UNIVERSITY OF CALIFORNIA

Los Angeles

Toll like receptor 2 and Toll like receptor 4 activation

and vitamin D effects in mouse osteoblasts

A dissertation submitted in partial satisfaction of the

requirements for the degree Doctor of Philosophy in

Oral Biology

by

Wareeratn Chengprapakorn

2016

©Copyright by

Wareeratn Chengprapakorn

2016 ABSTRACT OF THE DISSERTATION

Toll-like receptor 2 and Toll-like receptor 4 activation

and vitamin D effects on mouse osteoblast

by

Wareeratn Chengprapakorn

Doctor of Philosophy in Oral Biology

University of California, Los Angeles, 2016

Professor Ichiro Nishimura, Chair

The effects of vitamin D on bone health have been widely described, but the precise direct actions of vitamin D on bone forming osteoblasts have yet to be fully elucidated. Vitamin D can act to promote innate immune responses. Non-classical vitamin D functions are described in monocytes and macrophages. Stress and damage signals, as well as infection, may influence bone function and fracture healing. The current PhD project hypothesizes that innate immune response to vitamin D contributes to its effects on bone. Mouse osteoblasts obtained from primary calvaria and MC3T3-E1 cell lines were used and differentiated for 5 days, 14 days and

21 days to represent different stages of osteoblast differentiation. Cells were treated with vitamin

D metabolites 25(OH)D3 and 1,25(OH)2D3 to observe vitamin D effects on differentiated osteoblasts. Vitamin D metabolites did not alter vitamin D metabolism genes Vdr and Cyp27b1

ii in differentiation osteoblasts. In addition, LPS as Toll-like receptor 4 agonist and Pam3CysK as

Toll-like receptor 2 agonist were stimulated in differentiated osteoblasts to evaluate osteoblastic responses. Western blot and RT-PCR analyses confirmed expression of TLR2 and TLR4 and

TLR4-specific signaling factors TRIF at all stages of differentiation. Mouse osteoblasts displayed divergent responses to LPS and Pam3CysK stimulations in osteoblast differentiation markers and osteoclastogenesis markers. Although both LPS and Pam3CysK promote inflammatory cytokines, only Pam3CysK stimulation demonstrated potent modulation to promote osteoclastogenesis through increased expression of Rankl and suppression of Ocn.

TLR2 and TLR4 share an MyD88 signaling pathway to induce inflammation and osteoclastogeneis, but only TLR4 can signal via the intracellular TRIF/TRAM pathway.

TrifsiRNA was introduced to 14 days differentiated MC3T3-E1 cells. RT-PCR analysis showed suppression of Vdr, Alp, Opg and Il-6 by TrifsiRNA. In addition, TLRs signaling activates

MAPK phosphorylation, Pam3CysK induced higher phosphorylation of p38 MAPK and inhibition of p38 by TLCK (NF-κB inhibitor) neutralized suppression of osteoblast differentiation markers by Pam3CysK. In conclusion, TLR2 and TLR4 stimulation showed differential effects in osteoblasts possibly due to distinct MAPK phosphorylation pattern.

iii

The dissertation of Wareeratn Chengprapakorn is approved.

John Adams

Sotritis Tetradis

Sanjay Mallya

Ichiro, Nishimura, Committee Chair

University of California, Los Angeles

2016

iv Table of contents

Introduction 1

Vitamin D and disease 1

Vitamin D metabolism and enzymes 13

Vitamin D and antimicrobial and antiviral effects 30

Pattern recognition receptors 32

TLRs signaling 33

Vitamin D and osteoblasts 40

Vitamin D and osteoclasts 43

Vitamin D and chondrocytes 44

Hypothesis 46

Chapter 1

Objective 47

Materials and methods 47

Results 49

Discussion 61

Chapter 2

Objective 66

Materials and methods 66

Results 69

Discussion 101

Chapter 3

Objective 108

Materials and methods 108

v Results 110

Discussion 130

Bibliography 132

vi Biographical Sketch

Name: Wareeratn Chengprapakorn

Institution and location Degree Year Field of study

Chulalongkorn University, Thailand DDS. 1998-2004 Dentistry

Positions and Employment

2004-2010 Dentist, Maxillofacial prosthodontics Unit, Faculty of Dentistry, Chulalongkorn

University, Bangkok, Thailand

2010-present Junior lecturer, Prosthodontics Department, Faculty of Dentistry, Chulalongkorn

University, Bangkok, Thailand

Poster presentations

1. W. Chengprapakorn (2008). “Bacterial Colonization of maxillofacial prostheses”.

International Society for Maxillofacial Rehabilitation 8th meeting. Bangkok, Thailand

2. W. Chengprapakorn (2010). “Quality of life in hemimandibulectomy patients in Faculty

of Dentistry, Chulalongkorn University: Subjective and Objective analysis”. International

Society for Maxillofacial Rehabilitation 9th meeting. Sestri Levante, Italy

3. W. Chengprapakorn. A.Garcia, R.. Chun, I. Nishimura, M. Hewison (2013). American

Society for Bone and Mineral Research. Baltimore, USA.

vii Introduction

Vitamin D and Disease

Vitamin D roles in bone remodeling

Bony deformities or Rickets disease has been described before 1645. Daniel Whistler(1), an English physician is credited with earliest description of rickets ; a childhood disease that effect the growth of long bone which manifests in bow legs in his thesis presentation to

University of Leyden. 4 years later, Francis Glisson, professor from Cambridge University fully described rickets in young children(2). 90% of death of children before the age of 4 in 18th century had rickets according to Schmorl’s investigation(3). Armand Trousseau placed puppies in a dark basement and found that these animal developed advanced rickets in 4-5 months. This led him to postulate that deficiency in diet and less exposure to sunlight were causative to rickets(4). Initially, researchers believed that substance in cod liver oil presumably vitamin A cured rickets. Mellanby suspected the assumption that fat-soluble vitamin A and anti-rachitic factor are the same was wrong because of his experiment in growing dogs. The fastest growing dogs has little fat-soluble vitamin A in their diets(3). Prophylaxis experiment in Vienna showed that cod-liver oil given to infants could prevent rickets which has high prevalence during winter time. Also children with rickets, after 2-4 weeks of cod-liver oil added in their milk improved calcium deposition in bone as seen in radiographs(5). Furthermore, therapeutic exposure to sunlight was also included in experiment. They found that children who were exposure to sunlight as well as given cod-liver oil showed healed more rapidly compared to only cod-liver diets only and sunlight exposure only(5). These results promoted ideas of radiation from sunlight influence the synthesis of this vitamin in animal or human body. Disease in adults with similar symptoms of rickets was reported as osteomalacia. Disease was characterized by waddling gait, pain on body movement and pain of ribs on compression of thorax. They were treated with high

1 dose of cod-liver oil and showed similar improvement in bone function as in patients with rickets(5). However, not until 1922 that McCollum and co-worker found that substance found in certain fat was differed from fat-soluble vitamin A by oxidized cod-liver oil and showed that it lost its anti-xerophthalmic function but retained its calcium deposition function in bone. This factor has specific property to regulate metabolism of bones and it was called Vitamin D(6) since it was the fourth vitamin to be named.

Some rickets patients do not have vitamin D deficiency but instead dietary calcium are low and these group of patients have been found to have vitamin D receptor (VDR) polymorphism. FF allele was more frequently found in rachitic group in Nigerian population(7).

Hereditary vitamin D-dependent Rickets (HVDDR) or Vitamin D-dependent rickets type I, a rare autosomal-recessive disease is characterized by genetic defect in 1α-hydroxylase enzyme that convert 25 hydroxyvitamin D3 into 1,25 dihydroxyvitamin D3 active form of vitamin D(8). These patients exhibited low to undetectable levels of circulation 1,25(OH)2D3 level, early onset of hypocalcemia, hypophosphatemia, elevated serum alkaline phosphatase level and severe rachitic bone lesions. Familial hypophosphatemic osteomalacia also showed similar biochemistry findings with HVDDR. Vitamin D-dependent rickets type II (VDDR II), X-linked dominant disorder is characterized by similar symptoms of classic rickets. These patients have normal or elevated 1,25(OH)2D3 levels with absence of gastrointestinal malabsorption, renal or hepatic disease(9). Study from epidermal keratinocytes and dermal fibroblasts from patient with Vitamin

D dependent rickets type II displayed lack of binding to protein receptors(10). Target ablation of

VDR at second zinc finger of VDR’s DNA-binding domain in mice exhibited VDDR II characteristics(11) confirmed the defected VDR in patients with VDDR II.

In 1934, hypervitaminosis D rickets was reported. Long bones of rats feeding with overdose vitamin D were found to be much shorter than controlled group. Experimental group

2 showed thin epiphyseal plate, increased number of trabeculae and also large quantity of osteoid(12). With this experiment, they concluded that large amount of vitamin D inhibited normal calcification process in bone and vitamin D acts as intermediary in controlling serum calcium in parathyroid mechanism. Some patients in 1992 exhibited conditions of vitamin D intoxication. They were related with uncontrolled fortified vitamin D milk intakes(13).

Studies support the role of vitamin D as prohormone more than vitamin. Vitamin D3 is produced in skin of vertebrates and found in some fatty fish such as salmon (14). Vitamin D2 is found in plant sources such as plankton and mushroom. Source of vitamin D in diets are limited.

Therefore, fortification of vitamin D was introduced. Since 1930s, milk has been fortified with vitamin D in United States to prevent rickets. Fortified cereals and juices are also important sources of vitamin D(15).

Classical function of vitamin D acts by supplying calcium and phosphate to the calcification site. In 1984, Underwood showed that vitamin D is not directly responsible for bone mineralization by injecting calcium and phosphate solution in rats with severe vitamin D deficiency compared with injecting with vitamin D. The result showed that animals infused with calcium and phosphate had higher bone mineral concentration than vitamin D injection groups(16). Elegant experiment in 1987 by Clements, Johnson and Fraser showed Half-life of

25-hydroxyvitamin D in serum has linear relationship with log dietary calcium concentration.

For instance, half-life of 25(OH)D3 decreased with deprivation of calcium(17). Decreased

25(OH)D3 was utilized and converted to 1,25(OH)2D3. Also injection of 1,25(OH)2D3 in rats showed decreased in half-life of serum 25(OH)D3. These results suggested negative feedback control of plasma 25(OH)D3 level.

Osteoporosis defines as low bone mineral density associated with increased risk of fracture of vertebrae, hip, proximal femur(18) and distal forearm (Colles’ fracture). After the age

3 of 50, the incidence of hip fractures increased with age(19). This condition is prevalence in elderly female. In third decade of life, Bone mass reaches its peak density(20). Earlier guideline recommended bone mass compared to value of young adult man as diagnosis for osteoporosis. A value of more than 2.5SD below the young adult mean value suggests osteoporosis and more than 2.5SD suggests severe osteoporosis(21). Currently, bone mineral density and vertebrae imaging are diagnostic tools for osteoporosis. Osteporosis was categorized into type I

Postmenopausal osteoporosis and type II senile osteoporosis(22).

Type I osteoporosis occurs more common in women associated with fracture of vertebrae and distal forearms with some association of increased tooth loss. These patients showed decreased in secretion of parathyroid hormone, increased secretion of calcitonin and defect in 25-OHase activity that leads to decreased in calcium absorption(22). The rate of bone loss in trabecular bone is higher than cortical bone(23). Even though all post-menopausal women have estrogen deficiency, but not everyone exhibit type I osteoporosis(24) indicated that this condition is multifactorial disease. Type II osteoporosis is presented in both men and women and manifested with hip and vertebral fractures including other bones such as proximal humerus, proximal tibia and pelvis. This type showed proportionate loss of cortical and trabecular bone(25). Defect in calcium absorption results in hormonal adaptation and increase calcium resorption from bone and higher risk of osteoporosis.

Vitamin D receptor gene (VDR) polymorphisms of RFLP (Restriction fragment length polymorphisms detected by BsmI, EcoRV, and ApaI) markers are highly correlated with serum osteocalcin that related to bone density(26, 27). In Mexican-American women group, FokI polymorphism of VDR gene with ff alleles was found to correlate with significantly decreased bone mineral density(28). Secondary parathyroidism and increased bone turnover were found in

4 elderly women with serum 25(OH)D3 less than 30 nmol/l(29). In addition, women with osteoporosis often present with low serum 25(OH)D3(30).

Because of the strong relationship between vitamin D and bone mineral density, vitamin

D had been prescribed for patient with bone disease or with other osteoporosis drugs to balance calcium level in blood. 25 hydroxyvitamin D3 was used as a treatment for Rickets. It was found that this metabolite is far more potent and act more rapidly in stimulating calcium absorption in intestine(31). Studies with vitamin D supplementation showed that patients who had serum

25(OH)D3 levels between 71-90 nmol/l significantly found lower risks of fracture rates(32, 33).

Hence, optimal serum 25(OH)D3 was proposed to be higher than 75 nmol/l to lower risk of fracture(34). Nevertheless, recent Meta-analysis study showed vitamin D supplementation alone does not alter risk of having hip fracture incidence only when combine with calcium can help reduce the risk and suggested that using vitamin D supplementation for osteoporosis prevention in patients without vitamin D deficiency is inappropriate (35, 36).

In attempt to prevent osteoporosis, calcium intake of 1000 mg per day for men 50-70 years old and 1200 mg per day for women over 51 and men over 71 and also vitamin D intake

(800-1000 IU/day) if necessary are recommended(37). Current FDA-approved treatment for osteoporosis according to 2014 National Osteoporosis Foundation includes bisphosphonates, calcitonin, estrogen agonist/antagonist, estrogen or hormone therapy, tissue-selective estrogen complex, parathyroid hormone, RANKL inhibitor.

Vitamin D role in liver

In hepatic insufficiencies, osteodystrophy and higher requirements for vitamin D are observed. Vitamin D deficiency is also found in majority of patients (92%) with chronic liver disease especially in African-American females(38). Recent meta-analysis also showed the

5 relationship of low vitamin D in serum level and non-alcoholic fatty liver disease(39). VDR expression in hepatocytes is also negatively correlated with severity liver diseases such as steatohepatitis or chronic hepatitis C(40).

25 hydroxyvitamin D3 is produced in hepatic hydroxylation system. The conversion site takes place in liver microsomes and requires molecular oxygen. Hepatectomized rat by vascular

3 3 disconnection of liver could not convert vitamin D3- H into 25-hydroxyvitamin D- H(41). This early study shows 25-hydroxylase acts in liver. Treatment of anticonvulsant pheobarbitone to induced hepatic cytochrome P-450 enzymes associated with decreased plasma 25(OH)D3 level(17). Nevertheless, gene encoding this enzyme responsible for converting vitamin D to 25- hydroxyvitamin D3 has not been found until 30 years later. Cytochrome P450 was believed to act in hepatic drug detoxication system. Cytochrome P450 was named after its pigment peak at 450 nm spectra when reduced and bound to carbon monoxide. In 1984, with the advance in mRNA purification, the first cytochrome P450 was cloned from C57BL/6N mouse liver(42). Several cytochrome enzymes that expressed in liver were suggested as candidates for 25-hydroxylase gene. However, evolutionary conserved microsomal cytochrome P450 (CYP2R1) was identified with vitamin D 25-hydroxylase activity based on enzyme’s biochemical properties, conservation and expression pattern(43). Blood was drawn from patients who exhibit classic symptoms of vitamin D deficiency and analyzed. Transition mutation at exon of CYP2R1 gene was found in this patient. Cells transfected with this mutation plasmid and incubated with radiolabelled substrate, did not produce 25-hydroxyvitamin D3 when compared to controlled cells(44). These experiment confirmed that CYP2R1 encoded 25-hydroxylase in liver and play a major role in vitamin D metabolism.

Vitamin D role in kidney

6 In chronic renal failure, osteodystrophy change similar to osteomalacia of vitamin D deficiency was developed. In addition, hyperparathyroidism occurs in most patients with progression of chronic kidney disease (CKD). Low 1,25 dihydroxyvitamin D3 was found in chronic renal failure at all estimated glomerular filtration rate (eGFR)(45). Study showed that

97% of patients who undergone maintenance hemodialysis showed suboptimal level (<30 ng/ml) of serum 25 hydroxyvitamin D3(46). Attempt was made to treat patient with chronic kidney disease with various metabolites of vitamin D. However, 1,25 dihydroxyvitamin D3 was found to be the most potent metabolites in restoring serum calcium levels(47). Current guidelines for patients in stage 3 and 4 chronic kidney disease indicate incorporation of activated vitamin D treatment to maximize inhibitory effect of parathyroid gland growth and reduce PTH levels.

Meta-analysis study result of vitamin D compound treatment in patients with chronic kidney disease is inconclusive. Vitamin D compound treatment resulted in increased risks of hypercalcemia and hyperphosphatamia but did not reduce parathyroid hormone levels while newer vitamin D analogues reduced PTH levels however still associated with hypercalcemia but not hyperphosphatamia(48).

Experiment by Fraser and Kodicek in 1970 showed that small intestine and blood plasma

3 of nephrectomized rats that were given with H-cholecalciferol (25(OH)D3) found no trace of

1,25(OH)2D3 compared with control group(49). In addition, nephrectomy prevents 1,25(OH)2D3 appearance in intestine(50). In vitro kidney homogenates showed identical metabolites to that found in intestine after administration of 25 hydroxyvitamin D3(50). These early studies indicate that kidney is important for vitamin D conversion to active form(51). The enzyme locates in mitochondria and requires molecular oxygen for 1-hydroxylation. Enzyme involves in vitamin D conversion is 1α-hydroxylase (1α-OHase). Polyclonal antibody of 1α-OHase showed distribution of this enzyme in proximal tubules, distal tubules and collecting ducts of kidney(52).

7 Renal reabsorption of vitamin D involves endocytic receptors in renal proximal tubule.

Megalin and Cubilin are colocalized in the apical endocytic apparatus(53). These proteins are responsible for protein reabsorption in kidney and act in synergy(54). Megalin knockout mice have normal glomerular filtration but showed urine DBP (vitamin D binding protein). Megalin has high affinity to DBP and responsible for internalized of DBP/vitamin D3 complexes in proximal convoluted tubules of kidney(55). Kidney specific-megalin knockout mice excrete significant amount of 25-OH vitamin D3 correlated with reduction in vitamin D level in plasma.

(56). As a consequence, these mice exhibit hypocalcemia and osteomalacia. Renal uptake of vitamin D is central regulatory step in vitamin D and calcium homeostasis.

In kidney of rachitic chick and rats, 1,25(OH)2D3 has high affinity binding to cytosolic fraction(57, 58). These early studies suggested VDR response genes in kidney. 1,25(OH)2D3, calcitonin and PTH were found to regulate calcium absorption in distal nephron. This indicates autocrine and paracrine function of vitamin D in kidney(59-61).

Vitamin D conversion in kidney is regulated by rate of parathyroid hormone secretion and calcitonin levels and not by direct plasma calcium level(62). PTH stimulates 1-hydroxylase activity while 1,25(OH)2D3 production inihibits enzyme activity. In chick kidney cell culture,

1,25(OH)2D3 treatment stimulate the 25-OHase(63).

1,25 dihydroxyvitamin D3 can also repress the renin-angiotensin system and protect cardiovascular system independent of extracellular calcium or phosphorus in 1α- hydroxylase knockout mice compared with wildtype mice that developed hypertension, impaired cardiac function and upregulation of renin-angiotensin system(64).

Not only vitamin D is converted in to active form in kidney, but enzyme responsible in neutralization of vitamin D, 24-hydroxylase also regulated in kidney. In nephrectomized rat, serum 24,25(OH)2D3 was not detected indicates that kidney is a major organ for 24-

8 hydroxylase(65). 24-hydroxylase mRNA in kidney but not in intestine down-regulated with parathyroid hormone(66). 1,25(OH)2D3 also stimulates 24-OHase activity by induction of mRNA(67).

Vitamin D role in intestine

Patients who suffered from Rickets or hyperparathyroidism caused by defective in VDR can be prevented by high calcium intake(68). Infusion of calcium to maintain calcium and phosphate levels in vitamin D deficient rats showed similar bony phenotypes as normal control mice(69). This suggests that intestinal calcium absorption is important for vitamin D role in calcium homeostasis and primary target of vitamin D in bone remodeling located in intestinal rather than in bone or parathyroid gland.

Calcium absorption in intestine has been advocated by saturable and nonsaturable process. The saturable calcium absorption occurs involve calcium entry, transcellular movement of calcium and calcium extrusion. For nonsaturable process, calcium can also diffused within intercellular spaces(70).

In 1968, Calcium binding protein in intestine was discovered by administration of vitamin D to rachitic chicks that leads to formation of new protein that binds to Calcium(71) and involve in calcium transport system(72). Calcium-binding protein concentration in duodenal mucosa is directly correlated with ability of animal to absorb Calcium. Ca2+-binding protein concentration in duodenum is more than in jejunum and ileum(73). In rat’s upper intestine, vitamin D regulated intracellular absorption of calcium by rate limiting steps. This step becomes significant when calcium intakes are low and in absence of vitamin D, only half of ingested calcium can be absorbed(74). Later this cytosolic protein was named Calbindin. Calbindin-D9K is a major mammalian intracellular calcium binding protein. The relationship between calcium

9 absorption efficiency and intestinal Calbindin-D9K is normally in linear relationship(75).

Calbidin possibly facilitates cytosolic diffusion of calcium. Calbindin-D9K mRNA levels reduced in VDR null mice’s intestines compared to wildtype(76).

Others suggested that epithelial calcium channel and calcium transport protein are involved in vitamin D-dependent intestinal calcium absorption. Two intestinal calcium channels

ECaC (epithelial calcium channel) and CaT1 (calcium transport protein type 1) encoded by TRP-

V genes (TRPV5 and TRPV6) express in duodenum and mRNA levels are both strongly VDR- dependent(77). The mRNA expression of ECaC and CaT1 were also down-regulated in several strain of VDR knockout mice(78). High calcium diet reduced ECaC mRNA levels. These channels are suggested to constitutively open and Ca2+ influx is regulated by number of channels.

After absorption, plasma membrane calcium ATPase (PMCA1b) at basolateral of enterocyte is responsible in calcium extrusion into circulation. This process is also vitamin D dependent. Injection of 1,25 dihydroxyvitamin D3 intravenously in chicken with vitamin D deficiency increased mRNA expression of duodenal PMCA(79). Studies showed that low dietary calcium and 1,25(OH)2D3 stimulate calcium pumps expression.

Calcium can also be absorbed via paracellular pathway. This process is nonsaturable process through tight junctions and intercellular spaces. Most calcium is absorbed in ileum.

Claudins have been identified as important component of tight junction complexes for selective ions. These proteins are found in small intestine to large intestine. For example, Cld-8 are increasingly expressed from duodenum to distal colon(80). Studies implicated that Claudin-2 and

Claudin-12 are VDR response genes. VDR knockout mice were found to have down-regulated

Cldn-2 and Cldn-12. In human cell line; Caco-2, 1,25(OH)2D3 intervention increased Cldn-2 and

10 Cldn-12 proteins in time and dose dependent manners(81). These evidences suggested that paracellular calcium absorption in intestine also regulated by vitamin D.

Crohn’s disease is inflammatory bowel disease causes by inflammation of lining cells in digestive tract. Commonly, Crohn’s disease affects ileum and the beginning of colon.

Extraintestinal manifestations of Crohn’s disease can involve in musculoskeletal, dermatologic, hepatopancreatobiliary, renal and pulmonary systems. Musculoskeletal complication is the most common one and occurs in 9-53% of patients with inflammatory bowel disease(82). Prevalence of osteoporosis is increased in patient with Crohn’s disease(83). Reduced serum 25- hydroxyvitamin D3 in Crohn’s disease have been reported and related to disease activity(84-86).

Crohn’s disease patients who received vitamin D supplement and had normal serum 25- hydroxyvitamin D levels had more prevalent in increased in bone mineral density compared with patient who did not receive supplement(87). Moreover, VDR polymorphisms are associated with susceptibility of Crohn’s disease more than patient with ulcerative colitis(88). Recent study showed that 1,25(OH)2D3 induced expression of genes encoding antimicrobial peptide defensin

β2 (DEFB2/HBD2) in macrophages. This induction was absent in macrophage from patients with Crohn’s disease(89).

Vitamin D and Parathyroid hormone

There are two distinct types of hyperparathyroidism, Primary and secondary hyperparathyroidism. Primary hyperparathyroidism is characterized enlargement of parathyroid gland and cause overproduction of parathyroid hormone which leads to hypercalcemia.

