Ameloblast Differentiation and Function: Exploring the Effects of microRNA, Fluoride, and Iron by Michael Huan Le DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Oral and Craniofacial Sciences in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO Copyright by Michael Huan Le 2016 ii Dedication and acknowledgements This project would not have been possible without the mentorship of my advisor, Dr. Pamela Den Besten, who has provided me with significant advice, feedback, and wisdom in the scientist portion of my clinician scientist training. I have been able to do many things that I would have otherwise thought I was not capable of doing during my PhD training at UCSF. I would also like to thank Dr. Yukiko Nakano, whose guidance in laboratory techniques, data analysis, subject matter expertise, and limitless patience were crucial to obtaining the results I report in this dissertation. I would also like to thank Drs. Li Zhu, and Yan Zhang, and all those I worked with in the Den Besten laboratory for their support and encouragement over the past five years. I would also like to thank Drs. Wu Li, Noelle L’Etoile, and Stefan Habelitz for being a part of my dissertation committee, providing an additional source of insight, encouragement, and support. I would also like to thank my present and past peers in the both Oral and Craniofacial Sciences PhD program who have provided me with numerous opportunities to take a (brief) break from research and enjoy the many of little things that life offers daily, while making many incredible memories in the process. I would also like to thank the students who make up the UCSF School of Dentistry during my time as a PhD student at UCSF. They have welcomed me to be a part of their class, not only allowing me to stay engaged in clinical dentistry and provide opportunities to share my clinical wisdom during my PhD training, but also helping me maintain the clinician aspect of being a clinician scientist. Lastly, I would like to thank my family for their patience and understanding in this path I have taken, as I have been much too busy to talk and see them as often as they like during these past nine years. And my girlfriend, Cynthia, who has never ceased to be my greatest supporter these past ten years, especially in the last five years as we simultaneously underwent the trials, tribulations, and triumphs in earning a PhD in the biological sciences. iii Abstract During amelogenesis (dental enamel formation), ameloblast cells undergo several morphological and functional changes as they form dental enamel. Interference in the timing of these changes results in enamel of poor quality, affecting the psychosocial development of children at a crucial age of their development. However, the mechanisms that facilitate these transitions in ameloblasts are not well understood. Using rodent models as a basis for study, I used microRNA, fluoride, and iron as tools to carry out histological, immunochemical, and quantitative PCR experiments that investigate the mechanisms that regulate and define ameloblast differentiation and function. As ameloblasts differentiate from one phase to another during amelogenesis, many genes are simultaneously up- and down- regulated. MicroRNAs serve as one mechanism for rapid control of post- transcriptional expression of many genes. Amelogenin, the predominant protein found in the enamel matrix, is alternatively spiced and may serve as a source for microRNAs. I found that exon 4 of the amelogenin gene produces a microRNA that regulates expression of Runx2, a key transcription factor for when ameloblasts are secreting the enamel matrix. Ameloblasts express and secrete KLK4, a crucial enzyme that facilitates the final phase of amelogenesis. However, it is unclear how ameloblasts begin increasing Klk4 expression at the right time. I found that androgen receptor (AR) serves as an intracellular regulator of expression for Klk4 and that exposure to fluoride inhibits AR translocation to result in decreased Klk4 expression and synthesis. Normal rodent ameloblasts deposit iron onto the enamel surface, resulting in yellow-brown colored surface enamel. This pigment is lost in rodents sensitive to high levels of fluoride. Iron in the surface enamel is thought to increase wear resistance in the enamel, though it is unclear how ameloblasts acquire, manage, and transport iron for deposition into enamel. I found that fluoride exposure resulted in a significantly increased expression of ferroportin (iron-exporting protein), and significantly decreased expression of the heavy chain subunit of ferritin, which facilitates the safe storage of iron in cells. iv Furthermore, it appears that changes in these iron-related proteins by fluoride may be changing free intracellular iron levels to affect expression and synthesis of oxidative stress-related genes. These studies have shown that multiple mechanisms are responsible for ameloblast differentiation. The effects of fluoride on ameloblast differentiation appear to be largely due to cellular effects of fluoride rather than fluoride related changes in the enamel matrix. v Table of Contents Chapter 1 – Introduction ............................................................................................................................... 1 Chapter 2 – Amelogenin Directs Ameloblast Differentiation ....................................................................... 6 Chapter 3 – Fluoride Downregulates Klk4 Expression Through Inhibition of Androgen Receptor Activation ........................................................................................ 21 Chapter 4 – Tracing Iron Transport Through Ameloblasts in Rodent Incisor Pigmentation ...................... 34 Chapter 5 – Summary and Future Directions .............................................................................................. 47 References ................................................................................................................................................... 49 Appendix 1 – The Complex Regulation of Klk4 Expression by TGF-β and Androgen Receptor Signaling in Amelogenesis ................................................................................... 55 Appendix 2 – Genetic Influences on Fluoride’s Effect on Ameloblast Differentiation .............................. 64 Appendix 3 – Primers sequences used for all qPCR experiments .............................................................. 74 vi List of Tables Table 1: Primers sequences used for all qPCR experiments ....................................................................... 74 vii List of Figures Figure 1: Prediction of amelogenin exon4 miRNA ...................................................................................... 9 Figure 2: miRNA derived from exon4 (miR-exon4) is generated from amelogenin .................................. 10 Figure 3: miR-exon4 is present in the mouse enamel organ ....................................................................... 11 Figure 4: Expression of major splicing variants of amelogenin mRNAs without exon4 (LF-4 and SF-4) and its exon4 included variants ......................................................... 11 Figure 5: miR-exon4 was present in mouse developing enamel organ ....................................................... 12 Figure 6: Runx2 expression was up-regulated by miR-exon4 mimic in LS8 cells ..................................... 13 Figure 7: miRNA derived from exon4 (miR-exon4) is expressed in Amel knockout (KO) enamel organ ..................................................................................................... 14 Figure 8: Ameloblasts from amel knockout (KO) mice are similar to WT mice ........................................ 15 Figure 9: Expression of Runx2 is downregulated in Amel knockout (KO) enamel organ at the secretory stage (P5) ........................................................... 16 Figure 10: Downstream targets of Runx2 were downregulated in Amel KO enamel organ ...................... 17 Figure 11: Alignment of intron7 vs. exon4 and intron4 in mouse amelogenin gene (gene ID 11704) ....... 19 Figure 12: Fluoride decreased in vivo KLK4 activity and Klk4 expression ............................................... 26 Figure 13: ArTfm mice exhibit enamel matrix protein retention similar to fluoride exposed mice .......................................................................................................... 27 Figure 14: Fluoride altered expression CCND1 and TGFBR2, regulators of androgen receptor activation ................................................................................................................ 28 Figure 15: Fluoride does not alter Klk4 or Ar expression at near physiologic serum fluoride levels ................................................................................................. 29 Figure 16: Fluoride significantly upregulated ferroportin (Fpn) expression without affecting expression or synthesis of transferrin receptor (Tfrc). ........................................................... 38 Figure 17: Fluoride did not affect ferric iron levels in maturation ameloblasts .......................................... 39 Figure 18: Fluoride significantly affected expression of the heavy chain subunit of ferritin (Fth) ............ 40 Figure 19: Fluoride increased activation of NFκB and decreased SOD1 synthesis in maturation ameloblasts ....................................................................................................................