2005). Therefore, 1,25(OH)2D3 binding to the VDR can down-regulate PTH expression. VDR binding of

1,25(OH)2D3 can also mediate inhibition of proliferation of the parathyroid Chief cells which can lead to reduced PTH secretion.

1.2.2.2.3 Phosphorous- FGF23-KLOTHO-mediated regulation

Parathyroid hyperplasia and resulting hyperparathyroidism can lead to chronic kidney failure which is characterized by increased levels of plasma phosphorus

(hyperphosphatemia) (Cozzolino et al., 2005). Hyperphosphatemia leads to binding of plasma phosphorus to calcium which ultimately leads to decreased blood ionized calcium levels and triggers PTH secretion. Fibroblast growth factor 23 (FGF23) is

37 secreted by osteocytes in response to hyperphosphatemia. FGF23 acts on the kidney to inhibit reabsorption of phosphorus. FGF23 binds to its receptor couple

αKLOTHO and FGF1Rc (Hu et al., 2013). Interestingly, αKLOTHO and FGF1Rc are also expressed in parathyroid Chief cells. FGF23 acts through αKLOTHO and

FGF1Rc on the parathyroid Chief cells to inhibit PTH secretion (Silver and Naveh-

Many, 2012). The integrated actions of phosphorous, FGF23, αKLOTHO, and

FGF1Rc on the parathyroid gland Chief cells and kidney form an important physiological mechanism for calcium-phosphorous regulation and PTH regulation.

1.2.2.3 Metabolism of PTH

After secretion, PTH is quickly removed from the circulation by peripheral clearance. PTH has a very short half-time of approximately 4-8 minutes (Koshikawa et al., 2010). Peripheral clearance of PTH is achieved in liver (60-70%) and kidneys

(20-30%) (D'Amour et al., 2005; D'Amour, 2006). Hepatic clearance of PTH is mediated by the Kupffer cells through rapid and extensive cleavage and proteolysis

(D'Amour et al., 1996). Renal clearance is achieved by reabsorption of PTH from the urine followed by PTH cleavage in the renal tubules. The small degraded fragments of the PTH appears in the urine ultra-filtrate (Hruska et al., 1981). Rapid renal and hepatic clearance of PTH ensures that the available PTH in the receptor proximity is exclusively determined by the PTH secretory rate.

Metabolism of PTH also occurs within the parathyroid gland Chief cells. This intra-glandular metabolism involves cleavage in 33-43AA region of PTH generating biologically active N-terminal fragments. PTH proteolysis is regulated to generate N-

38 terminal fragments which might have biological functions (Nguyen-Yamamoto et al.,

2002). Small fraction of carboxyl- and mid-region fragments of degraded PTH, which lack classic PTH activity, reappears in the blood. Biologically inactive fragments of degraded PTH via intra-glandular mechanism are also released by the parathyroid

Chief cells. The biologic actions of carboxyl- and mid-region fragments of PTH may act on tissues via receptors other than classical PTH receptors (Bringhurst et al.,

1988).

1.2.2.4 PTH- and PTHLH-PTH1R

The endocrine effects of PTH and the paracrine actions of PTHLH are initiated by the same intrinsic plasma membrane receptor, PTH1R receptor (Mannstadt et al.,

1999). The PTH1R activates at least two major second-messenger pathways. PTH stimulates generation of the second messenger cyclic adenosine monophosphate

(cAMP). The components of the PTH/PTHLH signaling pathway include one or more receptors that bind PTH or PTHLH and the G proteins which stimulate the respective enzymes adenylyl cyclase (AC) and phospholipase C (PLC) (Cupp et al., 2013). The

PTH1R receptor is ubiquitously expressed and binds to PTH and PTHLH with equal affinity. On binding of either PTH or PTHLH, the PTH1R receptor activates G protiens leading to AC stimulation and cAMP generation. A second receptor, PTH2R that binds PTH but not PTHLH has already been identified (Rubin et al., 1999) with limited tissue distribution (Della Penna et al., 2003).

1.3 Calcium Sensing Regulator (CASR)

The CASR is a type C G protein-coupled receptor (GPCR) which form homodimers (Ward and Riccardi, 2012). The CASR contains a large N-terminal extracellular calcium-binding domain. The intracellular C-terminal domain is small and associate with signaling proteins. The CASR has a seven transmembrane domain. When calcium binds to CASR it leads to receptor activation and subsequent activation of heterotrimeric G proteins such as Gq (Magno et al., 2011). This results in intracellular calcium increase and cAMP decrease in parathyroid gland Chief cells.

Intracellular calcium release suppressed the PTH vesicle fusion with the parathyroid

Chief cell membrane and thereby decreased PTH secretion. Deletion of G proteins,

Gq and G i, in mice resulted in features similar to the NSHPT, which suggested a possible involvement of these heterotrimeric G proteins in CASR signaling

(Wettschureck et al., 2007).

1.3.1 Isolation of Calcium Sensing Receptor

Full-length bovine CASR was first cloned in Xenopus laevis oocytes (Brown et al., 1993). Human CASR cDNA was isolated from parathyroid gland Chief cells, parafollicular cells of thyroid gland and kidneys using hybridization-based approach

(Ruat et al., 1995). CASR was isolated from rabbit kidneys and chicken parathyroid glands (Brown and MacLeod, 2001). CASRs from various species exhibit sequence similarity suggesting a common ancestral origin for CASR gene. Genes comparable to CASR (~60-70% overlap) have been identified in fishes such as salmon and shark

(Nearing et al., 2002).

CASR exists as a dimer with a disulphide bond linking corresponding cysteine at 129 and 131 position in the extracellular domains. (Ray et al., 1999). Although the receptor is heavily glycosylated, glycosylation is not essential for functionality of

CASR (Brauner-Osborne et al., 1999). Intracellular loops of the seven transmembrane and cytoplasmic-tail of the human CASR have five protein kinase C sites. Phosphorylation at threonine 888 inhibits the CASR-mediated signaling thereby serving a feedback regulation (Davies et al., 2007).

Flask-shaped invaginations of the parathyroid Chief cell membrane harbor functional CASR dimers (Kifor et al., 1998). These invaginations are called,

‘caveolae’. In the caveolae, cholesterol binding protein, caveolin-1, binds to CASR

(Chakravarti et al., 2012). The C-tail of CASR associates with filamin-A, an actin binding protein. Filamin-A acts as a scaffold for other intracellular proteins (Pi et al.,

2002). The CASR’s ability to activate MAPK, ERK1 and ERK2 is dependent on its affinity to bind filamin-A. The resistance of CASR to desensitization on repetitive exposure to calcium is due to its binding to the cytoskeletal filamin-A. This resistance to desensitization of CASR is important for its calcium sensing function in various cell types including parathyroid gland Chief cells.

1.3.2 CASR gene

The human, rat and mouse CASR gene is located on chromosome 3 (3q13.3-

21), 11 and 16 respectively (Brown and MacLeod, 2001). There are seven exons in the CASR gene. The second exon contains the start codon while the last 5 exons code for the full-length CASR . Out of the last five exons, four exon codes for the

41 extracellular domain while the seventh exon codes for the transmembrane and intracellular C-terminus region.

1,25(OH)2D3 mediates upregulation of CASR expression in the parathyroid

Chief cells (Brown and MacLeod, 2001). High extracellular calcium also initiates upregulation of CASR expression in the parathyroid gland Chief cells. The high The upregulation of CASR expression might be responsible for the inhibition of PTH secretion mediated by calcium and 1,25(OH)2D3 (Brown and MacLeod, 2001).

1.3.3 Functions of CASR in tissues

1.3.3.1 Parathyroid gland

The parathyroid glands express highest levels of CASR . CASR stimulates proliferation of parathyroid Chief cells. Humans with homozygous or heterozygous

CASR inactivating mutations exhibit parathyroid hyperplasia. Similarly, mice homozygous for CASR inactivating mutation exhibited marked hyperparathyroidism and parathyroid hyperplasia (Brown and MacLeod, 2001; Liu et al., 2011).

1.3.3.2 Parafollicular cells

The parafollicular cells of thyroid gland secrete calcitonin (CT) in response to increased extracellular calcium. CT is responsible to reduce extracellular calcium through actions on bone, kidney, and intestine. CT mediates decrease in calcium absorption through intestine and inhibits osteoclast activity in bone. In kidneys, CT decreases calcium reabsorption. Extracellular calcium activates CASR in parathyroid gland Chief cells to increase intracellular calcium which inhibits PTH

42 exocytosis (Brown and MacLeod, 2001). In contrast, extracellular calcium-mediated activation of CASR on parafollicular cells stimulates increase in CT exocytosis.

Sequence similarity exist between CASR isolated form humans, rabbit and rat

(Quarles, 2003). CASR in parathyroid gland helps to normalize low ionized calcium in blood; whereas CASR on parafollicular cells helps to normalized increased blood ionized calcium levels. (Fudge and Kovacs, 2004).

1.3.3.3 Kidney

In the kidney, expression of CASR is predominantly seen in the cells of the ascending loop of Henle. The ascending loop of Henle is responsible for the reabsorption of cations under the control of PTH. The CASR is also expressed in the cells of the distal convoluted tubule, which is involved PTH-mediated calcium reabsorption. In the proximal convoluted tubule, the CASR activation inhibits PTH- induced phosphorus wasting and stimulates expression of VDR.

1.3.3.4 Intestine

PTH indirectly stimulate 1,25(OH)2D3 production which leads to increased calcium absorption through the intestine. The duodenum is the primary site for

1,25(OH)2D3-mediated, trans-cellular calcium absorption (Brown and MacLeod,

2001). The human intestine can absorbs high amounts of calcium by 1,25(OH)2D3- dependent and independent pathways.

The CASR regulates secretomotor functions of the GI tract to modulate GI motility. This regulation is seen due to the high or low levels of the calcium.

Activation of the CASR leads to marked reduction in fluid secretion in the colon

(Cheng, 2012).

1.3.3.5 Bone

The concentration of calcium in the bone fluctuates during remodeling.

Calcium underneath resorbing osteoclasts can be as high as 8-40mM (Lam et al.,

2011). High calcium concentrations may activate CASR resulting in increased chemotaxis of pre-osteoblasts to the recent bone resorption site in vitro . High calcium-mediated CASR activation also leads to proliferation of pre-osteoblasts and promotes their differentiation into mature osteoblasts (Sanchez-Fernandez et al.,

2008). Enhanced proliferation and differentiation increase the capacity of mature osteoblasts to mineralize bone proteins. However, these responses due to CASR activation are not documented in vivo .

1.3.4 CASR signaling

In the canonical pathway, high extracellular calcium leads to activation of CASR in bovine parathyroid Chief cells stimulating phosphorylation of PLC, phospholipase

A2 (PLA 2), and phospholipase D (PLD) (Breitwieser, 2013). Phosphorylation of PLC leads to increased production of inositol triphosphate (IP3) which binds to its receptor on the ER and release from the intracellular calcium (Chang et al., 2001).

High calcium also mediates a decrease in cAMP levels in parathyroid Chief cells by inhibiting AC (Shoback and McGhee, 1988). High calcium-mediated CASR

44 stimulation also leads to activation of MAPKs, including ERK1/2 and p38 MAPK

(Magno et al., 2011).

Ubiquitously expressed ( β-arrestin1 and β-arrestin2), bind to phosphorylated

C-terminal of CASR (Fig.1.5), resulting in both an uncoupling of the receptor from its cognate G proteins and intracellular signaling (non-canonical pathway) (Doucette et al., 2009). This β-arrestin recruitment by CASR will be discussed in detail in chapter

Figure 1.5: CASR signaling cascade 2+ PTH: parathyroid hormone, eCa : extracellular calcium, CASR: calcium sensing receptor, G αq11 : heterotrimeric G protein, PLC: phospholipase C, IP3: Inositol triphosphate, DAG: diacyl glycerol, PKC: protein kinase C, iCa 2+ : intracellular calcium, ERK: extracellular signal-regulated kinase, VDR: vitamin D receptor (complete lines: stimulation, dotted lines: inhibition).

1.3.5 CASR agonists

CASR agonists are classified as type I, II and III. Type I agonists are so called since they can stimulate CASR even in absence of calcium, a known ligand. Other

45 than calcium, CASR respond to various di- and tri-valent cations including Mg 2+ , Sr 2+ and Gd 3+ . CASR also interacts with organic polycations, such as spermine (Brown et al., 1993; Brown and MacLeod, 2001). Most of the ligand receptor interactions occur at the extracellular domain (‘venus fly trap’ structure) of the CASR. Ca 2+ , Mg 2+ and spermine, have the potential to be physiologically relevant CASR activators. In the microenvironments of intestine and CNS, where calcium concentrations are comparatively low, spermine levels are sufficiently high to activate the CASR.

Type II agonists of CASR are also known as positive allosteric modulators (PAM) of CASR. Type II CASR agonists such as R567, S567, R568, S568 and Cinacalcet

Hydrochloride are currently being used clinically to treat hyperparathyroidism. These are small hydrophobic molecules, which interact with transmembrane region of the

CASR and increases CASR affinity for calcium. Type II agonists of CASR are also called as calcimimetics since they mimic calcium and binds at a site other than calcium binding site. Calcimimetics activate CASR in presence of calcium and are referred as ‘modular’ rather than as ‘agonists’ (Nemeth et al., 2004). Calcimimetics, such as R568 hydrochloride and Cinacalcet Hydrochloride, decrease PTH synthesis

(Levi et al., 2006) and secretion (Fox et al., 1999; Nemeth et al., 1998; Nemeth et al., 2004), and reduce parathyroid cell proliferation (Colloton et al., 2005; Wada et al., 1997; Wada et al., 2000).

Type III CASR agonists are also called as negative allosteric modulators (NAM) of CASR. These NAM are used in clinical trials for treatment of osteoporosis. NAM reduces CASR sensitivity to calcium and act pharmacologically as antagonists of the

CASR. Hence they are also called ‘calcilytics’ (Nemeth and Shoback, 2013).