Secondary hyperparathyroidism is the condition causes by another disease that result in low plasma calcium such as calcium or vitamin D deficiency and chronic renal failure(90).

11 Parathyroid hormone (PTH) is secreted in response to decreasing in blood calcium or vitamin D levels. PTH stimulates formation of 1,25-hydroxylase enzyme in kidney. In chicken with vitamin D deficient diet, their parathyroid glands were significantly enlarged after 14 days of diet(91). Within 2 hours of 1,25(OH)2D3 administration, this steroid is accumulated in parathyroid gland of vitamin D-deficient chicks(92). These results implied interaction of

1,25(OH)2D3 and parathyroid gland that effect the change in parathyroid hormone level. Vitamin

D receptor was found in parathyroid gland and recognized to localize in to nucleus after binding with 1,25(OH)2D3 (93, 94).

Rats feeding with high phosphate diet decreased half-life of serum 25(OH)D3, however, after parathyroidectomy, half-life of serum 25(OH)D3 was restored(17). Rats also subcutanously infusing with 1,25(OH)2D3 caused increased in biliary excretion of 25(OH)D3 and decreased in half-life of 25(OH)D3 in serum. This showed PTH increased degradation of 25(OH)D3. 1α- hydroxylase activity in Vitamin D-depleted chicks after parathyroidectomy, was decreased similar to the level of vitamin-D treated group(95). 1α-hydroxylase promoter luciferase reporter gene construct transfected in pig’s kidney cell line showed direct dose dependent stimulation of promoter activity by Parathyroid hormone(96). Disappearance of parathyroid hormone from circulation results in reduction of renal 1α -hydroxylase activity(91).

1,25(OH)2D3 dose dependently suppressed production of parathyroid hormone from bovine parathyroid cells. This suppression is reversible in absence of 1,25(OH)2D3(97).

Furthermore, 1,25(OH)2D3 was also inhibit mitoses of parathyroid cells. Injection of

1,25(OH)2D3 in nephrectomized rat on the day of operation prevented parathyroid hyperplasia(98). In rats treated with 1,25(OH)2D3 their parathyroid glands were dissected and found that mRNA of parathyroid hormone was increased(99).

12 Increased in intestinal calcium absorption is found in patients with hyperparathyroidism.

Calcium-sensing receptors mRNAs are reduced in parathyroid glands of type I and type II hyperparathyroidism(100). This agreed with less efficient in control of PTH secretion in response to plasma calcium level in diseased patients. Mutations of Calcium sensing receptor genes were found to responsible for familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism(101). Serum PTH was negatively correlated with serum 25(OH)D3.

However, calcium absorption was not correlated with serum PTH and serum 25(OH)D3(102).

Injection of parathyroid hormone daily to osteoporosis patients was found to exerted anabolic effects on bone. Synthetic fragment of parathyroid (hPTH 1-34) was given to patients, positive in calcium levels and increased in bone formation was found in osteoporosis patients(103, 104). Teriparatide (rhPTH) has been used to increase bone mineral density and reduce vertebral fractures in women. Randomized clinical trials showed that teriparatide is more useful than alendronate for osteoporosis therapy(105, 106).

Vitamin D metabolism and Enzymes

Vitamin D exists in 2 main forms, Vitamin D2 (Ergocalciferol) and Vitamin D3

(Cholecalciferol). Vitamin D2 is obtained mainly from plant. Vitamin D3 is attained from oily fish, egg yolk and also produced in skin by exposure to sunlight.

Vitamin D3 is lipophilic molecule and require protein carrier for solubility in plasma. In human, Vitamin D3 starts metabolized in skin from 7-dehydroxycholesterol upon exposure to

UV-light(107). As a lipophilic molecule, Vitamin D, then bound to Vitamin D binding protein

(DBP) or albumin as chylomicron and transport in the plasma to peripheral tissues such as muscle and adipose tissue. The remaining Vitamin D uptakes by liver and get converted to

25(OH)D3 by 25-hydroxylases (microsomal cytochrome P450 enzyme, CYP2R1 or

13 mitochondrial cytochrome P450, CYP27A1). Subsequently, another cytochrome P450 enzyme,

CYP27B1, converts 25(OH)D3 to active form of vitamin D 1,25(OH)2D3 in proximal tubule of the kidneys. 1,25(OH)2D3 and 25(OH)D3 bind to α-globulin and Vitamin D binding protein

(DBP). Other proteins in plasma can also bind to vitamin D such as albumin. DBP binds to

Megalin, surface receptors of proximal tubular epithelium, the complex then endocytosis and broken down via lysosomal degradation inside the cells(108). 25(OH)D3 is released inside cytoplasm and converted to 1,25(OH)2D3 by 1α-hydroxylase. 1,25(OH)2D3 then releases into interstitial fluid and binds to DBP to transport throughout body. Active form of vitamin D

(1,25(OH)2D3) binds to Vitamin D receptor (VDR) which heterodimerize with retinoid receptor

(RXR) and can bind to Vitamin D response element (VDRE) in promoter region of target genes.

VDR is a member of ligand-activated receptor superfamily. This family includes receptors for thyroid hormone, vitamin A derivatives and steroid hormone such as glucocorticoids, progesterone, estrogen, androgen and mineralocorticoids(109). Vitamin D is located in chromosome 12(110). cDNA of human VDR isolated from human intestine showed that VDR is 4605 bp cdNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame and 3209 bp of 3’ noncoding sequence(111). VDR contains 2 zinc finger motifs serve as conserved DNA binding domain. VDR is predominantly nuclear protein even in unoccupied state according to in situ immunocytochemical localization studies(112, 113). VDR mediate the action of 1,25(OH)2D3 by controlling action of this hormone sensitive genes.

Mutation at zinc finger regions result in inactivation of 1,25(OH)2D3 binding to the VDR and leads to Hereditary vitamin D resistant rickets. Unliganded VDR in nucleus is proposed to bind to corepressors related to Silencing Mediator for Retinor and Thyroid hormone receptor (SMRT) or nuclear receptor co-repressor (NCoR)(114, 115). These corepressors involve in formation of multicomponent repressor complexes that contain histone deacetylases (HDACs) and cause

14 deactivation of transcription by chromatin compaction(116, 117). After VDR heterdimerizes with RXR, VDR-RXR complexes allow release of corepressors and recruit of coactivators for chromatin decompaction and initiation of transcription. Then VDR-RXR complexes bind to short

DNA sequences called Vitamin D response elements (VDREs) to exert the influence of

1,25(OH)2D3 on transcription. ChIP-chip analysis showed that RXR bind to DNA more than when activate with 1,25(OH)2D3. Furthermore, most of the binding sites were bound by RXR before 1,25(OH)2D3 activation and VDR bind to the sites(118). VDR-RXR can recruit both corepressors and coactivators in agonist-dependent manner, it was postulated that transcription activation by 1,25(OH)2D3 may be modulated by relative levels of corepressors and coactivators in different target cells(119). Binding affinity of VDR is 500 fold higher with 1,25(OH)2D3 than in 25(OH)D3(120). Several VDREs were found in promoter area of VDR target genes(121).

ChIP-seq analysis of VDR in lymphoblastoid cell lines (LCLs) identified 2776 genomic positions occupied by VDR and 229 genes expression changes in response to vitamin D.

According to data from GWAS (genome-wide association studies), VDR occupancies significantly enriched near autoimmune and cancer associated genes(122). Amount of VDR

+ binding in CD4 cells correlated with serum 25(OH)D3 level. Enriched VDR binding was found near genes associated with T-regulatory and T-helper cells in patient with sufficient vitamin D levels (25(OH)D3 ≥75 nml/L)(123). 1,25(OH)2D3 autoregulates VDR expression through 3 introns downstream of transcription start site that co-localizes with RXR and enhances entry of

RNA polymerase II(124). This indicates distant regulatory sites regulate VDR transcription.

VDR has been found in many cells. VDR was detected in intestine since 1975. VDR is localized in intestinal epithelial cells but not in goblet cells or lymphatic tissue of intestine(125).

VDR is widely expressed in kidney as well, especially in distal renal tubules and collecting ducts(126). Inconsistency of VDR expression in liver was reported. Evidence suggested

15 extremely low levels of VDR in hepatocytes(127). VDR can be detected in osteoblast and chondrocytes. Outer hair shafts and sebaceous glands of epidermis are widely expressed with

VDR(128). Anterior and posterior pituitary gland, parathyroid gland and breast are also found with VDR expression(128). In immune cells, activated T-lymphocytes have been shown with

VDR. CD8- T lymphocytes had highest concentration of VDR while CD4-T lymphocytes had relatively low level of VDR(129). B-lymphocytes were not found with VDR expression.

Monocytes and macrophages under pathologic condition showed greater VDR expression levels compared with normal group(130, 131). Dendritic cells are constitutively express VDR(132).

Enhanced osteoblastic VDR levels by using Osteocalcin promoter overexpression of human VDR in C57Bl6 mice suggesting that osteoblastic VDR mainly regulates local activities of bone remodeling and do not alter systemic Calcium or Phosphate levels(133). Both cortical and trabecular bone of VDR transgenic mice were greater than wild type mice. Adding

1,25(OH)2D3 these mice elevated VDR expression in both cuboidal and flattened osteoblast(134). Others suggested that overexpression of vdr in mouse osteoblasts without treating with 1,25(OH)2D3 caused reduction of RANKL and OPG in long bone and resulted in suppression of osteoclastogenesis(135).

CYP24A1 encoding 24-hydroxylase is responsible for initiation of inactivation of vitamin D metabolites. This enzyme is also tightly regulated(136). CYP24A1 originally was known to function as 25(OH)D3 convertor to 24,25(OH)2D3(137, 138). 24-hydroxylase was found to be mitochondrial cytochrome P450-containing enzyme and localize in renal mitochondria of chicken. This enzyme is inhibited by its own products 24,25(OH)2D3 and also

1,25(OH)2D3(138, 139). This attenuation effect is to help prevent vitamin D toxicity. mRNA of

24OHase was abundant in proximal convoluted tubules of kidney(126). CYP24A1 is also found to express in other tissue responsible in classical action of vitamin D such as bone and intestine.

16 Later in 1970s, it was found that 24 hydroxylase can also hydroxylase 1,25(OH)2D3 into

1,24,25(OH)3D3 from rat’s kidney homogenates(140). Inaddition, 1,25(OH)2D3 also found to be preferred substrate for 24-hydroxylase(141). Study from cyp24a1 knockout mice showed that this enzyme is responsible for hydroxylation of 1,25(OH)2D3 at C-23 and C-26(142). CYP24A1 promoter region contains VDREs, hence CYP24A1 is also regulated by vitamin D(143).

Parathyroid hormone suppresses CYP24A1 in kidney proximal tubule cells and intestine(66,

144). Serum 1,25(OH)2D3 levels were found to negatively correlated with renal CYP24A1 mRNA level(145). A rare genetic disease, idiopathic infantile hypercalcemia was found to have mutation in CYP24A1 resulted in undetectable level of 24,25(OH)2D3 in system(146).

Characteristics of this syndrome include hypercalcemia, failure to thrive, elfin facial apprearance, mental and motor retardation, supravulvular aortic stenosis, nephrocalcinosis and hypercholesterolemia(147).

Main organ that responsible in producing 1,25(OH)2D3 for vitamin D endocrine system is kidney. This metabolite is the most important biologically active form of vitamin D. CYP27B1 was found over-induced in kidney of mice lacking Vitamin D receptor(148). CYP27B1 expression is regulated by PTH, calcitonin and 1,25(OH)2D3. Rats was given PTH, 1,25(OH)2D3 and calcitonin then kidneys were extracted and electrophoresed with 1α-hydroxylase probe.

Results showed that PTH and calcitonin induced expression of cyp27b1 gene while 1,25(OH)2D3 suppressed the gene expression(148). CYP27B1 promoter is upregulated by PTH through cAMP-dependent mechanism and inhibit by activation of VDR and binding of VDR to negative vitamin D response element. Study shows that transcription control of CYP27B1 gene promoter regulated by dual epigenetic. Liganded VDR bind to VDR-interacting repressor (VDIR) which directly bind to negative Vitamin D response element (1αnVDRE) at CYP27B1 promoter region and switch from co-activator complex containing histone acetyltransferase (HAT) to histone

17 deactylase (HDAC) and repress the CYP27B1 gene expression(149). In addition, 1,25(OH)2D3 induces DNA methylation at CpGsites in the promoter region of CYP27B1 gene(150). CYP27B1 is also known to have alternative splicing. Short hairpin RNA to inhibit intron 2 of CYP27V1 was introduced to HKC-8 cells (human proximal tubule cells) and found to significantly increased CYP27V1 protein expression. This indicated that 1,25(OH)2D3 synthesis by 1α- hydroxylase may regulated by non-coding splice variant of CYP27B1(151).

Polymorphism of CYP27B1 promoter was found to associate with endocrine autoimmune disease such as Addison’s disease, Hashimoto’s thyroiditis, Graves’ disease and diabetes mellitus type 1 in German population(152). Single nucleotide polymorphism (SNP) in

CYP27B1 was associated with congestive heart failure(153). Patients with VDDR-I were found to have R453C and P474R mutations(154). These proteins are responsible for protein activity.

Extra-renal expression of 1α- hydroxylase has been found in basal keratinocytes and hair follicles in skin, granulomata in lymph nodes, epithelial cells and parasympathetic ganglia in colon, pancreas, adrenal medulla, brain and placenta (155). In 1981, a case-report from sarcoidosis patients with anephric showed extra-renal generation of 1,25(OH)2D3(156). Local production of 1,25(OH)2D3 by osteoblast and osteoclast have been advocated for paracrine function of vitamin D in promoting maturation of bone cells. 25(OH)D3 and also 1,25(OH)2D3 were shown to inhibit osteoblast proliferation and promote mineralization(157). Osteocyte cell line; MLO-A5 showed increased in expression of osteocalcin and opg/rankl ratio after incubated with 1,25(OH)2D3(158).

Knockout and transgenic mice: VDR, CYP27B1, CYP24A1

VDR null mice were created by 2 independent studies. The first one by targeting exon 2 of VDR gene which encodes zinc finger and the other one targeting exon 3 which encodes second zinc finger of VDR(11, 76). Even though, VDR expression is found abundantly during

18 embryonic development, VDR null mice are found without any major developmental impairment. VDR knockout mice were normal before weaning(76) but increasing in osteoid was found after 7 weeks old. More than 85% of VDR null mice’ bone surface covered by osteoid at

70 days of age(159). These mice have low bone mass with hypocalcemia, hypophosphatemia, hyperparathyroidism, elevated 1,25(OH)2D3 and extremely low 24,25(OH)2D3 level. Alkaline phosphatase positive osteoblasts were weaker and sparse in VDR knockout mice.

Hyperparathyroidism with increased in size of parathyroid gland were found in these mice. The bone stiffness of VDR null mice was decreased by 7-fold and increased in ultimate deformation compared with wildtype bone(159). However, when VDR null mice were fed with rescue diet of calcium, phosphorus and lactose balances before development of osteomalacia, no increased of osteoid was found(159). Increased in number of osteoblastic cells were found but number of osteoclasts appeared to be normal. Mouse osteoblasts require vdr to form osteoclasts from either

WT or VDR nulled spleen cells(160). There was increased in hypertrophic chondrocytes in the growth plates around 15 days of age. Unlike animal with vitamin D deficiency, around 4 weeks of age these animals also develop alopecia similar to patients with VDDRII. Furthermore, female homozygous VDR null mice appeared infertile from impaired folliculogenesis of ovaries but male reproductive organs appeared to be normal. VDR knockout mice developed larger granuloma after infected with S. mansoni compared with wild type mice. Both groups of mice showed low calcium levels, indicated that infection result in decreased serum calcium(161).

Cyp24a1 null mice survived past weaning. These mice presented with vitamin D intoxication with intramembranous ossification defect. Keratinocytes from these mice showed

3 3 failure to convert [1β- H]calcitroic acid from [1β- H]1α,25(OH)2D3 as in control group(142).

Cyp27b1 mRNA expression from kidney of Cyp24a1 knockout mice showed low but detectable

19 transcripts demonstrated that Cyp24a1 null mice may survive because of low 1,25(OH)2D3 biosynthesis in the kidney.

Transgenic Cyp24a1 mice showed significantly low level of 24,25(OH)2D3 in plasma.

After weaning, these mice presented with hyperlipidemia and albuminuria. All lipoprotein were elevated and artherosclerotic lesions were found in aorta(162). Results indicated the role of

Cyp24a1 in function other than vitamin D homeostasis.

Cyp27b1 knockout mice created by targeting exon 8 of gene was created and found to exhibit similar characteristics as patients with psuedovitamin D-deficiency rickets(163). These mice presented with hypocalcemic, hypophosphatemic, hyperparathyroidic and undetectable

1,25(OH)2D3. Furthermore, severe organization of growth plate architecture and significant osteomalacia was found. Mice with target ablation of Cyp27b1 gene are found with low

Osteocalcin level and poorly mineralized bone. Moreover, osteoclast numbers were reduced with ectopic lymph node(164). Chondrocyte specific knockout for Cyp27b1 by Cyp27b1 heterozygous mated with Col2-Cre mice exhibited increased width of hypertrophic zone of the growth plate, increased bone volume in neonatal long bone, and increased in Indian Hedgehog

(Ihh) and also PTH/PTHrP receptor expression. These mice presented with decreased Rankl expression and reduction in osteoclastogenesis(165). From the observation above implies that

1,25(OH)2D3 local production plays crucial roles in endochondral ossification and chondrocyte development.

However, in Cyp27b1 null mice fed with high vitamin D diet, serum 1,25(OH)2D3 increased dramatically. Nonetheless, the reduced trabecular bone quality and disruption of the trabecular structure was shown in these mice even with rescue diet(166).

Vitamin D and autoimmune disease

20 Traditionally, vitamin D is known for the crucial role as a hormone to maintain calcium and phosphate hemostasis. Several indications that vitamin D is associated with immune cells was evident earlier on. In 1966, myelofibrosis in rickets patients was reversible with vitamin D supplement(167). Rickets patients were found with increased susceptibility to infection.

Phagocytosis function was impaired in rickets patient(168). This can be reversible with treatment of 1,25(OH)2D3 as shown in mice with vitamin D deficiency(169). In 1980s the role of vitamin D in immunomodulatory function has been described with the action of 1,25(OH)2D3 in inducing human myeloid leukemia cell line (HL-60) to differentiate into mature myeloid cells(170). In mice inoculated with murine myeloid leukemia cells (M1), 1,25(OH)2D3 prolonged survival rate of these mice from leukemia(171). In 1983, human peripheral blood monocytes were found to express Vitamin D receptors but not in resting human peripheral blood T and B lymphocytes(172). However, T lymphocytes after activation with phytohemagglutinin can induce VDR expression within 24 hours of activation(173). 1,25(OH)2D3 was found to inhibit T lymphocyte mitogenesis through suppression of interleukin 2 (IL-2)(174).

Tuberculosis is an infection disease by Mycobacterium Tuberculosis, acid-fast bacilli.

Tuberculosis patients have low 25(OH)D3 serum level compared with healthy age-match group(175). Meta-analysis study also supported that low 25(OH)D3 levels are associated with higher risk of active tuberculosis(176). Before the discovery of antibiotic as early as 18th century, vitamin D from cod-liver oil had been prescribed for tuberculosis patients. In 1930 Spies used repeated large dose of irradiated ergosterol in rabbit suffering tuberculosis to induce calcification in caseous lesion(177). It was believed that calcification of lesions marked the curing of the disease. With this concept, cutaneous tuberculosis patients or lupus vulgaris were treated with large dose of vitamin D(178, 179). Attempt had been made to show the effect of vitamin D in treating tuberculosis. Monocytes primed with 1,25(OH)2D3 for three days and infected with M.

21 tuberculosis caused inhibition in [3H] uracil uptake by bacilli. However, incubation of

1,25(OH)2D3 alone with M. tuberculosis did not showed significant effect on proliferation of bacteria(180). This evidence suggested the role of vitamin D in immune response.

Polymorphism at VDR gene showed population with tt genotype may be resistant to

Tuberculosis infection(181). Polymorphism of VDR also found to associated with outcome of the treatment. TaqI Tt genotype showed faster sputum culture conversion when compared with

TT genotype(182). Recent meta-analysis study of polymorphism of VDR supports the hypothesis that vitamin D deficiency might be a risk factor in developmental of tuberculosis(183). Current treatment guidelines for tuberculosis patients include the use of antibiotic isoniazid, rifampin, ethambutol and pyrazinamide. 2007 double blinded randomized controlled trial of vitamin D supplement for tuberculosis patient showed no different in TB score and sputum smear conversion rates between treated and placebo group(184). 100,000 IU of cholecalciferol was used at start of the trial and 5 and 8 months after. The author suggested that this dose might be insufficient to display improvement in clinical outcome of the disease.

Patients with sarcoidosis, chronic granulomatous disorder, present with enhanced intestinal absorption of ion, hypercalciuria with or without hypercalcemia and may be associated with renal stones or even impaired renal function. With the success of treating Lupus vulgaris patients with high dose of vitamin D, in early 1900s, sarcoidosis was treated with similar intervention. Administration of calciferol to patient who have sarcoid lesion of skin glands and lungs was beneficial(185). Patients who were treated with large dose vitamin D were found to develop rapid hypercalcemia(185). Abnormal calcium level was suggested to be from increased sensitivity to vitamin D(186). Increased in circulating 1,25(OH)2D3 in sarcoidosis was proposed to cause abnormal in calcium absorption(187). It was shown that sarcoid lymph node homogenate can convert 25(OH)D3 into 1,25(OH)2D3 while nonsarcoid lymph nodes can

22 not(188). This experiment implied the up-regulation of 1α-hydroxylase activity in infected monocyte. Primary cultures of pulmonary alveolar macrophages from broncholavage of sarcoidosis patients were studied for production of 1,25(OH)2D3. Obtained cells from bronchoalveolar lavage (mainly macrophages) and blood mononuclear cells from patient with tuberculosis and sarcoidosis were able to produce 1,25(OH)2D3 including T lymphocytes from tuberculosis patients(189, 190). From these earlier studies denote the role of vitamin D in immune function.

Vitamin D deficiency associates with higher risks of cancers, cardiovascular disease, rheumatoid arthritis, multiple sclerosis, type 1 diabetes mellitus and HIV. These diseases show defects in immune response. Epidemiology studies showed the prevalence of cancers and multiple sclerosis in population in higher latitudes. As early as 1941, Apperly studied the solar radiation and skin cancer mortality rates in North America. He recommended exposure to adequate sunlight to reduce cancer death(191). Later, number of studies supported this correlation in breast cancer and colon cancer(192-194). Treatment of multiple sclerosis with cod- liver oil in 1986 displayed decreased relapse rate(195). Mouse model of multiple sclerosis was created and the administration of 1,25(OH)2D3 at the start of the symptom prevented the progression of encephalomyelitis(196). CD4+ T cells can transfer autoimmune encephalomyelitis and diabetes in naïve mice(197, 198). However, the result showed in animal could not extrapolate directly to human with multiple sclerosis. Increased production of inflammatory cytokines, TNF-α, IFN-γ and IL-2 and decreased in anti-inflammatory cytokines, TGF-β1 and

IL-13 are found in MS patients(199-201). In recent randomized double blind study reported 6 months of vitamin D supplementation significantly increased serum transforming growth factor

β1 (TGF-β1)(202). Prospective study of Vitamin D intake inversely correlated with risk of developing MS(203).

23 Studies showed that patients with human immunodeficiency virus (HIV) have below normal level of 25(OH)D and 1,25(OH)2D3 which correlated with disease progression and survival of disease(204-207). Evidences also suggested that low 25(OH)D levels in HIV-infected patients are associated with increased risk for Mycobacterium tuberculosis infection(208).

EuroSIDA study in 2011 showed 83% of patients who received antiretroviral therapy have vitamin D deficiency. Independently, patients who have low 25(OH)D associated with higher risks of mortality and AIDs events(209). Fok-I polymorphism of VDR associated with progression of AIDS. Patients carrying Ff genotype have higher risk to faster the disease to

AIDS(210). Monocytes from AIDS patients showed reduction in migration capability.

Incubation of 1,25(OH)2D3 with monocytes from AIDS patients showed increased in chemotactic responsiveness(211). HIV induces T cells apoptosis and results in aggravation of disease by induce hypermethylation of VDR and activate reactive oxygen species(212).

