Tracking Endogenous Amelogenin and Ameloblastin In Vivo

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling

Nephrocalcinosis (enamel renal syndrome) caused by autosomal recessive FAM20A mutations

Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood

Involvement of neural crest and paraxial mesoderm in oral mucosal development and healing

International audienceTissue engineering therapies using adult stem cells derived from neural crest have sought accessible tissue sources of these cells because of their potential pluripotency. In this study, the gingiva and oral mucosa and their associated stem cells were investigated. Biopsies of these tissues produce neither scarring nor functional problems and are relatively painless, and fresh tissue can be obtained readily during different chairside dental procedures. However, the embryonic origin of these cells needs to be clarified, as does their evolution from the perinatal period to adulthood. In this study, the embryonic origin of gingival fibroblasts were determined, including gingival stem cells. To do this, transgenic mouse models were used to track neural crest derivatives as well as cells derived from paraxial mesoderm, spanning from embryogenesis to adulthood. These cells were compared with ones derived from abdominal dermis and facial dermis. Our results showed that gingival fibroblasts are derived from neural crest, and that paraxial mesoderm is involved in the vasculogenesis of oral tissues during development. Our in vitro studies revealed that the neuroectodermal origin of gingival fibroblasts (or gingival stem cells) endows them with multipotential properties as well as a specific migratory and contractile phenotype which may participate to the scar-free properties of the oral mucosa. Together, these results illustrate the high regenerative potential of neural crest-derived stem cells of the oral mucosa, including the gingiva, and strongly support their use in cell therapy to regenerate tissues with impaired healing

Ameloblastin and amelogenin mRNA expression in murine tissues.

<p>Tissues were dissected from 15 week old WT mice (n = 6 with n = 3 females and n = 3 males) and subjected to RT-PCR (see Materials and methods). Resulting products were resolved on a 2% agarose gel. AMBN positive tissues show one amplicon band at 287 bp and AMELX positive tissues show at least one of the three amplicon bands at 415 bp, 373 bp and 303 bp corresponding to transcript variants encoding different isoforms of AMELX described in the literature; at 415 bp (M217 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Bartlett2" target="_blank">[67]</a>–M194 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Simmer1" target="_blank">[68]</a>), 373 bp (M203 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Li2" target="_blank">[69]</a>–M180 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Lau1" target="_blank">[70]</a>) and 303 bp (M179 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Li2" target="_blank">[69]</a>–M156 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Lau1" target="_blank">[70]</a>). +, PCR products were repeatedly obtained; +/−, not all samples were positive; -, no PCR products were visible. The overall high signal in mRNA levels (++) in mandibular mineralized tissues led to perform additional RT-qPCR analyses.</p

Western blot of AMBN and AMELX in 15 week old WT mice.

<p>Proteins were extracted from dental epithelial cells (EP) (positive control), alveolar bone (AB), basal bone (BB) under dissociative and non-dissociative conditions. Proteins were loaded at 10 µg per lane and blots probed with anti-AMBN and anti-AMELX antibodies. <b>A.</b> Anti-AMBN probing of dissociative extracts show cross reactive species with molecular weights ranging from 20–67 kDa for AMBN (the 67 kDa band corresponds to nascent amelobastin). 67 kDa AMBN is present at similar relative amounts in the EP and AB extracts but far less readily detectable in BB. <b>B.</b> A similar situation exists for non-dissociative extracts. <b>C.</b> Anti-AMELX probing of dissociative extracts show cross reactive species at 26 kDa and below in EP samples (higher molecular weight staining may be due to AMELX aggregation). Feint cross reactivity at ∼25 kDa is visible in AB samples with even less been detected in BB; the relative amount of AMELX present in bone samples is far less than that seen in EP samples. <b>D.</b> A similar situation exists for non-dissociative extracts; AMELX is detectable in AB but it is present in the extract in much lower amounts compared to EP extracts.</p

AMELX mRNA and protein distribution in mandible from 8 week old WT and AMELX KO mice.

<p><b>A-B. </b><i>In situ</i> hybridization was performed using AMELX oligonucleotidic probes. <b>B.</b> WT mouse shows strong AMELX mRNA level in ameloblasts (Ambl) and odontoblasts (Odb). AMELX mRNA is also detected in bone-lining cells (red arrows) and, with an apparent lower level, in dental follicle (DF) area. <b>C–D.</b> Immunohistodetection was performed using anti-AMELX antibody. <b>D.</b> AMELX protein expression in WT mouse was detected in ameloblasts, odontoblasts and bone-lining cells (red arrows). Strong and diffuse protein signal was also observed in dental follicle. <b>B–D.</b> No AMELX RNA and protein signal is detected in striated muscle (Myo), a negative control tissue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone-0099626-t001" target="_blank">Table 1</a>). <b>A–C.</b> Neither AMELX mRNA nor protein expression is detected in AMELX KO mice.</p

AMBN and AMELX protein expression in 1 week old WT mice.

<p><b>A.</b> AMBN protein (red signal) is strongly expressed in enamel (En) and in dental follicle (DF) and is detected in cells lining alveolar bone (white arrows). <b>B.</b> On serial sections, AMELX protein (green signal) shows similar localization pattern with expression in enamel, dental follicle and bone-lining cells (white arrows). In addition, a diffuse AMELX signal is also detected in periosteum (Po) and bone (Bo) (in particular in matrix of trabebular spaces (white asterisks)). No protein expression is detected in striated muscle (Myo), a negative control tissue. <b>C.</b> Higher magnification of AMELX protein expression shows AMELX-positive osteoblastic cells lining bone trabeculae (red arrows) (<b>Blue box</b>), strong expression in dental follicle area (<b>Yellow box</b>). Higher magnification shows no signal in striated muscle (<b>Orange box</b>). Ambl  =  ameloblast, Bo  =  bone, De  =  dentin, DF  =  dental follicle, En  =  enamel, Myo  =  striated muscle and Po  =  periosteum.</p

TLR7 stimulation in human plasmacytoid dendritic cells leads to the induction of early IFN-inducible genes in the absence of type I IFN

International audienceOn recognition of influenza virus (Flu) by TLR7, plasmacytoid dendritic cells (pDCs) produce type I IFN in significant amounts. Synthetic TLR7 ligands induce the maturation of pDCs, as evidenced by the expression of costimulatory molecules and the production of proinflammatory cytokines; however, they induce only low-level production of IFN-alpha. To dissect the TLR7 signaling in pDCs and how these different profiles are induced, we studied the effects of 2 TLR7 ligands (Flu and CL097) on the activation of blood-isolated pDCs and the human GEN2.2 pDC cell line. Type I IFN production by pDCs correlates with differential interferon regulatory factor 7 (IRF7) translocation into the nucleus induced by the 2 TLR7 ligands. Surprisingly, with both activators we nevertheless observed the rapid expression of the IFN-inducible genes mxa, cxcl10, and trail within 4 hours of stimulation. This expression, controlled by STAT1 phosphorylation, was independent of type I IFN. STAT1 activation was found to be strictly dependent on the PI3K-p38MAPK pathway, showing a new signaling pathway leading to rapid expression of IFN-inducible genes after TLR7 triggering. Thus, pDCs, through this unusual TLR7 signaling, have the capacity to promptly respond to viral infection during the early phases of the innate immune response