CHAPTER 2: SPECIFIC AIMS

Fluoride, due to its anabolic effect on osteoblasts, was once used for treatment of resorptive bone diseases such as osteoporosis (Modrowski et al., 1992; Sogaard et al., 1997). Fluoride is no longer a treatment option for osteoporosis due to inconsistent outcomes. While in many cases bone mass improved there was often lack of concomitant improvement in bone quality. Features of skeletal fluorosis observed in fluoride endemic areas range from osteoporosis, osteomalacia, and osteosclerosis. Pseudo-hyperparathyroidism along with increased serum PTH are common findings in skeletal fluorosis patients (Teotia et al., 1998; Teotia and Teotia,

1973; Wang et al., 1994). Similar findings were observed in C3H mice when they were exposed to 3 weeks of fluoride through drinking water. Fluoride exposures in

C3H mice led to increased osteoclastogenesis demonstrated by an increase in osteoclast number and osteoclast potential along with increase in serum osteoclast biomarkers (such as PTH, RANKL, TRAP5b and decreased ALP and OPG) (Yan et al., 2007; Yan et al., 2011). However, B6 mice demonstrated increased ALP activity suggestive of anabolic bone changes. Therefore, C3H and B6 mice may represent the subset of human population demonstrating osteoporosis and osteosclerosis type bone lesion respectively, after fluoride exposure. Effects of fluoride on humans at sub-lethal/ lethal doses have been extensively researched in toxicological studies

(Barbier et al., 2010; Dalamaga et al., 2008). However, the effects of fluoride at optimal/ therapeutic level to induce a catabolic response have not been studied. It is

47 also unclear what role PTH plays in fluoride-induced osteoclastogenesis in C3H mice. Understanding the impact of fluoride on PTH is critical due to the involvement of PTH in mineral homeostasis, which is tightly regulated by BPK axis (Bergwitz and

Juppner, 2010; Lavi-Moshayoff et al., 2010; Torres and De Brauwere, 2011).

Unfolding of the unknown mechanistic aspects of fluoride-mediated PTH modulation will help us to better define treatment strategies and develop drug for treatment of resorptive bone diseases.

To understand the pathophysiology of fluoride-induced osteoclastogenesis we hypothesize that:

Fluoride directly acts on parathyroid gland to modulate PTH secretion.

To address this hypothesis we proposed two specific aims focused to determine the effect of fluoride on PTH secretion and involvement of CASR in fluoride-mediated

PTH modulation.

Sub-hypothesis 1: Fluoride leads to increased PTH secretion .

Specific aim 1A: Determine a direct effect of fluoride exposure on PTH secretion using mice thyro-parathyroid gland organ culture (in vitro ). C3H and

B6 mice thyro-parathyroid gland organ culture were treated with fluoride. PTH levels in media supernatants in the fluoride treated samples were compared to untreated samples to determine the effect of fluoride on PTH secretion.

Specific aim 1B: Investigate PTH secretion in sera of C3H and B6 mice after single fluoride dose via oro-gastric gavage. C3H and B6 mice were given a single gavage dose via oro-gastric route with fluoride or distilled water and serum fluoride, calcium, phosphorus, magnesium, and PTH levels were measured at serial time points. Expression of suite of genes involved in BPK axis was determined to understand the effect of fluoride on PTH secretion.

Sub-hypothesis 2: Effect of fluoride on PTH secretion is mediated through

CASR.

Specific aim 2A: Determine the effect of fluoride on β-arrestin recruitment in

HTL cells transfected with human CASR cDNA. HTL cells were transfected with human CASR and treated with fluoride to determine arrestin recruitment in the transfected cells using a modification of Tango ® assay ( β-arrestin recruitment assay) to identify fluoride-mediated CASR activation.

Specific aim 2B: Screen cDNA library of human orphan GPCRs to identify possible fluoride targets other than CASR. HTL cells were transfected with orphan GPCRs from class A, B, or C followed by treatment with fluoride to measure and compare β-arrestin recruitment after fluoride treatment to determine fluoride- mediated receptor activation.

Studies focusing on experiments outlined in specific aim 1 (1A and 1B) and specific aim 2 (2A and 2B) are discussed in detail in Chapter 3 and 4 respectively.

CHAPTER 3: FLUORIDE MODULATES PARATHYROID HORMONE SECRETION

IN VITRO AND IN VIVO

FLUORIDE MODULATES PARATHYROID HORMONE SECRETION IN VIVO AND

IN VITRO

Chaitanya P. Puranik a, Kathleen A. Ryan b, Shijia Hu a, E. Angeles Martinez-Mier c

and Eric T. Everett d a Oral Biology PhD Curriculum, University of North Carolina at Chapel Hill. b Dental Research, University of North Carolina at Chapel Hill c Department of Preventive and Community Dentistry, Indiana University School of

Dentistry d Department of Pediatric Dentistry, Carolina Center for Genome Sciences,

University of North Carolina at Chapel Hill.

3.1 Abstract

Systemic fluoride induces osteoclastogenesis in C3H/HeJ (C3H) mice evidenced by increased serum intact parathyroid hormone (iPTH), soluble receptor activator of nuclear factor kappa-B ligand (sRANKL), and tartrate-resistant acid phosphatase 5b (TRAP5b) along with reduced levels of osteoprotegerin (OPG); whereas, in C57BL/6J (B6) mice fluoride’s anabolic actions are favored (Yan D. et al, Bone 2007). In addition, pseudo-hyperparathyroidism often accompanies endemic skeletal fluorosis in humans. Mechanisms that underlie fluoride’s actions on the parathyroid gland are not well understood. The objective of this study was to investigate fluoride’s effects on iPTH secretion . Thryo-parathyroid complexes (TPCs) from 5-6 week old C3H (n=18) and B6 (n=18) male mice were dissected, dispersed and cultured in medium pre-optimized for strain specific calcium, phosphorus, and magnesium serum levels. TPCs were treated with 0, 250 or 500µM sodium fluoride for 24hrs and secreted intact parathyroid hormone (iPTH) in the media assayed by

ELISA. 5-6 weeks old C3H (n=78) and B6 (n=78) male mice were gavaged once with distilled or with fluoride (0.001mg [F -]/g body weight) water. At serial time points

(0.5, 1, 3, 6, 12 or 24hrs) serum iPTH, fluoride, total calcium, phosphorus and magnesium levels were determined. Expression of genes involved in mineral regulation via bone-parathyroid-kidney (BPK) axis such as: Pth, Casr, Vdr, Pthlh,

Fgf23, αKlotho, Fgf1rc, Tnfs11, Pth1r, Slc34a1, Slc9a3r1, Clcn5 and Pdzk1 genes were determined in TPCs, hummerii and kidneys at 24hrs in both strains. A dose dependent decrease in iPTH was seen in vitro for C3H ( p<0.005) and B6 ( p<0.005) at 500µM. Single gavage led to an increase in serum fluoride peaking at 0.5hr. iPTH

52 also peaked at 0.5hr in both C3H ( p=0.002) and B6 ( p=0.01) followed by a decrease in C3H at 24hrs ( p=0.002) returning to baseline at 48hrs. At 12hrs there was an increase in iPTH in B6 ( p<0.001) which returned to baseline at 24hrs. No significant changes in serum total calcium, phosphorus and magnesium were observed. We conclude that fluoride modulates iPTH secretion in vitro and in vivo . While fluoride’s effects on TPC, in vitro , were equivalent between the two mouse strains, fluoride demonstrated an early strain dependent effect on iPTH secretion in vivo . C3H and

B6 mice demonstrated a difference in the expression of genes involved in BPK axis suggesting an underlying difference in physiologic handling of fluoride by the two strains.

3.2 Introduction

Fluoride has recognized actions on bone homeostasis (Everett, 2011). Yan et al investigated in two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), fluoride actions on bone homeostasis (Yan et al., 2007). Short term systemic exposure to fluoride for C3H resulted in increased osteoclastogenesis as evidenced by increases in serum osteoclast biomarkers: intact parathyroid hormone (iPTH), soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) and tartrate- resistant acid phosphatase 5b (TRAP5b) along with decrease in serum osteoprotegerin (OPG) levels (Yan et al., 2007). Osteoclast numbers along bone surfaces and osteoclast potential of bone marrow cells were increased in C3H as well. However, similar fluoride exposure in B6 mice favored anabolic responses with increases in serum alkaline phosphatase (ALP) activity, proximal tibia trabecular and vertebral bone mineral density.

PTH is responsible for calcium homeostasis and is released in response to hypocalcemia to normalize serum calcium levels (Potts, 2005). PTH is an important component of the bone-parathyroid-kidney (BPK) feedback loop responsible for mineral homeostasis (Bergwitz and Juppner, 2010; Torres and De Brauwere, 2011).

Skeletal fluorosis results from systemic exposures to fluoride leading to calcification of ligaments, bone deformities, fractures, functional limitations, disturbed mineral balance and occasional pseudo-hyperparathyroidism (Teotia et al., 1998; Teotia and

Teotia, 1973). The mechanisms underlying altered PTH levels due to fluoride exposure are not understood. We hypothesize that PTH secretion is influenced by direct effects of fluoride on the parathyroid gland. Our in vitro and in vivo

54 experiments were directed to investigate early events following fluoride exposure on

PTH secretion.

3.3 Materials and Methods

3.3.1 Animals

Ninety-six, 5-6 week old male C3H/HeJ (C3H) and C57BL/6J (B6) mice (The

Jackson Laboratory, Bar Harbor, ME, USA) were placed on a constant nutrition no fluoride diet #5861 (Test Diet®, Richmond, IN, USA; fluoride: 0ppm, calcium: 0.60%, phosphorus: 0.57%, Vit D3: 2.2 IU/g, and Energy: 4.09 kcal/g) and provided distilled drinking water, ad libitum, for one week to acclimatize. Mice were housed in The

Division of Lab Animal Medicine facility at The University of North Carolina at Chapel

Hill, an AAALAC accredited unit. Mice were placed in 12:12 light and dark cycles at

21 oC ambient temperature. All experimental procedures were approved by the

Institutional Animal Care and Use Committee at The University of North Carolina at

Chapel Hill.

3.3.2 Parathyroid gland organ culture

Mice were euthanized by CO 2 and ventral neck dissection was performed to isolate and remove thyro-parathyroid complex (TPC). TPCs were finely minced and transferred to 2ml centrifuge tube with digestion buffer containing 1mg/mL of collagenase (Stem Cell Technologies, Vancouver, BC, Canada) in dispase solution

(Stem Cell Technologies). TPC were incubated at 37 oC for 3hrs with intermittent pipetting for homogeneous dispersion of cells. Dispersed cells were incubated at

0 37 C at 5% CO 2 in 2ml pre-warmed alpha MEM (Sigma Aldrich, St. Louis, MO, USA) media with 10% FBS (Clontech Laboratories, Mountain View, CA, USA) and 1% antibiotic-antimycotic (Sigma-Aldrich, St. Louis, MO, USA). Total calcium, magnesium, and phosphorus levels in media were equivalent to that of serum in

56 each strain of mice: C3H (calcium: 10.6mg/dL, phosphorus: 8.7mg/dL, and magnesium: 2.1mg/dL) and B6 (calcium: 10.4mg/dL, phosphorus: 10.3mg/dL, and magnesium: 2.9mg/dL). The TPC from C3H and B6 were randomly divided into 3 groups (n=6) based on fluoride concentrations 0, 250 or 500µM. Media supernatants were collected at 24hrs and stored at -80 oC.

3.3.3 Gavage and sample collections

Following acclimatization, mice from both strains were randomly divided in 6 groups based on time of sacrifice after gavage dose. Mice were further divided into control and test group (n=6) based on fluoride levels (0 or 100ppm) in gavage. Both the groups of mice were sacrificed at 0.5, 1, 3, 6, 12 or 24hrs. At the time of gavage, mice were weighed and the gavage dose was calculated for each mouse (0.001mg

[F -]/g body weight). Mice were anesthetized with ketamine-HCl (90mg/kg) and xylazine (14mg/kg) i.p. 15mins prior to the determined time of sacrifice. Sera were collected from mice and stored at -80 oC until use. TPCs, kidneys, and humerii were harvested, snap-frozen in liquid nitrogen, and stored at -80 oC.

3.3.4 ELISA and biochemistry assays

Media supernatants and sera were analyzed for iPTH using 1-84 PTH ELISA kits (Immutopics, San Clemente, CA, USA). The intra- and inter-assay precision

(coefficient of variation) for PTH ELISA was 3.2 and 8.4, respectively. Serum levels of fluoride were determined using direct fluoride microanalysis method (Vogel et al.,

1990). In this method micropipettes pulled from glass capillary tubes are combined with a custom-made micro-pipette holder and used to dispense nanoliter-sized standards and serum samples (diluted 9:1 with TISAB III). Serum samples are

57 extracted over a light table using the micropipette/micropipette holder and dispensed onto the surface of an inverted fluoride electrode (F-ISE) under a layer of mineral oil to prevent loss of fluoride. Using a micromanipulator and microscope for viewing, the reference electrode is touched to the standard or sample drop, resulting in an electrometer mV reading. Both the F-ISE and hand-pulled reference electrode are connected to an electrometer and computer with plot program monitoring/recording software. Samples are run in triplicate, with periodic electrode conditioning and standard checks. Calcium, phosphorus, and magnesium were determined by the

Animal Clinical Laboratory, University of North Carolina at Chapel Hill and the

Clinical Pathology Laboratory, College of Veterinary Medicine at North Carolina

State University.

3.3.5 RNA extractions and cDNA preparation

RNA was extracted from 24hrs TPCs, kidneys, and humerii samples using

RNeasy Midi kit (Qiagen Inc., Valencia, CA, USA). Quantitative analysis of RNA sample was performed using Agilent Bioanalyzer using Nano Labchip at the

Genomic Core Facility, University of North Carolina at Chapel Hill. All RNA sample had RIN values 7.5-10 range. High Capacity Reverse transcription kit (Applied

Biosciences, Carlsbad, CA, USA) was used to prepare cDNA. After preparation, cDNA was stored at -80oC.

qPCR

cDNA samples free of genomic DNA contamination were subjected to qPCR for suite of genes including parathyroid hormone ( Pth ), calcium sensing receptor ( Casr ), alpha-Klotho ( αKlotho ), fibroblast growth factor receptor 1c ( Fgf1rc), vitamin D

58 receptor ( Vdr ), parathyroid hormone like hormone ( Pthlh ), tumor necrosis factor 11

(Tnfs11 ), fibroblast growth factor 23 ( Fgf23 ), parathyroid hormone receptor 1 ( Pth1r ), chloride channel 5 ( Clcn5 ), Solute carrier 9 member 3 regulator 1 ( Slc9a3r1 ), PDZ domain containing 1 ( Pdzk1 ) and Solute carrier family 34 member 1 ( Slc34a1 ).

Expression of these genes was normalized using two house-keeping genes comprising of beta actin ( Actb ) and glyceraldehyde 3-phosphate dehydrogenase

(Gapdh ). All the primers were ordered from SA Biosciences (Qiagen Inc., Valencia,

CA, USA). Data were analyzed using 2- Ct method as previously described (Livak and Schmittgen, 2001; Yuan et al., 2006).

3.3.6 Statistical analysis

Data were reported as mean ± SE. Statistical analyses were performed using one-way ANOVA followed by Bonferroni as a post hoc test. For the in vitro experiments, six groups were compared based on strain (C3H or B6) and levels of fluoride exposure (0, 250 or 500µM). For the in vivo experiments 28 groups were compared based on strain (C3H or B6), levels of fluoride exposure (0 or 100ppm) and time points (0, 0.5, 1, 3, 6, 12 and 24hrs). SPSS statistics 18 software (SPSS

Inc., Chicago, IL, USA) was used for statistical analysis. Adjusted p-values ≤0.05 were considered significant.

3.4 Results

3.4.1 Responsiveness of TPC organ culture model

The TPC organ culture model was utilized in order to determine the direct effects of fluoride on the parathyroid gland. Responsiveness of the organ culture model to changes in the extracellular calcium, a known PTH mediator was determined , was used to validate the culture model . TPCs cul tured in varying calcium concentrations (hypocalcemic: 1.1mg/dL, normocalcemic: 10.5mg/dL or hypercalcemic: 25mg/dL) demonstrated changes in iPTH secretion into the media .

Figure 3.1: iPTH levels in media supernatant (A) iPTH ± SE (pg/mL) in C3H TPCs at three calcium concentrations; 1.1, 10.5 or 25mg/dL (n=3 in each group). (B) iPTH ± SE (pg/mL) in C3H and B6 TPCs after 24hrs of incubation at three fluoride concentrations; 0, 250 or 500µM (grey bar=C3H and black bar=B6, n= 6 in each group; **p<0.005).

After 24hrs of incubation at hypocalcemic condition, iPTH secretion

(129.43±27.14pg/mL) was significantly increased ( p<0.001) as compared to the normocalcemic condition (49.28±6.21pg/mL) (Fig. 3.1A) . The hypercalcemic conditio ns lead to a modest iPTH decline. Although not shown, TPCs in culture remain responsive to changes in calcium for 3 days.

3.4.2 Effect of fluoride on TPCs

TPCs from B6 and C3H mice were then cultured in media normalized with calcium, phosphorus, and magnesium levels present in the sera for each strain followed by treatment with varying fluoride concentrations (0, 250 or 500µM). Media supernatants were collected at 24hrs and iPTH determined. TPCs from C3H demonstrated a significant (p<0.001) decrease in iPTH with increasing fluoride concentration from 0µM (128.53±10.60pg/mL) to 250µM (86.20±12.47pg/mL, p<0.001) or 500µM (52.33±8.94pg/mL, p<0.001). TPCs from B6 demonstrated less decrease in iPTH with increasing fluoride concentrations from 0µM

(64.37±14.78pg/mL) to 250µM (50.99±12.22pg/mL) or 500µM (38.12±5.19pg/mL) and reaching significance at 500µM fluoride concentration (p<0.001) (Fig. 3.1B).

3.4.3 Serum fluoride levels after gavage

A calculated dose of fluoride was delivered by oro-gastric gavage. Serum fluoride concentrations were measured at serial time points (0.5, 1, 3, 6, 12 or 24hrs) after a single gavage dose. The fluoride levels in the serum peaked in both strains,

C3H (61.05±5.67µM) and B6 (26.74±3.17µM), at 0.5hr (p<0.005) after gavage.

Fluoride levels reached baseline in C3H (8.75±1.76µM) and B6 (3.57±0.42µM) after

6-9hrs (Fig. 3.2). Both the strains, C3H and B6 demonstrated a similar pattern of fluoride kinetics. However, fluoride levels in B6 were lower than C3H at each serial time point ( p<0.005) examined. The peak fluoride concentration in C3H

(61.05±5.67µM) was nearly three times that compared to B6 (26.74±3.17µM).

Figure 3.2: Serum fluoride levels Mean serum fluoride (F) ± SE (µM) after 100ppm fluoride gavage in C3H and B6 mice sacrificed at serial time points; 0.5, 1, 3, 6, 12 or 24hrs compared to baseline (no gavage), (grey bar=C3H and black bar=B6) (n=6 in each group; **p<0.005).

3.4.4 Serum iPTH after single fluoride dose via oro-gastric gavage

Serum iPTH was measured at various time points (0.5, 1, 3, 6, 12 or 24hrs) in

C3H and B6 mice after fluoride gavage (0.001mg [F -]/g body weight) or gavage with an equal volume of 0ppm fluoride water. At 0.5hr after 100ppm fluoride gavage, serum iPTH levels increased significantly for C3H (358.44±19.44pg/mL, p=0.002) and B6 (149.91±21.24pg/mL, p=0.01) as compared to baseline untreated levels

(C3H : 196.15±21.45pg/mL and B6: 114.13±27.43pg/mL) or control levels (0ppm fluoride gavage) (C3H : 244.49±35.52pg/mL and B6 : 98.616±6.45pg/mL ) groups. In the C3H strain iPTH dropped below baseline at 24h rs (82.07±10.71pg/mL , p=0.002)

(Fig. 3.3A); whereas, in B6, iPTH increase d at 12hr (176.99±20.82pg/mL , p<0.001) and then returned to baseline at 2 4hrs (95.65±19.91pg/mL) (Fig. 3. 3B). The peak

62 iPTH value in C3H (358.44±19.44pg/mL ) was twice that of peak iPTH values in B6

(176.99±20.82pg/mL).

Figure 3.3: Serum iPTH levels 0-24 hrs Mean serum iPTH ± SE (pg/mL) after 0 or 100ppm fluoride gavage in C3H (A) and B6 (B) mice sacrificed at serial time points; 0.5, 1, 3, 6, 12 or 24hrs compared to untreated mice (hatched bar=untreated/ baseline group, white bar=0ppm group and black bar=100p pm group) (n=6 in each group; *p<0.05, **p<0.005).

Since th e iPTH levels in C3H dropped below baseline at 24hrs additional time points were collected . The serum iPTH in C3H after single 100ppm fluoride gavage returned to baseline at 48 hrs (Fig. 3.4). These data demonstrated a difference in fluoride’s effects on iPTH secretion in two mice strains; C3H and B6 .

Figure 3.4: Serum iPTH levels 48-96hrs Mean serum iPTH ± SE (pg/mL) after 0 or 100ppm fluoride gavage in C3H harvested at extended time points; 48, 72 or 96hr compared to untreated mice (hatched bar=untreated/ baseline group, white bar=0ppm group and black bar=100ppm group) (n=6 in each group; **p<0.005).

3.4.5 Serum total calcium , phosphorus, and magnesium levels

Serum total calcium levels were relatively consistent between 0 and 100ppm fluoride gavage mice in both C3H (Fig. 3.5A) and B6 mice (Fig. 3.5D) . We observed a significant reduction (C3H: p=0.013 and B6: p=0.001) in serum total calcium only at 0.5hr in 100ppm fluoride gavage group (C3H: 8.7±0.2mg/dL and B6:

8.5±0.2mg/dL) as compared to 0ppm control (C3H: 8.9±0.1mg/dL and B6:

9.5±0.2mg/dL) or untreated baseline group (C3H: 9.3±0.1mg/dL and B6:

9.2±0.2mg/dL).

Variations were relatively common for serum phosphorus levels for both C3H

(Fig. 3.5B) and B6 (Fig. 3.5E) mice. But there were no statistically significant differences between 0 and 100ppm fluoride gavage mice at all observed time points.

Figure 3.5: Serum calcium, phosphorus, and magnesium levels

Mean serum calcium (Ca) ± SE, phosphorus (Pi) ± SE and magnesium (Mg) ± SE (mg/dL) after 0 or 100ppm fluoride gavage in C3H (A, B and C respectively) and B6 (D, E and F respectively) harvested at serial time points; 0.5, 1, 3, 6, 12 or 24hrs (hatched bar=untreated/ baseline group, white bar=0ppm group and black bar=100ppm group) (n=6 in each group; *p<0.05, **p<0.005).

Although serum magnesium levels were relatively stable, minor variations were seen for both C3H (Fig. 3.5C) and B6 (Fig. 3.5F) mice. Similar to phosphorus, no statistically significant differences were observed in magnesium levels between 0 and 100ppm fluoride gavage mice at all observed time points.

3.4.6 qPCR: Expression of genes involved in BPK feedback loop

We investigated the expression of various genes involved in the BPK feedback loop using TPC, humerii and kidneys. Expression of the genes in the tissues obtained from 100ppm fluoride gavage C3H and B6 mice group at 24hrs were normalized with an average of two house-keeping genes ( Actb and Gapdh ).

Gene expression was further normalized to control 0ppm fluoride gavage group and fold difference of genes in TPC (Fig. 3.6A), humerii (Fig. 3.6B) and kidneys (Fig.

3.6C) were calculated for C3H and B6 mice using 2- Ct method. In TPC, Pth

(p=0.001), Casr (p=0.012), αKlotho (p=0.003), Fgf1rc (p=0.035), Vdr (p=0.001) and

Pthlh (p=0.03) were significantly up-regulated in C3H as compared to B6. In humerii, only Pthlh (p=0.001) was significantly up-regulated in C3H as compared to B6 mice.

In kidney, Pth1r (p=0.005), Clcn5 (p=0.003), αKlotho (p=0.05), Fgf1rc (p=0.004) and

Slc9a3r1 (p=0.04) were significantly down-regulated in C3H as compared to B6.

Figure 3.6: Expression of genes involved in BPK feedback loop Fold-difference ± SE in C3H and B6 100ppm fluoride gavage group normalized to control, 0ppm fluoride group and house -keeping gene pool ( Actb and Gapdh ) in TPCs (A) , humerii (B) and kidneys (C). (grey bar=C3H and black bar=B6) (n=6 in each group; *p<0.05, **p<0.001).

3.5 Discussion

Skeletal fluorosis is characterized by a spectrum of radiographic bone changes ranging from osteoporosis to osteosclerosis (Wang et al., 1994). Although, clinical cases of skeletal fluorosis are rare in the United States (Kurland et al., 2007; Whyte et al., 2008) in other parts of the world where skeletal fluorosis is more common patients often demonstrate pseudo-hyperparathyroidism (Gupta et al., 2001; Koroglu et al., 2011; Teotia et al., 1998; Teotia and Teotia, 1973; Xu et al., 2010).

Epidemiologic studies show that individuals of the population exposed to similar fluoride levels demonstrate variable skeletal features ranging from excessive bone formation to bone resorption (Harinarayan et al., 2006; Teotia et al., 1998).

Therefore, the mechanisms that underlie fluoride’s actions on the bone and PTH are not clearly understood. Yan et al demonstrated a link between fluoride exposure and iPTH levels using the C3H and B6 inbred strains of mice (Yan et al., 2007). Fluoride exposure in C3H led to enhanced osteoclastogenesis including elevated serum iPTH, whereas in B6 an anabolic action of fluoride was favored. Additionally these strains are widely studied for their differences in bone biology. C3H mice have high bone mass, peak bone density and alkaline phosphatase activity and low bone resorption capacity as compared to B6 mice (Chen and Kalu, 1999; Linkhart et al.,

1999; Turner et al., 2000; Turner et al., 2001).

Direct or indirect effects of fluoride on the parathyroid gland have not been investigated. We developed a murine TPC organ culture model using strains with a predictable iPTH secretion response to changes in calcium. Similar TPC organ culture models have been employed using bovine, human and rat parathyroid

68 tissues to study parathyroid hormone regulation (Nakajima et al., 2010; Nielsen et al., 1996; Wongsurawat and Armbrecht, 1987). Calcium, phosphorus, and magnesium are known to mediate changes in iPTH secretion and therefore, the levels of these in the media were kept equivalent with strain specific serum levels to prevent unwanted effects on iPTH secretion. In both C3H and B6 fluoride exposure resulted in dose dependent reductions in iPTH secretion into the media measured at

24hrs. This reduction was more evident for C3H which at baseline secretes almost twice the iPTH as compared to B6.

The fluoride kinetics observed for B6 and C3H strains were consistent with previous studies where serum fluoride reaches a peak within 20-30mins (Ekstrand et al., 1977; Whitford, 1994). It is generally accepted that after absorption, fluoride is incorporated in newly mineralized bone or is excreted in the urine resulting in decline of serum levels reaching baseline by 6-8hrs (Ekstrand et al., 1980). In order for fluoride to elicit a rapid iPTH response it is hypothesized that fluoride interacts with the parathyroid gland directly through modulating iPTH release from vesicles in the

Chief cells. The exact molecular mechanism of this response is not understood.

However, our in vitro findings suggest the presence of such a direct interaction of fluoride on the parathyroid glands.

In the current study, C3H mice have higher baseline serum iPTH compared to

B6. This is consistent with our TPC baseline values. C3H mice are more responsive to the single fluoride exposure perhaps because reaching a higher peak serum fluoride level. This enhanced response is shown after the gavage when the levels of iPTH were higher in C3H as compared to B6. At 24hrs iPTH levels were lower than

69 baseline for both strains. Both strains also showed bimodal responses to fluoride.

For C3H, iPTH peaks at 0.5hr and declines below the baseline at 12hrs reaching a nadir at 24hrs then over the next 24hrs returns to baseline and remains at baseline at 96hrs. B6 mice show a similar but lower magnitude of response.

The modulation of iPTH secretion as a response to fluoride exposure may also be mediated by changes in calcium levels. Decreased serum calcium levels after acute hydrofluoric acid inhalation has been documented in humans (Zierold and

Chauviere, 2012). Acute exposure to fluoride through injection of hydrofluoric acid

(1.6mg/Kg) in rats led to a decrease in serum ionized calcium and total calcium after

30 and 300mins respectively (Santoyo-Sanchez et al., 2013). Similar decrease in plasma calcium was observed in rats due to acute fluoride exposure (Imanishi et al.,

2009). The reduced serum calcium is hypothesized to be due to the formation of

CaF 2 complexes resulting into a net reduction in calcium levels leading to a hypocalcemic response. In our study, we observed a transient decrease in calcium in both strains of mice only at 0.5hr with return to baseline within 1hr. There were no significant changes in phosphorus and magnesium at any time point.

BPK feedback loop has been extensively studied to understand the interaction of various genes involved in mineral homeostasis. The cross-talk between the bone, parathyroid and kidney is responsible for effective regulation of minerals. The effect of fluoride on these individual tissues after a single fluoride dose in these two strains of mice will help us to understand the effect of fluoride on regulatory aspects of mineral homeostasis. Change in gene expression patterns due to fluoride exposure have been studied in the past (Li and DenBesten, 1993; Nair et al., 2011; Pei et al.,

2012) but our study has focused only on expression of the genes involved in calcium and phosphorus homeostasis and are part of BPK feedback loop. Fold differences in expression of Pth , Casr , αKlotho , Fgf1rc , Vdr and Pthlh in TPCs suggests that at the molecular level fluoride has a differential impact on parathyroid tissues in C3H and

B6. The expression of these genes reflects a compensatory mechanism to counter changes in iPTH secretion. It is clear that fluoride effects are not limited to Pth but extends to Pth homologues such as Pthlh . Expression of Fgf1rc -αKlotho (receptor couple for Fgf23 ) and Pth1r are suggestive of an effort to intercept conservation of calcium and excretion of phosphorus in the kidneys to achieve homeostasis after initial iPTH secretion. Overall, there exists a difference in expression of BPK genes in the two strains of mice. This suggests a difference in physiologic handling of fluoride by the two strains. It will be interesting to study the effect of repeated fluoride dose on expression of the key genes in C3H and B6.

Our study investigated the early events involved in fluoride interaction on parathyroid gland in vitro and in vivo . Fluoride can act on the parathyroid gland directly to modulate iPTH secretion in vitro and in vivo . It is still not clear if the decrease in total serum calcium level is responsible for fluoride-mediated iPTH modulation. The effect of fluoride on iPTH secretion is rapid and observable even after single fluoride dose. The overall effects of fluoride are not limited to parathyroid gland but changes in expression of genes involved in BPK feedback loop suggest a broader impact of fluoride on multiple aspects of bone metabolism.

CHAPTER 4: FLUORIDE DOES NOT TRIGGER BETA-ARRESTIN

RECRUITMENT (NON-CANONICAL SIGNALING) BY CASR

FLUORIDE DOES NOT TRIGGER BETA-ARRESTIN RECRUITMENT (NON-

CANONICAL SIGNALLING) BY CASR

Chaitanya P. Puranik a, Patrick Giguere b, Bryan Roth c and Eric T. Everett d a Oral Biology PhD Curriculum, University of North Carolina at Chapel Hill. b Research Associate, Pharmacology Department, University of North Carolina at

Chapel Hill c Pharmacology Department, School of Medicine, University of North Carolina at

Chapel Hill d Department of Pediatric Dentistry, Carolina Center for Genome Sciences,

University of North Carolina at Chapel Hill.

4.1 Abstract

Fluoride exposure in C3H and B6 mice led to a decrease in parathyroid hormone

(PTH) secretion in vitro and in vivo . (Puranik et al , Chapter 3) Fluoride both enhances polyphosphoinositide (PPI) breakdown and reduces agonist-stimulated cAMP accumulation, resulting in inhibition of PTH release in vivo (Blackmore et al ,

1986). Fluoride is known to activate signaling pathways including GPCR pathway.

Calcium sensing receptor (CASR), a GPCR, present on varieties of cells including

Chief cells of the parathyroid gland are responsible for extracellular calcium sensing and PTH regulation. It is not clear if the CASR is involved in fluoride-mediated PTH modulation. This study aims to investigate the role of CASR in fluoride-mediated

PTH modulation. We also screened 126 human orphan GPCRs, for which ligands have not been identified, to identify fluoride as plausible ligand for receptor activation. We used HTL cells transiently transfected with human CASR or 126 orphan GPCRs constructs. Cells were treated with sodium fluoride for 16hrs in increasing dilutions. Activation of the GPCRS was determined using a modified

Tango ® assay (β-arrestin recruitment assay). Calcimimetic compound R568 hydrochloride (R568) which is a positive allosteric modulator of CASR was used as a control. We demonstrated that in R568 exhibited inverse agonist activity through non-canonical pathway in HTL cells transfected with human CASR. Fluoride treatment of HTL cells transfected with CASR did not demonstrate any change in β- arrestin recruitment. Moreover, sodium fluoride did not alter R568-mediated response in HTL cells. Among the 126 screened orphan GPCRs from class A, B, or

C only three GPCRs: GPR111, GPR142, and GPRC5D demonstrated increased β-

73 arrestin recruitment (>200%) after fluoride treatment as compared to untreated cells.

The study concluded that the effect of fluoride to modulate PTH secretion is independent of CASR. Additionally, the study identified ubiquitously expressed 3 candidate GPCRs which might be involved in fluorides actions on the cells. Further investigation of the candidate orphan GPCRs will be valuable in understanding the mechanistic aspects involved in fluoride metabolism.

4.2 Introduction

It is important for the cellular functioning to respond promptly and efficiently to the changes in the extracellular environment. Transmembrane receptors are evolved to effectively relay the extracellular signals through the plasma membrane and generate intracellular signal. Fluoride is known to stimulate a rapid dose-dependent increase in IP3, a potent intracellular calcium mobilizing compound (Shoback and

McGhee, 1988). Fluoride also activates GTP-dependent processes in the parathyroid gland Chief cells which leads to the breakdown of PPI and accumulation of cAMP (Shoback and McGhee, 1988) which may lead to suppression of PTH secretion. Our study demonstrated that fluoride exposure leads to a decrease in

PTH in a murine model, in vitro and in vivo (Puranik et al , Chapter 3). The CASR, a membrane bound GPCR present on the Chief cells of the parathyroid glands, mediates intracellular signaling events required for decrease in PTH secretion

(Brown and MacLeod, 2001; Magno et al., 2011; Ward and Riccardi, 2012).

Evidence from other secretory systems suggests that intracellular calcium dependent exocytosis involves GPCRs (Berridge and Irvine, 1984). Fluoride is known as an activator of signaling pathways including GPCR pathway (Downs et al.,

1980). Fluoride both enhances PPI breakdown and reduces agonist-stimulated cAMP accumulation, resulting in inhibition of PTH release in vivo (Blackmore and

Exton, 1986). There are reports suggesting that fluoride stimulates PLC-mediated

PPI breakdown through G-protein activation in various types of cells, including parathyroid cells (Blackmore et al., 1985; Taylor et al., 1986). Whether fluoride stimulates the activation of CASR to decrease PTH secretion is not known. Similarly,

75 presence of any receptor for sensing extracellular fluoride has not been identified yet. GPCR represents the largest class of receptors. Out of known 900 GPCRs more than 133 GPCRs are termed orphan receptors due to unidentified ligands (Chung et al., 2008; Ozawa et al., 2010; Vardy and Roth, 2013). Deorphanization is the process of identifying the functions and ligands for a specific orphan GPCRs (Tang et al., 2012). In the study we investigated the role of human CASR in fluoride- mediated inhibition of PTH secretion. We also screened the library of human orphan receptors from class A, B, or C to determine fluoride interaction and intracellular signaling.

4.3 Material and Methods

4.3.1 CASR and orphan GPCRs constructs

Human CASR cDNA and cDNa library of class A, B, and C orphan receptors

(IUPHAR class A database; IUPHAR class B database; IUPHAR class C database) δ-OR constructs were codon optimized for mammalian expression and synthesized by Blue Heron Biotech (Bothell WA, USA) and inserted into the modular vector system (Barnea et al., 2008) with all constructs confirmed by automated dsDNA sequencing.

4.3.2 Cells and transfection

HEK293T-derived cell line which contains a stably integrated tTA-dependent luciferase reporter commonly known as HTL cells were used for these experiments

(HTL cells were generously provided by Dr. Richard Axel to the Roth Lab). HTL cells were cultured in the Dulbecco’s Modified Eagle Medium (Life Technologies, Grand

Island, NY, USA) which was supplemented with 1-2% dialyzed FBS (Life

Technologies, Grand Island, NY, USA) in a 15cm tissue culture dish (9X10 6 cells in

o 20mL) and incubated at 37 C at 5%CO 2. HTL cells were transiently transfected with expression plasmids encoding CASR or orphan GPCRs using calcium phosphate as described earlier (Cozens et al., 1992a; Cozens et al., 1992b; Gruenert et al., 1988;

Kingston et al., 2001). Briefly, 20 g of target receptor (CASR or orphan receptor) and 2 g of YFP control were mixed with CaCl 2 in 0.1X TE and 2X HEPES buffer and incubated. The mix was then added to the cells followed by overnight incubation in the media containing 1% dialyzed FBS. Cells were trypsinized and counted after washing with PBS. Cells were plated in Poly-L-Lys (PLL) coated 384-well white clear

77 bottom (2X10 4 cells/50 L/well) in 384 well plates (Greiner bio-one, Frickenhausen,

Germany). Sodium fluoride or calcimimetic, R568 hydrochloride (Tocris Bioscience,

Bristol, UK) ligand solutions were prepared in filtered assay buffer (20 mM HEPES,

1X Hanks’ balanced salt solution (HBSS), pH 7.40) at 3.5X and added to cells (20 l per well) for overnight incubation (16-20hrs) in medium containing desired calcium concentration

4.3.3 β-arrestin recruitment assay

β-arrestin recruitment assays were performed using previously described modifications of the original Tango ® assay (Barnea et al., 2008) as described previously (Allen et al., 2011; Carlsson et al., 2011). Briefly, the ligand binding mediates recruitment of arrestin-TEV protease complex leading to a proteolytic cleavage at the receptor c-terminus. The proteolytic cleavage frees tTA which then localizes to the nucleus and activate reporter genes. Most GPCRs demonstrate arrestin recruitment after ligand binding through non-canonical pathway and hence arrestin recruitment assay can serve as a functional assay to screen the receptor activation after ligand binding. After overnight incubation with ligand (16-20hrs), media and ligand solutions were removed and 20 l per well of Bright-Glo reagent

(Promega, Madison, WI, USA) was added (1:20 dilution in assay buffer). Plates were incubated for 20min at room temperature in the dark before being counted using a

TriLux luminescence reader (PerkinElmer, Waltham, MA, USA) at 1s/well speed.

4.3.4 Statistical analysis

Data of four independent experiments (n=4) performed in quadruplicate were presented as Relative Luminescence Unit (RLU). Data were subjected to non-linear

78 least-squares regression analysis using the sigmoidal dose-response function provided in GraphPad Prism 6.0 (La Jolla, CA, USA).

4.4 Results

4.4.1 Effect of fluoride on human CASR

Transiently transfected HTL cells with CASR were treated with increasing dilution of sodium fluoride in presence of five concentrations of media calcium (0, 2.5, 5, 10, or 20mM). It was noted that all media calcium concentrations, fluoride did not demonstrate any dose-dependent change in β-arrestin recruitment as witnessed by comparatively unchanged relative luminescence (Fig.4.1). There was a difference in

β-arrestin recruitment based on difference in media calcium concentration. However, the overall effect of fluoride at any given calcium concentration was not different.

Figure 4.1: Effect of fluoride on CASR HTLA cells transfected with human CASR were treated with fluoride under five calcium concentrations in media: 0mM (white), 2.5mM (red), 5mM (blue), 10mM (brown), and 20mM (yellow). Fluorescence measured in relative luminescence units (RLU±SE) from four experiments are shown in graph.

4.4.2 Effect of calcimimetic (R568) on human CASR

We noticed the effect of media calcium on the CASR β-arrestin recruitment assay. Therefore, we screened the effect of known agonist of CASR, R568

80 hydrochloride (R568), on β-arrestin recruitment by CASR through non-canonical pathway. R568 is a known positive allosteric modulator of CASR. We used R568 in increasing dilution at five different media calcium concentration (0, 2.5, 5, 10, or

20mM). R568 at all media calcium concentration displayed an inverse agonist activity on CASR (Fig. 4.2). The IC 50 concentration at five different media calcium concentrations (0, 2.5, 5, 10, or 20mM) was 1.59e-6M, 1.46e-6M, 2.21e-6M, 1.98e-

6M, and 3.35e-6M respectively.

Figure 4.2: Effect of calcimimetic (R568) on CASR HTLA cells transfected with human CASR were treated with R568 under five calcium concentrations in media: 0mM (white), 2.5mM (red), 5mM (blue), 10mM (brown), and 20mM (yellow). Fluorescence measured in relative luminescence units (RLU±SE) from four experiments are shown in graph.

4.4.4 Effect of fluoride on R568-mediated CASR response

We screened the effect of sodium fluoride on R568-mediated CASR response. Β- arrestin recruitment was detected in presence of R568 with increasing sodium

81 fluoride dilutions (0, 100, 250, 500, or 1000 M). Sodium fluoride at any concentration did not modulate the R568-mediated CASR response (Fig. 4.3). The

IC 50 concentrations at five different media fluoride concentrations (0, 100, 250, 500, or 1000 M) were 1.81e-6M, 1.57e-6M, 1.96e-6M, 1.85e-6M, and 1.75e-6M respectively.

Figure 4.3: Effect of fluoride on R568-mediated CASR response HTLA cells transfected with human CASR were treated with R568 under five fluoride concentrations in media: 0µM (white), 100µM (red), 250µM (blue), 500µM (brown), and 1000µM (yellow). Fluorescence measured in relative luminescence units (RLU±SE) from four experiments are shown in graph.

4.4.5 Effect of R568 on CASR is specific

R568 demonstrated an inverse agonist response on CASR therefore, we validated this response. CASR-unrelated GPCRs, dopamine receptor D2 and D3

(DRD2 and DRD3) were selected and treated with increasing dilution of R568 alone, quinpirol (known ligand for DRD2 and DRD3) alone or R568 and quinpirol both using

β-arrestin recruitment assay. Quinpirol demonstrated a predictable increase in

82 luminescence suggestive of β-arrestin recruitment in both DRD2 and DRD3.

However, R568 did not demonstrate any β-arrestin recruitment alone, suggesting that R568 response is valid and specific to CASR (Fig. 4.4).

Figure 4.4: Effect of R568 on CASR is specific

HTLA cells transfected with human DRD2 and DRD3 were treated with known agonist of DRD2 and DRD3, quinpirol alone, calcimimmetic R568 alone, and both quinpirol and R568 both. Fluorescence measured in relative luminescence units (RLU±SE) from four experiments are shown in graph.

4.4.5 Effect of R568 extends to CASR related GPCRs

GPRC6A, is ubiquitously expressed in the human body and is a membrane bound receptor involved in calcium sensing. We screened the effect of R568, a calcimimetic on GPRC6A. GPRC6A was treated with increasing dilution of R568 to determine the impact on β-arrestin recruitment at five different media calcium concentrations (0, 2.5, 5, 10, or 20mM). R568 at all media calcium concentrations displayed an inverse agonist activity on GPRC6A too (Fig. 4.5). The IC 50 concentration at five different media calcium concentrations (0, 2.5, 5, 10, or 20mM) was 8.40e-1M, 1.85e-6M, 1.23e-5M, 1.05e-5M, and 3.79e-1M respectively.

Figure 4.5: Effect of R568 on GPRC6A

HTLA cells transfected with human GPRC6A were treated for 16hrs with R568 under five calcium concentrations in media: 0mM (white), 2.5mM (red), 5mM (blue), 10mM (brown), and 20mM (yellow). Fluorescence measured in relative luminescence units (RLU±SE) from four experiments are shown in graph.

4.4.6 Effect of fluoride on 126 orphan GPCRs

We screened 126 GPCRs from class A, B, or C orphan GPCRs using β-arrestin recruitment assay. Relative luminescence values were determined from fluoride treated and untreated cells. Percentage RLU was calculated using the formula:

%RLU= RLU from fluoride treated wells/ RLU from fluoride untreated wells (Table

4.1). Percentage RLU values more than 200 were considered significant. After fluoride treatment, out of the 126 screened human orphan GPCRs, only three

GPCRs demonstrated percentage RLU >200. Additional information regarding the 3 orphan GPCRs demonstrating activation due to fluoride is outlined in Table 4.2.

