University of Southern California Dissertations and Theses

The effect of leucine-rich amelogenin peptide on mouse bone density by histological analysis

(USC Thesis Other)

The effect of leucine-rich amelogenin peptide on mouse bone density by histological analysis

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content THE EFFECT OF LEUCINE-RICH AMELOGENIN PEPTIDE ON MOUSE BONE DENSITY BY HISTOLOGICAL ANALYSIS by Yisu Li _________________________________________________________ A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (CRANIOFACIAL BIOLOGY) August 2012 Copyright 2012 Yisu Li ii TABLE OF CONTENTS List of Tables iii List of Figures iv Abstract v Scope of Work vii Introduction 1 Materials and Methods 8 Results 16 Conclusions and Discussion 23 References 29 iii LIST OF TABLES Table 1: PCR time and temperature profiles. 10 Table 2: PCR reaction set up. 11 Table 3: Hematoxylin and eosin (H&E) staining protocol. 13 Table 4: Results of four groups of comparisons. 22 iv LIST OF FIGURES Figure 1: A schematic diagram showing the mouse amelogenin gene 6 and leucien-rich amelogenin peptide. Figure 2: A schematic diagram of the transgene created for expression 7 of LRAP from mouse FABP4 promoter. Figure 3: Histological images of trabecular bone mass in distal femoral 18 head for the “gain-of-function” comparisons. Figure 4: Histological images of trabecular bone mass in distal femoral 21 head for the “loss-of-function” comparisons. Figure 5: Wnt signaling in regulation of osteoblast specification and 24 differentiation. v ABSTRACT Objective: The objective of this study was to evaluate the effect of Leucine-rich amelogenin peptide (LRAP) on osteogenesis in the mouse femur model. Study Design: In a “gain-of-function” study, LRAP was overexpressed in the bone marrow of transgenic mice. In a “loss-of-function” study, an engineered amelogenin cDNA was “knocked in” to the amelogenin locus, resulting in the lack of LRAP expression. Femora were taken from 12-week-old mice and 4 groups of comparisons were made to determine whether there are trabecular bone density differences. In the “gain-of-function” study: male LRAP transgenic mice were compared with male wild-type mice; female LRAP transgenic mice were compared with female wild-type mice. In the “loss-of-function” study: male delta- A hemizygous mice were compared with male wild-type mice; female delta-A homozygous mice were compared with female delta-A heterozygous mice. Results: No statistically significant bone density difference between the male LRAP transgenic and male wild-type mice was observed. Bone density of female LRAP transgenic mice was statistically higher than that of female wild-type mice. For both male and female mice, there were no statistically significant differences in bone density between the different delta-A genotypes and their corresponding femora. Conclusions: The osteogenesis effect of LRAP is more evident in the female LRAP transgenic mice. Male LRAP transgenic mice did not exhibit statistically vi significant bone mass density difference from their wild-type littermates. For the “loss-of-function” design, no statistically significant bone density difference was found for either comparison. Increased sample size is needed and micro-CT analysis is recommended for complete analysis of osteogenesis role of LRAP. vii SCOPE OF WORK LRAP has been discovered to induce osteogenesis in various cell types in vitro, including embryonic stem (ES) cells and mesenchymal stem cells (MSCs). Studies have found the mechanism is that LRAP promotes osteogenesis of ES cells and MSCs by activating canonical Wnt signaling pathway. I am interested in studying the effect of LRAP expressed by animal itself on bone mass density. In order to do that, a “gain-of-function” study and a “loss-of- function” study were designed. Driven by FABP4 promoter, LRAP is intentionally overexpressed in LRAP transgenic mice. Therefore, the trabecular bone density of LRAP transgenic mice and wild-type littermates were compared for “gain-of-function” study. I expected LRAP transgenic mice have higher trabecular bone density than wild-type littermates. An engineered A-domain-deleted amelogenin cDNA was “knocked in” to the amelogenin locus, resulting in the lack of LRAP expression in delta-A mice (Zhu et al., 2006). Therefore, delta-A mice were used for “loss-of-function” study. I expected delta-A hemizygous mice (X Δ A Y) have lower trabecular bone density than wild-type male (XY). At the same time, I want to see if there is trabecular bone density difference between delta-A homozygous mice (X Δ A X Δ A ) and delta-A heterozygous mice (X Δ A X). viii Since X-chromosome is randomly inactivated in females, heterozygous females are not the best controls for homozygous females but the main reason for using delta-A homozygous and delta-A heterozygous is because the mating scheme would not allow me to have homozygous and wild-type females in the same litter. 