Type1 diabetes is characterized by immune-mediated destruction of β-cells in the islets of

Langerhans in pancreas. These cells possess vitamin D receptor. Several populations was studied and showed the polymorphism in VDR associated with susceptibility to type 1 diabetes in

Indian, Taiwanese, German and Japanese(213-216). In addition, polymorphism of CYP27b1 gene was found to associate with type 1 diabetes in U.K(217). Similar results also found in other vitamin D metabolism genes such as CYP2R1 and CYP24A1(218). Vitamin D deficiency rats showed reduction in insulin secretion from pancreases(219). In mice with pancreatic islets specific VDR knockout showed reduced serum insulin and mRNA levels(220). Moreover, vitamin D deficiency was found to accelerate type 1 diabetes in non-obese diabetic (NOD) mice(221).

Vitamin D and monocytes

24 3 subtypes of Monocytes are found in human blood, CD14++CD16-, CD14+CD16++ and

CD14++CD16-. Macrophages and dendritic cells are monocytes that migrate from peripheral blood to other tissues. These cells are responsible for cytokine production, phagocytosis and antigen presentation. Monocyte/macrophage lineage expresses small amount of VDR which increased with activation with 1,25(OH)2D3(129). Monocytes can be differentiated to dendritic cells with the activation by GM-CSF and IL-4 in vitro(222, 223). Dendritic cells also constitutively express VDR(132). These Antigen presenting cells are crucial for initiation of the adaptive immune response.

10 nM of 1,25(OH)2D3 was found to be optimal concentration to increase proliferation of

3 monocytes observed by [ H]-thymidine incorporation(224). In 1983, 1,25(OH)2D3 was shown to inhibit induction of human promyelocytic leukaemia (HL60) cells differentiation into macrophages(225). This active metabolite also suppresses proliferation of murine granulocyte- macrophage progenitor cells(226). 1,25(OH)2D3 is a potent inhibitor of proliferation of human myelomonocytic cell line U937 in dose-dependent manner. This inhibition process was associated with differentiation of cells to mature macrophages that express CD14(227).

25(OH)D3 and 24,25(OH)2D3 metabolites did not exhibit inhibition effect on U937. Retinoic acid also showed proliferative inhibition effect but did not differentiate myeloid cells into monocytes(228). 1,25(OH)2D3 was also shown to induce differentiation of bone marrow progenitor cells to monocyte-macrophages(225). It is showed that vitamin D promote fusion of macrophages and formation of multinucleated giant cells. Mouse alveolar macrophages fusion was induced by several vitamin D derivatives especially 1,25(OH)2D3(229). Moreover, when

1,25(OH)2D3 was added into immature dendritic cells, impaired differentiation was observed(230). Furthermore, immunoglobulin- and complement-dependent phagocytosis of monocytes are increased with vitamin D in dose and time dependent manner(231).

25 Normal pulmonary alveolar macrophages significantly produced 1,25(OH)2D3 by IFN-γ stimulation(180, 232). Furthermore, in human blood monocytes, 1,25(OH)2D3 significantly inhibited the production of IL-1α, IL-6 and TNF-α(233). In 2003 Hewison showed that human monocyte-derived dendritic cells can synthesize 1,25(OH)2D3 in vitro following increased in 1α- hydroxylase activity. VDR expression in these cells is down-regulated with dendritic cells maturation(234) making these cells less responsive to vitamin D as they mature. 1,25(OH)2D3 induces the 24-hydroxylase cDNA in HL-60 cells. The 24 hours activation results in 30 fold increased in mRNA level(235). However, ability of cells to produce 24,25(OH)2D3 metabolites are suppressed with differentiation of monocytes(236). LPS or viral infections can also up- regulate CYP27b1 expression in macrophage(237). 1,25(OH)2D3 interferes with differentiation of monocyte to macrophages. The cell surface receptors profiles after administration of vitamin

D showed negative or low positive for CD1a but higher levels of CD14 when compared with control cells. MHC II expression by CD40 and CD86 molecules were decreased with vitamin D treated dendritic cells. CD83, CD80m CD86, CD40, MHC I and MHC II involve in Antigen presenting were unable to up-regulate in vitamin D-pretreated dendritic cells after exposed to

LPS(230). 1,25(OH)2D3 also alters ability of monocytes to induce antigen-dependent T cell proliferation(238). Dendritic cells primed with 1,25(OH)2D3 showed favorable towards Th2 phenotype and less ability towards Th1 and Th17 responses(239, 240). These primed cells also induce T cell hyporesponsiveness(241). In summary, dendritic cells are crucial in VDR response by support the development of T cells that resolve inflammation.

Vitamin D and lymphocytes

Lymphocytes can be divided into T cells, B cells and natural killer cells. Natural killer cells are part of innate immune response. T cells are major player in adaptive immune response.

They have ability to discriminate antigens. B cells are accountable for humoral immunity related

26 to production of antibodies. T cells can be categorized into helper T cells (CD4+), cytotoxic T cells (CD8+) and γδ T cells. Studies showed that CD8+ lymphocytes contain highest amount of

VDR while CD4+ lymphocytes contain reasonably amount of VDR. However, B lymphocytes do not show traceable amount of VDR(129). Previously CD4+ cells were divided into Th1 and Th2 subsets. Th1 express IFN-γ and contribute to pathogenesis of some autoimmune diseases while

Th2 mainly express IL-4. Many subsets of CD4+ T cells have been identified recently including

IL-17-secreting T cells (Th17), regulatory T cells (Tregs) and T follicular helper cells (TFH) that express IL-17, Foxp3 and IL-21, respectively. Th17 has been implicated in pathogenesis of some autoimmune diseases(242). Regulatory T cells are responsible for inhibiting induction of autoimmunity and limiting immune responses.

Naïve T cells did not express VDR, but VDR expression can be induced with activation by T cell antigen receptor (TCR) signaling via MAPK p38 pathway. This activation requires vitamin D and leads to upregulation of phospholipase C- γ1 (PLC-γ1) expression which correlates with TCR responsiveness(243). Murine Th2 cells constitutively express high levels of

VDR(244).

1,25(OH)2D3 inhibits lymphocytes proliferation by arresting cells at G1a-G1b border and may be mediated through inhibition of IL-2(245). Inhibition of proliferation by 1,25(OH)2D3 also showed in freshly isolated CD4+ T cells(246). In 1988, Tobler showed that inhibition of monocyte-secreted granulocyte-macrophage colony-stimulating factor (GM-CSF) by

1,25(OH)2D3 occur posttranscriptionally(247). 1,25(OH)2D3 mediated direct transcriptional repression of IL-2 by loss of NFATp/AP-1 complex binding via inclusion of VDR complex(248). VDR-RXR complex can bind to IFN- γ at promoter region and down-regulate

IFN- γ expression in T-cells(249). 1,25(OH)2D3 treated T cells suppress normal responsive T cells proliferation and express characteristics of adaptive regulatory T cells. Combination of IL-2

27 and 1,25(OH)2D3 showed increased in population of CTLA-4 and FoxP3 expressed cells and showed greater suppression of responder T cell proliferation(250).

CD4+ T cells from BALB/c and C57BL/6 mice were used to demonstrate effect of vitamin D on Th cell polarization. Vitamin D inhibit Th1 by inhibit IFN-γ production and stimulate Th2 development through production of IL-4, IL-5 and IL-10. This effect mainly mediated by IL-4(251). Study showed that 1,25(OH)2D3 incubation resulted in IL-6 producing

CD4+ and CD8+ cells that does not match Th1 or Th2 subset(252). These later suggested that these 1,25(OH)2D3-treated T-cells are Treg as they express FoxP3 and CTLA-4 and showed inhibition of IL-17 expression(250). IL-2 inflammatory cytokine production of CD4+ T cells is

+ inhibited by 1,25(OH)2D3. IL-2 is critical in concert with TGF-β for induction of Foxp3 iTreg(253). Previously, effects on T cells were showed to modulate by dendritic cells. However, vitamin D directly can inhibit effector cytokine production of T cells such as IL-2, IFN-γ and IL-

17(250). Vitamin D directs cell fate of T cells into regulatory T cells. FoxP3 and IL-10 are two phenotypic molecules for Regulatory T cells. 1,25(OH)2D3 increased both IL-10 mRNA expression and secretion including IL-10+ CD4+ T cells(250, 254). Similar induction also found with FoxP3 mRNA expression by 1,25(OH)2D3(250, 255). Furthermore, serum 25(OH)D3 level is found to positively correlated with Treg function in multiple sclerosis patients(256).

In CD8+ T cells, vitamin D induced proliferation of IL-6+ cells whereas IL-6 were rarely present without 1,25(OH)2D3. In addition, IL-4 augmented vitamin D induction effect by 5 fold(257). Cytotoxic activity of T-cells were diminished after incubated with 1,25(OH)2D3(258).

T cells constitutively express CYP27B1 and 1α-hydroxylase can metabolized 25(OH)D3 into 1,25(OH)2D3. This vitamin D metabolite increased number of CD4CD25 T regulatory cells by induced expression of indoleamine 2,3 dioxygenase(246). Also, evidence indicated the

28 polymorphisms in CYP27B1 associated with multiple sclerosis patients in Sweden(259) supports the role of intracrine function of vitamin D in immune response.

With the role of 1,25(OH)2D3 in suppression of immune response, direct differentiation of T cells toward Treg and low vitamin D status in patients with autoimmune diseases, support the role of vitamin D in prevention and treatment of autoimmune disease.

Freshly isolated B cells showed low level of VDR and CYP24A1 expression. CYP27B1 mRNA is constitutively expressed and up-regulated by stimulation but not 1,25(OH)2D3. Initial

B cell division is not effected by vitamin D. However, in presence of stimuli such as IgM/anti-

CD40, anti-CD40/IL-21 or anti-IgM/anti-CD40/IL-21, 1,25(OH)2D3 significantly suppressed B cell proliferation. During B-cell maturation, 1,25(OH)2D3 induces apoptosis of activated B-cells and inhibit differentiation into plasma cells. Moreover, 1,25(OH)2D3 reduced the secretion of

IgG(260).

Patients with chronic renal failure who undergo hemodialysis and hypophosphatemic vitamin D-resistant rickets have defects in immune response(261). Natural killer cell activity diminished with decreased in serum 1,25(OH)2D3 in hemodialysis patients(262).

Supplementation of vitamin D in VDRR patients significantly increased NK cell number and activity including decreased susceptibility of infection in these patients(263). 1,25(OH)2D3 in combination with dexamethasone induced IL-10 expression in Natural killer cells. These IL-10 +

NK cells suppressed proliferation of Ag-induced T-cell and secretion of IL-13 and IFN-γ(264).

These results suggested polarization of NK cells toward regulatory function by vitamin D.

Vitamin D and antimicrobial, antiviral effects

Bermudez in 1990 showed that active form of vitamin D inhibit growth or kill

Mycobacterium avium complex in human macrophages(265). In 1991, Denis’s experiment about

-7 ability of human monocytes in killing M. tuberculosis demonstrated that 1,25(OH)2D3 at 10 to

29 -9 -7 10 M did not alter initial bacterial uptake. Nonetheless, 10 M of 1,25(OH)2D3 reduced intramacrophage bacterial growth. Moreover, combination of TNF-α, IFN-γ and 1,25(OH)2D3 effectively kill large portion of bacteria(266). Toxic effects of reactive oxygen intermediates

(ROI) by phagocytes during the respiratory burst have been the focused on mechanism of killing

M.tuberculosis. In murine macrophages, the nitric oxide (NO) release was a robust anti-bacterial mechanism(267). Bacterial products or pathogen-associated molecular patterns (PAMPs) can be recognized by pattern recognition receptors (PRRs) such as TLR, NLR and RLR. M. tuberculosis fragments consist of lipoprotein. Toll like receptor can discriminate and recognize lipoprotein by

TLR2.

Antimicrobial peptides are crucial for innate immune responses. These peptides are regularly cationic and may be produced constitutively or with infection or injury. Human neutrophils contain at least 4 α-defensins in cytoplasmic granules. Epithelial cells produced β- defensin (HBD-1) and HBD-2 are produced in inflamed skin(268). Cathelicidin is also known as hCAP18/LL37/FALL-39 stored in neutrophil granules(269). CAMP is produced and secreted by tissues that expose to environmental microbes. CAMP is strongly up-regulated when administrated with 1,25(OH)2D3(270).

Study by Liu et al, in 2006 sparked excitement in the role of vitamin D in antimicrobial properties. Based on previous study results, activation of TLR2/1 diminished intracellular

M.tuberculosis viability in human monocyte and macrophage(271). They showed activation of

Toll like receptor (TLR) in human macrophages up-regulated VDR and CYP27B1 expression.

The up-regulation of CYP27B1 in monocytes requires IL-15(272). IL-15 is also indispensable to induction of cathelicidin (CAMP). Dimerization of TLR2/1 on macrophage surfaces permit internalization of DBP-25(OH)D3 inside the cells and can be used as substrate for 1α- hydroxylase. These propagations lead to increase in production of antimicrobial peptide

30 cathelicidin which responsible for killing Mycobacterium tuberculosis observed by co- localization of cathelicidin and bacteria containing vacuole. They also reported that in subjects with low serum 25(OH)D3 such as in African-American population had inability to promote increased in cathelicidin mRNA expression(273). However, there is no correlation between serum vitamin D metabolites both 25(OH)D3 and 1,25(OH)2D3 with human cathelicidin expression levels(274). Data showed that extracellular 25(OH)D3 was more potent in inducing cathelicidin expression compared with equimolar concentration 1,25(OH)2D3.

The regulation of cathelicidin by vitamin D is human/primate specific(275). CAMP mRNA in VDR-deficient murine cells are similar to wild-type cells. Vitamin D and VDR complex in human macrophages bound directly on CAMP promoter that contains vitamin D response element (VDRE)(270). In leprosy, hsa-mir-21 (microRNAs) was found in granuloma and upregulated in primary human monocytes from patients with leprosy. This miRNA downregulated TLR2/1-mediated induction of CYP27B1 and IL-1B mRNA expression. Besides, hsa-mir-21 inhibited antimicrobial peptides, cathelicidin (CAMP) and beta-defesin 4A

(DEFB4A)(276).

T cells release IFN-γ and activate autophagy, phagosomal maturation and production of antimicrobial peptides. Autophagy is essential host defense mechanism involve lysosome- dependent degradation and recycling of intracellular components in response to stress.

Active form of vitamin D induces the autophagy in monocytes by cathelicidin and activated transcription of autophagy-related genes, Beclin-1 and Atg5. Cathelicidin also mediated formation of mycobacterial phagosomes in human macrophages(277). Mycobacterial lipoproteins bind to TLR2/1/CD14 and activate antibacterial autophagy through vitamin D receptor dependent pathway. 1,25(OH)2D3 induced induction of CYP27B1 and cathelicidin antimicrobial peptide (CAMP) and autophagy. Increased in expression of CYP27B1 require

31 CCAAT/enhancer-binding protein (C/EBP)-β. Cathelicidin promotes fusion of autophagosomes with lysosomes and terminates intracellular mycobacteria(277). In HIV-infected persons,

1,25(OH)2D3 mediated the HIV replication inhibition through induction of autophagy and phagosomal maturation. By the use of RNA interference of CAMP showed that CAMP is important in these inhibition process(278). IFN-γ induces the antimicrobial activity of macrophages. Vitamin D supplement in macrophages from vitamin D deficiency serum patients restored activity induced by IFN-γ(279).

Pattern recognition receptors (PRRs)

Multicellular organisms have mechanism to protect the host and defend against infection by obliterate micro-organisms and neutralize virulence factors also known as innate immunity(280). Innate immune system recognizes invariant molecules of infectious agents.

These molecules are imperative to survival of microbes and shared by large group of pathogens.

Furthermore, innate immunity can discriminate between self and non-self to avoid destruction of self-tissues(281). Innate immune system utilizes Pattern recognition receptors (PRRs) to initiate the cascade of immune response by recognizing variety of ligands from microbial or even host cells’ particles or Pathogen Associated Molecular Patterns (PAMPs)(282, 283). PRRs are expressed in cells involving in innate immunity including antigen-presenting cells (APC) in adaptive immunity and surface epithelium(284).

There are two classes of Pattern Recognition Receptors; transmembrane PRR and cytosolic PRR. Transmembrane PRR acknowledges PAMPs in extracellular region including in phagosomes or endosomes. Toll like receptors and Dectin-1 are members of transmembrane

PRRs. Cytosolic PRRs recognize and distinguish between intracellular infections and extracellular infections. They are classified by their mechanism and activation(285). There are six families of PRRs classified by common structures and functional domains, Toll-like receptors

32 (TLRs), C-type lectin receptors (CLRs), RIGs-I-like receptors (RLRs), AIM-like receptors

(ALRs), Nod-like receptors (NLRs) and OAS-like receptors (OLRs)(286-290).

Toll-like receptors (TLRs)

Toll receptors first identified in Drosophila functions in establishing dorsoventral pattern in embryo. Transcription factor Dorsal is activated upon binding to its ligand Spatzle(291). Later also found that Toll receptor induces antifungal immune response in adult fly(292). Toll gene consists of extracellular transmembrane and cytoplasmic domain. Extracellular domain contains similar sequence homology to platelet glycoprotein 1b and other leucine-rich repeat protein.

Leucine-rich repeat (LRR) has been implicated in protein-protein interaction. Cytoplasmic domain of Toll related to human interleukin-1 receptor (IL-1R). IL-1R stimulation induces nuclear localization and NF-κB transcriptional activation(293). Toll like receptors are mammalian homologue of Drosophila Toll. Toll like receptors are conserved in both invertebrate and vertebrate lineages(294). Transfection of constitutively active human Toll in human cell lines showed activation of NF-κB and production of inflammatory cytokines IL-1,IL-6 and IL-

8(295).

TLRs are expressed mainly on antigen-presenting cells such as dendritic cells (DCs) and macrophages as well as on B cells. It has been suggested that cytosolic signaling by PRRs in non-antigen presenting cells is involved in facilitating DC-mediated adaptive immune responses.

TLRs expressed in many cell types both lymphocytes and some nonhematopoietic cells. The patterns of each TLRs expression are varied on cell types. TLRs are found to express in most of immune cells such as naïve B cells, memory B cells. However, epithelial cells also express

TLRs. Human dermal endothelial cells have been shown to express TLR4(296). Intestinal and uterine epithelial cells also express TLRs especially in inflammatory condition(297, 298).

33 Dendritic cells are also found to express TLRs(299). Currently, there are 13 mammalian TLRs,

10 of them are found in humans and 12 are found in mice.

TLRs also respond to endogenous molecules or damage-associated molecular patterns

(DAMPs). DAMPs can promote immune response in noninfectious inflammatory setting. These molecules also known as alarmins. Injuries to tissues by ripped, squashed or wounded by mechanical forces cause trauma and cell damages. Other trauma from heat, cold, chemical insults or radiation can also induce tissue damages. Cell injuries release endogenous molecules such as

High Mobility Group Box 1 (HMGB1), S100 proteins, heat shock protein (hsp), IL-1α or uric acid. These alarmins are able to ligand with TLRs and activate signaling cascades(300).

Activation of TLRs not only propagates innate immunity, but also induces T helper 1-cell (TH-1) response(301).

TLRs are divided in 2 subgroups. First group expressed on cell surfaces and recognize microbial components such as lipids, lipoproteins and proteins to ensure rapid and highly sensitive detection. The other group expressed exclusively in intracellular vesicles including endoplasmic reticulum (ER), endosomes and lysosomes.

TLR2 detects lipopeptides from bacteria, peptidoglycan (PG) and lipoteichoic acid (LTA) from Gram-positive bacteria. TLR2 can forms heterodimer with TLR1 or TLR6. Different heterodimers discriminate changes in lipid portion of lipoprotein(302, 303). TLR2/1 heterodimer recognizes triacylated lipopeptides and TLR2/6 detects diacylated lipopeptides. As shown in

TLR6-deficient mice, they are unresponsive to diacylated lipopeptide from mycoplasma.

Moreover, co-expression of TLR2 and TLR6 are absolutely required for this bacterial response.

Different lipid-binding pockets of TLR1 and TLR6 are accountable for discrimination between diacylated and triacylated lipoproteins. TLR2-deficient mice are greatly susceptible to Gram- positive bacterial infection such as Staphylococcus aureus(304). As well as polymorphisms in

34 TLR2 that has been reported to associate with reduced response to bacterial lipoprotein and septic shock especially in Staphylococcus septic shock(305). TLR2 heterodimerizes with TLR1 and can identify cell-wall-associated lipoprotein from M.Tuberculosis(271). TLR2/1 also strongly expressed on monocytes and dendritic cells of leprosy patients(306). TLR2 activation is also exhibited to initiate apoptosis. Tumor necrosis factor receptor 1(TNFR1) stimulated parallel activation of apoptosis and NF-κB activation(307).

TLR4 is receptor that responds to bacterial lipopolysaccharides (LPS) or endotoxin from cell walls of gram-negative bacteria. A lipid portion of LPS called lipid A causes most of the major pathological reaction such as septic shock. C3H/HeJ mouse strain showed hyporesponsiveness to LPS. In addition, macrophages from C3H/HeJ mice showed diminish inflammatory cytokines induction including, TNF-α, IL-1 and IL-6(308). These mice have point mutation in TLR4 gene. TLR4-/- mice showed similar hyporesponsive to LPS(309). Co- expression of αv-TLR and β5-TLR in 293T cells showed homodimers of TLR4 initiate stronger

NF-κB activation than TLR4 monomer(310). Immunoprecipitation showed MD-2 coprecipitated with TLR4. MD-2 is dispensable for TLR4 response to LPS(311). TLR4 forms complex with

MD2 on cell surface and serve as LPS binding component. LPS-binding protein (LBP) is acute- phase soluble plasma protein that binds LPS presented in bloodstream. CD14 is a glycosylphosphatidylinositol-linked, leucine-rich repeat-containing protein(312). However,

CD14 is not transmembrane protein and not be able to transduce cytoplasmic signals. Both of them are also involved in LPS binding. CD14 binds LPS and delivers LPS-LBP to TLR4-MD2 complex. TLR4/MD-2 dimerization and CD14 is required for induction of rapid activation of

NF-κB and p38(313). TLR4 can induce both type I interferon and inflammatory cytokines. Main activation of TLR4 is induction of NF-κB nuclear translocation in dose and time dependent manner.

35 TLR4 can identify viral DNA as well. Respiratory syncytial virus (RSV) one of human respiratory pathogen infected TLR4-deficient mice longer compared with wildtype mice and IL-

6 were diminished in macrophages of TLR4-deficent mice. TLR4 activation mediates by

CD14(314) and initiate innate immune response. Mouse mammary tumor virus (MMTV) activated TLR4 and induced up-regulation of CD71 on dendritic cells(315). These activations also lead to production of type 1 interferon and inflammatory cytokines.

Other TLRs recognize different molecules of bacteria and virus. Double-stranded RNA form replication intermediate for single-stranded RNA can be recognized by TLR3. TLR5 recognizes flagellin, protein from motility apparatus of bacteria Flagella. Single stranded RNA from virus and host can be identified by TLR7 and TLR8. TLR9 discriminates bacterial genomic

DNA and viral DNA from unmethylated CpG-dinucleotides. Protozoan-parasites components have been showed to recognize by TLR9 and TLR11(316).

TLRs signaling

TLRs signaling can be divided to 2 distinct pathways. First is MyD88 pathway. MyD88 adaptor molecules are recruited to TLRs and are used for all TLR signaling except TLR3.

MyD88 protein activates NF- κB and Mitogen activated protein kinase (MAPK) and result in production of inflammatory cytokines. Dominant negative form of MyD88 showed defect in NF-

κB activation of TLR2 and 5 but not TLR4(310). TLR4 is the only TLRs that activate both

MyD88 and TRIF-dependent pathways. MyD88 deficient mice showed delay MAPK activation following LPS stimulation indicated TLR4 have alternative signaling pathway. After ligand binding, TLRs undergo conformational change for recruitment of adapter proteins.

TLR4 initially recruits TIRAP at the plasma membrane and subsequently facilitates the recruitment of MyD88 to trigger the initial activation of NF-κB and MAPK. TLR4 then undergoes dynamin-dependent endocytosis and is trafficked to the endosome. TLR4

36 subsequently, forms a signaling complex with TRAM and TRIF. This signaling cascade leads to

IRF3 activations and late-phase activation of NF-κB and MAPK.

