Yoshiharu Tanaka

Growth and differentiation of periodontal ligament-derived cells in serum-free defined culture

SummaryWe have developed a serum-free medium for the growth and differentiation of periodontal ligament-derived cells (PLC). In addition, the expression of both fibroblast growth factor (FGF) and FGF receptor (FGFR) in the PLC was investigated by immunohistochemical examination, heparin affinity chromatography (HAC), and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Optimal growth of the cells was achieved in Iscove’s modified Dulbecco’s medium supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, and oleic acid in type-I collagen-coated dishes. Both FGF-1 and FGF-2 stimulated cell growth and inhibited differentiation as measured by inhibition of alkaline phosphatase activity of the cells. An immunohistochemical analysis of FGF-1 and FGF-2 revealed that immunoreactive FGF-1 and FGF-2 were detected predominantly in the cytoplasm of growing cells. In addition, perinuclear FGF-1 staining and nuclear FGF-2 staining were observed in the same growing cells. In contrast, a faint diffuse staining of FGF-1 and FGF-2 was detected in cytoplasm of the confluent differentiated cells. The 2.15 M NaCl eluate from HAC of the cell extracts exhibited growth-promoting activities for the PLC, and it also stimulated the growth of human umbilical vein-derived endothelial cells and inhibited binding of [125I]-FGF to its receptors, indicating the cells produced FGFs or FGF-like growth factors. RT-PCR analysis revealed that the cells expressed FGFR-1 mRNA but not mRNAs for FGFR-2, FGFR-3 and FGFR-4 mRNA. These results suggest that the FGF-FGFR-1 system plays an important role in the growth and differentiation of periodontal ligament-derived cells.

Isolation and serum-free culture of epithelial cells derived from epithelial rests of Malassez in human periodontal ligament

SummaryTo study biological character and function of epithelial rests of Malassez (ERM) in human periodontal ligament, we have developed a serum-free culture system of epithelial cells (ME) derived from ERM. The mitogenic effects of fibroblast growth factor (FGF)-1, FGF-2, and FGF-7/keratinocyte growth factor (KGF) on ME, human periodontal ligament-derived fibroblasts (PLF), human oral epithelial cells (OE), and human submandibular gland-derived epithelial cells (SGE) were investigated under a serum-free culture condition. FGF-1 and FGF-7/KGF stimulated the growth of both ME and SGE but FGF-2 had no effect. On the other hand, FGF-1, FGF-2, and FGF-7/KGF increased the OE proliferation. These results suggested that the divergent requirement of FGF ligands among these cells would be attributed to the different expression pattern of FGF receptor (FGFR) messenger ribonucleic acid (mRNA) isotypes. Therefore, we examined the expression of FGFR isotypes in these cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of ME- and SGE-derived mRNAs revealed the presence of mRNA encoding FGFR2-IIIb, a high affinity receptor for FGF-1 and FGF-7/KGF. However, no mRNAs for other FGFR isotypes were detected in both ME and SGE. On the contrary, OE expressed FGFR1-IIIc, FGFR3-IIIb, and FGFR4 mRNAs as well as FGFR2-IIIb. These results indicate that FGF binding sites on ME dominantly bind to FGF-1 and FGF-7/KGF, which transduce their signals via FGFR2-IIIb. Immunohistochemical analysis, PCR-Southern, ribonuclease protection assay (RPA), and Western blotting revealed that PLF expressed FGF-7/KGF mRNA and its peptide. These observations suggest that FGF-7/KGF might mediate epithelial-mesenchymal interactions between ME and PLF to maintain normal structure and function of periodontal ligament.

EXPRESSION OF FIBROBLAST GROWTH FACTOR RECEPTOR GENES IN HUMAN HEPATOMA-DERIVED CELL LINES

SummaryThe fibroblast growth factor (FGF) function has been considered to contribute to various human tumors and malignant growth of neoplasm. Hepatocellular carcinoma (HCC) is a typical hypervascular tumor, and it is suggested that FGF may be involved in hepatocarcinogenesis. Therefore, the relationship between the progression of HCC and expression of FGFs and FGF receptors (FGFRs) was evaluated in this study. We investigated the expression of messenger ribonucleic acids (mRNAs) of FGFs and FGFRs by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in eight human hepatoma-derived cell lines (Hep3B, HLE, HLF, HUH6, HUH7, KIM1, Li7, and PLC/PRF/5), one hepatoblastome-derived cell line (HepG2), and human primary hepatocytes. In addition, effects of FGF-1, FGF-2, and FGF-7 on the growth of hepatoma-derived cell lines were studied in serum-free defined culture conditions. An RT-PCR analysis revealed that all cell lines except PLC/PRF/5 expressed all FGFR MRNAs: FGF-R1 (IIIc),-R2 (IIIb),-R3 (IIIb),-R3 (IIIc), and-R4 mRNAs. In contrast, human primary hepatocytes expressed FGF-R1 (IIIc),-R3 (IIIc), and-R4 mRNAs but not mRNAs of FGF-R2 (IIIb),-R2 (IIIc), and-R3 (IIIb). All cell lines except HUH6 and HUH7 expressed FGF-1 and FGF-2 mRNAs. Addition of exogenous FGF-1 or FGF-2 (or both) to culture stimulated cell proliferation in several cell lines, but FGF-7 exhibited no growth stimulation in all cells. Hepatoma cells may posses a proliferation mechanism regulated by an autocrine mechanism, a paracrine mechanism, or both, which are mediated by FGF-1/FGFR or FGF-2/FGFR (or both). In addition, a gain of FGF-R2 (IIIb),-R2 (IIIc), and-R3 (IIIb) may be associated with malignant transformation of liver tumor and may eventually serve as useful diagnostic and prognostic indicators.