1 INTRODUCTION Dental enamel is the hardest tissue in the human body (Ten, 1994). Enamel differs markedly from detine and bone, in that mature enamel contains little to no matrix protein. However, the nucleation, growth and organization of enamel occur in a protein matrix composed predominantly of amelogenin and its cleavage products (Termine et al., 1980; Fincham et al., 1999). Amelogenin plays an indispensable role for proper development of enamel (Gibson et al., 2001) and specific mutations in the amelogenin gene have been shown to be associated with amelogenesis imperfecta (Hart et al., 2002; Wright et al., 2003). Amelogenesis imperfecta is an X-linked developmental defect affecting the formation of enamel (Hart et al., 2002). The teeth have a higher risk for dental cavities and are hypersensitive to temperature changes. In general, an amelogenin gene is located on the X chromosome but in some species, including human, bovine, and porcine, there is an amelogenin gene on the Y chromosome (Gibson et al., 1991; Veis 2003). In other veterbrates, such as mice, the amelogenin gene is restricted to only the X chromosome. Besides providing structural organization of enamel, amelogenin has recently been found to be related to cell signaling. In clinical dentistry, Emdogain was used with success to improve periodontal tissue repair by enhancing proliferation and differentiation of osteoblastic precursor cells (Hammartrom 1997; Hammarstrom et al., 1997; Jiang et al., 2006). Emdogain is composed in its majority by the alternatively 2 spliced full-length amelogenin, amelogenin splice products and leucine-rich amelogenin peptide (LRAP) (Maycock et al., 2002). The amelogenin gene consists of 7 exons and encodes several alternative splice products (Lau et al., 1992). The primary RNA transcript can be alternatively spliced to form at least 15 mature mRNAs (Hu et al., 1997), which are translated into various amelogenin protein isoforms. The full-length murine amelogenin is called M180, and its N-terminal “A domain” detected by the yeast two hybrid assay is involved in the molecular interactions for the formation of nanospheres. The M180 C-terminal “B-domain” contributes to the stability and homogeneity of the nanospheres (Moradian-Oldak et al., 2000). Leucine-rich amelogenin peptide (LRAP) is a 59-residue protein translated from exon 2, 3, 5, 6D and 7 of amelogenin mRNA (Fincham et al., 1983). It is comprised of N-terminal 33 residues and the C-terminal 26 residues of the full-length protein (Fig 1). The biological function of LRAP in enamel formation has only recently been established, however, LRAP has been proposed to be involved in both biomineralization (Gibson, 2008) and in cell signaling events (Veis et al., 2000; Boabaid et al., 2004; Warotayanont et al., 2008). Studies have shown that LRAP can induce osteogenesis in various cell types such as rat muscle fibroblasts (Veis et al., 2000), mouse cementoblasts (Boabaid et al., 2004), mouse oral mucosal cells (Lacerda-Pinheiro et al., 2006), mouse embryonic stem cells 3 (Warotayanont et al., 2008) and mouse mesenchymal stem cells (MSCs) (Wen et al., 2011). LRAP has been shown to increase mineral matrix formation and increase calcium accumulation in the matrix from both wild-type ES cells and amelogenin- null ES cells in vitro (Warotayanont et al., 2008). It has also been demonstrated that LRAP induces the expression of bone maker genes bone sialoprotein (BSP) and osterix (OSx) in osteogenic-induced ES cells (Warotayanont et al., 2008). Moreover, LRAP has been observed to influence the fate determination of primary mouse bone marrow mesenchymal stem cells (BMMSCs) by stimulating osteogenic and inhibiting adipogenic differentiation (Wen et al., 2011). In addition to in vitro culture, an in vivo animal model was used to study LRAP effect (Warotayanont, 2008). In this in vivo animal model, ex vivo expanded BMMSCs were subcutaneously transplanted in the presence of LRAP into immunocompromised host mice. LRAP has been shown to increase bone formation and hematopoiesis as well as to inhibit adipogenesis of human BMMSCs (Warotayanont, 2008). LRAP has been demonstrated to function as a signaling molecule, serving to induce osteoblastogenesis in BMMSCs (Warotayanont, 2008). The mechanism for osteogenesis effect of LRAP on ES cells and MSCs is that LRAP activates the canonical Wnt signaling pathway (Warotayanont et al., 2009; Wen et al., 2011). The Wnt family of secreted glyoproteins has critical role in cell 4 growth and differentiation (Cadigan et al., 1997). Upon Wnt stimulation, stabilized β-catenin accumulates in the cytosol and translocates to the nucleus, where it activates Wnt target genes (Molenaar et al., 1996). In human and mice alike, bone mass is strongly dependent on Wnt signaling pathway (Glass et al., 2006; Glass et al., 2005). Amelogenin expression can be detected in long bone, cartilage, epiphyseal growth plate and bone marrow (Haze et al., 2007). In the current experiment, I want to study the effect of LRAP expressed by animal itself on trabecular bone density. A “gain-of-function” study and a “loss-of-function” study were designed to evaluate the effect of LRAP on mouse femur density. For a “gain-of-function” study, LRAP transgenic mice were used. Transgenic mice contain additionally introduced genetic material in every cell. In LRAP transgenic mice, LRAP is intentionally overexpressed in the bone marrow and the overexpression is driven by FABP4 promoter (Fig 2). The mRNA transcription of LRAP was detected by