We report the full-length sequencing, cell type-specific expression, and immunolocalization of a novel gene expressed in rat incisors, which we have designated ameloblastin. Northern blot analysis of RNA from multiple rat and mouse tissues demonstrated high levels of expression of two distinct transcripts of approximately 2.0 and 1.6 kilobase pairs that were expressed only in teeth. In situ hybridization using a digoxigenin-labeled RNA probe showed that the tissue distribution of ameloblastin was limited to the ameloblast in rat incisors. Immunohistochemical staining of rat incisors using a polyclonal antibody raised against a fusion protein revealed a unique localization pattern. Ameloblastin was found to be expressed during the differentiation of inner enamel epithelium into ameloblasts, with intense localization in the Tomes' processes of secretory ameloblasts. In contrast to amelogenin, only modest amounts of ameloblastin were detected in enamel matrix. The ameloblastin gene encodes an open reading frame of 422 amino acids corresponding to a putative protein of 45 kDa. The predicted protein is acidic (pI = 5.54) and the most abundant amino acids are Pro (15.2%), Gly (9.9%), and Leu (9.9%). We have also mapped the ameloblastin gene, Ambn, to a locus on mouse chromosome 5 near other genes associated with mineralized tissues. Thus, ameloblastin represents a unique ameloblast-specific gene product that may be important in enamel matrix formation and mineralization.

Although apparently phenotypically normal at birth, mice heterozygous for inactivation of the gene encoding parathyroid hormone-related peptide (PTHrP) develop haplotype insufficiency by 3 months of age. In addition to histologic and morphologic abnormalities similar to those seen in homozygous mutants, heterozygous animals demonstrated alterations in trabecular bone and bone marrow. These included metaphyseal bone spicules which were diminished in volume, irregularly distributed, and less well developed than those seen in age-matched controls as well as bone marrow, which contained an inordinate number of adipocytes. A substantial reduction in PTHrP mRNA was detected in heterozygous tissue, while circulating parathyroid hormone (PTH) and calcium concentrations were normal. Thus, while a physiologic concentration of PTH was capable of maintaining calcium homeostasis, it was incapable of compensating for PTHrP haploinsufficiency in developing bone. In normal animals, both PTHrP and the PTH/PTHrP receptor were expressed predominantly in chondrocytes situated throughout the proliferative zone of the tibial growth plate. In the metaphysis, the PTH/PTHrP receptor was identified on osteoblasts and preosteoblastic cells situated in the bone marrow, while PTHrP was expressed only by osteoblasts. These observations indicate that postnatal bone development involves susceptible pathways that display exquisite sensitivity to critical levels of PTHrP and imply that the skeletal effects of PTH are influenced by locally produced PTHrP. Moreover, identification of both the ligand and its N-terminal receptor in metaphyseal osteoblasts and their progenitors suggests an autocrine/paracrine role for the protein in osteoblast differentiation and/or function. Impairment in this function as a consequence of PTHrP haploinsufficiency may critically influence the course of bone formation, resulting in altered trabecular architecture and perhaps low bone mass and increased bone fragility.

It has been suggested that the types of inflammatory round cell infiltrates and the divergence in the cytokine production profile by macrophages and helper T cells regulate the course of infectious or inflammatory diseases, including periodontitis and gingivitis. We examined the expression of IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6 and tumour necrosis factor‐alpha (TNF‐α) mRNA in the inflamed gingiva by in situ hybridization. The results of single‐cell analysis were used as data sets for statistical analyses. The density of cells expressing IL‐1α, IL‐4 and IL‐5 mRNA was higher in periodontitis than in gingivitis. IL‐2 mRNA‐expressing cells were almost absent in gingivitis specimens. Principal component analysis disclosed three factors explaining 84.8% of the variance: one accounting for 40.5% of the variance and mainly regulated by IL‐1α, IL‐1β, IL‐6 and TNF‐α, and two others, explaining 29.9% and 14.4% of the variance, describing the relationship between the types of cytokines derived from macrophages or Th2 type. These results suggest that the cytokines produced by inflammatory cells infiltrating in the gingival tissue are influential on the progression of gingivitis, an acute and reversible inflammatory condition, to chronic and destructive periodontitis. Thus, periodontal disease progression may be regulated by the local cytokine network, and the bias in this network towards a Th2‐type cytokine dominance could be an exacerbating factor.

The exact cell type and site(s) involved in interleukin-1 (IL-1) production during gingival inflammation was determined by combining immunohistochemistry and in situ hybridization. IL-1 messenger RNA (mRNA)-expressing cells in human inflamed gingiva were identified as macrophages. The rate of IL-alpha mRNA expression in these macrophages was the same as IL-1 beta mRNA expression. The rate of IL-1 mRNA expression was higher in connective tissue furthest from the pocket epithelium, although more macrophages were present at the connective tissue subjacent to the pocket epithelium. The IL-1 activity in gingival crevicular fluid (GCF) obtained from inflamed gingiva was higher than that from healthy gingiva and decreased after periodontal therapy. The IL-1 activity in GCF was almost completely abolished by the addition of anti-IL-1 alpha antibody but not by anti-IL-1 beta antibody, indicating that IL-1 alpha is the predominant form in GCF. However, the IL-1 activity in GCF was unrelated to the number of IL-1 mRNA-expressing macrophages in the same gingival site where the GCF was obtained at the same time. The results suggest that macrophages in the connective tissue subjacent to the oral epithelium contribute to the production of IL-1 but those in connective tissue subjacent to the pocket epithelium play a different role in the generation of gingival inflammation.

The formation of tooth organs is regulated by a series of developmental programs. We have initiated a genome project with the ultimate goal of identifying novel genes important for tooth development. As an initial approach, we constructed a unidirectional cDNA library from the non-calcified portion of incisors of 3- to 4-week-old rats, sequenced cDNA clones, and classified their sequences by homology search through the GenBank data base and the PIR protein data base. Here, we report partial DNA sequences obtained by automated DNA sequencing on 400 cDNA clones randomly selected from the library. Of the sequences determined, 51% represented sequences of new genes that were not related to any previously reported gene. Twenty-six percent of the clones strongly matched genes and proteins in the data bases, including amelogenin, alpha 1(I) and alpha 2(I) collagen chains, osteonectin, and decorin. Nine percent of clones revealed partial sequence homology to known genes such as transcription factors and cell surface receptors. A significant number of the previously identified genes were expressed redundantly and were found to encode extracellular matrix proteins. Identification and cataloging of cDNA clones in these tissues are the first step toward identification of markers expressed in a tissue- or stage-specific manner, as well as the genetic linkage study of tooth anomalies. Further characterization of the clones described in this paper should lead to the discovery of novel genes important for tooth development.