In MyD88 pathways

MyD88 (Myeloid differentiation primary response protein 88) contains death domain

(DD) and TIR (Toll/Il-1 receptor) domain and acts as adapter involving most TLR activations except TLR3. MyD88 pathway was characterized by utilizing MyD88 deficient mice with exposure of microbial components. These mice showed defect in production of TNF or IL-6. Mal

(MyD88-adapter-like) also known as TIRAP (TIR-domain adaptor protein) can form homodimers and heterodimers with MyD88. Dominant negative form of Mal suppressed NF-κB activation by TLR-4 or LPS(317). After TLR/Tl-1R stimulation, MyD88 is recruited to cytoplasmic TIR domain and facilitates IRAK4 (IL-1 receptor-associated kinases 4), a serine/threonine kinase association through hemophilic death domain interaction. This binding activates IRAK1 phosphorylation at kinase-activation loop. After phosphorylation, IRAK1 autophosphorylates residues in N-terminus and permits binding of TNF receptor-associated factor 6 (TRAF6) to this complex to form oligomers. Interaction between IRAK and TRAF6 is crucial for signaling cascades as showed by dominant negative form of TRAF6(318). TRAF6 is a RING domain E3 protein that act together with ubiquitin conjugating enzyme Ubc13 and Ubc- like protein Uev1A or E2 enzyme catalyze the synthesis of polyubiquitin chains at lysine-63 of ubiquitin at target proteins(319). This complex also called TRIKA1 (TRAF6-regulated IKK activators). Ubiquitination TRAF6 activates TRIKA2 consists of protein kinase TAK1 (TGF-β- activated kinase), TAB1 and TAB2 (TAK1-binding proteins 1 and 2)(320). TAB2 and TAB3 preferentially bind to polyubiquitin chains that link by Lys63. Ubiquitin-activated TAK1 then phosphorylates two serine residues in the activation loop of IKKβ initiate IKK activation. This leads to NF-κB activation via phosphorylation and subsequent degradation of IκB protein(319).

37 Ubiquitinated TAK1 simultaneously activates MAPKs (Erk1, Erk2, p38 and Jnk) by phosphorylation of MAPK kinases. This MAPK activation subsequently leads to activation of transcription factors AP-1.

NF-κB is dimeric transcription factors contains family of highly conserved REL– homology domain (RHD). RHD family members in mammals are RelA/p65, RelB, c-Rel, p50(NF-κB1) and p52(NF-κB2). RHD bind to promoter and enhancer region of various genes at the specific DNA sequence called κB site(321). NF-κB involves in control of cell proliferation, differentiation and survival in various system including skeletal and immune system. Activation of NF-κB is tightly regulated by IκB inhibitor protein. In resting state, NF-κB dimers are associated with IκB in cytoplasm. Phosphorylation of IκBs on conserved destruction box serine residues leading to induction of K48-linked polyubiquitination by E3 ligases(322). IκBs are degraded by proteasome and NF-κB dimers translocate into nucleus. There are two types of NF-

κB signaling, canonical and non-canonical pathway distinct by presence of IKKβ and NEMO. In predominant canonical pathway, NF-κB is triggered by TLR signaling. IKKβ phosphorylated

IκBα or IκBβ and leads to ubiquitination and degradation of IκB by proteasome(323). This canonical pathway results in transcription of genes in innate immune response. In non-canonical pathway, IKKα-IKKα homodimer phosphorylation activates p100-RelB complex. Subsequently, p100 is ubiquitinated and degraded(324).

TRIF dependent pathway

Study in MyD88-deficient mice with LPS exposure showed expression of IFN-inducible genes but not inflammatory cytokines such as TNF, IL-6 and IL-1β(325). However, all of these genes were completely abolished in TLR4-deficient macrophages. This indicates that IFN- inducible genes are produced in TLR4 dependent with MyD88-independent manner.

38 Activation of TRIF (Toll/IL-1 receptor domain-containing adaptor inducing IFN-β or

TICAM1) pathway leads up to activation of IRF3 and NF-κB. TRIF was identified as adapter molecule for TLR3 recognizing double stranded RNA or poly(I):poly(C) and activates IFN-β production(326). TRIF-deficient mice showed defect in both TLR3 and TLR4-mediated expression of IRF-3. MyD88 and TRIF double knockout mice showed complete loss of NF-κB activation in response to TLR4(327). TRIF-related adaptor molecule (TRAM) is requisite to

TLR4 signaling but not other TLR ligands. TRAM- deficient mice showed defect in cytokine productions response to TLR4 ligand(328). IL-1β stimulation activated both NF-κB and JNK1 activation similarly in wild type and TRAM deficient mice indicated that TRAM is not necessary in IL-1R signaling. In vitro experiment showed cytoplasmic portion of TLR4 binds directly to

TRAM(326). TRIF dependent pathway activates both NF-κB and IRF3 transcription factors.

Activation of NF-κB is initiated with recruitment of RIP1 adaptor protein to TRIF(329). RIP1 then undergoes K-63-linked ubiquitination allow TRADD to binds RIP1(330). Pellino-1, E3 ubiquitin ligase also required for RIP1 ubiquitination(331). Multiprotein complex consists of

TRIF, TRAF6, TRADD, Pellino-1 and RIP1 is requisite for TAK1 activation that leads to activation of NF- κB and MAPK pathway(287).

Additionally, TRIF pathway also activates IRF3 transcription factor. Study from TRAF3- deficient fibroblast showed defect in type I IFN response from TLR activation(332). TRAF3 is required for activation of TBK-IKKi, noncanonical IKKs. This activation catalyzes phosphorylation of IRF3 and triggers its nuclear translocation. IRF3 activation leads to interferon

–β transcription. Ubiquitination of TRAF3 also leads to TAK1 activation(333).

IRF-3 is a member of interferon regulatory factor and present as a monomer in cytoplasm of all cell types. IRF-3 expression is not altered by present of viral infection or IFN treatment.

IRF-3 protein binds specifically to IFN-stimulated response element (ISRE)(334). IRF-3 is

39 constitutively phosphorylated on serine residues. Activation of IRF-3 occurs with phosphorylation of additional serine residue and induces conformational changes leads to dimer formation for association with CREB binding protein (CBP)/p300(122). IRF-3 dimers with

CBP/p300 holocomplex strictly localize in nucleus and CBP/p300 is necessary for ISRE binding activity.

TLRs also respond to endogenous molecules or damage-associated molecular patterns

(DAMPs). Heat shock protein (Hsp) and high-mobility group box 1 (HMGB1) proteins are agonists for cell surface TLRs. HSP60 can bind to CD14 and activate TLR4 which leads to p38

MAPK activation(335).

1,25(OH)2D3 time and dose dependently suppresses the expression of both TLR2 and

TLR4 in human monocytes. This inhibition requires VDR also impaired NF-κB/RelA nuclear translocation and reduction of p38 and p42/44 phosphorylation(336).

Vitamin D and osteoblast

Osteoblast derived from mesenchymal stem cells (MSCs) from bone marrow. Study from rat calvaria cells demonstrated three distinct steps in osteoblast maturation(337). Initially, osteoblast is in active phase of proliferation. At this stage type I collagen mRNA are observed at maximum levels. Maturation period occurs after suppression of proliferation genes. During maturation stage, osteoblasts express alkaline phosphatase and establish lay down of extracellular matrix for mineralization. Osteocalcin and osteopontin mRNA expression elevation marked the onset of mineralization. Macroscopic phenotype of osteoblast maturation includes mineralization of extracellular matrix surrounding multilayered nodule of cells.

hMSCs can convert 25(OH)D3 with 1α-hydroxylase encoded by CYP27B1 gene. siRNA of CYP27B1 indicated the role of intracrine vitamin D function in inhibition of hMSCs

40 proliferation by reduced cyclinD1 results in cell cycle arrest. 1,25(OH)2D3 stimulate osteoblast differentiation from hMSCs as well(338). Exposure of both 25(OH)D3 and 1,25(OH)2D3 to osteoblast resulted in inhibition of proliferation and increase mineralization, in vitro(157). Runx2 or Osf2/CBfa1 is transcription factor regulate osteoblast differentiation(339). 1,25(OH)2D3 regulates Runx2 expression by suppression. Proximal promoter of Runx2 contains VDREs that regulate by vitamin D(340).

1,25(OH)2D3 alone induces little upregulation of CYP24A1 mRNA while PTH has no effect on CYP24A1. However, PTH was found to enhance 1,25(OH)2D3- mediated induction of

CYP24A1 in osteoblastic cells(341). Data from ChIP-chip analysis showed occupancy of VDR at VDRE in promoter regions of Cyp24a1 located at -165 and -265 bp upstream of transcription start site (TSS) and +35-41 kb of downstream from TSS(118). These regions act synergistically in transactivation of Cyp24a1 gene.

Osteoblastic VDR mainly regulates local activities of bone remodeling(133). ChIP-chip analysis of VDR/RXR in preosteoblastic MC3T3-E1 cell line found that upon 1,25(OH)2D3 stimulation, VDR bound to more than 6,000 RXR-prebound sites. Co-occupancy with RNA polymerase II and H4ac were found in these sites(118). Autoregulation of VDR was identified by ChIP-seq analysis as S1, S3 and U1 distal enhancer regions. Through CRISP/Cas9 genome editing of S1, S3 and U1 in UAMS-PM cells originated from stromal-osteoblastic cells, deletion of each region showed minor effect on basal expression of VDR. However, after activation of

1,25(OH)2D3 S1-deletion displayed strongly reduction but not eliminate expression of

VDR(342).

Cyp27b1 mRNA expression of bone was independent to kidney Cyp27b1 levels. Serum

1,25(OH)2D3 and PTH are negatively regulated Cyp27b1 mRNA expression in bone. Increased in Cyp27b1 promoter activity preceded up-regulation of alkaline phosphatase, osteocalcin and

41 VDR mRNA expression (343). CYP27B1 siRNA specific knockdown in HOS cells showed 80% in reduction of 1,25(OH)2D3 secretion and OCN and CYP24A1 mRNA expression(157). In conclusion, bone Cyp27b1 might play a role in regulation of bone formation.

Osteocalcin (OCN) promoter contains Vitamin D response element (VDRE)(344).

Vitamin D receptor polymorphisms contribute to circulating osteocalcin levels(26). 1 nM of

1,25(OH)2D3 increased osteocalcin and alkaline phosphatase protein levels(345). In addition,

25(OH)D3 at physiological level (100nM) up-regulated OCN and osteopontin (OPN) greater than

1nM of 1,25(OH)2D3(157). Alkaline phosphatase response to vitamin D are different depend on stage of differentiation and concentration of vitamin D(346). In immature osteoblast, vitamin D inhibits cell growth and increase ALP activity. However, in more mature cells, vitamin D suppresses ALP activity.

Osteoprotegerin (OPG) is important for inhibition of osteoclastogenesis. 10-7 M of

1,25(OH)2D3 increased 90% of (OPG) mRNA expression in fetal osteoblastic cell line (hFOB) and 50% in normal trabecular osteoblastic cells (hOB) detected by northern blot analysis(347).

RANKL regulates differentiation, activation and survival of osteoclasts secreted by osteoblasts and stromal cells. ChIP-chip analysis showed 1,25(OH)2D3 binds to 75 kb upstream to transcription start site that contain several VDREs(348). This region can bind to PTH as well.

Deletion of -74 distal enhancer region in Rankl of osteoblasts suppressed Rankl and displayed similar bone mineral density as observed in PTH-deficient mice(349).

Osseointegration is the term used to describe structural and functional contact between bone and titanium surface occur in dental implant placement. Progression of osteoblastic marker genes pattern at implant site is similar to the osteoblast differentiation(350). Experiment of vitamin D insufficient rats and osseointegration exhibited impaired bone to implant contact ratio

(BIC) and significantly lower implant push-in values compared with control group(351).

42 1,25(OH)2D3 treatment decreased cell numbers on implant surfaces but increased alkaline phosphatase and osteocalcin activity(352).

Study of LPS from P.gingivalis showed stimulation of RANKL production from osteoblasts modulated by MyD88 and NF-κB mechanism(353). Normal bone secretes human β defensing (HBD-3). HBD-3 is upregulated by TLR2 and TLR4 ligands in osteoblast but this effect was not observed in chronic infected bone(354). However, vitamin D effects to TLRs response in osteoblast still remain to be elucidated.

Vitamin D and osteoclast

Osteoclast is a multinucleated tissue-specific macrophage derived from hematopoietic stem cells. Monocyte/macrophage precursors cells near bone surface is stimulated by RANKL and M-CSF and undergone differentiation into mature osteoclasts that can express tartrate- resistant acid phosphatase (TRAP), cathepsin K (CATK) and calcitonin(355).

1,25(OH)2D3 can stimulate differentiation of osteoclast from monocyte-macrophage stem cell precursors in vitro and increase their TRAP activity. In addition, calcitonin can inhibit the

1,25(OH)2D3 dependent differentiation and also TRAP activity(356). However, from VDR null mice study showed normal osteoclasts number indicated that 1,25(OH)2D3 is not essential for the formation of the osteoclasts(76). PTH and IL-1α can generate osteoclasts from VDR null mice supported that 1,25(OH)2D3 and VDR mediate but not require in osteoclastogenesis. Over- expression of VDR in mature osteoblast co-culture with monocytes decreased formation of multinucleated cells and resorption activity induced by 1,25(OH)2D3(135).

Osteoclasts derived from peripheral blood monocytes (PBMC) express CYP27B1 gene and VDR. CYP27B1 and VDR mRNA expression are increased with osteoclast maturation from macrophage. 25(OH)D treated PBMC showed higher number of TRAP-positive and

43 multinucleated giant cells than 1,25(OH)2D3 treatment(357). This suggests autocrine function of vitamin D in regulating osteoclastogenesis.

RANK (receptor activator of NF-κB) expresses on surface of hematopoietic precursor cells and requires for osteoclast activation and differentiation. RANKL binds to RANK and plays crucial role in bone remodeling. The activation influences osteoclasts survival and function in bone degradation. RANK signaling involves binding to TRAF2, 5 and 6(358). TRAF-6 deficient-mice have osteopetrosis and defect in bone remodeling and tooth eruption. This finding was reported due to impaired osteoclast function(359). OPG secreted by osteoblast is also

TNFR-related protein acts as decoy ligand for RANK to regulate bone density and bone mass.

Because osteoclasts develop from similar lineage as monocyte and macrophage, TLRs are postulated to play a crucial role in osteoclast development. Osteoclasts were stimulated by various TLR ligands and induced up-regulation of NF-κB and TNF-α production(360).On the other hands, TLRs stimulation in osteoclast precursor cells inhibit their differentiation into multinucleated cells. TLRs suppress RANK expression and inhibit M-CSF that required for osteoclastogenesis(361). LPS from P.gingivalis increased differentiation and formation of mature osteoclasts leaded to matrix degradation and mineral release in the bone(353, 362).

Vitamin D and chondroblast and chondrocyte

Proliferating chondrocytes showed no VDR but low level of VDR can be detected in hypertrophic chondrocytes(363). PTH, Cyp27b1 double knockout mice showed narrow growth plate width with decreased in chondrocytes number and type X collagen-positive hypertrophic zone. Administration of exogenous 1,25(OH)2D3 rescued these phenotypes(364). Evidences suggested anabolic effect of vitamin D in endochondral bone formation.

ATDC 5 cell lines were used to demonstrate vitamin D metabolism in chondrocytes.

Results showed vdr gene expression was higher when treated with PTHrP (PTH-related protein)

44 than in control cells but not 1α and 24 hydroxylase genes. The activation increases sensitivity of chondrocyte to 1,25(OH)2D3. 1,25(OH)2D3 decreased PTHrP production display feedback loop autocrine control(365).

Sox9 (Sry-type high mobility group domain) transcription factor is expressed chondrocytes and responsible for induction of differentiation of stem cells to chondrocyte.

Chondrocytes display de-differentiation when culture in vitro with decrease in expression of

Sox9. 1,25(OH)2D3 showed protective effect to chondrocytes in vitro by significantly up- regulated Sox9(366).

MMP (matrix metalloproteinase) is produced by activated-chondrocytes and function as matrix-degradation enzyme. VDR positive chondrocytes often found co-localized with MMPs-1,

-3 and -9. MMP-3 production is increased with 1,25(OH)2D3(367). Up-regulation of MMP13 is found in osteoarthritis joint with articular cartilage destruction. This enzyme cleaves type I, II,

III, IV, X and XIV collagen and aggrecan. 1,25(OH)2D3 time and dose dependently increased

MMP13 expression through p38 pathway while down-regulated type II collagen and aggrecan(368). This finding indicates that vitamin D can potentiate the progression of cartilage erosion at the inflammation sites.

According to all information gathered, direct effect of vitamin D metabolites on differentiated osteoblasts have not been fully elucidated. Moreover, increasing evidences of non- classical action of vitamin D in innate immunity combines with emerging field of osteoimmunity suggested that osteoblast might play a role in response to infection and inflammation. Limited studies of TLRs activation in osteoblasts lead to current project to determine TLRs activation and vitamin D metabolism in mouse osteoblast.

45

Hypothesis:

The effects of vitamin D on bone health have been widely described, but the precise direct actions of vitamin D on bone forming osteoblasts have yet to be fully elucidated. Vitamin D can act to promote innate immune responses. Non-classical vitamin D functions are described in monocytes and macrophages. Stress and damage signals, as well as infection, may influence bone function and fracture healing. The current PhD project hypothesizes that innate immune response to vitamin D contributes to its effects on bone.

Working hypotheses:

1. Vitamin D supplementation to osteoblasts may activate vitamin D metabolism leading to

modulation of downstream osteoblastic activities

2. Microbial challenge to osteoblasts activates TLR pathways resulting in the activation of

vitamin D molecular pathway.

46 Chapter 1

Aim 1: To characterize effects of vitamin D metabolites and signaling system in mouse osteoblast cells with differentiation

1. Observe effects of vitamin D metabolites on vitamin D metabolism genes and their

regulations.

2. Observe effects of vitamin D metabolites on osteoblast marker genes.

3. Observe effects of vitamin D metabolites on TLRs and antimicrobial peptide in mouse

osteoblast.

Materials and methods

Pam3Cys-Ser-(Lys)4, Trihydrochloride or Pam3CSK4.3HCL is a synthetic triacylated lipopeptide that mimics bacterial lipopeptides. This molecule can activate proinflammatory transcription factor NF-κB mediated by TLR1 and TLR2.

MC3T3-E1 mouse osteoblasts were cultured in alpha-MEM, 10% FBS, 10 ml of

Penicillin-streptomycin-Glutamine and differentiated with 5mM beta-glycerophosphate and 5

µg/ml. ascorbic acid at 37 degree C and 5 % carbon dioxide. Reagents such as 25(OH)D3 and

-3 -6 1,25(OH)2D3 were prepared a stock solution at 10 M and 10 M respectively in absolute ethanol. Cells were cultured under differentiation medium for 5 days, 2 weeks and 3 weeks.

Vitamin D metabolites were added for 6 hours and 24 hours before the last day of experiment.

100nM of 25(OH)D3 and 1nM of 1,25(OH)2D3 were used in experiment. Cells were pretreated with TLR-ligands 6 hours prior to treatment with any vitamin D metabolites and harvested.

RNA isolation

RNA was isolated from cells treated with vitamin D metabolites and vehicle for different time periods of differentiation. RNA was extracted using Trizol (Invitrogen). RNAs were reverse-

47 transcribed using SuperScript III RT (Invitrogen) as described by the manufacturer. 150 ng of

RNA was used to reverse transcriptase to cDNA.

Quantitative real time RT-PCR amplification of cDNAs

Expression of mRNAs for Cyp27b1, Vdr, IL-6, Ocn, Alp1 and Opg were quantified using an

Applied Biosystems (ABI). All reactions were mulitplexed with the housekeeping 18S rRNA gene (Assays-on-Demand (ABI) primer and probe mix Hs999999901_s1). Data were obtained as

Ct values (the cycle number at which logarithmic PCR plots across a calculated threshold line), and used to determine delta Ct values. Ct of target gene cDNA was conducted using the following Tagman mouse gene expression assays: Cyp27b1, Vdr, IL-6, Ocn and Alp1. All cDNAs were amplified under following conditions: 50°C for 2 min; 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed in triplicate.

Visual representation of data was conducted by converting delta Ct values to 1/mean dCt expression.

Immunoblot

Whole cell lysates were prepared with lysis buffer (RIPA and a mixture of protease inhibitor) and were centrifuged at 10,000 rpm (Eppendorf centrifuge). Protein concentration was determined by nanodrop machine. Western blot analysis was performed as described previously.

In brief, proteins were resolved on 7% to 10% SDS-PAGE and transferred onto PVDF membrane. The membranes were blocked with 5% non-fat milk in TBST buffer (TBS buffer containing 0.1% of Tween-20) for 1 hour at room temperature and incubated overnight at 4 degree C with primary antibodies CYP27B1 (1:1000; Santa Cruz Biotechnology, Inc., Santa

Cruz, CA, USA), VDR (1:1000; Santa Cruz Biotechnology,Inc.), β-actin (1:10000, Santa Cruz

Biotechnology, Inc.). Unbounded antibodies were removed with three times wash for 20 minutes with TBST. Then, the membranes were incubated with horseradish peroxidase-conjugated

48 secondary antibodies (1:2000) for 1 hour at room temperature and washed three times for 20 minutes with TBST. The antibody-associated protein bands were revealed with ECL-plus

Western Blotting system (Amersham Biosciences).

CYP27B1 5’-flanking-luciferase reporter

5’-Deletion constructs of Cyp27b1 promoter region fused to firefly luciferase reporter gene in pGL3 vector have been designed and described previously(369). MC3T3-E1 was maintained in

MEM-alpha media supplemented with 10% fetal calf serum with differentiation media for 2 weeks. All transient transections were performed using Lipofectamine 2000 Reagent (Invitrogen) at a ratio of 1:1 (total DNA to Lipofectamine) according to manufacturer’s recommendation.

Cells were seeded into 6-well plates. 200 ng of each Cyp27b1 constructs with 50 ng of TK promoter directed Renilla luciferase plasmid (pRLTK-LUC) used to normalize transfection efficiency were transfected in triplicate at day 12. LPS and Pam3Cysk were treated 6 hours before harvesting. Luciferase activity was determined using Luciferase reporter assay kit

(Promega) and measured using FLUOstar Omega instrument (BMG Labtech, Cary, NC). 20 µl of each sample was transferred into a white base 96-well plate. The FLUOstar was programmed to inject 90 µl Stop&Glo. Transcriptional activity was expressed as luciferase activity relative to

Renilla activity. Results were expressed as fold-change over untreated cells by dividing the firefly/Renilla ratio of treated cells by the firefly/Renilla ratio of untreated cells. Each treatment was performed in triplicate.

* means statistically significant with P< 0.05

** means statistically significant with P<0.01

Results

Vitamin D metabolites treatment on vitamin D metabolism in differentiated mouse osteoblasts

49 We selected day 5 differentiated cells to represent less differentiated osteoblasts, day 14 cultures to represent intermediate differentiated osteoblast and day 21 cultures to represent more mature osteoblasts. Protein analysis of vitamin D metabolism proteins by western blot analysis showed that after 5 days of differentiation, MC3T3-E1 expressed low VDR protein and protein levels were increased with differentiation. Vitamin D metabolites (25(OH)D3 and 1,25(OH)2D3) attenuated VDR protein expression. 1α-hydroxylase enzyme encoded by Cyp27b1 gene responsible for conversion of 25(OH)D3 into 1,25(OH)2D3 increased with differentiation of

MC3T3-E1 cells. After 14 days of differentiation, added 25(OH)D3 increased 1α-hydroxylase protein expression. (Figure: 1)

Figure 1: Protein expression of VDR (top row), 1α-hydroxylase (middle row) and β-actin

(bottom row) from MC3T3-E1 differentiated in differentiation media for 5 days, 14 days and 21 days. Cells were treated with vitamin D metabolites (25(OH)D3 and 1,25(OH)2D3) 24 hours before harvesting.