GPCR %RLU GPCR %RLU GPCR %RLU GPCR %RLU

GPR1 149.86 GPR111 258.89 GPRC5B 134.51 SUCN1 97.54

GPR3 109.16 GPR113 171.54 GPRC5C 90.80 TA1 92.27

GPR4 120.95 GPR114 144.20 GPRC5D 226.05 TAAR2 79.60

GPR6 93.34 GPR115 138.48 GPRC6A 113.50 TARR5 64.35

GPR12 72.79 GPR116 131.96 HCA1 162.89 TAAR6 115.58

GPR15 121.36 GPR119 95.70 HCA2 102.64 TARR8 81.74

GPR17 96.57 GPR123 128.30 HCA3 67.12 TAAR9 88.59

GPR19 141.49 GPR124 116.51 LPA4 125.11 CCR1 55.96

GPR20 142.03 GPR125 153.57 MAS1 125.03 CCR2 115.92

GPR26 96.77 GPR126 106.69 MAS1L 118.75 CCR3 95.46

GPR32 110.23 GPR132 109.92 MRGPRD 111.31 CCR4 109.65

GPR34 86.18 GPR133 128.13 MRGPRE 176.81 CCR5 117.28

GPR35 108.01 GPR141 87.06 MRGPRF 98.36 CCR6 109.41

GPR37 116.41 GPR142 320.78 MRGPRG 107.04 CCR7 70.12

GPR45 118.93 GPR143 121.50 MRGPRX1 79.86 CCR8 111.60

GPR50 134.19 GPR150 91.57 MRGPRX2 76.74 CCRL2 126.35

GPR52 92.00 GPR152 102.72 MRGPRX3 146.27 CD97 101.64

GPR55 118.14 GPR156 108.99 MRGPRX4 150.01 CMKLR1 90.49

GPR56 130.35 GPR157 98.02 RXFP1 76.87 CX3CR1 105.89

GPR61 130.76 GPR158 166.57 RXFP2 147.83 CXCR1 111.50

GPR63 133.15 GPR160 111.66 RXFP3 118.72 CXCR2 114.56

GPR64 154.80 GPR161 111.91 RXFP4 157.24 CXCR3 97.31

GPR65 120.57 GPR171 179.72 NPBW1 74.40 CXCR4 117.57

GPR75 130.21 GPR173 129.56 NPBW2 118.71 CXCR5 112.26

GPR78 107.35 GPR174 78.17 NPFF1 65.46 CXCR6 168.99

GPR82 115.84 GPR182 149.48 NPFF2 75.08 FFA1 105.18

GPR83 105.56 BBA3 97.41 OPN5 106.91 FFA2 103.09

GPR84 115.82 C5A 75.63 OXGR1 107.12 GPBA 110.75

GPR87 105.22 CCR10 130.98 PKR1 102.71 GPER 110.93

GPR92 116.29 ELD1 126.05 PKR2 70.67 GAL3 98.13

GPR97 97.55 FPR3 99.43 PRRP 76.54

GPR101 99.93 GPRC5A 187.08 QRFP 105.65 Table 4.1: Percentage RLU for screened orphan GPCRs

HTL cells transfected with 126 human orphan GPCRs from class A, B, or C. Cells were treated with 0 or 1mM fluoride and relative luminescence units (RLU) were measured to calculate %RLU calculated using formula %RLU= RLU from fluoride treated wells/ RLU from fluoride untreated wells (IUPHAR class A database; IUPHAR class B database; IUPHAR class C database).

GPCR Class Human Chr. (AA) Mouse Chr. (AA) % RLU

GPR142 Class A 17q23 (462) 11 E2 (365) 321

GPR111 Class B 6p12.3 (642) 17 B3 (644) 259

GPRC5D Class C 12p13.3 (300) 6 G1 (345) 226

Table 4.2: Orphan GPCRs activated by fluoride

4.5 Discussion

GPCR’s forms the largest family and pharmacologically diverse group of mammalian membrane bound proteins. About 1/3 rd of the known drugs target GPCR functions which suggest the importance of GPCRs in therapeutics. (Tang et al.,

2012). GPCRs are of great interest due to its unique property to transduce extracellular signal into intracellular signaling events through heterotrimeric G proteins. GPCRS binds to a wide range of ligands including ions, small peptides, and organic compounds (Vardy and Roth, 2013). Phylogenetically human GPCRs are categorized in to five main families such as; Rhodopsin, Glutamate, Adhesion,

Secretin, and Frizzled/Taste 2. Rhodopsin forms the largest family of GPCRs members of which are widely studied (Tang et al., 2012).

Deorphanization of the orphan GPCRs can be challenging due to lack of knowledge about the functionality and ligand specificity of these orphan receptors.

(Chung et al., 2008). Part of the challenge arises due to that fact that most deorphanation assay involves detection of changes in the levels of second messengers (canonical pathway). However, it is important to understand that levels of the second messengers are regulated by the heterotrimeric G proteins and can be independent of GPCR of interest. Similarly lack of knowledge about intracellular signaling after orphan GPCR activation potentially limits the selection of proper positive and negative controls. The β-arrestin recruitment assay (non-canonical signaling) is independent of intracellular second messengers (Barnea et al., 2008), which distinguishes the β-arrestin recruitment assay from other assays that measure

GPCR function.

In the β-arrestin recruitment assay an orphan receptor is genetically modified to transduce an intracellular signal into a reporter gene transcription. Transcriptional activation of reporter gene provides a quantitative measure for GPCR activation. The reporter gene activity is independent of endogenous receptors and hence β-arrestin recruitment assay is sensitive (Allen et al., 2011).

Figure 4.5: Schematic of the β-arrestin recruitment assay In the β-arrestin recruitment assay when a genetically modified GPCR (with attached transcription factor via protease site) binds to its ligand it leads to the recruitment of the arrestin-TEV protease fusion. Proteolytic cleavage at protease site liberates the tethered transcription factor (tTA). The tTA translocates to the nucleus leading to transcription of reporter gene. Increase in the reporter gene activity is used as a quantitative measure for receptor activation (Barnea et al., 2008).

The β-arrestin recruitment assay monitors ligand binding-mediated receptor activation (non-canonical pathway). We screened human CASR and orphan GPCRs using this generality to identify receptor activation by fluoride through β-arrestin recruitment assay which provides a more quantitative assay, unaffected by endogenous receptor signaling (Tohgo et al., 2003).

Fluoride is known to activate various G-proteins, including the two regulatory proteins G s and G i of the AC system (Sternweis and Gilman, 1982). Fluoride mimics the effects of high calcium on several intracellular second messengers to mediate a time- and dose-dependent increase in the accumulation of IP3 in bovine parathyroid cells (Chen et al., 1988).

Fluoride does not influence β-arrestin recruitment by CASR in HTL cells which suggests that actions of fluoride on parathyroid gland to modulate PTH secretion

(non-canonical pathway) are independent of CASR. Altering the calcium concentration did not have any impact on the fluoride-mediated arrestin recruitment which further supports our finding. There was a difference in β-arrestin recruitment based on difference in media calcium concentration. The role of media calcium concentration on CASR signaling has been previously documented (Roussanne et al., 2001). High media calcium increased arrestin recruitment and thereby luminescence readout. In our study 0mM calcium in the media demonstrated maximum arrestin recruitment which is in accordance with previously reported findings (Almaden et al., 2009; Nemeth et al., 1998). These findings suggests that increase in arrestin recruitment may be is responsible for a low extra-cellular calcium mediated increase in the PTH secretion (non-canonical pathway).

The IC 50 concentration (1-2mM) determined in our experiments with R568 are comparable to those in previously documented study (Li et al., 2009). However

Nemeth el al reported much lover IC 50 values (~27nM) compared to our values

(Nemeth et al., 1998). It is interesting to note that using arrestin recruitment assay in our study, R568 demonstrated an inverse agonist activity on CASR rather than positive allosteric modulator activity as previously described (Li et al., 2009; Nemeth et al., 2004; Wada et al., 2000). Our study demonstrated that the activity of R568 was specific for CASR and also extended to other known calcium sensing receptor such as GPRC6A.

Three orphan receptors identified by the β-arrestin assay: GPRC5D, GPR111 and GPR142 (Brauner-Osborne et al., 2001; Fredriksson et al., 2003), which were activated by fluoride treatment, are ubiquitously expressed. However, functional aspects of these three GPCRs are unknown. In a study by Everett et al to determine genetic basis for fluoride susceptibility in two strains of mice (A/J and 129P3/J) detected quantitative trait loci (QTLs) associated with dental fluorosis on chromosome 2 and 11(Everett et al., 2011). It is interesting to know that in mice

GPR142 is localized on chromosome 11. Therefore, future studies will be directed to identify the role of GPR142, GPR111 and GPRC5D in fluoride mediated actions on hard tissues to understand the patho-physiology of dental and skeletal fluorosis.

The findings of the study can be summarized as follows: Fluoride can modulate

PTH secretion independent of CASR activation. The possibility of involvement of other GPCRs in fluoride-mediated modulation of PTH cannot be rejected. Screening of the orphan GPCRs for fluoride-mediated receptor activation identified three

90 candidates: GPRC5D, GPR111, and GPR142. Future study of these candidates will help us to understand their role in the patho-physiology of dental and skeletal fluorosis and fluoride’s action on cellular biology.

Bibliography

Abbink W, Flik G (2007). Parathyroid hormone-related protein in teleost fish. General and comparative endocrinology 152(2-3) :243-251.

Akosu TJ, Zoakah AI (2008). Risk factors associated with dental fluorosis in Central Plateau State, Nigeria. Community dentistry and oral epidemiology 36(2) :144-148.

Al-Nowaiser A, Roberts GJ, Trompeter RS, Wilson M, Lucas VS (2003). Oral health in children with chronic renal failure. Pediatric nephrology 18(1) :39-45.

Allen JA, Yost JM, Setola V, Chen X, Sassano MF, Chen M et al. (2011). Discovery of beta-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proceedings of the National Academy of Sciences of the United States of America 108(45) :18488-18493.

Allolio B, Lehmann R (1999). Drinking water fluoridation and bone. Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 107(1) :12-20.

Almaden Y, Rodriguez-Ortiz ME, Canalejo A, Canadillas S, Canalejo R, Martin D et al. (2009). Calcimimetics normalize the phosphate-induced stimulation of PTH secretion in vivo and in vitro. Journal of nephrology 22(2) :281-288.

Anderson NG, Kilgour E, Sturgill TW (1991). Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate. The Journal of biological chemistry 266(16) :10131-10135.

Augenstein WL, Spoerke DG, Kulig KW, Hall AH, Hall PK, Riggs BS et al. (1991). Fluoride ingestion in children: a review of 87 cases. Pediatrics 88(5) :907-912.

Awadia AK, Haugejorden O, Bjorvatn K, Birkeland JM (1999). Vegetarianism and dental fluorosis among children in a high fluoride area of northern Tanzania. International journal of paediatric dentistry / the British Paedodontic Society [and] the International Association of Dentistry for Children 9(1) :3-11.

Barbier O, Arreola-Mendoza L, Del Razo LM (2010). Molecular mechanisms of fluoride toxicity. Chemico-biological interactions 188(2) :319-333.

Barnea G, Strapps W, Herrada G, Berman Y, Ong J, Kloss B et al. (2008). The genetic design of signaling cascades to record receptor activation. Proceedings of the National Academy of Sciences of the United States of America 105(1) :64-69.

Bellows CG, Heersche JN, Aubin JE (1990). The effects of fluoride on osteoblast progenitors in vitro. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 5 Suppl 1(S101-105.

Bergwitz C, Juppner H (2010). Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Annu Rev Med 61(91-104.

Berridge MJ, Irvine RF (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312(5992) :315-321.

Bhargavi V, Khandare AL, Venkaiah K, Sarojini G (2004). Mineral content of water and food in fluorotic villages and prevalence of dental fluorosis. Biological trace element research 100(3) :195-203.

Blackmore PF, Bocckino SB, Waynick LE, Exton JH (1985). Role of a guanine nucleotide-binding regulatory protein in the hydrolysis of hepatocyte phosphatidylinositol 4,5-bisphosphate by calcium-mobilizing hormones and the control of cell calcium. Studies utilizing aluminum fluoride. The Journal of biological chemistry 260(27) :14477-14483.

Blackmore PF, Exton JH (1986). Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate. The Journal of biological chemistry 261(24) :11056-11063.

Boland CJ, Fried RM, Tashjian AH, Jr. (1986). Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption- stimulating hormones. Endocrinology 118(3) :980-989.

Boyle WJ, Simonet WS, Lacey DL (2003). Osteoclast differentiation and activation. Nature 423(6937) :337-342.

Brauner-Osborne H, Jensen AA, Sheppard PO, O'Hara P, Krogsgaard-Larsen P (1999). The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain. The Journal of biological chemistry 274(26) :18382-18386.

Brauner-Osborne H, Jensen AA, Sheppard PO, Brodin B, Krogsgaard-Larsen P, O'Hara P (2001). Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochimica et biophysica acta 1518(3) :237-248.

Breitwieser GE (2013). The calcium sensing receptor life cycle: Trafficking, cell surface expression, and degradation. Best practice & research Clinical endocrinology & metabolism 27(3) :303-313.

Briancon D, Meunier PJ (1981). Treatment of osteoporosis with fluoride, calcium, and vitamin D. The Orthopedic clinics of North America 12(3) :629-648.

Bringhurst FR, Stern AM, Yotts M, Mizrahi N, Segre GV, Potts JT, Jr. (1988). Peripheral metabolism of PTH: fate of biologically active amino terminus in vivo. The American journal of physiology 255(6 Pt 1) :E886-893.

Broadus AE, Mangin M, Ikeda K, Insogna KL, Weir EC, Burtis WJ et al. (1988). Humoral hypercalcemia of cancer. Identification of a novel parathyroid hormone-like peptide. The New England journal of medicine 319(9) :556-563.

Bronckers AL, Jansen LL, Woltgens JH (1984). Long-term (8 days) effects of exposure to low concentrations of fluoride on enamel formation in hamster tooth- germs in organ culture in vitro. Archives of oral biology 29(10) :811-819.

Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O et al. (1993). Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 366(6455) :575-580.

Brown EM, MacLeod RJ (2001). Extracellular calcium sensing and extracellular calcium signaling. Physiological reviews 81(1) :239-297.

Browne D, Whelton H, O'Mullane D (2005). Fluoride metabolism and fluorosis. Journal of dentistry 33(3) :177-186.

