Efficient regulation of branching morphogenesis via fibroblast growth factor receptor 2c in early-stage embryonic mouse salivary glands.

Salivary gland (SG) defects have a wide range of health implications, including xerostomia, bacterial infections, and oral health issues. Branching morphogenesis is critical for SG development. A clear understanding of the mechanisms underlying this process will accelerate SG regeneration studies. Fibroblast growth factor receptor 2 (FGFR2) interacts with multiple fibroblast growth factors (FGFs), which promote development. FGFR2 consists of two isoforms, FGFR2b and FGFR2c. FGFR2b is critical for SG development, but little is known about the expression and function of FGFR2c. We investigated the expression of all FGFR family members in fetal SGs between embryonic day 12.5 (E12.5) and E18.5. Based on RT-PCR, we observed an increase in the expression of not only Fgfr2b, but also Fgfr2c in early-stage embryonic mouse SGs, suggesting that FGFR2c is related to SG development. The branch number decreased in response to exogenous FGF2 stimulation, and this effect was suppressed by a mouse anti-FGFR2c neutralizing antibody (NA) and siRNA targeting FGFR2c, whereas FGFR2b signaling was not inhibited. Moreover, the expression of marker genes related to EMT was induced by FGF2, and this expression was suppressed by the NA. These results suggested that branching morphogenesis in SGs is regulated by FGFR2c, in addition to FGFR2b. Interestingly, FGFR2c signaling also led to increased fgf10 expression, and this increase was suppressed by the NA. FGFR2c signaling regulates branching morphogenesis through the activation of FGFR2b signaling via increased FGF10 autocrine. These results provide new insight into the mechanisms by which crosstalk between FGFR2b and FGFR2c results in efficient branching morphogenesis.

Involvement of the T-box transcription factor Brachyury in early-stage embryonic mouse salivary gland.

The mouse submandibular gland (SMG) is important organ for embryonic development, and branching morphogenesis is regulated by many molecules containing transcription factors. Real-time reverse transcriptase polymerase chain reaction revealed that the expression of Brachyury increased in the SMG and peaked between E12.5-E13.5, concomitant with the early stage of branching morphogenesis. The expression of Brachyury in SMG rudiments between E12.5-E13.5 was confirmed by western blotting. In addition, fibronectin and Btbd7 (regulated by fibronectin), which are both essential for cleft formation, were expressed strongly during the same period. The Sox2 and Wnt3a, which regulate cell growth, were also expressed strongly during E12.5-E13.5. On the other hand, cleft formation and branching morphogenesis was suppressed by knockdown of Brachyury gene, suggesting that Brachyury plays a central role in regulating cell growth and cleft formation in early-stage embryonic mouse salivary gland development.

Biological characterization and analysis of metastasis-related genes in cell lines derived from the primary lesion and lymph node metastasis of a squamous cell carcinoma arising in the mandibular gingiva.

Controlling metastatic lesions is an important part of improving cancer prognosis, in addition to controlling the primary lesion. There have been numerous histological studies on primary and metastatic lesions, but little basic research has been performed using cell lines from primary and metastatic lesions belonging to the same patient. In this study, we successfully established a cell line derived from lower gingival carcinoma (WK2) as well as a line derived from secondary cervical lymph node metastasis (WK3F) through primary cultures of tissue from a patient with oral squamous cell carcinoma. We then investigated the biological characteristics of the cancer cell lines from these primary and metastatic lesions and analyzed metastasis-related genes. Comparison of the biological characteristics in vitro showed that WK3F had higher cell proliferation ability and shorter cell doubling time than WK2. WK3F also had increased cell migratory ability and higher invasive and self-replication abilities. Heterotransplantation into nude mice resulted in high tumor formation rates in the tongue and high metastasis rates in the cervical lymph nodes. Changes in WK2 and WK3F gene expression were then comprehensively analyzed using microarrays. Genes with increased expression in WK3F compared to WK2 were extracted when the Z-score was ≥2.0 and the ratio was ≥5.0, while genes with reduced expression in WK3F compared to WK2 were extracted when the Z-score was ≤-2.0 and the ratio was ≤0.2; differences were found in 604 genes. From these, MAGEC1 (88.0-fold), MMP-7 (18.6-fold), SNAI1 (6.6-fold), MACC1 (6.2-fold), and HTRA1 (0.012-fold) were selected as metastasis-related candidate genes. The results suggest that these molecules could be important for clarifying the mechanisms that regulate metastasis and provide new therapeutic targets for inhibiting tumor invasion.