Next, Vitamin D metabolism gene expression were observed with qPCR analysis after MC3T3-

E1 were differentiated for 5 days, 14 days and 21 days. Vitamin D metabolites did not change

50 Vdr and Cyp27b1 mRNA expression during all stages of differentiation. (Figure: 2a, 2b)

Cyp24a1 mRNA expression was altered by vitamin D metabolites. 1,25(OH)2D3 induced induction of Cyp24a1 mRNA at all stages of differentiation (p <0.05). 25(OH)D3 showed no alteration of Cyp24a1 mRNA expression when compared with vehicle but showed significantly different from 1,25(OH)2D3 group (p < 0.01). (Figure: 2c)

Vdr

0.14

0.12

0.1

0.08

1/dCt 0.06

0.04

0.02

0 5 days 14 days 21 days

Vehicle 25(OH)D3 1,25(OH)2D3 a

Cyp27b1

0.08 0.07 0.06 0.05 0.04 1/dCt 0.03 0.02 0.01 0 5 days 14 days 21 days

Vehicle 25(OH)D3 1,25(OH)2D3 b

51

** Cyp24a1 ** ** ** 0.07 * ** 0.06

0.05

0.04

1/dCt 0.03

0.02

0.01

0 5 days 14 days 21 days

Vehicle 25(OH)D3 1,25(OH)2D3 c

Figure 2: Quantitative PCR analysis of Vdr, Cyp27b1 and Cyp24a1 mRNA expression (a, b and c respectively) from MC3T3-E1 cells at 5 days, 14 days and 21 days of differentiation. Cultures were treated with 0.1% ethanol for Vehicle group, 100nM of 25(OH)D3 and 1nM of

1,25(OH)2D3. Graph represents means of 1/dCt± SD.

Luciferase activity of vitamin D metabolites on Cyp27b1 promoter constructs

Previous study reported proximal promoter of CYP27B1 lied in -305 bp of 5’flanking region of human CYP27B1 gene. Three possible Ets protein binding sites (EBSs) were found in

ROS 17/2.8 osteoblast-like cells in enhancer region from -531 to -997(370). Also full 5’flanking region pCyp27b1(-1500) contains repressive region. Thus, these 3 different deletion constructs were fused with firefly luciferase gene as reporter and were transiently transfected into MC3T3-

E1 cells using lipofectamine as transfectant reagent. After cells were differentiated in differentiation media for 14 days and 21 days, 2 days before harvesting, Cyp27b1 deletion

52 constructs were introduced to culture for overnight incubation and treated with vehicle and vitamin D metabolites; 25(OH)D3 and 1,25(OH)2D3 24 hours before harvesting.

After 2 weeks and 3 weeks of differentiation, MC3T3-E1 showed greatest

Luciferase/Renilla expression in pCyp27b1(-305)-Luc constructs. Both pCyp27b1(-997)-Luc and pCyp27b1(-1500)-Luc constructs at 2 weeks of differentiation showed trend of less luciferase activities but without statistically significant. However, we observed discrete Cyp27b1 promoter activity according to osteoblast maturation stages. After 3 weeks of differentiation, MC3T3-E1 cells showed less overall luciferase activity especially on pCyp27b1(-997)-Luc enhancer contained deletion construct.

Baseline Luciferase expression in Vehicle group

0.25

0.2

0.15

0.1

0.05

0 pCyp27b1(-305)-Luc pCyp27b1(-997)-Luc pCyp27b1(-1500)-Luc Luciferase/Renilla expression

14 days 21 days

Figure 3: Expression of 5’-flanking regions of Cyp27b1 gene in MC3T3-E1 cells. Deletion constructs of -305, -997 and -1500 were transiently transfected into MC3T3-E1 cells. Mean of firefly luciferase:Renilla luciferase ratios of triplicate samples were shown with SD.

53 Vitamin D metabolites treatment did not significantly change luciferase activity on any promoter deletion constructs in MC3T3-E1 at both 2 weeks and 3 weeks of differentiation

(Figure 4a, 4b). However, after 14 days of differentiation, 1,25(OH)2D3 increased pCyp27b1(-

997)-Luc activity but without statistically significant. After 21 days of differentiation, there was a trends of increasing expression of proximal promoter regions by 25(OH)D3 and 1,25(OH)2D3.

Overall data indicated that vitamin D metabolites did not alter 5’flanking promoter regions of

Cyp27b1 genes after 14 days and 21 days of differentiation in MC3T3-E1 cells.

Cyp27b1 promoter luciferase -day 14

4.5

4

3.5

3

2.5

2

1.5

1

Relative luciferase/Renilla actiivty 0.5

0 pCyp27b1(-305)-Luc pCyp27b1(-997)-Luc pCyp27b1(-1500)-Luc Vehicle 25(OH)D3 1,25(OH)2D3 a

54 Cyp27b1 promoter luciferase -day 21

3

2.5

2

1.5

1

0.5 Relative lucirease/Renila activity 0 pCyp27b1(-305)-Luc pCyp27b1(-997)-Luc pCyp27b1(-1500)-Luc

Vehicle 25(OH)D3 1,25(OH)2D3 b

Figure 4: Expression of 5’-flanking region of Cyp27b1 gene in MC3T3-E1 cells at 2 weeks (a) and 3 weeks of differentiation (b). Deletion constructs of -305, -997 and -1500 were transiently transfected into MC3T3-E1 cells. Relative luciferase activity with vehicle treated cells infected with pCyp27b1(-305)-Luc construct means are displayed. Firefly luciferase: Renilla luciferase ratios of triplicate samples were used.

miRNA 21 expression in differentiating mouse osteoblasts

Small non-coding RNA of around 22 nucleotides function as RNA silencing and post transcriptional regulation of gene expression is called micro RNA (miRNA). miRNAs derive from short hairpins RNA. miRNAs are part of active RNA-induced silencing complex (RISC) which contained Dicer and other associated proteins. miRNAs contain sequence complementary to 3’UTR of target gene. Earlier study reported no correlation of VDR protein and mRNA levels suggesting post-transcriptional regulation in colon cancer cells(371). miR-21 was identified to

55 upregulated in Leprosy lesion. Transfection of hsa-miR-21 in human primary monocytes were found to decreased TLR-induced expression of CYP27B1 mRNA by 34%. Also has-miR-21 inhibited expression of antimicrobial peptides, CAMP and DEFB4A. Results showed that

Cyp27b1 mRNA expression did not change which correlated with Cyp27b1 promoter luciferase reporter assay. miR-21 will possibly show post-transcriptional regulation of vitamin D metabolites on MC3T3-E1 cells. Differentiated MC3T3-E1 cells showed no different in miR-21 levels after 5 days, 14 days and 21 days of differentiation. Vitamin D metabolites also did not alter miR-21 mRNA levels. However, after 14 days of differentiation, there was a trend that

1,25(OH)2D3 increased miR-21 mRNA expression but not statistically significant different.

miR-21

0.09 0.08 0.07 0.06 0.05

1/dCt 0.04 0.03 0.02 0.01 0 5 days 14 days 21 days

Vehicle 25(OH)D3 1,25(OH)2D3

Figure 5: Quantitative PCR analysis of miR-21 from MC3T3-E1 cells at 5 days, 14 days and 21 days of differentiation. Cultures were treated with 0.1% ethanol for Vehicle group, 100nM of

25(OH)D3 and 1nM of 1,25(OH)2D3. Graph represents mean 1/dCt (SD).

Vitamin D metabolites treatment effects on osteoblast markers

56 25(OH)D3 and 1,25(OH)2D3 reported to inhibit osteoblast proliferation and increase osteoblast differentiation(372). However, effects of vitamin D metabolites in differentiating osteoblasts are not fully elucidated. This experiment showed 1,25(OH)2D3 increased Alp mRNA expression at early stage of differentiation of MC3T3-E1 cells. However, 25(OH)D3 and

1,25(OH)2D3 treatment did not alter Ocn mRNA expression during all stages of differentiation.

(Figure: 6a, 6b)

* * Alp 0.1 0.09 0.08 0.07 0.06 0.05

1/dCt 0.04 0.03 0.02 0.01 0 5 days 14 days 21 days Vehicle 25(OH)D3 1,25(OH)2D3 a

Ocn

0.07

0.06

0.05

0.04

1/dCt 0.03

0.02

0.01

0 5 days 14 days 21 days Vehicle 25(OH)D3 1,25(OH)2D3 b

57 Figure 6: Quantitative PCR analysis of Alp and Ocn mRNA expression (a and b respectively) from MC3T3-E1 cells at 5 days, 14 days and 21 days of differentiation. Cultures were treated with 0.1% ethanol for Vehicle group, 100nM of 25(OH)D3 and 1nM of 1,25(OH)2D3. Graph represents means of 1/dCt (SD).

Vitamin D metabolites effects on Toll-like receptors

Toll-like receptors are expressed in immune cells and non-immune cells that involve in immune responses such as epithelial cells, endothelial cells and liver cells. Study showed that osteoblasts also express TLRs(373). MC3T3-E1 expressed TLR2 and TLR4. Tlr2 mRNA expression was increased with 25(OH)D3 treatment after 21 days of differentiation (p < 0.05) while 1,25(OH)2D3 showed no differences in Tlr2 mRNA expression when compared with vehicle group (Figure: 7a). Tlr4 mRNA expression were suppressed by 25(OH)D3 and

1,25(OH)2D3 after 5 days of differentiation (p< 0.05). Furthermore, 25(OH)D3 increased Tlr4 mRNA expression after 21 days of differentiation (p <0.05). (Figure: 7b)

Tlr2

0.4 * *

0.35

0.3

0.25

0.2 1/dCt 0.15

0.1

0.05

0 5 days 14 days 21 days

Vehicle 25(OH)D3 1,25(OH)2D3 a

58 Tlr4 * * 0.2 * 0.18 0.16 0.14 0.12 0.1 1/dCt 0.08 0.06 0.04 0.02 0 5 days 14 days 21 days Vehicle 25(OH)D3 1,25(OH)2D3 b

Figure 7: Quantitative PCR analysis of Tlr2 and Tlr4 mRNA expression (a and b respectively) from MC3T3-E1 cells at 5 days, 14 days and 21 days of differentiation. Cultures were treated with 0.1% ethanol for Vehicle group, 100nM of 25(OH)D3 and 1nM of 1,25(OH)2D3. Graph represents means of 1/dCt± SD.

Vitamin D metabolites treatment on cytokines

Non classical function of vitamin D has been described in immune cells. These cells express cytokines in response to Toll-like receptor ligands challenge. Also IL-6 has been implicated in

Interleukin-6 secreted as an downstream response from NF-κB signaling. Expression of IL-6 mRNA were varied through differentiation period. 1,25(OH)2D3 increased IL-6 mRNA expression after 14 days of differentiation (p < 0.001). (Figure 8)

59 Il-6 0.14 **

0.12

0.1 * 0.08

1/dCt 0.06

0.04

0.02

0 5 days 14 days 21 days Vehicle 25(OH)D3 1,25(OH)2D3

Figure 8: Quantitative PCR analysis of IL-6 from MC3T3-E1 cells at 5 days, 14 days and 21 days of differentiation. Cultures were treated with 0.1% ethanol for Vehicle group, 100nM of

25(OH)D3 and 1nM of 1,25(OH)2D3. Graph represents relative expression to vehicle group at 5 days in fold change ± SD.

Antimicrobial peptides in mouse osteoblasts

25(OH)D3 is important in immune function of monocytes and macrophages. Activation of antimicrobial peptides require 25(OH)D3 and CYP27B1 converts inactive form of vitamin D into active form of vitamin D (1,25(OH)2D3). 1,25(OH)2D3 then binds to VDR and activate induction of Cathelicidin production to fight against microbials. MC3T3-E1 mouse osteoblasts also expressed Cathelicidin (Camp) mRNA. Vitamin D metabolites treatment did not change

Camp mRNA expression level.

60 Camp -3 weeks MC3T3-E1

0.045

0.044

0.043

0.042

1/dCt 0.041

0.04

0.039

0.038 Vehicle 25(OH)D3 1,25(OH)2D3

Figure 9: Quantitative PCR analysis of Camp mRNA from MC3T3-E1 cells after 21 days of differentiation. Cultures were treated with 0.1% ethanol for Vehicle group, 100nM of 25(OH)D3 and 1nM of 1,25(OH)2D3. Graph represents in 1/dCt (SD).

Discussion

MC3T3-E1 cell line under ascorbic acid and β-glycerophosphate can differentiate and and display sequential characteristics similar to in vivo bone formation(374). MC3T3-E1 was selected for the experiment for its stability and ability to mineralize. Cell lines are also easy to culture and simple to transfect. MC3T3-E1 cells were cultured and differentiated for period of time to represent different stages of osteoblast mimicking normal physiological environment.

Cells were differentiated under differentiation cocktails for 5 days to represent pre-osteoblast stage. 14 days of differentiation represented pre-mineralization stage and 21 days of differentiation represented mineralization stage of osteoblasts.

MC3T3-E1 cells exhibited functional vitamin D metabolism system by virtue of expression of Cyp27b1, Cyp24a1 and Vdr mRNA and protein. Regulations of vitamin D in

61 osteoblasts are differ from renal vitamin D metabolism. CYP27B1 gene is mainly expressed in kidney and responsible for determining circulating levels of 1,25(OH)2D3. In renal, biologically active vitamin D, 1,25(OH)2D3 is tightly controlled by feedback regulation of renal 1α – hydroxylase and 24-hydroxylase enzymes. 1,25(OH)2D3 formed complex with vitamin D receptor (VDR) bind to VDR interacting repressor (VDIR) and suppress gene by involving methylation of specific CpG sites in the promoter region(375). Non-classical action of CYP27B1 has been described in many tissues including bone. Osteoblasts display autocrine and paracrine effects of vitamin D metabolism(376). Calcium absorption and adequate osteoblastogenesis and osteoclastogenesis are modulated by 1,25(OH)2D3/VDR system(377).

Western blot protein analysis indicated that Vdr and 1α- hydroxylase proteins were increased with osteoblast maturity. Data from mRNA expression showed Vdr and Cyp27b1 mRNA did not display increased with maturity. Moreover, vitamin D metabolites including

25(OH)D3 and 1,25(OH)2D3 did not alter mRNA expression levels. Luciferase-promoter reporter constructs were transfected in differentiated MC3T3-E1 cells. Previous experiment by Turner et al, showed regulation of CYP27B1 gene in ROS17/2.8 osteoblast-like cells(370). Baseline luciferase-promoter expression showed less activity in more mature osteoblasts compared with osteoblast with 14 days of differentiation. Anderson(343) showed Cyp27b1-luc expression was highest at day 12 and decline at day 15. Also this induction of Cyp27b1 preceded marked increase in alkaline phosphatase, osteocalcin and vitamin D receptor. After treated with vitamin

D metabolites, luciferase promoter activities in 3 groups were not changed. However, after 14 days of differentiation, 1,25(OH)2D3 seemed to show more pCyp27b1(-997)-Luc activity than other group.

Discrepancy between protein and mRNA levels could be due to post-transcription regulation. MicroRNAs control gene expression through specific site at the 3’-UTR of target

62 mRNA resulting in translational repression or degradation(378). Several microRNAs are implicated in process of proliferation, differentiation and apoptosis. Mir-21 was showed to modulate Cyp27b1 expression by Toll-like receptor transduction(276). Moreover, increasing mir-21 in aorta is also associated with vitamin D deficiency and contributing to development of atherosclerosis and osteoporosis(379). Results also showed no discrepancy between maturations and vitamin D metabolites treatments.

Cyp24a1 is important in maintaining physiological levels of intracellular 1,25(OH)2D3 and 25(OH)D3. Cyp24a1, 1,25(OH)2D3 responsive gene is a marker for 1,25(OH)2D3 catabolism.

Upregulation of Cyp24a1 mRNA by 1,25(OH)2D3 treatment found here is similar to previous study by Atkins(157). However, they also described that Cyp24a1 is altered by 25(OH)D3 which in this study showed no significant different from control group. Across differentiation time points, Cyp24a1 mRNA expression showed increasing in expression with osteoblast maturation.

Evidence suggested that vitamin D especially 1,25(OH)2D3 can inhibit the growth of several cancer cells(380). Upregulation of Cyp24a1 mRNA expression possibly prevent antiproliferative effect of 1,25(OH)2D3. DNA Methylation status of CYP24 were differ even in endothelial cells isolated from different sources(381).

Vitamin D has been implicated in bone remodeling. Higher bone mass and decreased risk of fracture are associated with adequate vitamin D status or as indicated by level of serum level of 25-hydroxyvitamin D. This effect of vitamin D is classified as classical effect of vitamin

D by endocrine action of 1,25(OH)2D3 in maintaining calcium homeostasis. However, direct effects of vitamin D on osteoblasts are still unclear. Hormone responsiveness was previously described to attribute to stage of differentiation in rat and chick-derived osteoblasts. Vitamin D have biphasic effects on osteoblast phenotypic effects depend on hormone treatment and basal levels of gene expression(382). Previous studies using chronic treatment of vitamin D and

63 observe long term effects of vitamin D in cultured osteoblasts and suggested that vitamin D promote differentiation of osteoblasts. Current study using short term treatment of vitamin D to observe the immediate effects of vitamin D in differentiated osteoblasts. Results showed that vitamin D metabolites including active form 1,25(OH)2D3 and non-active form 25(OH)D3 did not change osteoblast markers during differentiation in normal culture condition except at earlier differentiation stage. After 5 days of differentiation, 1,25(OH)2D3 enhanced induction of alkaline phosphatase expression levels similar to effects showed in osteosarcoma cell line(383).

Local production of 1,25(OH)2D3 in late osteoblastic cells (MLO-A5) were found to increase Dmp1 and Phex mRNA expression(158). Knockdown of Cyp27b1 using siRNA in human osteoblast cells resulted in reduction in transcription of osteocalcin and CYP24A1(157,

384). Osteocalcin is the 1,25(OH)2D3 response gene. We anticipated that Ocn mRNA level should upregulated with 1,25(OH)2D3 treatment. However, current results showed no alteration of osteocalcin expression across differentiation time point and vitamin D metabolite treatment.

This could be due to mutation of cell line resulted in different gene response.

Toll-like receptors (TLRs) plays important roles in host defense against pathogens. TLRs were identified in immune cells such as monocytes and macrophages. Periodontal disease and osteomyelitis are inflammatory disease affected bone. In these conditions, osteoblasts possibly expose to microbial and have effects in cells. Osteoclasts and bone marrow macrophages express

TLR4 mRNA and also CD14 component of TLR4 in detecting LPS(385). MC3T3-E1 osteoblasts expressed Tlr2 and Tlr4 as earlier as 5 days of differentiation and continue to express throughout maturation periods. Study by Liu et al, reported that TLR1/2 ligands stimulated induction of

CYP27B1 and result in increased in production of 1,25(OH)2D3(273). However, study related to vitamin D metabolites effects on TLRs are limited. TLR9 is down-regulated by vitamin D whereas TLR3 is unaffected in human monocytes(386). Current study showed that 25(OH)D3

64 and 1,25(OH)2D3 suppressed Tlr4 at early stage of differentiation but increased Tlr4 expression at late stage of differentiation. This result might indicate that differential responses to vitamin D metabolites depend on stages of differentiation of osteoblasts.

IL-1 increased osteoclast formation and function in osteoblast by induce matrix metalloproteinases in mouse calvaria(387). IL-6 transduces signal through gp130 similar to IL-1.

IL-6 plays a crucial role in acute phase response, systemic responses to local injury and haemopoiesis. IL-6 also induces osteoclast formation in cocultures of mouse bone marrow cells and osteoblast in the presence of soluble IL-6 receptor(388). Current study showed 1,25(OH)2D3 enhance Il-6 expression after 14 days of differentiation. 1,25(OH)2D3 did not enhance IL-6 mRNA in primary osteoblast and MC3T3-E1 in earlier studies(389). In human osteoblast cells,

1,25(OH)2D3, IL-6 and PTH exerted no effects to IL-6 mRNA expression(390).

Innate immune cells produce cationic antimicrobial peptides including α – and β – defensins and cathelicidins to combat against pathogens. Several tissue were found to have enhance human cathelicidin antimicrobial peptides (CAMP) such as, acute myeloid leukemia, immortalized keratinocyte, colon cancer cell lines and normal human bone marrow derived macrophages. The induction of cathelicidins occurs through vitamin D response element

(VDRE) in the CAMP promoter(270) but not found in mice. Cathelicidin asides from its antimicrobial properties has also been shown to regulate cell-specific apoptosis such as in periodontal ligament(391), neutrophil and osteoblasts (MG-63)(392). Current study showed mature osteoblasts express CAMP but expression level was not altered by vitamin D metabolites similar to previously reported that VDRE in the CAMP promoter of primates was absent in mouse, rat and canine genomes(274).

65 Chapter 2

Aim 2:

To characterize the effects of TLR ligands on vitamin D metabolism and signaling in differentiating mouse osteoblasts.

Objectives

1. Observe Tlrs responses with TLR ligands in mouse osteoblasts (primary calvaria and

MC3T3-E1)

2. Observe effects of TLR ligands on vitamin D metabolism

3. Observe effects of TLR ligands on osteoblast markers and osteoclast markers

4. Observe effects of TLR ligands on cytokines production

Materials and methods

Cells and reagents

MC3T3-E1 mouse osteoblasts were cultured in alpha-MEM, 10% FBS, 10 ml of Penicillin- streptomycin-Glutamine and differentiated with 5mM beta-glycerophosphate and 5 µg/ml. ascorbic acid at 37 degree C and 5 % carbon dioxide. Reagents such as 25(OH)D3 and

-3 -6 1,25(OH)2D3 were prepared a stock solution at 10 M and 10 M respectively in absolute ethanol. Cells were cultured under differentiation medium for 5 days, 2 weeks and 3 weeks.

Vitamin D metabolites were added for 6 hours and 24 hours before the last day of experiment.

100nM of 25(OH)D3 and 1 nM of 1,25(OH)2D3 were used in experiment. Vehicle group was added with 0.1% ethanol. LPS (Lipopolysaccharide) or Toll-like receptor 4 ligands and

Pam3CysK or Toll-like receptor 2 ligands were used to induce the immune response of osteoblastic cells at the concentration of 1 ng/ml. and 100 ng/ml. respectively. Cells were

66 pretreated with TLR-ligands 6 hour prior to treatment with any vitamin D metabolites and harvested.

RNA isolation

RNA was isolated from cells treated with vitamin D metabolite and vehicle for different time periods of differentiation. RNA was extracted using Trizol (Invitrogen). RNAs were reverse- transcribed using SuperScript III RT (Invitrogen) as described by the manufacturer. 150 ng of

RNA was used to reverse transcriptase to cDNA.

Quantitative real time RT-PCR amplification of cDNAs

Expression of mRNAs for Cyp27b1, Vdr, IL-6, Ifn-β, Ocn, Alp1, Rankl and Opg were quantified using an Applied Biosystems (ABI). All reactions were mulitplexed with the housekeeping 18S rRNA gene (Assays-on-Demand (ABI) primer and probe mix Hs999999901_s1). Data were obtained as Ct values (the cycle number at which logarithmic PCR plots across a calculated threshold line), and used to determine delta Ct values. Ct of target gene cDNA was conducted using the following Tagman mouse gene expression assays: Cyp27b1, Vdr, IL-6, Ifn-β, Ocn,

Alp1, Rankl and Opg All cDNAs were amplified under following conditions: 50°C for 2 min;

95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed in triplicate. Visual representation of data was conducted by 1/mean dCt.

25D metabolism assays

Cells were cultured and differentiated for 5 days, 14 days and 21 days and incubated for 5 hours

3 at 37°C and 5% carbon dioxide with radiolabeled H-25D3 in serum-free media. The reaction was terminated by freezing at -20°C. Cell lysis and protein precipitation was done by adding

1000 µl of nitrite and 1000 µl of K2HPO4 and centrifuge at 3,000 rpm for 10 min at 4 °C.

Vitamin D metabolites were extracted by elution on C18-OH columns according to manufacturer’s instructions (Diasorin). The eluent was resuspended in elution solvent

67 hexane:methanol:isopropanol (90:5:5) and individual metabolites separated by HPLC using a

Beckham Gold system with an Agilent Technologies Zobax Sil normal phase column (Agilent

Technologies) eluted at a rate of 1.5 ml/min for 20 min. Elution profiles for standard vitamin D metabolites (25(OH)D3,1,25(OH)2D3) were determined by UV absorbance at 264 nm. Lauralite 3 software (LabLogic) was used to quantitate peaks of radioactivity corresponding to 25(OH)D3 or

1,25(OH)2D3 and 25(OH)D3 to 24,25(OH)2D3.