Burgener D, Bonjour JP, Caverzasio J (1995). Fluoride increases tyrosine kinase activity in osteoblast-like cells: regulatory role for the stimulation of cell proliferation and Pi transport across the plasma membrane. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 10(1) :164-171.

Buzalaf MA, Whitford GM (2011). Fluoride metabolism. Monographs in oral science 22(20-36.

Carlsson J, Coleman RG, Setola V, Irwin JJ, Fan H, Schlessinger A et al. (2011). Ligand discovery from a dopamine D3 receptor homology model and crystal structure. Nature chemical biology 7(11) :769-778.

Carvalho JG, Leite AL, Yan D, Everett ET, Whitford GM, Buzalaf MA (2009). Influence of genetic background on fluoride metabolism in mice. Journal of dental research 88(11) :1054-1058.

Caverzasio J, Imai T, Ammann P, Burgener D, Bonjour JP (1996). Aluminum potentiates the effect of fluoride on tyrosine phosphorylation and osteoblast replication in vitro and bone mass in vivo. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 11(1) :46-55.

Caverzasio J, Palmer G, Suzuki A, Bonjour JP (1997). Mechanism of the mitogenic effect of fluoride on osteoblast-like cells: evidences for a G protein-dependent tyrosine phosphorylation process. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 12(12) :1975-1983.

Chakravarti B, Chattopadhyay N, Brown EM (2012). Signaling through the extracellular calcium-sensing receptor (CaSR). Advances in experimental medicine and biology 740(103-142.

Chang W, Pratt S, Chen TH, Bourguignon L, Shoback D (2001). Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation. The Journal of biological chemistry 276(47) :44129-44136.

Chen C, Kalu DN (1999). Strain differences in bone density and calcium metabolism between C3H/HeJ and C57BL/6J mice. Bone 25(4) :413-420.

Chen CJ, Anast CS, Brown EM (1988). Effects of fluoride on parathyroid hormone secretion and intracellular second messengers in bovine parathyroid cells. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 3(3) :279-288.

Cheng SX (2012). Calcium-sensing receptor inhibits secretagogue-induced electrolyte secretion by intestine via the enteric nervous system. American journal of physiology Gastrointestinal and liver physiology 303(1) :G60-70.

Chung S, Funakoshi T, Civelli O (2008). Orphan GPCR research. British journal of pharmacology 153 Suppl 1(S339-346.

Collier DR (1980). Fluorine: an essential element for good dental health. Journal of public health dentistry 40(3) :296-300.

Colloton M, Shatzen E, Miller G, Stehman-Breen C, Wada M, Lacey D et al. (2005). Cinacalcet HCl attenuates parathyroid hyperplasia in a rat model of secondary hyperparathyroidism. Kidney international 67(2) :467-476.

Cozens AL, Yezzi MJ, Chin L, Simon EM, Finkbeiner WE, Wagner JA et al. (1992a). Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells. Proceedings of the National Academy of Sciences of the United States of America 89(11) :5171-5175.

Cozens AL, Yezzi MJ, Yamaya M, Steiger D, Wagner JA, Garber SS et al. (1992b). A transformed human epithelial cell line that retains tight junctions post crisis. In vitro cellular & developmental biology : journal of the Tissue Culture Association 28A(11- 12) :735-744.

Cozzolino M, Brancaccio D, Gallieni M, Galassi A, Slatopolsky E, Dusso A (2005). Pathogenesis of parathyroid hyperplasia in renal failure. Journal of nephrology 18(1) :5-8.

Cupp ME, Nayak SK, Adem AS, Thomsen WJ (2013). Parathyroid hormone (PTH) and PTH-related peptide domains contributing to activation of different PTH receptor-mediated signaling pathways. The Journal of pharmacology and experimental therapeutics 345(3) :404-418.

D'Amour P, Rousseau L, Rocheleau B, Pomier-Layrargues G, Huet PM (1996). Influence of Ca2+ concentration on the clearance and circulating levels of intact and carboxy-terminal iPTH in pentobarbital-anesthetized dogs. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 11(8) :1075-1085.

D'Amour P, Brossard JH, Rousseau L, Nguyen-Yamamoto L, Nassif E, Lazure C et al. (2005). Structure of non-(1-84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism. Kidney international 68(3) :998-1007.

D'Amour P (2006). Circulating PTH molecular forms: what we know and what we don't. Kidney international Supplement 102) :S29-33.

Dalamaga M, Karmaniolas K, Nikolaidou A, Papadavid E (2008). Hypocalcemia, hypomagnesemia, and hypokalemia following hydrofluoric acid chemical injury. Journal of burn care & research : official publication of the American Burn Association 29(3) :541-543.

Davies SL, Ozawa A, McCormick WD, Dvorak MM, Ward DT (2007). Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity. The Journal of biological chemistry 282(20) :15048-15056.

Debinski A, Nowicka G (2004). [Effect of sodium fluoride on ectopic induction of bone tissue]. Annales Academiae Medicae Stetinensis 50 Suppl 1(23-27.

Della Penna K, Kinose F, Sun H, Koblan KS, Wang H (2003). Tuberoinfundibular peptide of 39 residues (TIP39): molecular structure and activity for parathyroid hormone 2 receptor. Neuropharmacology 44(1) :141-153.

Dimai HP, Linkhart TA, Linkhart SG, Donahue LR, Beamer WG, Rosen CJ et al. (1998). Alkaline phosphatase levels and osteoprogenitor cell numbers suggest bone formation may contribute to peak bone density differences between two inbred strains of mice. Bone 22(3) :211-216.

Doucette C, Vedvik K, Koepnick E, Bergsma A, Thomson B, Turek-Etienne TC (2009). Kappa opioid receptor screen with the Tango beta-arrestin recruitment technology and characterization of hits with second-messenger assays. Journal of biomolecular screening 14(4) :381-394.

Downs RW, Jr., Spiegel AM, Singer M, Reen S, Aurbach GD (1980). Fluoride stimulation of adenylate cyclase is dependent on the guanine nucleotide regulatory protein. The Journal of biological chemistry 255(3) :949-954.

Ekstrand J, Alvan G, Boreus LO, Norlin A (1977). Pharmacokinetics of fluoride in man after single and multiple oral doses. Eur J Clin Pharmacol 12(4) :311-317.

Ekstrand J (1978). Relationship between fluoride in the drinking water and the plasma fluoride concentration in man. Caries research 12(3) :123-127.

Ekstrand J, Ehrnebo M, Whitford GM, Jarnberg PO (1980). Fluoride pharmacokinetics during acid-base balance changes in man. Eur J Clin Pharmacol 18(2) :189-194.

Ekstrand J, Spak CJ, Ehrnebo M (1982). Renal clearance of fluoride in a steady state condition in man: influence of urinary flow and pH changes by diet. Acta pharmacologica et toxicologica 50(5) :321-325.

Ekstrand J, Ziegler EE, Nelson SE, Fomon SJ (1994). Absorption and retention of dietary and supplemental fluoride by infants. Advances in dental research 8(2) :175- 180.

Everett ET, McHenry MA, Reynolds N, Eggertsson H, Sullivan J, Kantmann C et al. (2002). Dental fluorosis: variability among different inbred mouse strains. Journal of dental research 81(11) :794-798.

Everett ET, Yan D, Weaver M, Liu L, Foroud T, Martinez-Mier EA (2009). Detection of dental fluorosis-associated quantitative trait Loci on mouse chromosomes 2 and 11. Cells, tissues, organs 189(1-4) :212-218.

Everett ET (2011). Fluoride's effects on the formation of teeth and bones, and the influence of genetics. Journal of dental research 90(5) :552-560.

Everett ET, Yin Z, Yan D, Zou F (2011). Fine mapping of dental fluorosis quantitative trait loci in mice. European journal of oral sciences 119 Suppl 1(8-12.

Farge P, Ranchin B, Cochat P (2006). Four-year follow-up of oral health surveillance in renal transplant children. Pediatric nephrology 21(6) :851-855.

Farley JR, Wergedal JE, Baylink DJ (1983). Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone-forming cells. Science 222(4621) :330-332.

Farley JR, Tarbaux N, Hall S, Baylink DJ (1988). Evidence that fluoride-stimulated 3[H]-thymidine incorporation in embryonic chick calvarial cell cultures is dependent on the presence of a bone cell mitogen, sensitive to changes in the phosphate

97 concentration, and modulated by systemic skeletal effectors. Metabolism: clinical and experimental 37(10) :988-995.

Farley JR, Tarbaux N, Hall S, Baylink DJ (1990). Mitogenic action(s) of fluoride on osteoblast line cells: determinants of the response in vitro. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 5 Suppl 1(S107-113.

Finazzi D, Cassel D, Donaldson JG, Klausner RD (1994). Aluminum fluoride acts on the reversibility of ARF1-dependent coat protein binding to Golgi membranes. The Journal of biological chemistry 269(18) :13325-13330.

Fordyce FM, Vrana K, Zhovinsky E, Povoroznuk V, Toth G, Hope BC et al. (2007). A health risk assessment for fluoride in Central Europe. Environ Geochem Health 29(2) :83-102.

Fox J, Lowe SH, Petty BA, Nemeth EF (1999). NPS R-568: a type II calcimimetic compound that acts on parathyroid cell calcium receptor of rats to reduce plasma levels of parathyroid hormone and calcium. The Journal of pharmacology and experimental therapeutics 290(2) :473-479.

Fredriksson R, Hoglund PJ, Gloriam DE, Lagerstrom MC, Schioth HB (2003). Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives. FEBS letters 554(3) :381-388.

Fudge NJ, Kovacs CS (2004). Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release in vivo. BMC physiology 4(5.

Gensure RC, Ponugoti B, Gunes Y, Papasani MR, Lanske B, Bastepe M et al. (2004). Identification and characterization of two parathyroid hormone-like molecules in zebrafish. Endocrinology 145(4) :1634-1639.

Gensure RC, Gardella TJ, Juppner H (2005). Parathyroid hormone and parathyroid hormone-related peptide, and their receptors. Biochemical and biophysical research communications 328(3) :666-678.

Grant MP, Stepanchick A, Breitwieser GE (2012). Calcium signaling regulates trafficking of familial hypocalciuric hypercalcemia (FHH) mutants of the calcium sensing receptor. Molecular endocrinology 26(12) :2081-2091.

Greco RJ, Hartford CE, Haith LR, Jr., Patton ML (1988). Hydrofluoric acid-induced hypocalcemia. The Journal of trauma 28(11) :1593-1596.

Gruenert DC, Basbaum CB, Welsh MJ, Li M, Finkbeiner WE, Nadel JA (1988). Characterization of human tracheal epithelial cells transformed by an origin-defective

98 simian virus 40. Proceedings of the National Academy of Sciences of the United States of America 85(16) :5951-5955.

Guerreiro PM, Renfro JL, Power DM, Canario AV (2007). The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish. American journal of physiology Regulatory, integrative and comparative physiology 292(2) :R679-696.

Gunther T, Chen ZF, Kim J, Priemel M, Rueger JM, Amling M et al. (2000). Genetic ablation of parathyroid glands reveals another source of parathyroid hormone. Nature 406(6792) :199-203.

Gupta SK, Khan TI, Gupta RC, Gupta AB, Gupta KC, Jain P et al. (2001). Compensatory hyperparathyroidism following high fluoride ingestion - a clinico - biochemical correlation. Indian Pediatr 38(2) :139-146.

Gutknecht J, Walter A (1981). Hydrofluoric and nitric acid transport through lipid bilayer membranes. Biochimica et biophysica acta 644(1) :153-156.

Habener JF, Kemper B, Potts JT, Jr. (1975). Calcium-dependent intracellular degradation of parathyroid hormone: a possible mechanism for the regulation of hormone stores. Endocrinology 97(2) :431-441.

Hall BK (1987). Sodium fluoride as an initiator of osteogenesis from embryonic mesenchyme in vitro. Bone 8(2) :111-116.

Harinarayan CV, Kochupillai N, Madhu SV, Gupta N, Meunier PJ (2006). Fluorotoxic metabolic bone disease: an osteo-renal syndrome caused by excess fluoride ingestion in the tropics. Bone 39(4) :907-914.

Heard K, Delgado J (2003). Oral decontamination with calcium or magnesium salts does not improve survival following hydrofluoric acid ingestion. Journal of toxicology Clinical toxicology 41(6) :789-792.

Hogan BM, Danks JA, Layton JE, Hall NE, Heath JK, Lieschke GJ (2005). Duplicate zebrafish pth genes are expressed along the lateral line and in the central nervous system during embryogenesis. Endocrinology 146(2) :547-551.

Hruska KA, Korkor A, Martin K, Slatopolsky E (1981). Peripheral metabolism of intact parathyroid hormone. Role of liver and kidney and the effect of chronic renal failure. The Journal of clinical investigation 67(3) :885-892.

Hu MC, Shiizaki K, Kuro-o M, Moe OW (2013). Fibroblast growth factor 23 and Klotho: physiology and pathophysiology of an endocrine network of mineral metabolism. Annual review of physiology 75(503-533.

Ibarra-Santana C, Ruiz-Rodriguez Mdel S, Fonseca-Leal Mdel P, Gutierrez-Cantu FJ, Pozos-Guillen Ade J (2007). Enamel hypoplasia in children with renal disease in a fluoridated area. The Journal of clinical pediatric dentistry 31(4) :274-278.

Imanishi M, Dote T, Tsuji H, Tanida E, Yamadori E, Kono K (2009). Time-dependent changes of blood parameters and fluoride kinetics in rats after acute exposure to subtoxic hydrofluoric acid. J Occup Health 51(4) :287-293.

Ismail AI, Hasson H (2008). Fluoride supplements, dental caries and fluorosis: a systematic review. Journal of the American Dental Association 139(11) :1457-1468.

IUPHAR class A database.

IUPHAR class B database.

IUPHAR class C database.

Jarnberg PO, Ekstrand J, Ehrnebo M (1983). Renal excretion of fluoride during water diuresis and induced urinary pH-changes in man. Toxicology letters 18(1- 2) :141-146.

Judex S, Garman R, Squire M, Donahue LR, Rubin C (2004). Genetically based influences on the site-specific regulation of trabecular and cortical bone morphology. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 19(4) :600-606.

Kanbur M, Eraslan G, Silici S, Karabacak M (2009). Effects of sodium fluoride exposure on some biochemical parameters in mice: evaluation of the ameliorative effect of royal jelly applications on these parameters. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 47(6) :1184-1189.

Kao WF, Dart RC, Kuffner E, Bogdan G (1999). Ingestion of low-concentration hydrofluoric acid: an insidious and potentially fatal poisoning. Annals of emergency medicine 34(1) :35-41.

