Multiple functional involvement of thymosin beta-4 in tooth germ development.

Thymosin beta-4 (Tß4) is known to be ubiquitously involved in the actin monomer sequestering on the cytoskeleton. Our previous study showed specific temporal and special in situ expression pattern of Tß4 mRNA in dental epithelial and mesenchymal cells in the developing tooth germ of the mouse lower first molar. In this study, we examined the functional implications of Tß4 in the developmental course of the mouse lower first molar. An inhibition assay using Tß4 antisense sulfur-substituted oligodeoxynucleotide (AS S-ODN) in cultured embryonic day 11.0 (E11.0) mandibles showed a significant growth inhibition of the tooth germ. However, no growth arrest of the cultured E15.0 tooth germ was observed by using Tß4 AS S-ODN. The Tß4 knockdown led to significantly decreased expression levels of type II/III runt-related transcription factor 2 (Runx2) and nucleolin (Ncl) in the cultured E11.0 mandibles. Since our previous studies proved that the inhibition of type II/III Runx2 and Ncl translations resulted in the developmental arrest of the tooth germ in the cultured E11.0 mandible, Tß4 appears to play roles in tooth germ development via the regulation of the type II/III Runx2 and Ncl expressions. Tß4 knockdown also resulted in decreased secretion of matrix metalloproteinase (Mmp)-2, a reduced cell motility activity and upregulation of E-cadherin in dental epithelial mDE6 cells. These results suggest that Tß4 plays multiple functional roles in odontogenic epithelial cells in the early stages of tooth germ development by regulating the expression of odontogenesis-related genes.

Induction of dental epithelial cell differentiation marker gene expression in non-odontogenic human keratinocytes by transfection with thymosin beta 4.

Previous studies have shown that the recombination of cells liberated from developing tooth germs develop into teeth. However, it is difficult to use human developing tooth germ as a source of cells because of ethical issues. Previous studies have reported that thymosin beta 4 (Tmsb4x) is closely related to the initiation and development of the tooth germ. We herein attempted to establish odontogenic epithelial cells from non-odontogenic HaCaT cells by transfection with TMSB4X. TMSB4X-transfected cells formed nodules that were positive for Alizarin-red S (ALZ) and von Kossa staining (calcium phosphate deposits) when cultured in calcification-inducing medium. Three selected clones showing larger amounts of calcium deposits than the other clones, expressed PITX2, Cytokeratin 14, and Sonic Hedgehog. The upregulation of odontogenesis-related genes, such as runt-related transcription factor 2 (RUNX2), Amelogenin (AMELX), Ameloblastin (AMBN) and Enamelin (ENAM) was also detected. These proteins were immunohistochemically observed in nodules positive for the ALZ and von Kossa staining. RUNX2-positive selected TMSB4X-transfected cells implanted into the dorsal subcutaneous tissue of nude mice formed matrix deposits. Immunohistochemically, AMELX, AMBN and ENAM were observed in the matrix deposits. This study demonstrated the possibility of induction of dental epithelial cell differentiation marker gene expression in non-odontogenic HaCaT cells by TMSB4X.

STAT3 signal transduction through interleukin-22 in oral squamous cell carcinoma.

Interleukin (IL)-22 is a member of the IL-10 family. Its main targets are epithelial cells, not immune cells. We examined IL-22 signal transduction in oral squamous cell carcinoma (OSCC) cells. Immunohistochemical staining revealed that IL-22R was expressed more highly in OSCC compared to normal regions. An IL-22R signal was also observed in metastatic OSCC cells in the lymph node. RT-PCR showed that the human OSCC cell lines MISK81-5, HSC-3, HSC-4, SAS and SQUU-B expressed IL-22 receptor chains. Immunoblotting showed that IL-22 induced a transient tyrosine phosphorylation of STAT3 (pY705-STAT3) in MISK81-5 cells. The change in the serine phosphorylation of STAT3 was subtle during the examination periods. Simultaneously, pY705-STAT3 activation in HSC-3 cells was undetectable after IL-22 stimulation. Immunocytochemistry demonstrated that IL-22 induced the translocation of phosphorylated STAT3 into the nucleus of MISK81-5 cells. IL-22 temporarily upregulated the expression of anti-apoptotic and mitogenic genes such as Bcl-x, survivin and c-Myc, as well as SOCS3. IL-22 transiently activated ERK1/2 and induced a delayed phosphorylation of p38 MAP kinase, but negligibly involved the activation of NF-κB in MISK81-5 cells. MISK81-5 and SQUU-B cells treated with IL-22 showed mild cellular proliferation. MISK81-5, HSC-4 and SAS cells treated with IL-22 downregulated the keratinocyte differentiation-related genes compared with unstimulated cells. Conversely, STAT3 suppression by STAT3 siRNA strongly disrupted the downregulation of these genes by IL-22, but it did not significantly affect the activation of ERK1/2 by IL-22. The OSCC cells used in this study upregulated the expression of SERPINB3/4 (SCCA1/2), well-known SCC markers, following treatment with IL-22. These results indicate that IL-22 differentially activates the STAT3 signaling system depending on the type of OSCC. IL-22 may therefore play a role in tumor growth, cell differentiation and progression through STAT3-dependent and -independent pathways.