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Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts.

ABSTRACT: In the mouse tooth organ, shortly after birth, ameloblasts acquire their secretory phenotype, which is characterized by the prominent expression and subsequent secretion of two isoforms of amelogenin, M180 and M59 (LRAP, [A-4]). Amelogenin deposition into the ameloblast extracellular matrix promotes enamel biomineralization. A complex set of intercellular signaling events, reciprocal communications between the developing oral epithelium and its underlying dental mesenchyme, guide the expression of amelogenin mRNA, and limit it to a defined period of tooth development. In tooth germ organ culture, addition of the [A-4] isoform, lacking amelogenin exon 4 and exon 6 segments a, b, c, was shown to affect ameloblast development. To understand the basis for this regulatory activity, we have studied the effects of r[A-4] on ameloblast-like LS8 cells, and the role of the putative [A-4] cell surface receptor, LAMP1, as well as the related receptor LAMP3. In the LS8 cells, the expression of the spliced isoforms of amelogenin, LAMP1, and LAMP3 were identified by RT-PCR, and real-time PCR semi-quantitative analysis assessed the modulation of M180 message. M180 mRNA was up-regulated by exogenous [A-4], and this was further increased by blockade of LAMP1, suggesting additive effects between the intracellular signaling pathways activated by the discrete agonists. Immunofluorescence staining identified the patterns of [A-4] and LAMP1 localization in LS8 cells. Internalized r[A-4] was co-localized with LAMP1 in late endosomal/lysosomal compartments. Thus, the LAMP1 and [A-4] intracellular sorting pathways are interrelated. The nitric oxide (NO) signaling pathway was activated by exogenous [A-4]. [A-4] modulated inducible nitric oxide synthase (iNOS, NOS2) and endothelial nitric oxide synthase (eNOS, NOS3) expression, albeit, to different extents. NOS2 was significantly up-regulated after 4 h, while NOS3 increased slightly after 24 h. Co-treatment of LS8 cells with r[A-4] and anti-LAMP1 antibodies further enhanced NOS2 expression. Anti-LAMP1 antibodies did not abrogate NO production in LS8 cells treated for 4 h with r[A-4], but the iNOS inhibitor, l-Nil, down-regulated both NO production and the expression of M180 mRNA. These data suggest that [A-4] modulates M180 mRNA expression, partly, via the NO signaling pathway.

SUBMITTER: Iacob S

PROVIDER: S-EPMC2442712 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts.

Iacob Stanca S Veis Arthur A

Bone 20080215 6

In the mouse tooth organ, shortly after birth, ameloblasts acquire their secretory phenotype, which is characterized by the prominent expression and subsequent secretion of two isoforms of amelogenin, M180 and M59 (LRAP, [A-4]). Amelogenin deposition into the ameloblast extracellular matrix promotes enamel biomineralization. A complex set of intercellular signaling events, reciprocal communications between the developing oral epithelium and its underlying dental mesenchyme, guide the expression ...[more]

PMID: 18394981

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Project description:BACKGROUND: The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns. RESULTS: Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DDelta7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dDelta3 (deleted for exon 3) and Rad51dDelta5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dDelta3 being detected in each tissue except ovary and Rad51dDelta5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10. CONCLUSION: These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

Project description:BACKGROUND:Although adult human tissue-derived epidermal stem cells are capable of differentiating into enamel-secreting ameloblasts and forming teeth with regenerated enamel when recombined with mouse dental mesenchyme that possesses odontogenic potential, the induction rate is relatively low. In addition, whether the regenerated enamel retains a running pattern of prism identical to and acquires mechanical properties comparable with human enamel indeed warrants further study. METHODS:Cultured human keratinocyte stem cells (hKSCs) were treated with fibroblast growth factor 8 (FGF8) and Sonic hedgehog (SHH) for 18 h or 36 h prior to being recombined with E13.5 mouse dental mesenchyme with implantation of FGF8 and SHH-soaked agarose beads into reconstructed chimeric tooth germs. Recombinant tooth germs were subjected to kidney capsule culture in nude mice. Harvested samples at various time points were processed for histological, immunohistochemical, TUNEL, and western blot analysis. Scanning electronic microscopy and a nanoindentation test were further employed to analyze the prism running pattern and mechanical properties of the regenerated enamel. RESULTS:Treatment of hKSCs with both FGF8 and SHH prior to tissue recombination greatly enhanced the rate of tooth-like structure formation to about 70%. FGF8 and SHH dramatically enhanced stemness of cultured hKSCs. Scanning electron microscopic analysis revealed the running pattern of intact prisms of regenerated enamel is similar to that of human enamel. The nanoindentation test indicated that, although much softer than human child and adult mouse enamel, mechanical properties of the regenerated enamel improved as the culture time was extended. CONCLUSIONS:Application of FGF8 and SHH proteins in cultured hKSCs improves stemness but does not facilitate odontogenic fate of hKSCs, resulting in an enhanced efficiency of ameloblastic differentiation of hKSCs and tooth formation in human-mouse chimeric tooth germs.

Dataset Information

Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts.

Publications

Identification of the functional activity of the [A-4] amelogenin gene splice product in newborn mouse ameloblasts.

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