Kassem M, Mosekilde L, Eriksen EF (1993). 1,25-dihydroxyvitamin D3 potentiates fluoride-stimulated collagen type I production in cultures of human bone marrow stromal osteoblast-like cells. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 8(12) :1453-1458.

Kassem M, Mosekilde L, Eriksen EF (1994). Effects of fluoride on human bone cells in vitro: differences in responsiveness between stromal osteoblast precursors and mature osteoblasts. European journal of endocrinology / European Federation of Endocrine Societies 130(4) :381-386.

100

Kawase T, Ishikawa I, Suzuki A (1988). NaF-induced Ca2+ mobilization is dependent upon the culture density in a parathyroid hormone-responsive osteoblast- like cell line. Life sciences 43(26) :2241-2247.

Khandare AL, Kumar PU, Shanker RG, Venkaiah K, Lakshmaiah N (2004). Additional beneficial effect of tamarind ingestion over defluoridated water supply to adolescent boys in a fluorotic area. Nutrition 20(5) :433-436.

Khandare AL, Harikumar R, Sivakumar B (2005). Severe bone deformities in young children from vitamin D deficiency and fluorosis in Bihar-India. Calcified tissue international 76(6) :412-418.

Khokher MA, Dandona P (1990). Fluoride stimulates [3H]thymidine incorporation and alkaline phosphatase production by human osteoblasts. Metabolism: clinical and experimental 39(11) :1118-1121.

Kifor O, Diaz R, Butters R, Kifor I, Brown EM (1998). The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells. The Journal of biological chemistry 273(34) :21708-21713.

Kim K, Lee SH, Ha Kim J, Choi Y, Kim N (2008). NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP). Molecular endocrinology 22(1) :176-185.

Kingston RE, Chen CA, Okayama H (2001). Calcium phosphate transfection. Current protocols in immunology / edited by John E Coligan [et al] Chapter 10(Unit 10 13.

Kleerekoper M, Peterson EL, Nelson DA, Phillips E, Schork MA, Tilley BC et al. (1991). A randomized trial of sodium fluoride as a treatment for postmenopausal osteoporosis. Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA 1(3) :155-161.

Koch MJ, Buhrer R, Pioch T, Scharer K (1999). Enamel hypoplasia of primary teeth in chronic renal failure. Pediatric nephrology 13(1) :68-72.

Koroglu BK, Ersoy IH, Koroglu M, Balkarli A, Ersoy S, Varol S et al. (2011). Serum parathyroid hormone levels in chronic endemic fluorosis. Biological trace element research 143(1) :79-86.

Koshikawa M, Nishiguchi K, Yorifuji S, Shimazu K, Takaori K, Mori K et al. (2010). Amino terminal cleavage of PTH(1-84) to PTH(7-84) is regulated by serum calcium concentration via calcium-sensing receptor in hemodialysis patients. Clinical and experimental nephrology 14(3) :233-238.

101

Kurland ES, Schulman RC, Zerwekh JE, Reinus WR, Dempster DW, Whyte MP (2007). Recovery from skeletal fluorosis (an enigmatic, American case). J Bone Miner Res 22(1) :163-170.

Lam BS, Cunningham C, Adams GB (2011). Pharmacologic modulation of the calcium-sensing receptor enhances hematopoietic stem cell lodgment in the adult bone marrow. Blood 117(4) :1167-1175.

Lau KH, Freeman TK, Baylink DJ (1987). Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix. The Journal of biological chemistry 262(3) :1389-1397.

Lau KH, Farley JR, Freeman TK, Baylink DJ (1989). A proposed mechanism of the mitogenic action of fluoride on bone cells: inhibition of the activity of an osteoblastic acid phosphatase. Metabolism: clinical and experimental 38(9) :858-868.

Lau KH, Baylink DJ (1998). Molecular mechanism of action of fluoride on bone cells. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 13(11) :1660-1667.

Lavi-Moshayoff V, Wasserman G, Meir T, Silver J, Naveh-Many T (2010). PTH increases FGF23 gene expression and mediates the high-FGF23 levels of experimental kidney failure: a bone parathyroid feedback loop. American journal of physiology Renal physiology 299(4) :F882-889.

Levi R, Ben-Dov IZ, Lavi-Moshayoff V, Dinur M, Martin D, Naveh-Many T et al. (2006). Increased parathyroid hormone gene expression in secondary hyperparathyroidism of experimental uremia is reversed by calcimimetics: correlation with posttranslational modification of the trans acting factor AUF1. Journal of the American Society of Nephrology : JASN 17(1) :107-112.

Leyte A, Barr FA, Kehlenbach RH, Huttner WB (1992). Multiple trimeric G-proteins on the trans-Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation. The EMBO journal 11(13) :4795-4804.

Li H, Ruan G, Li Z, Liu Z, Zheng X, Zheng H et al. (2009). The calcimimetic R-568 induces apoptotic cell death in prostate cancer cells. Journal of experimental & clinical cancer research : CR 28(100.

Li R, DenBesten PK (1993). Expression of bone protein mRNA at physiological fluoride concentrations in rat osteoblast culture. Bone Miner 22(3) :187-196.

Linkhart TA, Linkhart SG, Kodama Y, Farley JR, Dimai HP, Wright KR et al. (1999). Osteoclast formation in bone marrow cultures from two inbred strains of mice with different bone densities. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 14(1) :39-46.

102

Litovitz TL, Klein-Schwartz W, Rodgers GC, Jr., Cobaugh DJ, Youniss J, Omslaer JC et al. (2002). 2001 Annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. The American journal of emergency medicine 20(5) :391-452.

Liu J, Lv F, Sun W, Tao C, Ding G, Karaplis A et al. (2011). The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH. PLoS genetics 7(9) :e1002294.

Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4) :402- 408.

Lyaruu DM, de Jong M, Bronckers AL, Woltgens JH (1986). Ultrastructural study of fluoride-induced in-vitro hypermineralization of enamel in hamster tooth germs explanted during the secretory phase of amelogenesis. Archives of oral biology 31(2) :109-117.

Magno AL, Ward BK, Ratajczak T (2011). The calcium-sensing receptor: a molecular perspective. Endocrine reviews 32(1) :3-30.

Mannstadt M, Juppner H, Gardella TJ (1999). Receptors for PTH and PTHrP: their biological importance and functional properties. The American journal of physiology 277(5 Pt 2) :F665-675.

Marquis RE (1995). Antimicrobial actions of fluoride for oral bacteria. Canadian journal of microbiology 41(11) :955-964.

Martinez-Mier EA, Soto-Rojas AE, Urena-Cirett JL, Katz BP, Stookey GK, Dunipace AJ (2004). Dental fluorosis and altitude: a preliminary study. Oral health & preventive dentistry 2(1) :39-48.

Matsuo S, Inai T, Kurisu K, Kiyomiya K, Kurebe M (1996). Influence of fluoride on secretory pathway of the secretory ameloblast in rat incisor tooth germs exposed to sodium fluoride. Archives of toxicology 70(7) :420-429.

McCann HG (1953). Reactions of fluoride ion with hydroxyapatite. The Journal of biological chemistry 201(1) :247-259.

Mertz W (1981). The essential trace elements. Science 213(4514) :1332-1338.

Messer HH, Armstrong WD, Singer L (1973). Fluoride, parathyroid hormone and calcitonin: effects on metabolic processes involved in bone resorption. Calcified tissue research 13(3) :227-233.

103

Messer HH, Ophaug R (1991). Effect of delayed gastric emptying on fluoride absorption in the rat. Biological trace element research 31(3) :305-315.

Messer HH, Ophaug RH (1993). Influence of gastric acidity on fluoride absorption in rats. Journal of dental research 72(3) :619-622.

Metzger M, Houillier P, Gauci C, Haymann JP, Flamant M, Thervet E et al. (2013). Relation Between Circulating Levels of 25(OH) Vitamin D and Parathyroid Hormone in Chronic Kidney Disease: Quest for a Threshold. The Journal of clinical endocrinology and metabolism 98(7) :2922-2928.

Mittal M, Flora SJ (2006). Effects of individual and combined exposure to sodium arsenite and sodium fluoride on tissue oxidative stress, arsenic and fluoride levels in male mice. Chemico-biological interactions 162(2) :128-139.

Modrowski D, Miravet L, Feuga M, Bannie F, Marie PJ (1992). Effect of fluoride on bone and bone cells in ovariectomized rats. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 7(8) :961- 969.

Mornstad H, Hammarstrom L (1978). Morphologic changes in the rat enamel organ following a single intraperitoneal injection of sodium fluoride. Scandinavian journal of dental research 86(4) :211-220.

Mousny M, Banse X, Wise L, Everett ET, Hancock R, Vieth R et al. (2006). The genetic influence on bone susceptibility to fluoride. Bone 39(6) :1283-1289.

Mullett T, Zoeller T, Bingham H, Pepine CJ, Prida XE, Castenholz R et al. (1987). Fatal hydrofluoric acid cutaneous exposure with refractory ventricular fibrillation. The Journal of burn care & rehabilitation 8(3) :216-219.

Mullins ME, Warden CR, Barnum DW (1998). Pediatric death and fluoride-containing wheel cleaner. Annals of emergency medicine 31(4) :524-525.

Murray TM, Rao LG, Divieti P, Bringhurst FR (2005). Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl- terminal ligands. Endocrine reviews 26(1) :78- 113.

Nair M, Belak ZR, Ovsenek N (2011). Effects of fluoride on expression of bone- specific genes in developing Xenopus laevis larvae. Biochem Cell Biol 89(4) :377- 386.

104

Nakajima K, Okazaki T, Okamoto T, Kimura H, Takano K, Sato K (2010). Genes up- or down-regulated by high calcium medium in parathyroid tissue explants from patients with primary hyperparathyroidism. Endocr J 57(2) :153-159.

Nearing J, Betka M, Quinn S, Hentschel H, Elger M, Baum M et al. (2002). Polyvalent cation receptor proteins (CaRs) are salinity sensors in fish. Proceedings of the National Academy of Sciences of the United States of America 99(14) :9231- 9236.

Nemeth EF, Steffey ME, Hammerland LG, Hung BC, Van Wagenen BC, DelMar EG et al. (1998). Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proceedings of the National Academy of Sciences of the United States of America 95(7) :4040-4045.

Nemeth EF, Heaton WH, Miller M, Fox J, Balandrin MF, Van Wagenen BC et al. (2004). Pharmacodynamics of the type II calcimimetic compound cinacalcet HCl. The Journal of pharmacology and experimental therapeutics 308(2) :627-635.

Nemeth EF, Shoback D (2013). Calcimimetic and calcilytic drugs for treating bone and mineral-related disorders. Best practice & research Clinical endocrinology & metabolism 27(3) :373-384.

Nguyen-Yamamoto L, Rousseau L, Brossard JH, Lepage R, Gao P, Cantor T et al. (2002). Origin of parathyroid hormone (PTH) fragments detected by intact-PTH assays. European journal of endocrinology / European Federation of Endocrine Societies 147(1) :123-131.

Nielsen PK, Feldt-Rasmussen U, Olgaard K (1996). A direct effect in vitro of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells. Nephrol Dial Transplant 11(9) :1762-1768.

Nopakun J, Messer HH, Voller V (1989). Fluoride absorption from the gastrointestinal tract of rats. The Journal of nutrition 119(10) :1411-1417.

Okuda A, Kanehisa J, Heersche JN (1990). The effects of sodium fluoride on the resorptive activity of isolated osteoclasts. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 5 Suppl 1(S115-120.

Olympio KP, Bardal PA, Cardoso VE, Oliveira RC, Bastos JR, Buzalaf MA (2007). Low-fluoride dentifrices with reduced pH: fluoride concentration in whole saliva and bioavailability. Caries research 41(5) :365-370.

Ozawa A, Lindberg I, Roth B, Kroeze WK (2010). Deorphanization of novel peptides and their receptors. The AAPS journal 12(3) :378-384.

105

Pak CY, Sakhaee K, Piziak V, Peterson RD, Breslau NA, Boyd P et al. (1994). Slow- release sodium fluoride in the management of postmenopausal osteoporosis. A randomized controlled trial. Annals of internal medicine 120(8) :625-632.

Parnell C, Whelton H, O'Mullane D (2009). Water fluoridation. European archives of paediatric dentistry : official journal of the European Academy of Paediatric Dentistry 10(3) :141-148.

Pei J, Li B, Gao Y, Wei Y, Zhou L, Yao H et al. (2012). Fluoride decreased osteoclastic bone resorption through the inhibition of NFATc1 gene expression. Environmental toxicology .

Pessan JP, Toumba KJ, Buzalaf MA (2011). Topical use of fluorides for caries control. Monographs in oral science 22(115-132.

Pi M, Spurney RF, Tu Q, Hinson T, Quarles LD (2002). Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor. Endocrinology 143(10) :3830-3838.

Pizzo G, Piscopo MR, Pizzo I, Giuliana G (2007). Community water fluoridation and caries prevention: a critical review. Clinical oral investigations 11(3) :189-193.

Poll C, Kyrle P, Westwick J (1986). Activation of protein kinase C inhibits sodium fluoride-induced elevation of human platelet cytosolic free calcium and thromboxane B2 generation. Biochemical and biophysical research communications 136(1) :381- 389.

Pontigo-Loyola AP, Islas-Marquez A, Loyola-Rodriguez JP, Maupome G, Marquez- Corona ML, Medina-Solis CE (2008). Dental fluorosis in 12- and 15-year-olds at high altitudes in above-optimal fluoridated communities in Mexico. Journal of public health dentistry 68(3) :163-166.

Post GR, Brown JH (1996). G protein-coupled receptors and signaling pathways regulating growth responses. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 10(7) :741-749.

Potts JT (2005). Parathyroid hormone: past and present. The Journal of endocrinology 187(3) :311-325.

Quarles LD (2003). Extracellular calcium-sensing receptors in the parathyroid gland, kidney, and other tissues. Current opinion in nephrology and hypertension 12(4) :349-355.

Rao HV, Beliles RP, Whitford GM, Turner CH (1995). A physiologically based pharmacokinetic model for fluoride uptake by bone. Regulatory toxicology and pharmacology : RTP 22(1) :30-42.

106

Ray K, Hauschild BC, Steinbach PJ, Goldsmith PK, Hauache O, Spiegel AM (1999). Identification of the cysteine residues in the amino-terminal extracellular domain of the human Ca(2+) receptor critical for dimerization. Implications for function of monomeric Ca(2+) receptor. The Journal of biological chemistry 274(39) :27642- 27650.

Reed BY, Zerwekh JE, Antich PP, Pak CY (1993). Fluoride-stimulated [3H]thymidine uptake in a human osteoblastic osteosarcoma cell line is dependent on transforming growth factor beta. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 8(1) :19-25.

Richards A, Mosekilde L, Sogaard CH (1994). Normal age-related changes in fluoride content of vertebral trabecular bone--relation to bone quality. Bone 15(1) :21- 26.