reverse transcription polymerase chain reaction (RT- PCR). Here we test the hypothesis that LRAP transgenic mice have higher trabecular bone mass density (TBV/TV) than wild-type littermates. Female and male LRAP transgenic mice are compared separately. For a “loss-of-function” study, delta-A mice were used. A domain of amelogenin gene is the amino-terminal conserved domain defined by residues 1-42. For delta-A mice model, an engineered A-domain-deleted amelogenin cDNA was 5 “knocked in” to the amelogenin locus, resulting in the lack of LRAP expression (Zhu et al., 2006). To determine whether lacking of LRAP will result a decrease in trabecular bone mass density, male delta-A hemizygous mice (X Δ A Y) were compared with male wild-type mice (XY). Female delta-A homozygous mice (X Δ A X Δ A ) were compared with female heterozygous mice (X Δ A X). Since X- chromosome is randomly inactivated in females, heterozygous females are not the best controls for homozygous females. But the main reason for using homozygous and heterozygous females is that mating scheme cannot produce homozygous females and wild-type females in the same litter. At the same time, the altered enamel architecture is found only where the engineered amelogenin protein is expressed and that normal enamel architecture is “rescued” when the engineered amelogenin is silent in delta-A heterozygous females (Zhu et al., 2006), suggesting that delta-A heterozygous females and delta-A homozygous females exhibit different phenotypes. In mice, bone modeling regulated by osteoblasts and osteoclasts ensures functional bone morphology and bone mass accrual throughout the first 3 months of life until peak bone mass is reached (Kramer et al., 2010). There are no dramatic trabecular bone mass changes after the first 12 weeks in mice; therefore femora were taken from 12-week-old mice for trabecular bone density comparison. 6 Fig 1. A schematic diagram showing the mouse amelogenin gene and leucine-rich amelogenin peptide (LRAP). LRAP is a 59-residue protein translated from exon 2, 3, 5, 6D and 7 of amelogenin mRNA. It is comprised of N-terminal 33 residues and the C-terminal 26 residues of the full-length protein. 7 Fig 2. A schematic of the transgene created for expression of LRAP from mouse FABP4 promoter. A transgene is an artificial gene and transgene design must incorporate all appropriate elements critical for gene expression. Each transgene contains a promoter, a protein coding sequence, an intron, and transcriptional stop sequence. Fatty acid-binding protein 4 (FABP4) promoter was used to facilitate the transcription of LRAP. IRES-GFP was included to monitor LRAP expression, however, due to high background green fluorescence in bone marrow, we were not able to utilize it as planned. LoxP sites were included as an option to remove the IRES-GFP cassette. Inclusion of an intron in a transgene construct results in a significantly greater percentage of active transgene. Each transgene must also contain a transcriptional stop signal to match the start signal included in the promoter. Eukaryotic transcriptional stop signal include a polyA additon sequence (AAAUAA) as well as hundrends of downstream nucleotides. The most convenient arrangement is to include a gene or gene fragment at the end of the coding sequence with both an intron and transcriptional stop sequence. 8 MATERIALS AND METHODS 1. Primer pairs and PCR Assay. Primers for detecting LRAP transgenic mice are called LRAPtg DIG for and LRAPtg DIG rev (Table 1). LRAP is intentionally overexpressed driven by FABP4 promoter in C57BL/DBA mice. LRAP transgenic will show a 610 bp amplicon band on agarose gel, wild-type will demonstrate no amplicon on agarose gel. Primer sequences from 5’ to 3’ are: LRAPtg DIG for: ACA ACC ATG GTG AGC A LRAPtg DIG rev: TGC TGG ATG AAG TGG TAC TC Delta-A mice are originally from mouse ES cell line 129/RW4 (Zhu et al., 2006). Two sets of primer pairs are needed for genotyping (Table 1). Primer 289 and 290 were used to detect the wild-type amelogenin gene, which will reveal a 900 bp band on agarose gel. Primer sequences from 5’ to 3’ are: SN289: GAA TGC AGA GCA CAC AAT CTT GG SN290: GCC GCA CCT TCT TTT TGA TTA GC Primer 289 and 502 were used to detect the delta-A knocked-in amelogenin cDNA and it presents a 500 bp band on agarose gel. Primer sequences from 5’ to 3’ are: SN289: GAA TGC AGA GCA CAC AAT CTT GG 9 SN502: TCC ATC TGC ACG AGA CTA GTG AGA CG Therefore, hemizygous male will show one 500 bp band on agarose gel and wild- type male will show one 900 bp band on agarose gel. Homozygous female will show one 500 bp band on agarose gel and heterozygous female will show one 500 bp band and one 900 bp band on agarose gel. 2. Genotyping procedures. a. Obtain tail tissue from mice after they were weaned. b. Lyse tissue to release DNA. Each tail tissue is dissolved in a mixed solution of 100 µl tail buffer (DirectPCR Lysis Reagent from Viagen Biotech Inc.) and 10 µl Proteinase K (20 mg/ml). Samples were incubated in 55 °C oven overnight. c. For Proteinase K inactivation samples were incubated at 85 °C for 45 min. d. Set up the reaction for each set of primers, total volume for each set is 25µl (Table 2). e. Run PCR cycle following each protocol. f. Run electrophoresis agarose gel and obtain genotype for each mouse. 