Abraham L A, Chinni C, Jenkins A L, Lourbakos A, Ally N, Pike R N, Mackie E J
School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
Bone. 2000 Jan;26(1):7-14. doi: 10.1016/s8756-3282(99)00237-9.
Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca2+]i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca2+]i. Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca2+]i followed by a sustained plateau; [Ca2+]i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.
成骨细胞表达蛋白酶激活受体-1（PAR-1），该受体可被凝血酶或与PAR-1经受体裂解产生的新“拴系配体”N端对应的合成肽激活。凝血酶和人PAR-1激活肽均可刺激人SaOS-2成骨样细胞系中[Ca2+]i升高，但该肽刺激受体介导的Ca+内流，而凝血酶则不然。大鼠PAR-1激活肽对大鼠原代成骨样细胞增殖的刺激作用大于凝血酶。由于现在已知PAR-1激活肽可激活PAR-2，因此开展本研究以调查成骨细胞是否表达该受体，若表达，这是否可解释成骨细胞对凝血酶和PAR-1激活肽反应中观察到的差异。逆转录聚合酶链反应（RT-PCR）和免疫细胞化学研究证明大鼠颅骨成骨样细胞原代培养物中存在PAR-2表达。在胚胎小鼠骨骼的免疫组织化学研究中，成骨细胞显示PAR-2阳性染色。PAR-2的激活剂包括胰蛋白酶、肥大细胞类胰蛋白酶、牙龈蛋白酶-R以及与PAR-2拴系配体序列对应的合成肽。用大鼠PAR-2激活肽（SLIGRL）处理大鼠原代成骨样细胞，或用人PAR-2激活肽（SLIGKV）处理SaOS-2细胞，可导致[Ca2+]i呈剂量依赖性增加。胰蛋白酶或牙龈蛋白酶-R也可诱导细胞内钙浓度升高，并引起相互交叉脱敏。PAR-2激活剂导致[Ca2+]i出现尖峰，随后是持续的平台期；用乙二醇双四乙酸（EGTA）处理后，[Ca2+]i恢复到基线水平。体外用SLIGRL处理大鼠成骨样细胞不影响胸苷掺入或内源性碱性磷酸酶活性。本文给出的结果表明成骨细胞表达PAR-2，且这种表达能够解释凝血酶和PAR-1激活肽在引发钙内流能力方面观察到的差异，但不能解释增殖反应方面的差异。
