Vasoactive intestinal polypeptide-like immunoreactivity in the bovine heart: high degree of coexistence with neuropeptide Y-like immunoreactivity

Summary Recent studies have accomplished the establishment of a collagenous fiber-fringe matrix upon dental root surfaces in vitro. The present study was undertaken to follow the development of such a matrix in vitro and to test the possible effects of root surface treatments upon this matrix. Periodontal ligament cells, 0.1 to 0.2-mm thick dental root discs, and alveolar bone cells were derived after extraction from four partially erupted third molars and the accompanying interradicular bony septa of 1 male patient. Autologous serum was obtained by venipuncture. Cultures were initiated by delivering a 1-ml suspension of 50000 tritiated thymidine-labeled periodontal ligament cells and 50000 alveolar bone cells onto each of 42 culture sets. The following day, demineralized or non-demineralized root discs treated with autologous serum, fibronectin or complete medium were placed in pairs, separated by a 0.1–1.0 mm gap, upon the initial cell layer. Representative cultures were terminated after 2, 3, 4, 5 and 6 weeks, and processed for light- and electron microscopy, morphometric analysis and autoradiography. An outstanding feature of the early cultures (2, 3 and 4 weeks) was a patchwise, random distribution of matrix making a precise developmental study impossible, although collagen fibrils were produced within the first 2 weeks. Some 3-week cultures already demonstrated a mature fiber-fringe characterized electron-microscopically as oriented, densely packed collagen fibrils closely abutting the cementum-lined root discs. The treatments (including autologous serum) used in this study had no appreciable morphologic or morphometric effect upon the fiber-fringe formed. Because none of the cultures in the present or past studies have demonstrated a true cementoid matrix, this model may not be suitable for the in-vitro study of cementum formation.     

Rescue of odontogenesis in Dmp1-deficient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a 3-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor-null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting a different effect of DMP1 on mineralization. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects in mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression of Dmp1 in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation

TGF-β subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-β signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-β signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2^fl/fl;Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-β2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-β signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.     

Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1)

We recently identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene induced in adventitial fibroblasts after arterial injury. Cthrc1 is a 30 kDa secreted protein that has the ability to inhibit collagen matrix synthesis. Cthrc1 is also glycosylated and retains a signal sequence consistent with the presence of Cthrc1 in the extracellular space. In injured arteries and skin wounds, we have found Cthrc1 expression to be associated with myofibroblasts and sites of collagen matrix deposition. Furthermore, we demonstrated that Cthrc1 inhibits collagen matrix deposition in vitro. Using in situ hybridization and immunohistochemistry, we characterized the expression domains of Cthrc1 during murine embryonic development and in postnatal tissues. In mouse embryos, Cthrc1 was expressed in the visceral endoderm, notochord, neural tube, developing kidney, and heart. Abundant expression of Cthrc1 was observed in the developing skeleton, i.e., in cartilage primordia, in growth plate cartilage with exclusion of the hypertrophic zone, in the bone matrix and periostium. Bones from adults showed expression of Cthrc1 only in the bone matrix and periostium while the articular cartilage lacked expression. Cthrc1 is typically expressed at epithelial-mesenchymal interfaces that include the epidermis and dermis, basal corneal epithelium, airway epithelium, esophagus epithelium, choroid plexus epithelium, and meninges. In the adult kidney, collecting ducts and distal tubuli expressed Cthrc1. Collectively, the sites of Cthrc1 expression overlap considerably with those reported for TGF-beta family members and interstitial collagens. The present study provides useful information towards the understanding of potential Cthrc1 functions.     

Candidate-gene exclusion in a family with inherited non-syndromic dental disorders

Objectives

Amelogenesis imperfecta, dentinogenesis imperfecta, and dentin dysplasia are the most common non-syndromic dental disorders. In this study, we analysed and localised the gene(s) responsible for inherited non-syndromic dental disorders in a Chinese family.

Methods

This study identified and researched non-syndromic dental disorders in a four-generation Chinese family, including four affected individuals whose clinical phenotype was atypical. Linkage analysis with seven polymorphic markers that localise to six different autochromosomes showed that the family was linked through chromosome 4q. All exons and exon–intron boundaries of dentin sialophosphoprotein (DSPP), enamelin (ENAM), and ameloblastin (AMBN), which are located on chromosome 4q, were sequenced in nine of the family members.

Results

Direct DNA sequence analysis revealed the existence of a G to A transversion in exon 4 (g.13081786G > A, c.727G > A, p.Asp243Asn, based on reference sequences NM_014208.3) of the DSPP gene, and this sequence variation correlated exactly with the presence of the disease.

Conclusion

These results indicate that mutation p.Asp243Asn is a highly probable cause of non-syndromic dental disorder in this Chinese family. The presence of symptom heterogeneity is possible, as the clinical classification system is hampered by the lack of close correlation between the subtype and the molecular defect.     

Dorsoventral patterning of the C. elegans postembryonic mesoderm requires both LIN-12/Notch and TGFbeta signaling

The heparin binding molecules MK and HB-GAM are involved in the regulation of growth and differentiation of many tissues and organs. Here we analyzed the expression of MK and HB-GAM in the developing mouse incisors, which are continuously growing organs with a stem cell compartment. Overlapping but distinct expression patterns for MK and HB-GAM were observed during all stages of incisor development (initiation, morphogenesis, cytodifferentiation). Both proteins were detected in the enamel knot, a transient epithelial signaling structure that is important for tooth morphogenesis, and the cervical loop where the stem cell niche is located. The functions of MK and HB-GAM were studied in dental explants and organotypic cultures in vitro. In mesenchymal explants, MK stimulated HB-GAM expression and, vice-versa, HB-GAM upregulated MK expression, thus indicating a regulatory loop between these proteins. BMP and FGF molecules also activated expression of both cytokines in mesenchyme. The proliferative effects of MK and HB-GAM varied according to the mesenchymal or epithelial origin of the tissue. Growth, cytodifferentiation and mineralization were inhibited in incisor germs cultured in the presence of MK neutralizing antibodies. These results demonstrate that MK and HB-GAM are involved in stem cells maintenance, cytodifferentiation and mineralization processes during mouse incisor development.     

The effects of phosphoproteins on collagen self-assembly in tail tendon and incision dentin from rats

Phosphoproteins retard the rate at which collagen molecules undergo self-assembly into fibrils. The inhibition appears to be dependent on the amount of phosphoprotein present, with increasing phosphoprotein concentrations resulting in greater inhibition. Prior treatment of the phosphoprotein with calcium markedly increases the resultant inhibitory effect. Dentin phosphoproteins are considerably more effective than phosvitin in retarding collagen self-assembly, with retardation times for these hard tissue extracellular matrix proteins being 25–30 times greater than control values.     

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.