Expression of the urokinase receptor regulates focal adhesion assembly and cell migration in adenoid cystic carcinoma cells.

Adenoid cystic carcinoma (AdCC) cell lines (ACCS and ACCT) showed higher migration responses and adhesion to the extracellular matrix (ECM), especially types I and IV collagen, than did the oral squamous cell carcinoma (SCC) lines (NA and TF). The response to collagens was largely and exclusively inhibited by anti-alpha(2) integrin antibody. Moreover, AdCC cell lines expressed higher surface levels of urokinase-type plasminogen activator receptor (uPAR) than did SCC cell lines. When AdCC cells were plated on collagen, the surface level of uPAR was increased, and numerous focal adhesions consisting of uPAR, vinculin, and paxillin were assembled; whereas collagen-stimulated SCC cell counterparts or AdCC cells plated on other types of ECM, such as fibronectin, failed to assemble such definite focal adhesions. In order to elucidate the association of uPAR with collagen-induced events, an ACCS-AS cell line transfected with a vector expressing antisense uPAR RNA was established and shown to have reduced uPAR (about 10% that of parental ACCS at both the protein and mRNA levels). ACCS-AS showed a strong reduction of collagen-stimulated migration and focal adhesion assembly of alpha(2) integrin, vinculin, and paxillin. These findings suggest that AdCC has a proclivity for migrating to types I and IV collagens due to the overexpression of uPAR, which plays a key role in focal adhesion assembly and migration.

Involvement of hepatocyte growth factor in branching morphogenesis of murine salivary gland.

We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5-12 (E11.5-E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real-time reverse transcriptase-polymerase chain reaction showed that the expression of the c-met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14-E15.5. Although strong expression of the HGF and c-met transcripts was observed in the tongue muscles, this expression was limited at E13.5-E14.5. Serum-free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme-free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme-free epithelium failed to grow. When E13.5 or E14 mesenchyme-free epithelium and a recombinant human HGF (rh-HGF) -soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh-HGF and anti-HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh-HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh-HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c-met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c-Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme-derived HGF and c-met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage.

Calcium induces differentiation of primary human salivary acinar cells.

Cultivation of human parotid glands in serum-free medium (Ca(2+) concentration, 0.2 mM) with growth supplements resulted in isolation of a homogeneous population of epithelial cells without any mesenchymal cells. The isolated cells showed an undifferentiated phenotype with scant cytoplasmic organelles, and low levels of alpha-amylase expression. The cells remained viable and undifferentiated for up to 24 passages when subcultured at 80% confluence in 0.2 mM Ca(2+) medium with a 1:3 split ratios. There was little cell-cell contact. A Ca(2+) switch from 0.2 to 1 mM induced cell-cell contact with translocation of desmosomal proteins from the cytoplasm to the cell membrane, and sequential differentiation of serous acinar cells with a glandular arrangement, well-developed cytoplasmic organelles and an increased level of alpha-amylase expression. These morphological changes and desmosome assembly were blocked by treatment with non-specific PKC inhibitor. Moreover, the addition of PKC activator, tetradecanoylphorbol 13-acetate (TPA), to 0.2 mM Ca(2+) medium caused transient assembly of desmosome-like structure, but did not induce cell-cell contact or morphological differentiation. Cultivation of the cells in 1.5 mM Ca(2+) medium resulted in increased stratification of the cells and reduced alpha-amylase expression. These findings provide the first demonstration that continuous cultivation in 1.0 mM Ca(2+) medium is required for cellular differentiation of salivary gland acinar cells, and maintenance of the differentiated state.