Ameloblastin Functions in Enamel, Periodontium and Long Bone
thesisposted on 2016-02-25, 00:00 authored by Xuanyu Lu
Though first introduced from developing enamel matrix, there is increasing evidence that ameloblastin (AMBN), first known as an adhesion molecule, is localized in other tissues such as bone, where it plays significant roles associated with mineral metabolism. To study the effect of AMBN in enamel formation, we overexpressed AMBN using a keratin 14 promoter and removed amelogenin (AMELX) by crossbreeding with AMELX null (amelx-/-) mice. Enamel coverings of amelx-/- mice and of the squamate Iguana were used for comparison. SEM analysis documented that AMBN transgenic (AMBN TG) × amelx-/- mouse molars were covered by a thin 'enameloid' layer resembling that the Iguana squamate. TEM revealed that the enamel of developing AMBN TG × amelx-/- mouse molars contained short and randomly oriented crystals. These studies illustrate that Ameloblastin-rich enamel matrix favors short and randomly oriented apatite crystals. To investigate AMBN function in the periodontium, we characterized the phenotype of the mandibular bone of AMBN overexpressor. Transgenic mice suffered from root cementum resorption. In an in vitro model of osteoclastogenesis, AMBN modulated osteoclast differentiation from bone marrow derived monocytes (BMMCs), demonstrated by dramatically increased osteoclast numbers and resorption pits, doubled BMMCs adhesion, accelerated cell spreading, and promoted actin ring formation. These effects were associated with elevated ERK1/2 phosphorylation as well as higher expression of osteoclast activation related genes. Blocking integrin α2β1 and ERK 1/2 pathways alleviated the effects of AMBN on osteoclast differentiation. These data indicate that Ameloblastin modulates osteoclastogenesis through the integrin/ERK pathway. Third part of our studies, we employed an AMBN-deficient mouse model (AMBNΔ5-6) to investigate AMBN function in long bone. AMBN mutants were delayed femur growth during the first 6. µ-CT documented decreases in bone mass which corresponded to the changes in femur biomechanical. Histological studies showed that AMBN mutation resulted in a shortened growth plate with less proliferating chondrocytes. In vitro studies indicated that bone marrow stromal cells (BMSCs) isolated from AMBN mutant had defects in cell proliferation and differentiation potential. PCR array and qRT-PCR analyses demonstrated down-regulation of several osteogensis-related growth factors in AMBN-deficient BMSCs. Together, our data indicate that AMBN is not only involved in tooth amelogenesis, but also functions outside of enamel in periodontal tissue homeostasis and long bone development and fracture healing.