Boaz Shay

Amelogenin expression in long bone and cartilage cells and in bone marrow progenitor cells

The amelogenin protein is considered as the major molecular marker of developing ectodermal enamel. Recent data suggest other roles for amelogenin beyond structural regulation of enamel mineral crystal growth. Here we describe our novel discovery of amelogenin expression in long bone cells, in cartilage cells, in cells of the epiphyseal growth plate, and in bone marrow stromal cells. Anat Rec, 2007.

Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis.

Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth‐supporting tissues, alveolar bone, periodontal‐ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM+), induced in vivo regeneration of all tooth‐supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM+ induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio‐temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM+.

The Human Tuftelin Gene and the Expression of Tuftelin in Mineralizing and Nonmineralizing Tissues

Tuftelin has been suggested to play an important role during the development and mineralization of enamel, but its precise function is still unclear. This article reviews major milestones in the discovery, structural characterization, expression, localization, and conservation of tuftelin in different vertebrate species. It focuses on the structure of the human tuftelin gene, which has recently been deciphered [12]. It describes the exon-intron organization, sizes and structure, the promoter structure, and the newly discovered alternatively spliced human tooth-bud tuftelin mRNA transcripts. It also examines information on the structural motifs in the human-derived tuftelin protein and how they relate to tuftelin from other species. It reviews our recent results on the transcription of tuftelin mRNA and protein expression in several nonmineralizing soft tissues, using reverse-transcription polymerase chain reaction (RT-PCR) followed by DNA cloning and sequencing, indirect immunohistochemistry, immunohistochemistry combined with confocal microscopy, and in situ hybridization. These results and earlier Northern blot results show that tuftelin, in addition to being expressed in the developing and mineralizing tooth, is also expressed in several nonmineralizing soft tissues, suggesting that tuftelin has a universal function and/or a multifunctional role.

Amelogenin in cranio-facial development: the tooth as a model to study the role of amelogenin during embryogenesis

The amelogenins comprise 90% of the developing extracellular enamel matrix proteins and play a major role in the biomineralization and structural organization of enamel. Amelogenins were also detected, in smaller amounts, in postnatal calcifying mesenchymal tissues, and in several nonmineralizing tissues including brain. Low molecular mass amelogenin isoforms were suggested to have signaling activity; to produce ectopically chondrogenic and osteogenic-like tissue and to affect mouse tooth germ differentiation in vitro. Recently, some amelogenin isoforms were found to bind to the cell surface receptors; LAMP-1, LAMP-2 and CD63, and subsequently localize to the perinuclear region of the cell. The recombinant amelogenin protein (rHAM(+)) alone brought about regeneration of the tooth supporting tissues: cementum, periodontal ligament and alveolar bone, in the dog model, through recruitment of progenitor cells and mesenchymal stem cells. We show that amelogenin is expressed in various tissues of the developing mouse embryonic cranio-facial complex such as brain, eye, ganglia, peripheral nerve trunks, cartilage and bone, and is already expressed at E10.5 in the brain and eye, long before the initiation of tooth formation. Amelogenin protein expression was detected in the tooth germ (dental lamina) already at E13.5, much earlier than previously reported (E19). Application of amelogenin (rHAM(+)) beads together with DiI, on E13.5 and E14.5 embryonic mandibular mesenchyme and on embryonic tooth germ, revealed recruitment of mesenchymal cells. The present results indicate that amelogenin has an important role in many tissues of the cranio-facial complex during mouse embryonic development and differentiation, and might be a multifunctional protein.

The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation

The tuftelin protein isoforms undergo post‐translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down‐regulation of HIF1α mRNA blocked hypoxia‐induced HIF1α expression, and reduced by 89% hypoxia‐induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF‐mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA‐signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the “hypoxic genome,” and NGF‐induced neurotrophic and angiogenic effects. J. Cell. Physiol. 226: 165–172, 2010.

Localization, Quantification, and Characterization of Tuftelin in Soft Tissues

Tuftelin was initially found in the developing and mature extracellular enamel. Here we describe our novel discovery of tuftelin cellular distribution (protein and mRNA) in six soft tissues. The expression levels of tuftelin mRNA were significantly higher in mouse kidney and testis, in which oxygen levels are hovering closely to hypoxia under normal conditions. Anat Rec, 2007.

High yield expression of biologically active recombinant full length human tuftelin protein in baculovirus-infected insect cells

Tuftelin is an acidic protein expressed at very early stages of mouse odontogenesis. It was suggested to play a role during epithelial-mesenchymal interactions, and later, when enamel formation commences, to be involved in enamel mineralization. Tuftelin was also detected in several normal soft tissues of different origins and some of their corresponding cancerous tissues. Tuftelin is expressed in low quantities, and undergoes degradation in the enamel extracellular matrix. To investigate the structure and function of tuftelin, the full length recombinant human tuftelin protein was produced. The full length human tuftelin cDNA was cloned using Gateway recombination into the Bac-to-Bac system compatible transfer vector pDest10. This vector adds a hexahistidine tag to the N-terminus of the expressed protein, enabling one-step affinity purification on nickel column. The recombinant human tuftelin protein was transposed into the bacmid and expressed in Spodoptera frugiperda (Sf9) insect cells. The yield of the purified, his-tagged recombinant full length human Tuftelin (rHTuft+) was 5-8 mg/L culture. rHTuft+ was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, restriction mapping and MS/MS sequencing. The availability of the purified, full length recombinant human tuftelin protein opened up the possibility to investigate novel functions of tuftelin. Application of rHTuft+ agarose beads onto embryonic mouse mandibular explants caused changes in the surrounding epithelial cells, including morphology, orientation and spatial organization. Further studies using DiI labeling, revealed that rHTuft+, placed on the tooth germ region, brought about recruitment of adjacent embryonic mesenchymal cells. These findings support the hypothesis that tuftelin plays an important role during embryogenesis.