10 Table 1. PCR time and temperature profiles. PCR Time / Temperature Profiles 94°C, 4 minutes Denature 94°C, 30 seconds 59°C, 30 seconds 72°C, 30 seconds 35 cycles corresponding to denature, annealing and extension Primer pair: LRAPtg DIG for LRAPtg DIG rev 72°C, 5 minutes Extension 94°C, 4 minutes Denature 94°C, 30 seconds 59°C, 30 seconds 72°C, 1 minute 35 cycles corresponding to denature, annealing and extension Primer pair: 289, 290 72°C, 5 minutes Extension 94°C, 4 minutes Denature 94°C, 30 seconds 56°C, 30 seconds 72°C, 30 seconds 35 cycles corresponding to denature, annealing and extension Primer pair: 289, 502 72°C, 5 minutes Extension 11 Table 2. PCR reaction set up. DNA 1 µl Primer #1 (10 ρmol/µl) 0.5 µl Primer #2 (10 ρmol/µl) 0.5 µl dNTP (10 ρmol/µl) 0.5 µl 10 x Buffer 2.5 µl Distilled H2O 19.75 µl Taq enzyme 0.25 µl 12 3. Histological procedures. a. At week 12, mice femora were obtained and fixed in freshly made 4% paraformaldehyde in PBS overnight at room temperature. b. Samples were decalcified in 10% EDTA at room temperature for 10 to 14 days. 10% EDTA solution was changed daily. c. To remove H2O from tissue, samples were transferred through baths of progressively more concentrated ethanol. d. This is followed by the clearing agent xylene to remove ethanol, and finally the infiltration agent paraffin was used to replace xylene. e. After samples have been dehydrated, cleared and infiltrated, they were ready for embedding. Samples were placed into molds along with liquid paraffin. Embedded samples were air dried and then stored at -20°C freezer for 1-2 days. f. The tissue samples were sectioned at 8 µm thickness and followed with hematoxylin and eosin (H&E) staining (Table 3). 13 Table 3. Hematoxylin and eosin (H&E) staining protocol. Xylene 2-3 minutes with dipping Xylene 2-3 minutes with dipping Xylene 2-3 minutes with dipping 100% ETOH 1 minute with dipping 100% ETOH 1 minute with dipping 100% ETOH 1 minute with dipping 95% ETOH 20-30 dipping 95% ETOH 20-30 dipping 95% ETOH 20-30 dipping Deion. Water 20-30 dipping Deion. Water 20-30 dipping Lerner Hematoxylin 3-5 minutes Acid rinse 8-10 dipping Deion. water 20-30 dipping Lerner Bluing solution 1 minute Deion. water 20-30 dipping Deion. water 20-30 dipping 95% ETOH 20-30 dipping 95% ETOH 20-30 dipping Lerner Eosin-Y 15-30 seconds 14 Table 3, continued 95% ETOH 20-30 dipping 95% ETOH 20-30 dipping 95% ETOH 20-30 dipping 100% ETOH 20-30 dipping 100% ETOH 20-30 dipping 100% ETOH 20-30 dipping Xylene 20-30 dipping Xylene 20-30 dipping Xylene Leave slides at all time 15 4. Imaging and statistical analysis. The slides were viewed and photos were taken with an Olympus SZX 12 microscope at 32X magnification. Photographs were analyzed using Photoshop CS 5 software. For each femur, multiple images were taken. For each picture, an area of interest of 0.8-millimeter width x 1.6-millimeter height was selected 0.3- millimeter distance from epiphyseal line of the distal femoral head. Trabecular bone volume was selected and software provided total trabecular bone volume (TBV) as in pixels, and total volume (TV) of selected area was given by software as image size in pixels (Schenk et al., 1980, Can et al., 2011). For each femur, multiple TBV/TV were averaged to obtain a mean TBV/TV value. Independent t-test was used to assess trabecular bone mass difference for each comparison. A P-value of less than 0.05 was considered significant. 16 RESULTS Four groups of comparisons were made: (1) Male transgenic mice and male wild-type mice (Fig 3). The mean TBV/TV for 5 male transgenic mice were: 0.191, 0.178, 0.171, 0.188, and 0.159. The mean TBV/TV for 4 male wild-type mice were: 0.172, 0.141, 0.171, and 0.214. H0: There is no statistically significant TBV/TV difference between male LRAP transgenic mice and male wild-type mice. H1: The TBV/TV of male LRAP transgenic mice is greater than that of male wild- type mice. N1= 5 =0.1774 S1= 0.013 N2=4 =0.1745 S2= 0.03 Sp=0.02197 t=0.1968 df=7 P>0.1 Therefore, there is no statistically significant TBV/TV difference between male LRAP transgenic mice and male wild-type mice. (2) Female LRAP transgenic mice and female wild-type mice (Fig 3). 17 The mean TBV/TV for two female LRAP transgenic mice were: 0.138 and 0.158. The mean TBV/TV for three female wild-type mice were: 0.091, 0.082, and 0.118. H0: There is no statistically significant TBV/TV difference between female LRAP transgenic mice and female wild-type mice. H1: The mean TBV/TV of female LRAP transgenic mice is greater than that of female wild-type mice. N1=2 = 0.148 S1 =0.014 N2=3 = 0.097 S2=0.137 Sp=0.0173 t= 3.22 df= 3 0.01<P<0.025 Therefore, the mean TBV/TV of female LRAP transgenic mice is statistically significantly higher than that of female wild-type mice. 18 Fig 3. A—LRAP transgenic male. B—Wild-type male. C--LRAP transgenic female. D—Wild-type female. 