Morimoto Y, Morimoto H, Okamura H, Nomiyama K, Nakamuta N, Kobayashi S, Kito S, Ohba T, Haneji T
Department of Dental Radiology, Kyushu Dental College, Kokurakita-ku, Kitakyushu 803-8580, Japan.
Arch Oral Biol. 2000 Aug;45(8):657-66. doi: 10.1016/s0003-9969(00)00038-8.
Fas receptor is a member of a superfamily of receptors characterized by cysteine-rich motifs in the extracellular domain of the molecule. Binding of Fas ligand to the receptor leads to receptor activation and the induction of intracellular signals that result in apoptotic cell death. In the present study, the expression of mRNA and proteins of Fas receptor and Fas ligand were examined in human submandibular gland ductal (HSG) cells treated with okadaic acid by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis. Six hundred and eighty-two bp of the PCR product of Fas receptor mRNA was detected in HSG cells and a protein with an estimated molecular weight of 58,000 was expressed in HSG cells. Treatment of HSG cells with an agonistic anti-Fas monoclonal antibody resulted in death of HSG cells, indicating that the functional Fas receptor protein is expressed in HSG cells. Fas receptor protein expression stimulated by okadaic acid was elevated in a dose- and time-dependent manner, with maximal expression at 20 nM and 48 h treatment. Fas ligand mRNA was also detected constitutively in HSG cells by RT-PCR. Okadaic acid stimulated the expression of Fas ligand protein in HSG cells in a time-dependent manner, while the expression of the ligand was low in untreated HSG cells. The molecular weight of Fas ligand was estimated as 68,000. An antagonistic anti-Fas ligand monoclonal antibody prevented okadaic acid-induced death in HSG cells in a dose-dependent fashion as determined by WST-1 assay. The results indicate that the expression of Fas receptor and ligand is regulated by protein phosphatase(s) sensitive to okadaic acid and is involved in okadaic acid-induced apoptosis in HSG cells. The results also suggest that the Fas receptor-ligand system might regulate apoptosis in HSG cells.
Fas受体是受体超家族的成员，其特点是分子的细胞外结构域中有富含半胱氨酸的基序。Fas配体与受体结合导致受体激活，并诱导细胞内信号，从而导致凋亡性细胞死亡。在本研究中，通过逆转录聚合酶链反应（RT-PCR）和免疫印迹分析，检测了用冈田酸处理的人下颌下腺导管（HSG）细胞中Fas受体和Fas配体的mRNA和蛋白质表达。在HSG细胞中检测到Fas受体mRNA的PCR产物为682bp，并且在HSG细胞中表达了一种估计分子量为58,000的蛋白质。用激动性抗Fas单克隆抗体处理HSG细胞导致HSG细胞死亡，表明功能性Fas受体蛋白在HSG细胞中表达。冈田酸刺激的Fas受体蛋白表达以剂量和时间依赖性方式升高，在20 nM处理48小时时表达最高。通过RT-PCR也在HSG细胞中组成性地检测到Fas配体mRNA。冈田酸以时间依赖性方式刺激HSG细胞中Fas配体蛋白的表达，而在未处理的HSG细胞中配体的表达较低。Fas配体的分子量估计为68,000。如通过WST-１测定所确定的，拮抗性抗Fas配体单克隆抗体以剂量依赖性方式阻止了冈田酸诱导的HSG细胞死亡。结果表明，Fas受体和配体的表达受对冈田酸敏感的蛋白磷酸酶调节，并参与冈田酸诱导的HSG细胞凋亡。结果还表明，Fas受体-配体系统可能调节HSG细胞中的凋亡。
