Transforming growth factor-ß3 (TGF-ß3) knock-in ameliorates inflammation due to TGF-ß1 deficiency while promoting glucose tolerance.

Three homologues of TGF-ß exist in mammals as follows: TGF-ß1, TGF-ß2, and TGF-ß3. All three proteins share high homology in their amino acid sequence, yet each TGF-ß isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-ß proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-ß knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-ß1 ligand with a sequence from TGF-ß3 using targeted recombination to create chimeric TGF-ß1/3 knock-in mice (TGF-ß1(Lß3/Lß3)). In the TGF-ß1(Lß3/Lß3) mouse, localization and activation still occur through the TGF-ß1 latent associated peptide, but cell signaling is triggered through the TGF-ß3 ligand that binds to TGF-ß receptors. Unlike TGF-ß1(-/-) mice, the TGF-ß1(Lß3/Lß3) mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-ß3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-ß1 deficiency. However, the TGF-ß1(Lß3/Lß3) mice have a shortened life span and display tooth and bone defects, indicating that the TGF-ß homologues are not completely interchangeable. Remarkably, the TGF-ß1(Lß3/Lß3) mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-ß isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-ß pathway in human disease.

Leucine rich amelogenin peptide prevents ovariectomy-induced bone loss in mice.

Amelogenins, major extra cellular matrix proteins of developing tooth enamel, are predominantly expressed by ameloblasts and play significant roles in the formation of enamel. Recently, amelogenin has been detected in various epithelial and mesenchymal tissues, implicating that it might have distinct functions in various tissues. We have previously reported that leucine rich amelogenin peptide (LRAP), one of the alternate splice forms of amelogenin, regulates receptor activator of NF-kappa B ligand (RANKL) expression in cementoblast/periodontal ligament cells, suggesting that the amelogenins, especially LRAP, might function as a signaling molecule in bone metabolism. The objective of this study was to identify and define LRAP functions in bone turnover. We engineered transgenic (TgLRAP) mice using a murine 2.3kb α1(I)-collagen promoter to drive expression of a transgene consisting of LRAP, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) to study functions of LRAP in bone formation and resorption. Calvarial cell cultures from the TgLRAP mice showed increased alkaline phosphatase (ALP) activity and increased formation of mineralized nodules compared to the cells derived from wild-type (WT) mice. The TgLRAP calvarial cells also showed an inhibitory effect on osteoclastogenesis in vitro. Gene expression comparison by quantitative polymerase chain reaction (Q-PCR) in calvarial cells indicated that bone formation makers such as Runx2, Alp, and osteocalcin were increased in TgLRAP compared to the WT cells. Meanwhile, Rankl expression was decreased in the TgLRAP cells in vitro. The ovariectomized (OVX) TgLRAP mice resisted bone loss induced by ovariectomy resulting in higher bone mineral density in comparison to OVX WT mice. The quantitative analysis of calcein intakes indicated that the ovariectomy resulted in increased bone formation in both WT and TgLRAP mice; OVX TgLRAP appeared to show the most remarkably increased bone formation. The parameters for bone resorption in tissue sections showed increased number of osteoclasts in OVX WT, but not in OVX TgLRAP over that of sham operated WT or TgLRAP mice, supporting the observed bone phenotypes in OVX mice. This is the first report identifying that LRAP, one of the amelogenin splice variants, affects bone turnover in vivo.

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts.

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

TGF-ß regulates enamel mineralization and maturation through KLK4 expression.

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.

Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III.

Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels.