Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human Xchromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca 2؉ uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. Annexins are a family of proteins that has in common the ability to bind to acidic phospholipids in the presence of Ca 2ϩ (1, 2). The family consists of at least 12 members, and three of them, annexins II, V, and VI, are highly expressed in calcifying cartilage and bone (3, 4). Annexin II and V each contain four 70 -80 amino acids repeats with an annexin consensus sequence. Annexin VI contains eight such repeats. These four or eight repeats form the conserved core region, which is responsible for the Ca 2ϩ -dependent binding of the proteins to phospholipids. In contrast, the N-terminal regions of the annexins are highly variable and may contribute to the specific functions of the various annexins (1, 2).Annexins II, V, and VI are major components of matrix vesicles (MV), 1 which are particles that, after being released from the plasma membrane of hypertrophic chondrocytes or osteoblasts, have the critical role of initiating the mineralization process in cartilage and perhaps in bone (3, 5). Three independent lines of evidence indicate that annexin II, V, and VI exhibit distinct Ca 2ϩ ion channel properties. First, when inserted into artificial phosphatidylserine bilayers they form voltage-dependent Ca 2ϩ ion channels (6 -8). Second, the crystal structures of these annexins are largely ␣-helical with parallel barrels of ␣-helical domains forming a hydrophilic, charged pore through the center of the protein (6, 8, 9). Third, annexin II, V, and VI are able to mediate Ca 2ϩ influx into artificial liposomes (10;11). It was shown that annexin-mediated Ca 2ϩ influx into liposomes is rapid during the first 20 min and then reaches a plateau after 20 min (10, 11).The initial phase of MV-mediated mineralization is characterized by the uptake of mineral ions by these particles and the formation and growth of the first mineral phase inside the vesicles (5). Because MV are enclosed by a membrane, channel proteins are required to mediate the influx of mineral ions into these particles. Previous findings from our and other laboratories, showed that chymotrypsin treatment, which removes most of the annexins from MV, and zinc treatment, which inhibits annexin-mediated Ca 2ϩ influx into phosphatidylserine (PS)-enriched liposomes, diminished MV Ca 2ϩ uptake (12-15), suggesting that annexins II, V, and VI serve as ion channels in MV, enabling Ca 2ϩ loading of the vesicles during the initial phase of mineralization.Previous studies have revealed that collagen types II and X are associated with the outer surface of MV (16). We...

Annexin V is a major component of matrix vesicles and has a role in mediating the influx of Ca2+ into these vesicles, thus promoting the initiation of hypertrophic cartilage matrix mineralization. However, the mechanisms and factors regulating annexin V-mediated Ca2+ influx into these vesicles are not well understood. Since the lipid composition of matrix vesicles differs from that of the plasma membrane of chondrocytes and is rich in phosphatidylserine, we asked whether the lipid composition may regulate annexin V function. We prepared liposomes containing different concentrations of phosphatidylserine and determined how the lipid composition affected (a) the interactions between annexin V and liposomes and (b) annexin V-mediated Ca2+ influx into the liposomes. We found that annexin V was able to bind to every liposome tested. However, we observed the most prominent increases in tryptophan 187 emission intensity, a measure of the degree of interactions between annexin V and lipid bilayers, only with liposomes containing a high concentration of phosphatidylserine. In addition, a significant fraction of annexin V associated with phosphatidylserine-rich liposomes was not extractable by EDTA treatment but required a detergent, indicating that annexin V inserts into bilayers of these liposomes. Chemical cross-linking analysis revealed that matrix vesicles and phosphatidylserine-rich liposomes induced the formation of the annexin V hexamer. Interestingly, a significant Ca2+ influx in the presence of annexin V occurred only in liposomes containing a high phosphatidylserine content. Moreover, annexin V-mediated Ca2+ influx into these liposomes was inhibited (i) by anti-annexin V antibodies and (ii) by treatment with zinc and cadmium, indicating the essential role of the protein in Ca2+ influx. The results of this study indicate that phosphatidylserine-rich bilayers induce the formation of a hexameric annexin V, possibly leading to a Ca2+-dependent insertion of annexin V into the bilayer and establishment of annexin V-mediated Ca2+ influx into matrix vesicles or liposomes. The phosphatidylserine-rich membrane of matrix vesicles in vivo may thus offer an ideal specialized environment in which the biological function of annexin V is optimized, leading to rapid Ca2+ influx, intralumenal crystal growth, and cartilage matrix mineralization.

We propose that toxin-induced Ca 2 + fluxes mobilize LFA-1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells.

No abstract