CYP27B1 5’-flanking-luciferase reporter

5’-Deletion constructs of Cyp27b1 promoter region fused to firefly luciferase reporter gene in pGL3 vector have been designed and described previously(369). MC3T3-E1 was maintained in

MEM-alpha media supplemented with 10% fetal calf serum with differentiation media for 2 weeks. All transient transections were performed using Lipofectamine 2000 Reagent (Invitrogen) at a ratio of 1:1 (total DNA to Lipofectamine) according to manufacturer’s recommendation.

Cells were seeded into 6-well plates. 200 ng of each Cyp27b1 constructs with 50 ng of TK promoter directed Renilla luciferase plasmid (pRLTK-LUC) used to normalize transfection efficiency were transfected in triplicate at day 12. LPS and Pam3CysK were treated 6 hours before harvesting. Luciferase activity was determined using Luciferase reporter assay kit

(Promega) and measured using FLUOstar Omega instrument (BMG Labtech, Cary, NC). 20 µl of each sample was transferred into a white base 96-well plate. The FLUOstar was programmed to inject 90 µl Stop&Glo. Transcriptional activity was expressed as luciferase activity relative to

Renilla activity. Results were expressed as fold-change over untreated cells by dividing the firefly/Renilla ratio of treated cells by the firefly/Renilla ratio of untreated cells. Each treatment was performed in triplicate.

Alkaline phosphatase assay

68 After 2 weeks of differentiation, MC3T3-E1 cells were fixed with formaldehyde for 60 seconds and wash with PBS. BCIP/NBT (5- Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) tablets were dissolved in deionized water (1 tablet/10 ml) to make substrate solution. PBS was carefully aspirated and cells were stained with BCIP/NBT solution and incubate in dark for 15 minutes. Then cells were evaluated for staining results.

Results

TLRs expression with TLR2 and TLR4 ligands stimulation

Mouse osteoblast exhibited functional Toll-like receptors. Stimulation of TLR ligands on differentiated cells showed that LPS or TLR4 ligand increased Tlr2 mRNA expression after 21 days of differentiation. Pam3CysK suppressed Tlr2 mRNA expression after 5 days of differentiation. Moreover, responses to LPS and Pam3CysK by MC3T3-E1 cells were statistical different after 5 days and 14 days of differentiation. Similar trends were found in Tlr4 mRNA expression. Pam3CysK suppressed Tlr4 mRNA expression throughout differentiation (5 days, 14 days and 21 days) with statistically significant at p<0.01. LPS also suppressed Tlr4 mRNA expression after 21 days of differentiation but increased Tlr4 mRNA after 14 days of differentiation. MC3T3-E1 showed divergent responses of Tlr4 mRNA expression to LPS and

Pam3CysK except after 21 days of differentiation.

69

Tlr4 ** ** 0.16000 * * * ** 0.14000 ** 0.12000

0.10000

0.08000 1/dCt 0.06000

0.04000

0.02000

0.00000 5 days 14 days 21 days Vehicle LPS Pam3CysK b

Figure 10: Toll like receptor ligands challenged in mouse osteoblast cell line (MC3T3-E1) showed functional TLRs in osteoblast. Tlr2 mRNA (a) and Tlr4 mRNA (b) expression in

70 MC3T3-E1 cells after culture in differentiation media for 5 days, 14 days and 21 days. Data showed in mean 1/dCt (SD).

In primary Calvaria

Tlr2 and Tlr4 mRNA expressions were slightly different in primary calvaria compared to

MC3T3-E1 cells. After 14 days and 21 days of differentiation, Pam3CysK stimulation significantly increased Tlr2 mRNA expression at p<0.01. LPS did not alter Tlr2 mRNA expression when compared with vehicle treated group (Figure 11a). However, Pam3CysK suppressed Tlr4 mRNA expression after 14 days of differentiation at p<0.05 while LPS did not alter Tlr4 mRNA expression after 14 days and 21 days of differentiation (Figure 11b).

** Tlr2 ** ** ** 0.14000

0.12000

0.10000

0.08000

1/dCt 0.06000

0.04000

0.02000

0.00000 14 days 21 days

Vehicle LPS Pam3CysK a

71 * Tlr4 0.08200 * 0.08000 0.07800 0.07600 0.07400

1/dCt 0.07200 0.07000 0.06800 0.06600 0.06400 14 days 21 days

Vehicle LPS Pam3CysK

Figure 11: Toll like receptor ligands challenged in mouse primary calvaria cells showed functional TLRs in osteoblast. Tlr2 mRNA (a) and Tlr4 mRNA (b) expression in primary calvaria cells after culture in differentiation media for 5 days, 14 days and 21 days. Data showed in mean 1/dCt (SD).

Adaptor protein TRIF/TRAM expression in primary calvaria

Toll-like receptor signaling mainly transduce through MyD88 adaptor protein. However,

TLR3 and TLR4 can also signal via MyD88 independent pathway using TRIF/TRAM adaptor proteins. Previous study reported non-detectable of Tram mRNA in mouse osteoblast. In this experiment, Trif and Tram mRNA expression was measured in 14 days and 21 days differentiated primary calvaria cells with qPCR analysis. Both LPS and Pam3CysK did not alter

Trif and Tram mRNA expression during all stages of differentiation (Figure: 12 a, b).

72 Trif

0.11500

0.11000

0.10500

0.10000 1/dCt 0.09500

0.09000

0.08500 14 days 21 days

Vehicle LPS Pam3CysK a

Tram

0.08800 0.08600 0.08400 0.08200 0.08000 0.07800

1/dCt 0.07600 0.07400 0.07200 0.07000 0.06800 0.06600 14 days 21 days

Vehicle LPS Pam3CysK b

Figure 12: Toll like receptor ligands challenged in mouse primary calvaria cells showed functional TLRs in osteoblast. Trif mRNA (a) and Tram mRNA (b) expression in primary calvaria cells after culture in differentiation media for 5 days, 14 days and 21 days. Data showed in mean 1/dCt (SD).

73 Autocrine, paracrine function of vitamin D on osteoblast

Osteoblasts contain functional Cyp27b1 gene responsible for local production of

1,25(OH)2D3(118). Toll like receptor ligands altered vitamin D metabolism genes. Pam3CysK significantly suppressed Cyp27b1 mRNA expression after 5 days and 14 days of differentiation at p<0.01 (Figure: 13a). However, after 21 days of differentiation Pam3CysK increased Cyp27b1

(p<0.01). LPS did not altered Cyp27b1 mRNA levels during all stages of differentiation compared with vehicle treated group. 1,25(OH)2D3 binds to Vitamin D receptor (Vdr) and exert its effects. Vdr mRNA expression was increased by LPS stimulation after 14 days of differentiation at p < 0.001. After 14 days and 21 days of differentiation, LPS and Pam3CysK

also significantly altered in Vdr mRNA expression at p<0.001. (Figure: 13b)

*

Cyp27b1 ** 0.10000 ** ** ** 0.09000 0.08000 0.07000 0.06000 0.05000 1/dCt 0.04000 0.03000 0.02000 0.01000 0.00000 5 days 14 days 21 days

Vehicle LPS Pam3CysK a

74 Vdr 0.250 ** **

0.200

* 0.150 1/dCt 0.100

0.050

0.000 5 days 14 days 21 days

Vehicle LPS Pam3CysK b

Figure 13: Toll like receptor ligands challenged in mouse osteoblast cell line (MC3T3-E1).

Cyp27b1 mRNA (a) and Vdr mRNA (b) expression in MC3T3-E1 cells after culture in differentiation media for 5 days, 14 days and 21 days. Data showed in mean 1/dCt(SD).

However, in primary calvaria cells, both LPS and Pam3CysK did not alter Cyp27b1 mRNA (Figure 14a) and Cyp24a1 mRNA (Figure 14c) expression levels at both after 14 days and 21 days of differentiation. After 21 days of differentiation, Pam3CysK increased Vdr mRNA expression at p < 0.01(Figure 14b) but not after 14 days of differentiation.

75 Cyp27b1

0.09000 0.08000 0.07000 0.06000 0.05000

1/dCt 0.04000 0.03000 0.02000 0.01000 0.00000 14 days 21 days

Vehicle LPS Pam3CysK a

Vdr ** ** 0.10000 0.09000 0.08000 0.07000 0.06000 0.05000

1/dCt 0.04000 0.03000 0.02000 0.01000 0.00000 14 days 21 days b Vehicle LPS Pam3CysK

76 Cyp24a1

0.05000

0.04800

0.04600

0.04400

1/dCt 0.04200

0.04000

0.03800

0.03600 14 days 21 days

Vehicle LPS Pam3CysK c

Figure 14: Toll like receptor ligands challenged in primary calvaria cells. Cyp27b1 mRNA (a),

Vdr mRNA (b) and Cyp24a1 mRNA (c) expression in primary calvaria cells after culture in differentiation media for 5 days, 14 days and 21 days. Data showed in mean 1/dCt(SD).

HPLC analysis LPS and Pam3Cysk treatment on Cyp27b1 and Cyp24a1 activities

To verify the function of Cyp27b1 and Cyp24a1 genes in mouse osteoblast, MC3T3-E1 were cultured for 5 days, 14 days and 21 days with differentiation media and given with

3 radiolabelled [ H]25(OH)D3 to observe local production of 1,25(OH)2D3 and 24,25(OH)2D3 measured by high performance liquid chromatography (HPLC). Results showed that at 5 days of differentiation, LPS showed suppression but Pam3CysK increased 1,25(OH)2D3 production with no statistical significant. After 14 days of differentiation, LPS significantly increased production of when compared to control group (p < 0.05). However, after 21 days of differentiation, LPS significantly suppressed 1,25(OH)2D3 production (p<0.05) (Figure 15).

77 1(OH)ase activity 3.000 * *

2.500

2.000

1.500

1.000

0.500

0.000 5 days 14 days 21 days Vehicle LPS Pam3CysK

3 Figure 15: HPLC analysis of [ H] 1,25(OH)2D3 production by 1(OH)ase enzyme in MC3T3-E1 cells after 5 days, 14 days and 21 days of differentiation.

HPLC analysis also showed local production of 24,25(OH)2D3 in MC3T3-E1 cells. After

5 days and 21 days of differentiation, LPS and Pam3CysK did not altered production of

24,25(OH)2D3. Nonetheless, after 14 days of differentiation, Pam3CysK significantly increased

24,25(OH)2D3 production in MC3T3-E1. (Figure 16)

78 24(OH)ase activity

35.000

30.000

25.000

20.000 *

15.000

10.000

5.000

0.000 5 days 14 days 21 days

Vehicle LPS Pam3CysK

3 Figure 16: HPLC analysis of [ H] 24,25(OH)2D3 production by 24(OH)ase enzyme in MC3T3-

E1 cells after 5 days, 14 days and 21 days of differentiation.

Luciferase activity of Cyp27b1 promoter constructs

Using previously described deletion of 5’ flanking region of Cyp27b1-promoter constructs(370),

MC3T3-E1 showed disparate luciferase activity responses to LPS and Pam3CysK. After 2 weeks of differentiation, pCyp27b1(-305)-Luc when treated with Pam3Cysk showed significant increased luciferase activity compared to vehicle treated group. (Figure: 17)

79 Cyp27b1 promoter luciferase -14 days

4 *

3.5

3

2.5

2

1.5

1

0.5 Relative Luciferase/Renilla expression 0 -305 -997 -1500

Vehicle LPS Pam3CysK

Figure 17: Expression of the 5’flanking region of Cyp27b1 gene containing -305, -997 and -

1500bp of 5’ flanking region were transiently transfected in MC3T3-E1 cells after 14 days of differentiation and treated with TLR ligands; LPS and Pam3CysK. The relative luciferase activity of firefly luciferase: Renilla luciferase ration showed represent the mean±S.D.

After 21 days of differentiation, LPS and Pam3CysK did not show statistically different in luciferase activity across all pCyp27b1-promoter constructs. However, LPS and Pam3CysK showed different trends of pCyp27b1(-997)-Luc and pCyp27b1(-1500)-Luc activities.

Pam3CysK showed reduced trends of luciferase activities of both pCyp27b1(-997)-Luc and pCyp27b1(-1500)-Luc when compared to vehicle group while LPS showed higher pCyp27b1(-

997)-Luc activity with no statistically significant. (Figure: 18)

80 Cyp271b1 promoter luciferase -day 21

4

3.5

3

2.5

2

1.5

1

0.5

Relative Luciferase/Renilla expression 0 -305 -997 -1500

Vehicle LPS Pam3CysK

Figure 18: Expression of the 5’flanking region of Cyp27b1 gene containing -305, -997 and -

1500bp of 5’ flanking region were transiently transfected in MC3T3-E1 cells after 21 days of differentiation and treated with TLR ligands; LPS and Pam3CysK. The relative luciferase activity of firefly luciferase: Renilla luciferase ration showed represent the mean±S.D.

MC3T3-E1 were treated with vitamin D metabolites 24 hours prior to harvest and 6 hours prior to harvest, Toll like receptor 2 and 4 ligands were administered to cultured cells. Further analysis of luciferase activity of Cyp27b1 promoter constructs with added vitamin D metabolites showed that after 14 days of differentiation, Pam3CysK and Pam3Cysk with both vitamin D metabolites added showed significantly different in pCyp27b1(-305)-Luc activity when compared with vehicle control and LPS treated groups. (Figure 19)

Further analysis of added vitamin D metabolites showed that there was no statistically different in pCyp27b1(-305)-Luc activity after 21 days of differentiation. In pCyp27b1(-997)-Luc

81 construct activities, only in LPS added with 25(OH)D3 group showed statistically significant different activity with Pam3CysK, Pam3CysK with 25(OH)D3 and Pam3CysK with 1,25(OH)2D3 groups (p < 0.05). (Figure 19)

mean Luciferase/Renilla-promoter 305 16.00 means standardized to vehicle=1

8.00 day 14 4.00

2.00

1.00

0.50

0.25

82 mean Luciferase/Renilla-promoter 997 means standardized to vehicle=1

35.000 30.000 25.000 20.000 15.000 10.000 5.000 0.000 -5.000

mean Luciferase/Renilla-promoter 1500 means standardized to vehicle=1

20.000

15.000

10.000

5.000

0.000

-5.000

day 14 day 21

Figure 19: Expression of the 5’flanking region of Cyp27b1 gene containing -305, -997 and -

1500bp of 5’ flanking region were transiently transfected in MC3T3-E1 cells after 21 days of

83 differentiation. Cells were treated with vitamin D metabolites and TLR ligands; LPS and

Pam3CysK. The relative luciferase activity of firefly luciferase: Renilla luciferase ration showed represent the mean±S.D.

Effects of TLR ligands on osteoblast markers

Osteoblast is important in bone remodeling. Alkaline phosphatase (ALP) expresses during maturation stage of osteoblast and responsible for extracellular matrix establishment.

Osteocalcin (OCN) elevates at onset of mineralization. Effects of TLR ligands on Alp and Ocn mRNA expression were also observed throughout differentiation in MC3T3-E1 cells. Pam3CysK or TLR2 ligand significantly suppressed Alp mRNA expression during all stages of differentiation at p < 0.01. LPS did not change Alp mRNA expression after 5 days and 21 days of differentiation but significantly increased Alp mRNA expression after 14 days of differentiation with p <0.05. LPS and Pam3CysK also expressed disparate effects on Alp mRNA expression in

MC3T3-E1 cells at all stages of differentiation (p < 0.01). (Figure)

Alp ** 0.12000 ** * ** ** ** 0.10000 ** 0.08000

0.06000 1/dCt

0.04000

0.02000

0.00000 5 days 14 days 21 days

Vehicle LPS Pam3CysK a

84

Alp 0.12000

0.10000

0.08000

0.06000 1/dCt 0.04000

0.02000

0.00000 14 days 21 days

Vehicle LPS Pam3CysK b

Figure 20: Expression of Alp mRNA in MC3T3-E1 (a) and primary calvaria (b) cells after 5 days, 14 days and 21 days of differentiation. Cells were treated with LPS (TLR4 ligand) and

Pam3CysK (TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

ALP staining

MC3T3-E1 cells were cultured and differentiated for 14 days with differentiation media. Cells were treated with Vehicle, Pam3CysK and LPS 6 hours and 24 hours before harvesting to observe TLR-ligands effect on ALP function. Result from alkaline phosphatase staining showed no different between groups. (Figure 21)

85

Figure 21: Alkaline phosphatase activity of 14 days differentiated MC3T3-E1 cells treated with

TLR ligands (Pam3CysK and LPS) 6 hours (top row) and 24 hours (bottom row) before harvesting.

Another mature osteoblast marker, osteocalcin is detected in mineralized osteoblast. Osteocalcin mRNA expression were altered by both LPS and Pam3CysK after 5 days of differentiation of

MC3T3-E1 cells. LPS and Pam3CysK significantly suppressed Ocn mRNA expression at p

<0.001. Pam3CysK also suppressed Ocn mRNA expression after 14 days and 21 days of differentiation (p <0.05) while LPS did not alter Ocn mRNA expression. (Figure 22)

86 ** Ocn ** * 0.08000 ** ** **

0.07000

0.06000

0.05000

0.04000 1/dCt 0.03000

0.02000

0.01000

0.00000 5 days 14 days 21 days

Vehicle LPS Pam3CysK

* Ocn 0.05200

0.05000

0.04800

0.04600

0.04400 1/dCt 0.04200

0.04000

0.03800

0.03600 14 days 21 days

Vehicle LPS Pam3CysK b

Figure 22: Expression of Ocn mRNA in MC3T3-E1 (a) and primary calvaria (b) cells after 5 days, 14 days and 21 days of differentiation. Cells were treated with LPS (TLR4 ligand) and

Pam3CysK (TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

87 In addition to TLR ligands, vitamin D metabolites were added in 14 days differentiated

MC3T3-E1 cells. Ocn mRNA levels was increased by both 25(OH)D3 and 1,25(OH)2D3 in LPS and Pam3CysK treated group but with no statistically significant. However, there were statistically different between LPS with vitamin D metabolites and Pam3CysK with vitamin D metabolites group at p <0.05.

Ocn

0.08 0.07 0.06 0.05 0.04 1/dCt 0.03 0.02 0.01 0

Figure 23: Expression of Ocn mRNA in MC3T3-E1 cells after 14 days of differentiation. Cells were pretreated with 25(OH)D3 and 1,25(OH)2D3 24 hours before harvesting and treated with

LPS (TLR4 ligand) and Pam3CysK (TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

Effects of TLR ligands on osteoclast differentiation markers

Osteoblasts involve in bone remodeling in both axes. Osteoblasts express RANKL

(Receptor activator of NF-κB ligand) to bind and activate RANK (Receptor activator of NF-κB)

88 on osteoclast precursor cells and activate differentiation into osteoclasts(393). Osteoprotegerin

(OPG) protects bone from excessive resorption by acting as a decoy receptor binding to

RANKL. Opg mRNA expression in differentiated MC3T3-E1 cells were altered by Pam3CysK.

Pam3CysK significantly suppressed Opg mRNA expression during all differentiation stages (p <

0.001) while LPS did not change Opg mRNA expression when compared with vehicle group.

(Figure 24)

Opg ** 0.35000 ** ** ** 0.30000 ** ** 0.25000

0.20000

1/dCt 0.15000

0.10000

0.05000

0.00000 5 days 14 days 21 days

Vehicle LPS Pam3CysK

Figure 24: Expression of Opg mRNA in MC3T3-E1 cells after 5 days, 14 days and 21 days of differentiation. Cells were treated with LPS (TLR4 ligand) and Pam3CysK (TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

Rankl mRNA expression could not be detected in MC3T3-E1 cell. Data from mouse primary calvaria cells showed that Opg mRNA expression were increased by LPS after 21 days of differentiation (p < 0.05). In addition, Rankl mRNA expression were increased by Pam3CysK after 14 days and 21 days of differentiation (p< 0.001). Similar differential effects of LPS and

Pam3CysK also observed in Rankl mRNA expression (p<0.01). (Figure 25)

89 Opg *

0.14000

0.12000

0.10000

0.08000

1/dCt 0.06000

0.04000

0.02000

0.00000 14 days 21 days

Vehicle LPS Pam3CysK

Rankl 0.14000 **

0.12000 **

0.10000

0.08000

1/dCt 0.06000

0.04000

0.02000

0.00000 14 days 21 days

Vehicle LPS Pam3CysK

Figure 25: Expression of Opg and Rankl mRNA in primary calvaria cells after 5 days, 14 days and 21 days of differentiation. Cells were treated with LPS (TLR4 ligand) and Pam3CysK

(TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

90 Effect of TLRs on cytokines production

Toll like receptors signal through MyD88 pathway and activate NF-κB translocation into nucleus. This activation leads to secretion of several cytokines including IL-6 (Interleukin-6).

Moreover, TLR4 can also signal through TRIF/TRAM pathway and activate IFN-β. MC3T3-E1 cells showed functional TLRs and IL-6 can be detected in mouse osteoblast. LPS increased IL-6 mRNA expression during all stages of osteoblast differentiation (p < 0.01). Pam3CysK increased

IL-6 mRNA expression only after 21 days of differentiation (p< 0.001). (Figure 26a) LPS also increased Ifn-β mRNA expression after 14 days and 21 days of differentiation. However, after 21 days of differentiation, Pam3CysK also increased Ifn-β mRNA expression (p< 0.05). (Figure

26b)

Il-6 ** ** ** 0.09 0.08 ** 0.07 0.06 0.05

1/dCt 0.04 0.03 0.02 0.01 0 5 days 14 days 21 days

Vehicle LPS Pam3CysK a

91 Ifn-β *

0.1 ** ** * 0.09 0.08 0.07 0.06 0.05 1/dCt 0.04 0.03 0.02 0.01 0 5 days 14 days 21 days

Vehicle LPS Pam3CysK b

Figure 26: Expression of IL-6 and Ifn-β mRNA (a and b respectively) in MC3T3-E1 cells after 5 days, 14 days and 21 days of differentiation. Cells were treated with LPS (TLR4 ligand) and

Pam3CysK (TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

Effect of TLRs on cathelicidin (Camp) in osteoblasts

Primary calvaria cells were differentiated for 14 days and 21 days, Camp mRNA was upregulated by Pam3CysK after 14 days of differentiation at p < 0.01. Also LPS and Pam3CysK stimulation showed differential responses from primary calvaria cells. LPS treatment showed similar Camp mRNA expression levels to vehicle group at both after 14 days and 21 days of differentiation.

92 ** Camp 0.12 * **

0.1

0.08

0.06 1/dCt

0.04

0.02

0 14 days 21 days

Vehicle LPS Pam3CysK

Figure 27: Expression of Camp mRNA in primary calvaria cells after 14 days and 21 days of differentiation. Cells were treated with LPS (TLR4 ligand) and Pam3CysK (TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

Vitamin D metabolites role in TLRs challenging MC3T3-E1

Vitamin D metabolites did not alter of vitamin D metabolism genes, osteoblast marker or osteoclastogenesis marker genes in normal differentiation environment. In this experiment, vitamin D metabolites were added 24 hours previous to TLRs ligand challenging to observe the effect of vitamin D supplement in compromised environment. Results showed that MC3T3-E1 constitutively expressed Vdr, Cyp27b1 mRNA at comparable or higher levels when induced than

J744 monocytes.

Vdr mRNA levels were suppressed by 25(OH)D3 in mouse monocytes J744 cells but not statistically significant. 25(OH)D3 supplement suppressed Vdr mRNA expression in LPS treated group after 5 days and 14 days of differentiation at p <0.05 (Figure 28). Vitamin D metabolites

93 treatment in LPS treated group after 14 days of differentiation of MC3T3-E1 also showed significantly increased in Cyp27b1 mRNA levels (Figure 29b) correlated with early functional experiment that LPS group showed higher local conversion of 25(OH)D3 to 1,25(OH)2D3.

Cytokines production were also measured by expression of Il-11 and Il-6 mRNA expression. MC3T3-E1 showed increased expression of Il-11 mRNA with 24,25(OH)2D3 treatment after 5 days of differentiation in both vehicle and LPS treated group (Figure 30b).

MC3T3-E1 also showed comparable expression levels of Il-11 and Il-6 mRNA as J744.

However, after stimulation of LPS, J744 induced higher expression level of Il-6 mRNA.

25(OH)D3 did not suppress Il-6 expression level in J744 but significantly suppressed Il-6 expression in 14 days differentiated MC3T3-E1 at p < 0.05 (Figure 31b).