19 (3) Delta-A hemizygous male (X Δ A Y) and wild-type male (XY) (Fig 4). The average TBV/TV for 10 male wild-type mice were: 0.098, 0.079, 0.091, 0.098, 0.108, 0.073, 0.083, 0.097, 0.109, and 0.094. The average TBV/TV for 5 male delta-A hemizygous mice were: 0.088, 0.115, 0.110, 0.095, and 0.102. H0: There is no statistically significant TBV/TV difference between male wild-type and male delta-A hemizygous mice. H1: The TBV/TV of male wild-type mice is greater than that of male delta-A hemizygous mice. N1=10 = 0.093 S1=0.012 N2=5 =0.102 S2= 0.011 Sp=0.0115 t=-1.426 df=13 P>0.05 Therefore, there is no statistically significant TBV/TV difference between male wild-type mice and male delta-A hemizygous mice. (4) Female delta-A heterozygous (XX Δ A ) and female delta-A homozygous (X Δ A X Δ A ) (Fig 4). The average TBV/TV for 2 delta-A heterozygous females were 0.145 and 0.074. 20 The average TBV/TV for 3 delta-A homozygous females were 0.077, 0.065, and 0.082. H0: There is no statistically significant TBV/TV difference between female delta-A heterozygous mice and female delta-A homozygous mice. H1: The mean TBV/TV of female delta-A heterozygous mice is greater than that of female delta-A homozygous mice. N1=2 = 0.1095 S1 = 0.05 N2=3 =0.07467 S2=0.087 Sp=0.02985 t= 1.2782 df= 3 P>0.1 Therefore, there is no statistically significant TBV/TV difference between female delta-A heterozygous and female delta-A homozygous mice. 21 Fig 4. E—Wild-type male. F—Delta-A hemizygous male. G—Delta-A heterozygous female. H—Delta-A homozygous female. 22 Table 4. Results of four groups of comparisons. Group Genotype Gender N Mean TBV/TV SD Range P value Transgenic / M 5 0.1774 0.013 0.159-0.191 1 Wild-type / M 4 0.1745 0.03 0.141-0.214 P>0.1 Transgenic / F 2 0.148 0.014 0.138-0.158 2 Wild-type/ F 3 0.097 0.137 0.082-0.118 0.01<P <0.025 Wild-type / M 10 0.093 0.012 0.073-0.109 3 Hemizygous / M 5 0.102 0.011 0.088-0.115 P>0.05 Heterozygous / F 2 0.1095 0.05 0.074-0.145 4 Homozygous / F 3 0.07467 0.0087 0.065-0.082 P>0.1 23 CONCLUSIONS AND DISCUSSION Osteoblasts are multipotent cells that derived from mesenchymal stem cells (MSCs) and are responsible for bone formation. They give rise to osteoblasts, chrondrocytes, adipocytes, myoblasts, and tendon cells while maintaining undifferentiated reservoirs of cells for maintenance and regeneration (Aubin, 2001). Two transcription factors, Runx2 and Osx, are required for osteoblast differentiation (Schroeder et al., 2005). Studies have shown that both Runx2 knockout mice and Osx knockout mice exhibit defects in bone formation (Komori et al., 1997, Nakashima et al., 2002), but Osx acts downstream of Runx2 (Nakashima et al., 2002) (Fig 5). Wnt signaling pathways play important roles in the regulation of osteoblast differentiation. Inactivation of β-catenin in osteoprogenitor cells inhibits osteoblast differentiation and shifts the cell differentiation from osteoblast to chondrocyte in long bones (Day et al., 2005; Hill et al., 2005; Hu et al., 2005). It has also been suggested that β-catenin is required for an early step of osteoblast differentiation since the development of osteoprogenitor cells is arrested in the absence of β- catenin (Hu el al., 2005) (Fig 5). 24 Fig 5. Wnt signaling in regulation of osteoblast specification and differentiation. Osteoblasts arise from osteoprogenitor cells and are responsible for bone formation. Runx2 and Osx are required for osteoblast differentiation and maturation. Wnt signaling pathway plays an important role in the regulation of osteoblast differentiation. When binds to its receptor, β-catenin can increase Runx2 expression. 25 Leucine-rich amelogenin peptide (LRAP) has been shown to induce osteogenesis in various cell types (Veis et al., 2000; Boabaid et al., 2004; Warotayanont et al., 2008; Lacerda-Pinheiro et al., 2006; Wen et al., 2011) in vitro. Previous study has demonstrated that LRAP induces osteogenesis of mouse ES cells by activation of the Wnt pathway (Warotayanont et al., 2009). β- catenin protein level was increased and the Wnt reporter was increased following LRAP treatment, suggesting the canonical Wnt pathway is activated during the early events leading to osteogenic differentiation (Warotayanont et al., 2009). In addition, it has also been shown that LRAP promotes osteogenesis of MSCs at the expense of adipogenesis through upregulating Wnt10b expression to activate Wnt signaling (Wen et al., 2011). In the current experiment, I focused on the effect of LRAP expressed by animal itself on bone density. I used trabecular bone volume / total volume (TBV/TV) as an indicator for mice femoral bone density. In a “gain-of-function” study, LRAP transgenic mice were compared with wild-type littermates, females and males were compared separately. In a “loss-of-function” study, male delta-A hemizygous mice were compared with male wild-type mice; female delta-A homozygous mice were compared with female delta-A heterozygous mice. In the “gain-of-function” study, there is no statistically significant trabecular bone density difference between male LRAP transgenic mice and male wild-type mice. However, female LRAP transgenic mice have statistically significantly higher 26 trabecular bone density than female wild-type littermates. The fact that LRAP transgenic females have increased bone mass compared with wild-type littermates is consistent with previous finding about the osteogenesis function of LRAP. This gender-specific response to enhanced LRAP level is also consistent with previous findings about bone mass phenotype. It