Riggs BL, Hodgson SF, Hoffman DL, Kelly PJ, Johnson KA, Taves D (1980). Treatment of primary osteoporosis with fluoride and calcium. Clinical tolerance and fracture occurrence. JAMA : the journal of the American Medical Association 243(5) :446-449.

Riggs BL, Hodgson SF, O'Fallon WM, Chao EY, Wahner HW, Muhs JM et al. (1990). Effect of fluoride treatment on the fracture rate in postmenopausal women with osteoporosis. The New England journal of medicine 322(12) :802-809.

Riggs BL, O'Fallon WM, Lane A, Hodgson SF, Wahner HW, Muhs J et al. (1994). Clinical trial of fluoride therapy in postmenopausal osteoporotic women: extended observations and additional analysis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 9(2) :265-275.

Ritter CS, Brown AJ (2011). Direct suppression of Pth gene expression by the vitamin D prohormones doxercalciferol and calcidiol requires the vitamin D receptor. Journal of molecular endocrinology 46(2) :63-66.

Robinson C, Connell S, Kirkham J, Brookes SJ, Shore RC, Smith AM (2004). The effect of fluoride on the developing tooth. Caries research 38(3) :268-276.

Roussanne MC, Lieberherr M, Souberbielle JC, Sarfati E, Drueke T, Bourdeau A (2001). Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate. European journal of clinical investigation 31(7) :610-616.

Ruat M, Molliver ME, Snowman AM, Snyder SH (1995). Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals. Proceedings of the National Academy of Sciences of the United States of America 92(8) :3161-3165.

107

Rubin DA, Hellman P, Zon LI, Lobb CJ, Bergwitz C, Juppner H (1999). A G protein- coupled receptor from zebrafish is activated by human parathyroid hormone and not by human or teleost parathyroid hormone-related peptide. Implications for the evolutionary conservation of calcium-regulating peptide hormones. The Journal of biological chemistry 274(33) :23035-23042.

Sampaio FC, Levy SM (2011). Systemic fluoride. Monographs in oral science 22(133-145.

Sanchez-Fernandez MA, Gallois A, Riedl T, Jurdic P, Hoflack B (2008). Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling. PloS one 3(10) :e3537.

Santoyo-Sanchez MP, del Carmen Silva-Lucero M, Arreola-Mendoza L, Barbier OC (2013). Effects of acute sodium fluoride exposure on kidney function, water homeostasis, and renal handling of calcium and inorganic phosphate. Biol Trace Elem Res 152(3) :367-372.

Selz T, Caverzasio J, Bonjour JP (1991). Fluoride selectively stimulates Na- dependent phosphate transport in osteoblast-like cells. The American journal of physiology 260(6 Pt 1) :E833-838.

Shoback DM, McGhee JM (1988). Fluoride stimulates the accumulation of inositol phosphates, increases intracellular free calcium, and inhibits parathyroid hormone release in dispersed bovine parathyroid cells. Endocrinology 122(6) :2833-2839.

Silver J, Naveh-Many T (2012). FGF23 and the parathyroid. Advances in experimental medicine and biology 728(92-99.

Sogaard CH, Mosekilde L, Thomsen JS, Richards A, McOsker JE (1997). A comparison of the effects of two anabolic agents (fluoride and PTH) on ash density and bone strength assessed in an osteopenic rat model. Bone 20(5) :439-449.

Song I, Kim JH, Kim K, Jin HM, Youn BU, Kim N (2009). Regulatory mechanism of NFATc1 in RANKL-induced osteoclast activation. FEBS letters 583(14) :2435-2440.

Spak CJ, Berg U, Ekstrand J (1985). Renal clearance of fluoride in children and adolescents. Pediatrics 75(3) :575-579.

Steingrimsdottir L, Gunnarsson O, Indridason OS, Franzson L, Sigurdsson G (2005). Relationship between serum parathyroid hormone levels, vitamin D sufficiency, and calcium intake. JAMA : the journal of the American Medical Association 294(18) :2336-2341.

108

Sternweis PC, Gilman AG (1982). Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proceedings of the National Academy of Sciences of the United States of America 79(16) :4888-4891.

Stremski ES, Grande GA, Ling LJ (1992). Survival following hydrofluoric acid ingestion. Annals of emergency medicine 21(11) :1396-1399.

Strewler GJ, Stern PH, Jacobs JW, Eveloff J, Klein RF, Leung SC et al. (1987). Parathyroid hormonelike protein from human renal carcinoma cells. Structural and functional homology with parathyroid hormone. The Journal of clinical investigation 80(6) :1803-1807.

Strnad CF, Wong K (1985). Calcium mobilization in fluoride activated human neutrophils. Biochemical and biophysical research communications 133(1) :161-167.

Suva LJ, Winslow GA, Wettenhall RE, Hammonds RG, Moseley JM, Diefenbach- Jagger H et al. (1987). A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science 237(4817) :893-896.

Talmage RV, Roycroft JH, Anderson JJ (1975). Daily fluctuations in plasma calcium, phosphate, and their radionuclide concentrations in the rat. Calcified tissue research 17(2) :91-102.

Tanaka S, Miyazaki T, Fukuda A, Akiyama T, Kadono Y, Wakeyama H et al. (2006). Molecular mechanism of the life and death of the osteoclast. Annals of the New York Academy of Sciences 1068(180-186.

Tang XL, Wang Y, Li DL, Luo J, Liu MY (2012). Orphan G protein-coupled receptors (GPCRs): biological functions and potential drug targets. Acta pharmacologica Sinica 33(3) :363-371.

Taylor CW, Merritt JE, Putney JW, Jr., Rubin RP (1986). A guanine nucleotide- dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland. Biochemical and biophysical research communications 136(1) :362- 368.

Teotia M, Teotia SP, Singh KP (1998). Endemic chronic fluoride toxicity and dietary calcium deficiency interaction syndromes of metabolic bone disease and deformities in India: year 2000. Indian J Pediatr 65(3) :371-381.

Teotia SP, Teotia M (1973). Secondary hyperparathyroidism in patients with endemic skeletal fluorosis. Br Med J 1(5854) :637-640.

Thomas AB, Hashimoto H, Baylink DJ, Lau KH (1996). Fluoride at mitogenic concentrations increases the steady state phosphotyrosyl phosphorylation level of

109 cellular proteins in human bone cells. The Journal of clinical endocrinology and metabolism 81(7) :2570-2578.

Tohgo A, Choy EW, Gesty-Palmer D, Pierce KL, Laporte S, Oakley RH et al. (2003). The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. The Journal of biological chemistry 278(8) :6258-6267.

Torres PA, De Brauwere DP (2011). Three feedback loops precisely regulating serum phosphate concentration. Kidney international 80(5) :443-445.

Turner CH, Hsieh YF, Muller R, Bouxsein ML, Baylink DJ, Rosen CJ et al. (2000). Genetic regulation of cortical and trabecular bone strength and microstructure in inbred strains of mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 15(6) :1126-1131.

Turner CH, Hsieh YF, Muller R, Bouxsein ML, Rosen CJ, McCrann ME et al. (2001). Variation in bone biomechanical properties, microstructure, and density in BXH recombinant inbred mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 16(2) :206-213.

Usdin TB, Paciga M, Riordan T, Kuo J, Parmelee A, Petukova G et al. (2008). Tuberoinfundibular Peptide of 39 residues is required for germ cell development. Endocrinology 149(9) :4292-4300. van Biesen T, Luttrell LM, Hawes BE, Lefkowitz RJ (1996). Mitogenic signaling via G protein-coupled receptors. Endocrine reviews 17(6) :698-714.

Vardy E, Roth BL (2013). Conformational ensembles in GPCR activation. Cell 152(3) :385-386.

Vieira AP, Hancock R, Limeback H, Maia R, Grynpas MD (2004). Is fluoride concentration in dentin and enamel a good indicator of dental fluorosis? Journal of dental research 83(1) :76-80.

Vieira AP, Hancock R, Dumitriu M, Limeback H, Grynpas MD (2006). Fluoride's effect on human dentin ultrasound velocity (elastic modulus) and tubule size. European journal of oral sciences 114(1) :83-88.

Villa A, Anabalon M, Zohouri V, Maguire A, Franco AM, Rugg-Gunn A (2010). Relationships between fluoride intake, urinary fluoride excretion and fluoride retention in children and adults: an analysis of available data. Caries research 44(1) :60-68.

110

Vogel GL, Carey CM, Chow LC, Ekstrand J (1990). Fluoride analysis in nanoliter- and microliter-size fluid samples. J Dent Res 69 Spec No(522-528; discussion 556- 527.

Wada M, Furuya Y, Sakiyama J, Kobayashi N, Miyata S, Ishii H et al. (1997). The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. Control of parathyroid cell growth via a calcium receptor. The Journal of clinical investigation 100(12) :2977-2983.

Wada M, Nagano N, Furuya Y, Chin J, Nemeth EF, Fox J (2000). Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. Kidney international 57(1) :50-58.

Wang Y, Yin Y, Gilula LA, Wilson AJ (1994). Endemic fluorosis of the skeleton: radiographic features in 127 patients. AJR Am J Roentgenol 162(1) :93-98.

Ward DT, Brown EM, Harris HW (1998). Disulfide bonds in the extracellular calcium- polyvalent cation-sensing receptor correlate with dimer formation and its response to divalent cations in vitro. The Journal of biological chemistry 273(23) :14476-14483.

Ward DT, Riccardi D (2012). New concepts in calcium-sensing receptor pharmacology and signalling. British journal of pharmacology 165(1) :35-48.

Wergedal JE, Lau KH, Baylink DJ (1988). Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures. Clinical orthopaedics and related research 233) :274-282.

Wettschureck N, Lee E, Libutti SK, Offermanns S, Robey PG, Spiegel AM (2007). Parathyroid-specific double knockout of Gq and G11 alpha-subunits leads to a phenotype resembling germline knockout of the extracellular Ca2+ -sensing receptor. Molecular endocrinology 21(1) :274-280.

Whitford GM, Pashley DH, Stringer GI (1976). Fluoride renal clearance: a pH- dependent event. The American journal of physiology 230(2) :527-532.

Whitford GM, Pashley DH (1984). Fluoride absorption: the influence of gastric acidity. Calcified tissue international 36(3) :302-307.

Whitford GM (1994). Intake and metabolism of fluoride. Advances in dental research 8(1) :5-14.

Whitford GM (1996). The metabolism and toxicity of fluoride. Monographs in oral science 16 Rev 2(1-153.

Whitford GM (1997). Determinants and mechanisms of enamel fluorosis. Ciba Foundation symposium 205(226-241; discussion 241-225.

111

Whitford GM, Thomas JE, Adair SM (1999). Fluoride in whole saliva, parotid ductal saliva and plasma in children. Archives of oral biology 44(10) :785-788.

Whitford GM, Sampaio FC, Pinto CS, Maria AG, Cardoso VE, Buzalaf MA (2008). Pharmacokinetics of ingested fluoride: lack of effect of chemical compound. Archives of oral biology 53(11) :1037-1041.

Whyte MP, Totty WG, Lim VT, Whitford GM (2008). Skeletal fluorosis from instant tea. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 23(5) :759-769.

Wilson AC, Bawden JW (1991). Salivary fluoride concentrations in children with various systemic fluoride exposures. Pediatric dentistry 13(2) :103-105.

Wiren KM, Ivashkiv L, Ma P, Freeman MW, Potts JT, Jr., Kronenberg HM (1989). Mutations in signal sequence cleavage domain of preproparathyroid hormone alter protein translocation, signal sequence cleavage, and membrane-binding properties. Molecular endocrinology 3(2) :240-250.

Wongsurawat N, Armbrecht HJ (1987). Comparison of calcium effect on in vitro calcitonin and parathyroid hormone release by young and aged thyroparathyroid glands. Exp Gerontol 22(4) :263-269.

Wright JT, Chen SC, Hall KI, Yamauchi M, Bawden JW (1996). Protein characterization of fluorosed human enamel. Journal of dental research 75(12) :1936-1941.

Wu LW, Yoon HK, Baylink DJ, Graves LM, Lau KH (1997). Fluoride at mitogenic doses induces a sustained activation of p44mapk, but not p42mapk, in human TE85 osteosarcoma cells. The Journal of clinical endocrinology and metabolism 82(4) :1126-1135.

Xu H, Liu QY, Zhang JM, Zhang H, Li GS (2010). Elevation of PTH and PTHrp induced by excessive fluoride in rats on a calcium-deficient diet. Biological trace element research 137(1) :79-87.

Yamaguchi DT, Hahn TJ, Iida-Klein A, Kleeman CR, Muallem S (1987). Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels. The Journal of biological chemistry 262(16) :7711-7718.

Yan D, Gurumurthy A, Wright M, Pfeiler TW, Loboa EG, Everett ET (2007). Genetic background influences fluoride's effects on osteoclastogenesis. Bone 41(6) :1036- 1044.

112

Yan D, Willett TL, Gu X, Martinez-Mier EA, Sardone L, McShane L et al. (2011). Phenotypic Variation of Fluoride Responses between Inbred Strains of Mice. Cells Tissues Organs .

Yavropoulou MP, Yovos JG (2008). Osteoclastogenesis--current knowledge and future perspectives. Journal of musculoskeletal & neuronal interactions 8(3) :204- 216.

Yoder KM, Mabelya L, Robison VA, Dunipace AJ, Brizendine EJ, Stookey GK (1998). Severe dental fluorosis in a Tanzanian population consuming water with negligible fluoride concentration. Community dentistry and oral epidemiology 26(6) :382-393.

Yolken R, Konecny P, McCarthy P (1976). Acute fluoride poisoning. Pediatrics 58(1) :90-93.

Yoshihara A, Sakuma S, Kobayashi S, Miyazaki H (2001). Antimicrobial effect of fluoride mouthrinse on mutans streptococci and lactobacilli in saliva. Pediatric dentistry 23(2) :113-117.

Yuan JS, Reed A, Chen F, Stewart CN, Jr. (2006). Statistical analysis of real-time PCR data. BMC Bioinformatics 7(85.

Zero DT, Raubertas RF, Fu J, Pedersen AM, Hayes AL, Featherstone JD (1992). Fluoride concentrations in plaque, whole saliva, and ductal saliva after application of home-use topical fluorides [published eerratum appears in J Dent Res 1993 Jan;72(1):87]. Journal of dental research 71(11) :1768-1775.

Zerwekh JE, Morris AC, Padalino PK, Gottschalk F, Pak CY (1990). Fluoride rapidly and transiently raises intracellular calcium in human osteoblasts. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 5 Suppl 1(S131-136.

Zierold D, Chauviere M (2012). Hydrogen fluoride inhalation injury because of a fire suppression system. Mil Med 177(1) :108-112.

113