Constitutively expression levels of Tlr-2 in MC3T3-E1 are slightly lower than in J744 cells. 25(OH)D3 suppressed Tlr-2 mRNA in LPS treated-5 days differentiated MC3T3-E1 but not at other time point at p<0.01 (Figure 32b). LPS treatment suppressed Tlr-4 mRNA expression in J744 cells and vitamin D supplement did not change expression levels (Figure

33a). In 14 days differentiated-MC3T3-E1, 1,25(OH)2D3 suppressed Tlr-4 mRNA expression in

Pam3CysK treated group (Figure 33b). Also 24,25(OH)2D3 suppressed Tlr-4 mRNA expression in LPS treated group at p <0.001.

94 Vdr in J744

0.062 0.060 0.058 0.056 0.054 0.052 0.050 0.048 0.046

a

Vdr in MC3T3-E1

0.300

0.250

0.200

0.150 1/dCt 0.100

0.050

0.000 day 5 day 14 day 21

Vehicle 25(OH)D3 1,25(OH)2D3

24,25(OH)2D3 LPS LPS+25(OH)D3

LPS+1,25(OH)2D3 LPS+24,25(OH)2D3 Pam3CysK b Pam3CysK+25(OH)D3 Pam3CysK+1,25(OH)2D3

Figure 28: Expression of Vdr mRNA in J744 (a) and MC3T3-E1(b) cells after 5 days, 14 days and 21 days of differentiation. Cells were pretreated with 25(OH)D3, 1,25(OH)2D3 and

24,25(OH)2D3 24 hours before harvesting and treated with LPS (TLR4 ligand) and Pam3CysK

(TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt

95 Cyp27b1 in J744

0.052 0.05 0.048 0.046

1/dCt 0.044 0.042 0.04 0.038

a

Cyp27b1 in MC3T3-E1

0.250

0.200

0.150

1/dCt 0.100

0.050

0.000 day 5 day 14 day 21 mean mean mean

Vehicle 25(OH)D3 1,25(OH)2D3

24,25(OH)2D3 LPS LPS+25(OH)D3

LPS+1,25(OH)2D3 LPS+24,25(OH)2D3 Pam3CysK b Pam3CysK+25(OH)D3 Pam3CysK+1,25(OH)2D3

Figure 29: Expression of Cyp27b1 mRNA in J744 (a) and MC3T3-E1(b) cells after 5 days, 14 days and 21 days of differentiation. Cells were pretreated with 25(OH)D3, 1,25(OH)2D3 and

24,25(OH)2D3 24 hours before harvesting and treated with LPS (TLR4 ligand) and Pam3CysK

(TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt. 96 IL-11 in J744

0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0

a

IL-11 in MC3T3-E1

0.080 0.070 0.060 0.050 0.040 1/dCt 0.030 0.020 0.010 0.000 day 5 day 14 day 21

Vehicle 25(OH)D3 1,25(OH)2D3

24,25(OH)2D3 LPS LPS+25(OH)D3

LPS+1,25(OH)2D3 LPS+24,25(OH)2D3 Pam3CysK b

Pam3CysK+25(OH)D3 Pam3CysK+1,25(OH)2D3

Figure 30: Expression of Il-11 mRNA in J744 (a) and MC3T3-E1(b) cells after 5 days, 14 days and 21 days of differentiation. Cells were pretreated with 25(OH)D3, 1,25(OH)2D3 and

24,25(OH)2D3 24 hours before harvesting and treated with LPS (TLR4 ligand) and Pam3CysK

(TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

97 IL-6 in J744

0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000

a

IL-6 in MC3T3-E1

0.140

0.120

0.100

0.080

1/dCt 0.060

0.040

0.020

0.000 day 5 day 14 day 21

Vehicle 25(OH)D3 1,25(OH)2D3

24,25(OH)2D3 LPS LPS+25(OH)D3 LPS+1,25(OH)2D3 LPS+24,25(OH)2D3 Pam3CysK b Pam3CysK+25(OH)D3 Pam3CysK+1,25(OH)2D3

Figure 31: Expression of Il-6 mRNA in J744 (a) and MC3T3-E1(b) cells after 5 days, 14 days and 21 days of differentiation. Cells were pretreated with 25(OH)D3, 1,25(OH)2D3 and

24,25(OH)2D3 24 hours before harvesting and treated with LPS (TLR4 ligand) and Pam3CysK

(TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

98 Tlr2 in J744

0.120 0.100 0.080 0.060 0.040 0.020 0.000

a

Tlr2 in MC3T3-E1

0.350 0.300 0.250 0.200 0.150 0.100 0.050 0.000 day 5 day 14 day 21

Vehicle 25(OH)D3 1,25(OH)2D3

24,25(OH)2D3 LPS LPS+25(OH)D3 LPS+1,25(OH)2D3 LPS+24,25(OH)2D3 Pam3CysK b Pam3CysK+25(OH)D3 Pam3CysK+1,25(OH)2D3

Figure 32: Expression of Tlr-2 mRNA in J744 (a) and MC3T3-E1(b) cells after 5 days, 14 days and 21 days of differentiation. Cells were pretreated with 25(OH)D3, 1,25(OH)2D3 and

24,25(OH)2D3 24 hours before harvesting and treated with LPS (TLR4 ligand) and Pam3CysK

(TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

99 Tlr4 in J744

0.090 0.085 0.080 0.075 0.070 0.065 0.060

a

Tlr4 - in MC3T3-E1

0.450 0.400 0.350 0.300 0.250 0.200 0.150 0.100 0.050 0.000 day 5 day 14 day 21

Vehicle 25(OH)D3 1,25(OH)2D3

24,25(OH)2D3 LPS LPS+25(OH)D3

LPS+1,25(OH)2D3 LPS+24,25(OH)2D3 Pam3CysK b Pam3CysK+25(OH)D3 Pam3CysK+1,25(OH)2D3

Figure 33: Expression of Tlr-4 mRNA in J744 (a) and MC3T3-E1(b) cells after 5 days, 14 days and 21 days of differentiation. Cells were pretreated with 25(OH)D3, 1,25(OH)2D3 and

24,25(OH)2D3 24 hours before harvesting and treated with LPS (TLR4 ligand) and Pam3CysK

(TLR2 ligand) 6 hours before harvesting. Data represent mean + S.D. of 1/dCt.

100 Discussion

Periodontal disease and osteomyelitis are infection of bone. Most common causative pathogens of osteomyelitis have been reported as Staphylococcus or Gram-positive bacteria(394). However, pathogens associated with severe periodontal disease are mainly Gram- negative bacteria including Aggregatibacter actinomycetemcomitan and Porphyromonas gingivalis(395, 396). Therefore, both TLR2 (Gram –positive bacteria) and TLR4 (Gram-negative bacteria) are implicated as causative agents of bony infection. Immune cells are considered first line of defense against microbes. These immune cells express pattern recognition receptors

(PRRs) such as Toll-like receptors (TLRs) and recognize microbial particles lipopolysaccharides

(LPS), or component of bacterial outer membranes. The liganded receptors trigger inflammatory responses from release of proinflammatory cytokines. These process is crucial for activation of other immune cells in clearance of pathogens. TLR2 can discriminate gram positive bacteria through heterodimerization. TLR2/1 discriminate bacterial lipoprotein (triacylated) while

TLR2/6 recognize mycoplasmal lipoprotein (diacylated)(397).

Some studies suggested that both TLR2 and TLR4 are involved in LPS signaling but are differently regulated in various cells(398, 399). These results could be from contamination of highly endotoxin protein from LPS. Mouse primary osteoblasts and osteoblast cell line MC3T3-

E1 constitutively expressed TLR2 and TLR4. Pam3CysK and LPS upregulated TLR2, TLR4 and

TLR9 mRNA in bronchoalveolar cells and monocytes(400). In current study, LPS also upregulated TLR2 and TLR4 mRNA but at different stage of differentiation of MC3T3-E1.

Pam3CysK suppressed TLR4 mRNA in both primary calvaria and MC3T3-E1 cells. Tissue- specific TLR expression patterns are considered to represent distinct adaptation of each tissue to immune response. Different stages of osteoblasts might need different protection levels from pathogens and result in dissimilar responses of TLR2 and TLR4 mRNA expression. LPS

101 suppressed Tlr4 mRNA expression after 21 days of differentiation of MC3T3-E1 might be explained by desensitization of LPS similar to response in human monocytes to prevent against excessive host responses toward LPS(401).

TLRs are activated by ligand induced homodimerization in TLR4 and heterodimerization in TLR1/2 and TLR2/6 and cause conformational changes in receptor leading to activation of other signaling domains. Toll-IL-1 resistance (TIR) domain-containing adapter proteins including MyD88, TIR domain-containing adapter inducing IFN-β (TRIF) and TRIF-related adapter molecule (TRAM) are important for TLR2 and TLR4 signaling. In current study, primary calvaria’s Trif and Tram mRNA can be detected after 14 days and 21 days of differentiation in contrast to previous reported that Tram was not found in osteoblast(373).

Non-classical function of vitamin D in innate immunity has been widely described mainly in monocytes and macrophages. TLR1/2 activation in human macrophages upregulates

CYP27B1 and VDR leading to enhance expression of cathelicidin and killing intracellular

M.tuberculosis(273). However, roles of TLRs in vitamin D metabolism system in osteoblasts are not elucidated. Current study showed that TLR2 and TLR4 ligands modulated vitamin D system in mouse osteoblasts. LPS induced Vdr mRNA expression after 14 days of differentiation and

Cyp27b1 also showed similar increased in mRNA but with no statistically significant in MC3T3-

E1 cells. In primary calvaria, Cyp27b1 expression was differ from in MC3T3-E1 which could be reflected from different in chromatin architecture similar to differential Cyp27b1 gene in HEK-

293 and MCF-7 cells(402). However, functionality experiment using HPLC analysis showed that after 14 days of differentiation, LPS stimulated significantly more conversion of 25(OH)D3 to

1,25(OH)2D3 which also support roles of extra-renal 1α-hydroxylase in osteoblasts(376). These findings suggested that TLR4-modulated vitamin D system is more responsive in mineralizing osteoblasts.

102 CYP24A1 gene encodes 24-hydroxylase enzyme responsible for neutralization of vitamin

D for excretion. In some cancer cells, CYP24A1 expression is controlled by 5’-CpG island methylation(403). Both LPS and Pam3CysK did not change Cyp24a1 mRNA levels in primary calvaria cells but LPS and Pam3CysK showed differential responses from osteoblasts.

Pam3CysK significantly increased production 24,25(OH)2D3 metabolites in 14 days differentiated MC3T3-E1 cells by HPLC analysis. Study showed that injury triggers increased

TLR2, CYP24A1 in human keratinocytes via vitamin D metabolism system(404). Osteoblast might have similar reaction as keratinocytes to TLR2 ligand.

On account of differential responses from LPS and Pam3CysK in Cyp27b1 transcription and function, Cyp27b1-promoter luciferase analysis was done to explore promoter binding activity of TLR2 and TLR4 ligands. Regulation of CYP27B1 promoter has been reported by parathyroid hormone (PTH)(96), TGF-β(370) and DNA methylation(375, 405). TGF-β repressed

CYP27B1 promoter at -531 and -305 bp in ROS 17/2.8 osteoblast cells. However, another study showed strong upstream repressive region from -997 to -1100 in osteoblasts(406). LPS and

Pam3CysK showed differential activities of Cyp27b1-promoter-Luc constructs. Pam3CysK significantly upregulated pCyp27b1(-305)-luc activity after 14 days of differentiation.

Maturation time also showed distinct regulation on Cyp27b1 promoter. Current study showed novel differential regulation of Cyp27b1 promoter in osteoblast depend on maturation stages.

Vitamin D metabolites were added into cultures to observe the role of vitamin D in regulation of CYP27B1 promoter in inflammation environment. Vitamin D did not alter

Cyp27b1-promoter activity in LPS treated group. However, both 25(OH)D3 and 1,25(OH)2D3 increased pCyp27b1(-305)-luc and pCyp27b1(-1500)-luc activities but not pCyp27b1(-997)-luc that contains enhancer region. However, results from mRNA expression showed 25(OH)D3 and

103 1,25(OH)2D3 increased Cyp27b1 expression levels in LPS treated group. This might indicate post-transcriptional regulation of vitamin D on Cyp27b1 in TLR4 signaling pathway.

In monocytes cultured with 100nM vitamin D3 TLR2, TLR4 and TLR9 expression was downregulated in time dependent manner(386). 1,25(OH)2D3 also suppresses expression of

TLR2 and TLR4 protein and mRNA in human monocytes(336). ChIP-seq with 1,25(OH)2D3 treatment showed VDR-binding sites were enriched near autoimmune genes identified from genome-wide association studies (GWAS)(407).

Range of differentiation/mineralization potentials were found in various subclonal of

MC3T3-E1 cells(408). Highly differentiating subclones displayed strong correlation between osteocalcin mRNA and transcriptional activity. In primary calvaria, LPS and Pam3CysK did not alter Alp expression levels. However, in MC3T3-E1, Pam3CysK significantly suppressed Alp expression levels during all differentiation time points. The reason why this suppression did not display in alkaline phosphatase staining of MC3T3-E1 cells possibly due to timing of exposure of 6 hours and 24 hours were not enough for protein machinery to produce changes in function of cells. Pam3CysK also suppressed Ocn mRNA expression during all differentiation time points in MC3T3-E1 cells. Additional of vitamin D metabolites did not alter Ocn expression levels but emphasize on differential regulation of TLR2 and TLR4 ligands on osteoblast marker genes.

TLRs were showed to affect osteogenic potential of human mesenchymal stromal cells depending on their tissue origin. TLR3 and TLR4 activation increased osteogenic differentiation process of adipose derived mesenchymal stem cells(409). Moreover, Oncostatin M (OSM) secreted from LPS-activated macrophages can stimulate osteoblast differentiation from mesenchymal stem cells(410). Recent study also showed that weak inflammatory response of

TLR2 and TLR4 ligands could enhance osteogenesis(411). Current study used 1nM of LPS and

104 100nM of Pam3CysK. The concentration used possibly favor weak inflammatory response in

TLR4 ligands but generate higher level of inflammation in TLR2 ligand group.

LPS and Pam3CysK are part of Toll-like receptor ligands. However, their effects on mouse osteoblast yielded contrast responses. RANKL with M-CSF stimulate differentiation of osteoclast precursors to osteoclasts. LPS has been showed to increase RANKL mRNA in osteoblast cell lines(362). LPS also promotes survival of osteoclasts via TLR4(412). Current study showed Pam3CysK suppressed Opg expression in MC3T3-E1 and LPS increased Opg levels in primary calvaria cells. Human gingival fibroblasts OPG were upregulated by LPS and result in inhibition of osteoclastogenesis(413). Moreover, mice injected with LPS showed significantly down-regulation of RANKL whereas serum OPG levels were upregulated(414,

415). In contrast to rheumatoid arthritis synoviocytes, TLR2 and TLR4 were both stimulated upregulation of RANKL(416). Nonetheless, Pam3CysK also enhanced Rankl mRNA levels in primary calvaria cells. These results encouraged that Pam3CysK or TLR2 ligand advocates for osteoclastogenesis effect in mouse osteoblasts.

Monocytes and macrophages but not osteoclasts produce cytokines in response to LPS including IL-1, TNF-α and IL-6(385). Mouse osteoblast infected with Porphyromonas gingivalis showed induced production of interleukin-6(417). IL-6 is important for inflammatory acute- phase response after tissue damage or infection(418). It is also showed to induce bone resorption(389) and osteoclast differentiation(419). TLR9-induced IL-6 production was impaired in monocytes cultured with vitamin D3(386). Some study indicated that IL-6 can stimulate mesenchymal progenitor cells toward osteoblastic lineage(420). LPS induced more IL-6 mRNA production than Pam3CysK after 14 days of osteoblast differentiation in MC3T3-E1 cells. This could be due to IL-6 is activated by both MyD88 dependent and TRIF/TRAM pathway in LPS treated group. Similar increased in IL-6 but decreased in OPG mRNA was found in primary

105 osteoblast under mechanical loading(421). Other factors that modulate IL-6 in osteoblasts are including estrogen(422)

TLRs activation through TRIF/TRAM adaptor proteins induced IRF-3 transcription factor and resulted in production of type I interferon or IFN-β. IFN-β treatment in peripheral blood lymphocytes resulted in marked decreased of osteoclastogenesis(423).In addition, RANKL induces IFN-β gene in osteoclast precursor(424). IFN-β also increased in osteoclast progenitor cells with 1,25(OH)2D3 treatment(425) and interfere with osteoclastogenesis. Mice deficient in

IFN-β exhibited osteopenia and increased in osteoclastogenesis(426). IFN-β is suggested to play critical roles in immune responses and bone homeostasis in both physiological and pathological conditions. Current study showed LPS significantly upregulated Ifn-β expression after 14 days and 21 days of differentiation in MC3T3-E1. Pam3CysK also upregulated Ifn-β after 21 days of differentiated MC3T3-E1 cells. Interferon production can suppress tumors. IFN induced osteosarcoma inhibition and replaced by mature normal bone tissue(427).

Heat shock protein 70 (Hsp70) induces inflammatory cytokines production by utilizing both TLR2 and TLR4(428). These combinations were showed to have synergistic effects on NF-

κB activity. Heat shock proteins also bind to pattern recognition receptors (PRRs) such as TLRs,

NODs and RLRs. DAMPs or danger associated molecular patterns can be signaled by injury of host cells such as fracture or even from orthodontics tooth movement(300). These signaling can also trigger upregulation of cytokines(429).

Interest in cationic antimicrobial peptides and TLRs was sparked by elegant study by Liu et al, that redefine the world of vitamin D(273). Cationic antimicrobial peptides are expressed in immune cells and epithelial cells. Their ability against pathogen included bacteria, fungi and viruses. Cathelicidin production by TLR1/2 stimulation responsible in autophagy of bacteria.

Current study showed that primary osteoblast constitutively express Camp mRNA. Camp

106 expression was modulated by Pam3CysK or TLR1/2 ligands similar to macrophages respond to

M.tuberculosis. LPS did not increase Camp expression. TLR3 and TLR4 agonists have been showed to suppress vitamin D-induced Camp expression via inhibition of retinoid-Xreceptor α

(RXRα) expression(430). On the contrary, LPS enhanced expression of cathelicidin-related antimicrobial peptide (CRAMP) in osteoblasts and neutralize action of LPS(431). In addition to antibacterial property, CAMP also reported to stimulated differentiation of monocytes into a novel bone forming cells(432). Induction of Camp by TLR2/1 may also play a role in bone remodeling.

Differential responses of TLR2 and TLR4 in osteoblast could be due to several factors.

Highly conserved structure among TIR domains is located in the loop connecting the second β strand with the second helix or “BB loop”(433). Differential in BB loops between TLR2 and

TLR4 may serve distinct roles in function of both TLRs(434). Responses of tissue to LPS also depend on doses and duration. Tolerogenic doses of LPS induced antimicrobial activity of macrophages while lethal doses of LPS induces different responses(435). Increased TLR2 and

TLR4 expression in monocytes are found in patients with type 1 diabetes which could contribute to proinflammatory state of disease(436). Similar contribution to proinflammation may also explain in mouse osteoblasts stimulated by TLR2/1 agonist.

Institute of Medicine and the Endocrine Society have made recommendation for vitamin

D status. Vitamin D deficiency is defined as a 25(OH)D3 below 20 ng/ml and vitamin D insufficiency is defined as 21-29 ng/ml(437). Study about relationship of vitamin D supplementation and methylation status of vitamin D metabolism genes suggested that CYP2R1 gene responsible for metabolizing vitamin D to 25(OH)D3 methylation levels are associated with amount of increased serum 25(OH)D3 from vitamin D supplementation(438).

107

Chapter 3

Aim 3: To determine molecular mechanism of TLRs and observe innate immune response of TLRs in MC3T3-E1 cells

Toll-like receptor signaling through MyD88 pathway. However, TLR4 also contain another adaptor proteins TRIF/TRAM. TRIF/TRAM was reported to activate IRF and result in secretion of IFN-β in monocytes and macrophage cells. Previous aim showed the differential regulation of

TLR2 and TLR4 in mouse osteoblasts. Osteoblast also exhibits functional TLRs, this study will explore signaling pathway of TLR2 and TLR4 and their effectors in mouse osteoblast.

Materials and methods

MC3T3-E1 mouse osteoblasts were cultured in alpha-MEM, 10% FBS, 10 ml of

Penicillin-streptomycin-Glutamine and differentiated with 5mM beta-glycerophosphate and 5

µg/ml. ascorbic acid at 37 degree C and 5 % carbon dioxide. Reagents such as 25(OH)D3 and

-3 -6 1,25(OH)2D3 were prepared a stock solution at 10 M and 10 M, respectively in absolute ethanol. Cells were cultured under differentiation medium for 24 hours, 3 days, 5 days, 2 weeks and 3 weeks. Vitamin D metabolites were added for 6 hours and 24 hours before the last day of experiment. 100nM of 25(OH)D3 and 1 nM of 1,25(OH)2D3 were used in experiment. Vehicle group was added with 0.1% ethanol. LPS (Lipopolysaccharide) or Toll-like receptor 4 ligands and Pam3CysK or Toll-like receptor 2 ligands were used to induce the immune response of osteoblastic cells at the concentration of 1 ng/ml. and 100 ng/ml. respectively. Cells were pretreated with TLR-ligands 6 hour prior to treatment with any vitamin D metabolites and harvested. Tosyl-L-lysyl-chloromethane hydrochloride (TLCK) 1 µM and 10 µM were added 6 hours prior to TLR-ligands challenged. siRNA transfection 108 MC3T3-E1 cells were plated in 6 well-plate and cultured in differentiation media. After 12 days of differentiation, cells were transfected with annealed double-stranded siRNA specific for TRIF

(Ticam 1) and TLR4 from Invitrogen at 50 pmol and 100 pmol with Silencer negative control siRNA (Invitrogen) 75 pmol using Lipofectamine RNAiMAX according to manufacturer’s instruction. After 24 hours, cells were treated with Pam3CysK 1 µg/ml and LPS 1ng/ml for 6 hours.

RNA isolation

RNA was isolated from cells treated with vitamin D metabolite and vehicle for different time periods of differentiation. RNA was extracted using Trizol (Invitrogen). RNAs were reverse- transcribed using SuperScript III RT (Invitrogen) as described by the manufacturer. 150 ng of

RNA was used to reverse transcriptase to cDNA.

Quantitative real time RT-PCR amplification of cDNAs

Expression of mRNAs for Cyp27b1, Vdr, Il-6, Ocn, Alp1 and Opg were quantified using an

Applied Biosystems (ABI). All reactions were mulitplexed with the housekeeping 18S rRNA gene (Assays-on-Demand (ABI) primer and probe mix Hs999999901_s1). Data were obtained as

Ct values (the cycle number at which logarithmic PCR plots across a calculated threshold line), and used to determine delta Ct values. Ct of target gene cDNA was conducted using the following Tagman mouse gene expression assays: Cyp27b1, Vdr, Il-6, Ocn, Alp1 and Opg. All cDNAs were amplified under following conditions: 50°C for 2 min; 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed in triplicate.

Visual representation of data was conducted by mean of 1/dCt.

Immunoblot

Whole cell lysates were prepared with lysis buffer (RIPA and a mixture of protease inhibitor) and were centrifuged at 10,000 rpm (Eppendorf centrifuge). Protein concentration was

109 determined by nanodrop machine. Western blot analysis was performed as described previously.

In brief, protein was resolved on 7% to 10% SDS-PAGE and transferred onto PVDF membrane.

The membranes were blocked with 5% non-fat milk in TBST buffer (TBS buffer containing

0.1% of Tween-20) for 1 hour at room temperature and incubated overnight at 4 degree C with primary antibodies CYP27B1 (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA),

VDR (1:1000; Santa Cruz Biotechnology,Inc.), beta-actin (1:10000, Santa Cruz Biotechnology,

Inc.). Unbounded antibodies were removed with three times wash for 20 minutes with TBST.

Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000) for 1 hour at room temperature and washed three times for 20 minutes with

TBST. The antibody-associated protein bands were revealed with ECL-plus Western Blotting system (Amersham Biosciences).

Statistical analysis

Differences between groups were compared using ANOVA. A P value less than 0.05 was considered significant.

Results

Previous result in aim 2 showed that MC3T3-E1 cells exhibit functional TLRs and response to

LPS and Pam3CysK stimulation. TIR domain-containing adaptor inducing IFN-β (Trif) or

Ticam1 is adaptor protein for TLR4. MC3T3-E1 cells were differentiated for 5 days, 14 days and

21 days then treated with LPS and Pam3CysK before harvesting. qPCR analysis showed LPS significantly induced Trif mRNA expression during all stages of differentiation (p < 0.05) and especially after 14 days of differentiation (p< 0.001). Pam3CysK treatment showed no different in Trif mRNA expression except after 14 days of differentiation, Pam3CysK suppressed Trif mRNA (p < 0.01)

110 Trif ** * * 0.20000 ** ** * 0.18000 0.16000 0.14000 0.12000 0.10000

1/dCt 0.08000 0.06000 0.04000 0.02000 0.00000 5 days 14 days 21 days

Vehicle LPS Pam3CysK

Figure 34: Quantitative PCR analysis of Trif mRNA expression from MC3T3-E1 cells at 5 days,

14 days and 21 days of differentiation. Cultures were treated with 0.1% ethanol for Vehicle group, LPS and Pam3CysK. Graph represents means of 1/dCt (SD).

Trif siRNA on TLRs treatment

Since Trif is essential adaptor to MyD88 and contribute to late phase activation of NF-κB and MAPK(439) and Trif mRNA is induced by LPS stimulation. Trif siRNA was transiently transfected in 14 days differentiated MC3T3-E1 cells. Trif siRNA was successfully suppressed

Trif mRNA induced by Vehicle and LPS (p < 0.05). Trif siRNA did not suppress Trif mRNA in

Pam3CysK treated group (Figure 35).

111 * Trif * 0.1400 **

0.1200

0.1000

0.0800

1/dCt 0.0600

0.0400

0.0200

0.0000 Vehicle LPS Pam3CysK

Negative 50pMol 100pMol

Figure 35: Quantitative PCR analysis of Trif mRNA expression from MC3T3-E1 cells at 14 days of differentiation. Cells were transiently transfected with Trif siRNA at 50 pMol and 100 pMol.

Cultures were treated with 0.1% ethanol for Vehicle group, LPS and Pam3CysK. Graph represents means of 1/dCt (SD).

Trif siRNA did not suppress Tlr2 and Tlr4 mRNA expression since Trif is downstream to

TLRs.

14 days differentiated MC3T3-E1 cells were transient transfected with Trif siRNA 2 days before harvesting and cultures were treated with LPS and Pam3CysK 6 hours before harvesting.

Trif siRNA did not suppressed Tlr2 and Tlr4 mRNA expression but exhibited some trends of upregulation of Tlr2 and Tlr4 mRNA expression with no statistically significant different.

Because Trif is adaptor protein of Tlr4, inhibition of Trif did not alter its upstream receptors.

112 Tlr2

0.08

0.07

0.06

0.05

0.04 1/dCt 0.03

0.02

0.01

0 Scramble 50pmol 100pmol Scramble 50pmol 100pmol LPS Pam

Tlr4

0.07

0.06

0.05

0.04

1/dCt 0.03

0.02

0.01

0 Scramble 50pmol 100pmol Scramble 50pmol 100pmol LPS Pam

Figure 36: Quantitative PCR analysis of Tlr2 (a) and Tlr4 (b) mRNA expression from MC3T3-

E1 cells at 14 days of differentiation. Cells were transiently transfected with Trif siRNA at 50 pMol and 100 pMol. Cultures were treated with LPS and Pam3CysK. Graph represents means of

1/dCt (SD).

113 Trif siRNA on vitamin D metabolism gene expression

LPS or TLR4 ligand upregulated Vdr mRNA expression in both monocytes/macrophages and 14 days differentiated MC3T3-E1 cells while Pam3CysK did not alter Vdr mRNA levels. Trif siRNA was introduced in 14 days differentiated MC3T3-E1 cells and found to suppress Vdr mRNA expression levels induced by LPS at p < 0.01. Moreover, Trif siRNA also silenced Vdr mRNA expression in vehicle and Pam3CysK treated group at p < 0.01.

** Vdr ** ** ** 0.1200 **

** 0.1000

* 0.0800

0.0600 1/dCt

0.0400

0.0200

0.0000 Vehicle LPS Pam3CysK

Negative 50pMol 100pMol

Figure 37: Quantitative PCR analysis of Vdr mRNA expression from MC3T3-E1 cells at 14 days of differentiation. Cells were transiently transfected with Trif siRNA at 50 pMol and 100 pMol.

Cultures were treated with LPS and Pam3CysK. Graph represents means of 1/dCt (SD).

Trif siRNA on osteoblast and osteoclast markers

MC3T3-E1 cells were also tested for Alp mRNA expression levels after Trif siRNA treatment.

Trif siRNA suppressed Alp mRNA expression in all treatment groups at p < 0.01.

114 ** Alp ** ** ** ** ** 0.1200

0.1000

0.0800

0.0600 1/dCt 0.0400

0.0200

0.0000 Vehicle LPS Pam3CysK

Negative 50pMol 100pMol

Figure 38: Quantitative PCR analysis of Alp mRNA expression from MC3T3-E1 cells at 14 days of differentiation. Cells were transiently transfected with Trif siRNA at 50 pMol and 100 pMol.

Cultures were treated with LPS and Pam3CysK. Graph represents means of 1/dCt (SD).

Osteoprotegerin (Opg) mRNA expression in 14 days differentiated MC3T3-E1 cells were also suppressed by Trif siRNA treatment at p < 0.01 in all treatment groups.

Opg ** ** ** ** 0.2000 ** 0.1800 ** 0.1600 0.1400 ** 0.1200 0.1000

1/dCt 0.0800 0.0600 0.0400 0.0200 0.0000 Vehicle LPS Pam3CysK

Negative 50pMol 100pMol

115 Figure 39: Quantitative PCR analysis of Opg mRNA expression from MC3T3-E1 cells at 14 days of differentiation. Cells were transiently transfected with Trif siRNA at 50 pMol and 100 pMol. Cultures were treated with LPS and Pam3CysK. Graph represents means of 1/dCt (SD).

Trif siRNA and cytokines production

IL-6 production was described in TLR signaling through MyD88 pathway. Trif is part of MyD88 independent pathway. However, Trif siRNA suppressed Il-6 mRNA expression in vehicle, LPS treated and Pam3CysK treated MC3T3-E1 cells at p < 0.01.

** Il-6 ** 0.1800 ** ** 0.1600 ** ** ** ** 0.1400 0.1200 0.1000 **

1/dCt 0.0800 0.0600 0.0400 0.0200 0.0000 Vehicle LPS Pam3CysK

Negative 50pMol 100pMol

Figure 40: Quantitative PCR analysis of Il-6 mRNA expression from MC3T3-E1 cells at 14 days of differentiation. Cells were transiently transfected with Trif siRNA at 50 pMol and 100 pMol.

Cultures were treated with LPS and Pam3CysK. Graph represents means of 1/dCt (SD).

Protein analysis of Mitogen-activated protein kinases (MAPKs)

MAPK is one of downstream signaling cascades in MyD88 pathway. TRAF6 bind to

MyD88 and act with ubiquitinase conjugating enzyme to ubiquitin TAK1. TAK1 then phosphorylates IKKβ and initiates NF-κB activation. TAK1 also activates MAPK and result in

AP-1 activation. NF-κB and AP-1 activations result in activation of inflammatory cytokine 116 genes. MC3T3-E1 cells were differentiated for 14 days and treated with LPS and Pam3CysK with addition of TLCK. Protein analysis from western blot showed Pam3CysK increased phosphor-P38 protein while LPS did not showed activation in phosphor-p38. TLCK inhibitor used for NF-κB inhibition suppressed phosphorylation of p38 in both Pam3CysK and LPS stimulations. (Figure)

Figure 41: Protein expression of Phosphor-p38 (top row), p38 (middle row) and β-actin (bottom row) from MC3T3-E1 differentiated in differentiation media 14 days. Cells were treated with

TLCK 24 hours and treated with LPS and Pam3CysK 6 hours before harvesting.

ERK1/2 or p42/44 MAPK is also one of TAK1 downstream cascades. LPS and

Pam3CysK showed similar phosphor-ERK1/2 protein levels. However, addition of TLCK increased phosphorylation of ERK1/2 in vehicle, LPS and Pam3CysK treated group. 25(OH)D3 appeared to suppress phosphorylation of ERK1/2 (Figure).

117

Figure 42: Protein expression of Phosphor-ERK1/2 (top row), ERK1/2 (middle row) and β-actin

(bottom row) from MC3T3-E1 differentiated in differentiation media 14 days. Cells were treated with TLCK and 25(OH)D3 for 24 hours and treated with LPS and Pam3CysK 6 hours before harvesting.

NF-κB inhibitor on TLRs treatment

NF-κB is important for both MyD88 dependent and MyD88 independent pathway. Alkaline phosphatase staining of 14 days differentiated MC3T3-E1 pretreated with TLCK showed no change of Alkaline phosphatase activity after stimulated with LPS and Pam3CysK.

118

Figure 43: Alkaline phosphatase activity of 14 days differentiated MC3T3-E1 cells treated with

TLR ligands (Pam3CysK and LPS) 6 hours (top row) and 24 hours (bottom row) before harvesting.

TRIF/TRAM responses to TLCK

MC3T3-E1 cells were cultured in differentiation media for 14 days and 21 days and pretreated with TLCK 24 hours before harvesting. Cells were also treated with LPS and Pam3CysK 6 hours before harvesting. TLCK did not alter Trif mRNA expression in Vehicle and Pam3CysK treated group after both 14 days and 21 days of differentiation. However, TLCK significantly suppressed Trif mRNA expression after 21 days of differentiation. After 14 days of differentiation, TLCK also showed suppression of Trif mRNA in LPS treated group but without statistically significant (Figure b). Signaling cascades of TLR4 also involve TRAM in monocytes and macrophages. After 14 days of differentiation, 1 µM and 10 µM TLCK increased Tram mRNA expression in LPS and Pam3CysK treated group at p < 0.05. In addition, 1 µM and 10

µM TLCK also increased Tram mRNA expression in all treated groups at p < 0.01.

119 Trif

0.350000

0.300000

0.250000 **

0.200000 **

1/dCt 0.150000

0.100000

0.050000

0.000000 Vehicle LPS Pam3CysK Vehicle LPS Pam3CysK 14 days 21 days No TLCK 1µM TLCK 10 µM TLCK a

Figure 44: Quantitative PCR analysis of Trif (a) and Tram (b) mRNA expression from MC3T3-

E1 cells at 14 days and 21 of differentiation. Cells were treated with 1µM and 10µM TLCK

120 inhibitor. Cultures were treated with LPS and Pam3CysK 6 hours before harvesting. Graph represents means of 1/dCt (SD).

TLCK responses in vitamin D metabolism genes

After 14 days of differentiation, TLCK suppressed upregulation of Vdr mRNA expression by

LPS (p < 0.05 in 1 µM and p < 0.01 in 10 µM). However, after 21 days of differentiation, TLCK increased Vdr mRNA expression at p < 0.01 (Figure a). For Cyp27b1 mRNA expression, TLCK only increased Cyp27b1 mRNA expression after 14 days of differentiation (p < 0.05 in 1 µM and p < 0.01 in 10 µM) (Figure b).

** Vdr * 0.250000

0.200000

** 0.150000 ** 1/dCt 0.100000

0.050000

0.000000 Vehicle LPS Pam3CysK Vehicle LPS Pam3CysK 14 days 21 days No TLCK 1µM TLCK 10 µM TLCK a

121 Cyp27b1

0.140000 ** 0.120000 *

0.100000

0.080000

1/dCt 0.060000

0.040000

0.020000

0.000000 Vehicle LPS Pam3CysK Vehicle LPS Pam3CysK 14 days 21 days No TLCK 1µM TLCK 10 µM TLCK b

Figure 45: Quantitative PCR analysis of Vdr (a) and Cyp27b1 (b) mRNA expression from

MC3T3-E1 cells at 14 days and 21 days of differentiation. Cells were treated with 1µM and

10µM TLCK inhibitor. Cultures were treated with LPS and Pam3CysK 6 hours before harvesting. Graph represents means of 1/dCt (SD).

TLCK responses on osteoblast markers

TLCK rescued Alp mRNA suppression by LPS and Pam3CysK after 14 days of differentiation but did not alter Alp mRNA expression in Vehicle treated group. Similar upregulation of Alp mRNA expression by TLCK also found in MC3T3-E1 after 21 days of differentiation (Figure a).

On the other hand, TLCK did not change Ocn mRNA expression after 14 days and 21 days of differentiation of MC3T3-E1 cells except in 14 day- differentiate MC3T3-E1 with Pam3CysK treated group. 1µM and 10µM TLCK rescued suppression of Ocn mRNA by Pam3CysK at p <

0.01 (Figure b). TLCK can also rescued Dmp1 mRNA suppression by Pam3CysK after 14 days and 21 days of differentiate MC3T3-E1 cells at p < 0.01 (Figure c). 122

123

Figure 46: Quantitative PCR analysis of Alp (a), Ocn (b) and Dmp1 (c) mRNA expression from

MC3T3-E1 cells at 14 days and 21 days of differentiation. Cells were treated with 1µM and

10µM TLCK inhibitor. Cultures were treated with LPS and Pam3CysK 6 hours before harvesting. Graph represents means of 1/dCt (SD).

TLR4 siRNA on TLRs treatment

Tlr4 siRNA effects

Previous study reported that Toll-like receptor signaling in osteoblast does not require Trif/Tram, and Tram does not exist in osteoblast. Tlr4 siRNA was transiently transfected in 14 days differentiated MC3T3-E1 cells. Vitamin D metabolites, 25(OH)D3 is also given to cultured cells to observe any effects of vitamin D in TLRs challenged environment. Tlr2 and Camp mRNA was not altered by Tlr4siRNA and vitamin D treatment (Figure a, c). However, Ifn-β mRNA

124 expression was significantly increased by 25(OH)D3 when compared with Tlr4 siRNA treatment only group.

Tlr2

0.140

0.120

0.100

0.080

1/dCt 0.060

0.040

0.020

0.000 Vehicle LPS Pam3CysK scramble siRNA Tlr4siRNA siRNA+25(OH)D3 a

Ifn-β * 0.080

0.070

0.060

0.050

0.040 1/dCt 0.030

0.020

0.010

0.000 Vehicle LPS Pam3CysK scramble siRNA Tlr4siRNA siRNA+25(OH)D3 b

125 Camp

0.07

0.06

0.05

0.04

1/dCt 0.03

0.02

0.01

0 Vehicle LPS Pam3CysK scramble siRNA Tlr4siRNA siRNA+25(OH)D3 c

Figure 47: Quantitative PCR analysis of Tlr2 (a), Ifn-β (b) and Camp (c) mRNA expression from

MC3T3-E1 cells at 14 days of differentiation. Cells were treated with scramble siRNA,

Tlr4siRNA and TLR4siRNA and 25(OH)D3. Cultures were treated with vehicle, LPS and

Pam3CysK 6 hours before harvesting. Graph represents means of 1/dCt (SD).

Discussion

Results from chapter 2 showed that there were differential regulations between TLR2 and

TLR4 stimulation in mouse osteoblasts. These two receptors signaling cascades share common

MyD88 pathway but TLR4 also transduces signal through TRIF/TRAM pathway. MyD88- dependent response mediates induction of proinflammatory cytokines and RANKL production in osteoblasts(373). TRIF-dependent response is important for induction of interferons and interferon related genes.

TLR4 signaling is unique in that all other interferon activators induced activation of IRF3 or IRF7 at the intracellular levels but TLR4 senses bacterial cell wall and activate from plasma

126 membrane of cells. TRIF functions as downstream to both TLR3 and TLR4 signaling pathway, but TRAM is only restricted to TLR4 pathway from dominant negative experiment in monocytes(440). TRIF does not directly bind to TLR4 but TRAM transmit the LPS-TLR4 signal to TRIF and activates IRF-3 similar to Mal/TIRAP adapter to MyD88(441). Recent study indicated that osteoblast response to LPS and TLR1/2 stimuli by IL-6 production solely through

MyD88 pathway and lack of TRIF-related adaptor molecule (TRAM) were showed in osteoblast(373). Current study from chapter 2 contradicted the statement by indicated constitutively expressed Tram mRNA expression in primary calvaria cells. IFN-β effector cytokines from TRIF/TRAM pathway was also induced in LPS treated osteoblasts. Therefore, detection of TRAM in osteoblasts suggested roles of interferon responses in this cells. Moreover, differential responses from TLR2 and TLR4 might become from TRIF/TRAM dependent pathway of TLR4 signaling. Also, study showed differences in ubiquitination of TRAF3 of TLRs are critical to selective production of proinflammatory cytokines or interferons(333). In addition,

IFN-β reported to inhibit mineralization in osteoblasts(442). In this chapter, Trif siRNA was conducted to observe the role of Trif in mouse osteoblast.

According to predicted structures, Mal binds to TLR4 homodimer with closer proximity than TRAM does. This could explain Mal/MyD88 trigger early NF-κB expression and

TRAM/TRIF trigger late NF-κB expression(443). It was proposed that TLR4 might first induces

MyD88 signaling at the plasma membrane and then endocytosed and activates TRIF/TRAM signaling from early endosomes. It might be possible that all type I interferon responses are from intracellular location(444). TrifsiRNA inhibited Trif mRNA in vehicle control and LPS treated group but not in Pam3CysK treated group. Inactivation of Trif mRNA in Pam3CysK could be due to that Pam3CysK signaling cascade does not require Trif. Therefore, TrifsiRNA was not able to inhibit already low levels of Trif in Pam3CysK treated osteoblast. TrifsiRNA did not

127 inhibit both Tlr2 and Tlr4 mRNA in LPS and Pam3CysK treated osteoblasts. Tlr2 mRNA showed trends of increasing in mRNA with TrifsiRNA. TrifsiRNA altered expression of Vdr,

Alp. Opg and Il-6 mRNA in 14 days differentiated MC3T3-E1. These results suggested that Trif might responsible for intracellular signaling important for osteoblast maturation and cytokines production.

Toll-like receptors after engagement by their ligands activate two major kinase-mediated signaling pathway; MAPK and IκB kinase (IKK) complexes which activate AP-1 and NF-κB transcription factors. Activity of IκB is modulated by phosphorylation and proteolysis. MAPK pathway in TLRs signaling involve ERK, p38 and JNK pathway(445). MyD88 recruit members of IL-1 receptor-associated kinase (IRAK) family upon activation and phosphorylate TNF receptor-associated factor 6 (TRAF6) to activate MAPK kinase kinase (MAPKKK) called TAK-

1 which phosphorylate kinase upstream to p38 and JNK. TAK-1 can also activate IκBα kinase complex leading to release of NF-κB and translocate into nucleus.

In mouse osteoblasts, p38 MAPK along with ERK is activated as osteoblast differentiates(446, 447). Osteoblast with dominant negative p38 MAPK displayed delay in ALP and mineral deposition(448). Inhibition of p38 in mouse osteoblast by SB203580 inhibitor decreased alkaline phosphatase (ALP) activity(449). Moreover, PTH activates p38 MAPK which also resulted in upregulation of ALP and play major roles in bone matrix development(450).

ERK and p38 MAPK also important for calcium sensing receptor-stimulated mitogenic responses in mouse osteoblasts(451). Fibroblast growth factor (FGF) activates p38 MAPK and resulted in IL-6 production in MC3T3-E1 cells(452). Divergent roles of p44/42 and p38 MAPK displayed in osteoblasts. p38 MAPK exhibit positive effect in osteocalcin synthesis while p44/42

MAPK acts as a negative regulator(453). This previous finding suggested differential role of p38 and p44/42 MAPK in osteoblasts. Current study showed increased in phosphorylation of p38 by

128 Pam3CysK compared with vehicle and LPS groups. This result emphasizes divergent roles of p38 and p44/42 in mouse osteoblasts. In mature dendritic cells, p38 inhibitor suppresses more cytokine production and surface antigen expression when compared with ERK inhibitor(454).

Blockage of p38 activity inhibit upregulation of Cyp27b1 by TLR2/1 in human monocytes(455) support the role of p38 in TLR2/1 signaling. Mouse osteoblast might display similar p38 activation as monocytes.

Bone marrow stem cells displayed protective effects on LPS-induced lung injury via inhibition of TLR3 which leads to inhibition of TRIF and MAPK as well as NF-κB in rats(456).

TLCK (Nα-p-Tosyl-L-lysyl-chloromethyl ketone) is inhibitor of trypsin and trypsin-like serine proteases. It has been used as inhibitor for cytotoxic T lymphocytes(457), their cytotoxicity(458), nitric oxide production in β-cells(459) and of proliferation of transformed mouse fibroblasts(460). Recent study showed TLCK could inhibit mature caspases in cells exposed to pro-apoptotic stimuli(461). Studies also showed NF-κB inhibitory effect of TLCK.

TLCK suppresses NF-κB/p65 nuclear translocation in LPS-stimulated THP-1 cell(462). Also

TLCK inhibits phosphorylation of IκB leading to inhibition of nuclear translocation of NF-

κB(463, 464). In this study, TLCK also suppressed induction of Vdr expression by LPS suggesting role of NF-κB signaling in Vdr activation in mouse osteoblasts. TLCK also neutralized suppression of osteoblast marker genes including Alp, Ocn and Dmp1 expression by

Pam3CysK. TLCK also inhibit increased in phosphorylation of p38 by Pam3CysK which could shift MAPK phosphorylation profile of activated osteoblasts towards TLR4 stimulation. Current study suggested that different levels of MAPK phosphorylation could responsible for differential regulation of osteoblasts by TLR2 and TLR4 ligands activation.

Stimulation of TLR1/2 in monocytes not only activates CYP27B1 and VDR upregulation but also enhance cytokines production such as IL-15 and IL-1β. These monokines act in

129 autocrine fashion to promote antimicrobial peptides production(465). 1,25(OH)2D3 treated phagocytes showed impaired NF-κB/RelA translocation into nucleus and reduced p38 and p42/44 phosphorylation(336).

Studies suggested LPS response could activate through both TLR2 and TLR4(398, 399).

This chapter employed Tlr4siRNA to observe effect of LPS and Pam3CysK on mouse osteoblast through TLR2. Results showed that LPS did not increase Tlr2 expression with Tlr4siRNA treatment. In addition, IFN-β did not increase in LPS treated group. This finding indicates that

LPS requires TLR4 to upregulated production of IFN-β in mouse osteoblasts. Interferon-β (IFN-

β) produces by osteocytes exhibit osteoclastogenesis inhibition capacity to osteoclast precursor cells(466). In early phase of osteoblast differentiation, IFN-β inhibits extracellular matrix synthesis and mineralization leading to delayed bone formation(467). Cathelicidin is important for autophagy in monocytes via 1,25(OH)2D3(277). Autophagy is important for degradation of cytosolic components and organelle turnover(468). Tlr4siRNA did not change Camp mRNA levels in mouse osteoblasts. This result validate role of TLR2 signaling in upregulation of Camp expression.

Future experiments with knockdown of vitamin D system with TLR2 and TLR4 challenges might help elucidate the role of vitamin D metabolism and innate immune function in osteoblasts. Furthermore, specific MAPK inhibitors will help unravel differential regulation of

TLR2 and TLR4.

Conclusion

This study showed novel differential responses of TLR2 and TLR4 in osteoblasts and also demonstrated that mouse osteoblasts constitutively express Trif and Tram throughout osteoblast differentiation period. Study from TrifsiRNA displayed that Trif/Tram dependent

130 TLR4 signaling might play important role in osteoblast differentiation. Apart from different TLR adaptor proteins, differential in MAPK phosphorylation might be the reason why mouse osteoblasts showed divergent effects of TLR2 and TLR4 ligands stimulation. Phosphorylation of p38 upon TLR2 ligand stimulation might activate suppression of osteoblast markers and upregulate osteoclastogenesis. Short term stimulation of LPS did not change osteoclastogenesis effect of osteoblast but increase vitamin D metabolism genes and increase conversion of

25(OH)D3 to 1,25(OH)2D3.

TLR1 TLR2 TLR4 MyD8 TLR4 8 MyD8 8 TRIF/ NF-�B TRAM

P-p38 P-ERK1/2 Cyp27b1 25(OH) D Camp

1,25(OH) D 2 3 Vdr IL-6

Ifn-β

Dmp1 Ocn ↓ Alp↓ ↓

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