has been demonstrated that genetic alterations in components of the Wnt pathway appear to affect female mice more than male mice in vivo (Noh et al., 2009). Since LRAP influences bone mass by activating Wnt pathway (Warotayanont et al., 2009; Wen et al., 2011), it is reasonable that females were more responsive than males to LRAP and female bone mass phenotype is more affected. At the same time, it should be noticed that this is a pilot study and the sample size is not large enough to draw conclusion. Increased sample size is recommended. In the “loss-of-function” study, there was no observed statistically significant bone mass difference for either comparison. Bone mass was unaffected by lacking of LRAP in male mice is reasonable since females are more responsive than males to Wnt pathways (Noh et al., 2009). I suggest two reasons for female’s unaffected response. Firstly, it could be due to the fact that female LRAP transgenic and their wild-type controls are from the same litter, whereas female delta-A heterozygous and delta-A homozygous are from a mixture of 2 litters. Differences between litters could be a contributing factor especially when sample size is so small. To detect whether there is really no trabecular bone density difference between delta-A heterozygous and delta-A homozygous females, 27 increased sample size is needed. Secondly, because of X-chromosome inactivation, it is possible that the expression of wild-type allele is not enough to “rescue” the expression of delta-A allele in heterozygous females. In addition to increasing sample size number, I would also suggest to compare the trabecular bone density of wild-type females (XX) and heterozygous females (X Δ A X). In this experiment, I studied the effect of LRAP on 12-week-old mice. Since bone density decrease with age, it would also be interesting to study bone density in aged mice. In a “gain-of-function” design, I would compare aged wild-type mice with aged LRAP transgenic mice to see if LRAP transgenic mice have delayed bone loss. Females and males are to be compared separately. In a “loss-of- function” design, I would compare aged delta-A males with wild-type males as well as aged heterozygous females with homozygous females to see if lacking of LRAP would caused more severe bone loss. Although two-dimensional histological analysis of bone density offers high spatial resolution and high image contrast, there are some limitations to this method. One disadvantage of this method is its destructive nature. Serial sectioning prevents specimen being used for other measurements such as mechanical testing since samples are destroyed during preparation. Moreover, although histological analysis of trabecular bone density is inexpensive, micro-computed topographic (µCT) analysis is a more accurate tool to derive primary parameters such as TBV/TV, BS/TV (bone surface area / tissue volume), Tb.Th (trabecular 28 thickness), Tb.Sp (trabecular separation) (Muller et al., 1998) as well as Bone Mineral Density (BMD) value, which is the golden standard for studying bone density (Masud et al., 2000). Therefore, the most appropriate way is to do micro-CT 3D analysis first, followed with any mechanical tests needed, and finally do the histological analysis. In this way, more data is collected and high contrast histological pictures and 3D images can supplement each other. The discoveries that LRAP stimulate osteogenesis by activating Wnt/β-catenin signaling pathway and that osteogenic stimulation may be gender-specific make LRAP available for novel therapies for bone mass disease as well as enamel problems in clinical dentistry. 29 REFERENCES: Aubin JE (2001). Regulation of osteoblast formation and function. Rev Endocr Metab Disord 2, 81-94. Boabaid F, Gibson CW, Keuhl MA, Berry JE, Snead ML, Nociti FHJ, Katchburian E, Sommerman MJ (2004). Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis. J Periodontol 75, 1126-1136. Cadigan KM, Nusse R (1997). Wnt signaling: a common theme in animal development. Genes Dev 11, 3286-3305. Cao J, Wang L, Lei DL, Liu YP, Du ZJ, Cui FZ (2011). Local injection of nerve growth factor via a hydrogel enhances bone formation during mandibular distraction osteogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011 Mar 31. Day TF, Guo X, Garrett-Beal L, Yang Y (2005). Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell8, 739-750. Fincham AG, Belcourt AB, Termine JD, Butler WT, Cothran WC (1983). Amelogenin sequence homologies in enamel-matrix proteins from three mammalian species. J Biochem 211,149-154. Fincham AG, Moradian-Oldak J, Shimmer JP (1999). The structural biology of the developing dental enamel matrix. J Struct Biol 126, 270-299. 30 Gibson C, Gobub E, Herold R, Risser M, Ding W, Shimokawa H, Young M, Termine J, Rosenbloom J (1991). Structure and expression of the bovine amelogenin gene. Biochemistry 30, 1075-1079. Gibson CW (2008). The amelogenin “enamel proteins” and cells in the periodontium. Cell Tissues Organ 18, 345-360. Gibson CW, Yuan ZA, Hall B, Longenecker G, Chen E, Thyagarajan T, Sreenath T, Wright JT, Decker S, Piddington R, Harrison G, Kulkarn AB (2001). Amelogenin-deficient mice display an amelogenesis imperfect phenotype. J Biol Chem 276, 31871-31875. Glass DA, 2 nd , Bialek P, Ahn JD, Starbuck M, Patel MS, et al. (2005). Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev Cell 8, 751-764. Glass DA, 2 nd , Karsenty G (2006). Molecular bases of the regulation of bone remodeling by the canonical Wnt signaling pathway. Curr Top Dev Biol 73,43-84. Hammarstrom L (1997). The role of enamel matrix proteins in the development of cementum and periodontal tissues. Ciba Found Symp 205, 246-255. Hammarstrom L, Heijl L, Gestrelius S (1997). Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. J Clin Periodontol 24, 669-677. 31 Hart PS, Hart TC, Simmer JP, Wright JT (2002). A nomenclature for X-linked amelogenesis imperfecta. Arch Oral Biol 47, 255-260. Haze A, Taylor AL, Blumenfeld A, Rosenfeld E, Leiser Y, Dafni L, et al. (2007). Amelogenin expression in long bone and cartilage cells and in bone marrow progenitor cells. Anat Rec (Hoboken) 290, 455-460. Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C (2005). Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 8, 727-738. Hu CC, Ryn OH, Qian Q, Zhang CH, Simmer JP (1997). Cloning, characterization, and heterologous expression of exon-4-containing amelogenin mRNAs. J Dent Res 76(2), 641-7 Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F (2005). Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development 132, 49- 60. Jiang J, Goodarzi G, He J, Li H, Safavi KE, Spangberg LS, Zhu Q (2006). Emdogain-gel stimulates proliferation of odontoblasts and osteoblasts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 102, 698-702. 32 Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, et al. (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755-764. Kramer I, Halleux C, Keller H, Pegurri M, Gooi JH, Weber PB, Feng JQ, Bonewald LF, Kneissel M (2010). Osteocyte Wnt/β-catenin signaling is required for normal bone homeostasis. Mol Cell Biol 30(12), 3071-3085. Lacerda-Pinheiro S, Jegat N, Septier D, Priam F, Bonnefoix M, Bitard J, Kellermann O, Tompkins K, Veis A, Goldberg M, Poliard A (2006). Early in vivo and vitro effects of amelogenin gene splice products on pulp cells. Eur J Oral Sci 114, 232-238. Lau EC, Simmer JP, Bringas P Jr, Hsu DD, Hu CC, Zeichner-David M (1992). Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity. Biochem Biophys Res Commun 188, 1253-1260. Masud T, Francis RM (2000). The increasing use of peripheral bone densitometry. Br Med J 321, 396–398. Maycock J, Wood SR, Brookes SJ, Shore RC, Robinson C, Kirkham J (2002). Characterization of a porcine amelogenin preparation. EMDOGAIN, a biological treatment for periodontal disease. Connet Tissue Res 43, 472-476. 33 Molenaar M, Wetering M, Oosterwegel M, Clevers H (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391-399. Moradian-Oldak J, Paine ML, Lei YP, Fincham AG, Snead ML (2000). Self- Assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy. J Structure Biol 131, 27-37. Muller R, Van Campenhout H, Van Damme B, Van Der Perre G, Dequeker J, Hildebrand T and Ruegsegger P (1998). Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro- computed tomography. Bone 23, 59-66. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B (2002). The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17- 29. Noh T, Gabet Y, Cogan J, Shi Y, Tank A, Frenkel B (2009). Lef1 haploinsufficient mice display a low turnover and low bone mass phenotype in a gender- and age- specific manner. PLoS ONE 4(5), e5438. 34 Schenk RK, Olah AJ (1980). Histomorphometrie. In: Kuhlencordt F and Bartelheimer H, Eds Handbuch der inneren Medizin VI/1A, Knochen Gelenke Muskeln. Berlin: Springer 1980, 437-494. Schroeder TM, Jensen ED, Westendorf JJ (2005). Runx2: a master organizer of gene transcription in developing and maturing osteoblastes. Birth Defects Res C Embryo Today 75, 213-225. Ten Cate AR (1994). Oral Histology: Development, Structure, and Function, 4 th Ed. St. Louis, Mosby. Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen MU (1980). Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. J Biol Chem 255, 9760-9768. Veis A (2003). Amelogenin gene splice products: potential signaling molecules. Cell Mol Life Sci 60, 38-55. Veis A, Tompkinds K, Alvares K, Wei K, Wang L, Wang S, Brown AG, Jengh SM, Healy KE (2000). Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo. J Biol Chem 275, 41263-41272. Warotayanont R (2008). Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells. PhD Dissertation. 35 Warotayanont R, Frenkel, B, Snead ML, Zhou Y (2009). Leucine-rich amelogenin peptide induces osteogenesis by activation of the Wnt pathway. Biocehmical and Biophysical Research Communications 387,558-563. Warotayanont R, Zhu D, Snead ML, Zhou Y (2008). Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells. Biochem Biophys Res Commum 367,1-6. Wen X, Cawthorn WP, MacDougald OA, Stupp SI, Snead, ML, Zhou Y (2011). The influence of Leucine-rich amelogenin peptide on MSC fate by inducing Wnt10b expression. Biomaterials 32, 6478-6486. Wright JT, Hart PS, Aldred MJ, Seow K, Crawford PJ, Hong SP (2003). Relationship of phenotype and genotype in X-linked amelogenesis imperfecta. Connect Tissue Res 44, 72-78. Zhu DH, Paine ML, Luo W, Bringas P, Snead ML (2006). Altering biomineralization by protein design. J Biol Chem 281, 21173-21182.

