Mechanisms of Parathyroid Hormone Receptor Signaling in Mouse Salivary Gland Development.

本研究旨在阐明甲状旁腺激素受体（PTHR）信号在唾液腺发育中的作用。采用定性PCR、原位杂交和免疫组织化学方法检测小鼠唾液腺中PTHR及其配体甲状旁腺激素相关肽（PTHrP）的表达。PTHrP在发育中的小鼠颌下腺中表达量在E13.5时达到峰值，随后逐渐降低。另一方面，PTHR的表达量在E14.5 ~ E15.5达到峰值，随后逐渐降低。PTHrP和PTHR主要在上皮细胞而非间充质细胞中表达。PTHR的细胞内信号通路包括g和Gq两条g蛋白通路。g亚基和Gq亚基均在唾液腺上皮细胞中表达。为了评估PTHR信号在唾液腺发育中的作用，我们分析了PTHR缺失小鼠的唾液腺。在E12.5 ~ E15.5期间，pthr基因缺失小鼠的下颌骨腺发育正常，但在发育过程中，下颌骨腺发育不正常。此外，通过BrdU染色评估，与野生型小鼠相比，pthrr -null小鼠在E15.0时唾液腺上皮细胞中BrdU并入细胞核的数量减少。这些结果表明，抑制唾液腺上皮细胞的细胞增殖导致芽发育减少。为了研究细胞内信号通路是gs级联还是gq级联刺激了唾液腺的萌芽发育，我们用特异性的刺激类似物刺激了唾液腺外植体培养中的次级信使。gq -磷脂酶C的刺激以剂量依赖的方式促进唾液腺芽的发育。另一方面，刺激gs -腺苷酸环化酶- camp减少芽发育，促进导管发育。这些发现表明PTHR信号通过Gq-PKC级联刺激唾液腺芽发育。在唾液腺发育过程中，受体酪氨酸激酶受体信号通路EGF、FGF和HGF激活PKC信号通路，刺激芽发育。然而，PTHR配体对EGF或FGF诱导的芽发育没有影响，这表明PTHR使用不同于受体酪氨酸激酶信号通路的pkc信号通路。少

This study has focused on clarifying the roles of Parathyroid hormone receptor (PTHR) signaling in salivary gland development.Expression of PTHR and its ligand, Parathyroid hormone related peptide (PTHrP), in developing mouse salivary gland, was examined by qualitative PCR, in situ hybridization and immunohistochemistry. Expression level of PTHrP was peaked at E13.5,and then gradually decreased in developing mouse submandibular gland. On the other hand, expression level of PTHR peaked at E14.5 to E15.5, and then gradually decreased. PTHrP and PTHR were dominantly expressed in epithelial cells rather than mesenchymal cells. Intracellular signaling of PTHR utilizes 2 G-protein pathways, Gs and Gq. Both Gs and Gq subunit were expressed in epithelial cells of salivary gland.To assess the roles of PTHR signaling in salivary gland development, we have analyzed the salivary glands of PTHR-null mice. Submandibular glands in PTHR-null mice developed normally during E12.5 to E15.5,and then faile … More d to develop buds, as compared to those of litter mate wild-type mice. Furthermore, BrdU incorporation into cellular nuclei was decreased in salivary gland epithelial cells of PTHR-null mice at E15.0, compared to that of wild type mice, when assessed by BrdU staining. These findings suggest that inhibited cellular proliferation of salivary gland epithelial cells resulted in reduced bud development.To examine which intracellular signaling pathway, Gs-cascade or Gq-cascade, stimulate budding development of salivary gland, we have stimulated those secondary messenger in salivary glands explants culture by those specific stimulating analogs. Stimulation of Gq-phospholipase C stimulated bud development of salivary glands in a dowse dependent manner. On the other hand, stimulation of Gs-adenylcyclase-cAMP reduced bud development and stimulated duct development. These findings suggest that PTHR signaling stimulate bud development of salivary glands via Gq-PKC cascade.In salivary gland development, receptor tyrosine kinase receptor signaling, i.e. EGF, FGF and HGF, activate PKC signaling and stimulate bud development. However, there was no effect of PTHR ligands on EGF or FGF induced-bud development, suggesting that PTHR use distinct PKC-signaling pathway from receptor tyrosine kinase signaling. Less