Abstract (if available)

Abstract Objective: The objective of this study was to evaluate the effect of Leucine-rich amelogenin peptide (LRAP) on osteogenesis in the mouse femur model. ❧ Study Design: In a “gain-of-function” study, LRAP was overexpressed in the bone marrow of transgenic mice. In a “loss-of-function” study, an engineered amelogenin cDNA was “knocked in” to the amelogenin locus, resulting in the lack of LRAP expression. Femora were taken from 12-week-old mice and 4 groups of comparisons were made to determine whether there are trabecular bone density differences. In the “gain-of-function” study: male LRAP transgenic mice were compared with male wild-type mice

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells

PDF

Amelogenin peptides for biomimetic remineralization of enamel and dentin

PDF

Global analysis of the molecular activities defining maturation-stage amelogenesis

PDF

Modulation of C/EBP alpha in the regulation of mouse amelogenin transcription during tooth formation

PDF

Dynamics of amelogenin self-assembly during in vitro proteolysis by Mmp-20

PDF

Antibody mediated osseous regeneration

PDF

Ameloblastin-protein interactions pattern enamel matrix

PDF

Chitosan‐amelogenin hydrogel‐saliva interactions: towards optimization of a protocol for enamel repair

PDF

A Western blot analysis and immunohistochemical study of 4-day old mice teeth over-expressing amelotin during amelogenesis

PDF

Functionalization of scaffolds with anti-BMP-2 antibody: role in antibody mediated osseous regeneration (AMOR)

PDF

Marginal bone response of implants placed in post-extraction sites following ridge preservation with bovine anorganic bone

PDF

Healing of extraction sockets treated with anorganic bovine bone minerals: a micro-CT analysis

PDF

Amelogenin peptide - chitosan for remineralization of artificial enamel lesions: a QLF analysis

PDF

Human histological analysis of autogenous block grafts

PDF

Enamelysin (MMP20) and Kallikrein 4 (KLK4) functions during enamel formation

PDF

Remineralization of deminrealized dentin by amelogenin peptide P26

PDF

Cone beam computed tomographic measurements of buccal alveolar bone widths overlying the maxillary premolars

PDF

Amelogenin-ameloblastin protein interaction and function in dental enamel formation

PDF

Alveolar process inclination as related to tooth inclination on near normal patients -- in three dimensional space

PDF

The effect of vertical level discrepancy of adjacent dental implants on crestal bone resorption: a retrospective radiographic analysis

Asset Metadata

Creator Li, Yisu (author)

Core Title The effect of leucine-rich amelogenin peptide on mouse bone density by histological analysis

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 07/25/2012

Defense Date 06/12/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag bone density,leucine-rich amelogenin peptide,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Paine, Michael L. (committee chair), Oldak, Janet (committee member), Snead, Malcolm L. (committee member), Zhou, Yan (committee member)

Creator Email yisu.li88@gmail.com,yisuli@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-65480

Unique identifier UC11288346

Identifier usctheses-c3-65480 (legacy record id)

Legacy Identifier etd-LiYisu-990.pdf

Dmrecord 65480

Document Type Thesis

Rights Li, Yisu

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA