Brochhausen Christoph, Neuland Pia, Kirkpatrick C James, Nüsing Rolf M, Klaus Günter
Institute of Pathology, Johannes Gutenberg-University, Mainz, Germany.
Arthritis Res Ther. 2006;8(3):R78. doi: 10.1186/ar1948. Epub 2006 Apr 28.
Prostaglandin E2 (PGE2) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE2 pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE2 receptors) is essential. We therefore examined the production of PGE2 in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE2 on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE2 receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE2 synthesis was determined by mass spectrometry, cell proliferation by DNA [3H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE2 into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE2-dependent proliferation. Exogenously added PGE2 stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE2 was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE2 release, which stimulates cell proliferation via the EP1 receptor.
前列腺素E2（PGE2）在骨骼发育和代谢中起重要作用。然而，要在治疗上干预PGE2途径，了解相关酶（环氧化酶）和受体（PGE2受体）至关重要。因此，我们检测了体外培养的生长板软骨细胞中PGE2的产生以及外源性添加PGE2对细胞增殖的影响。此外，我们分析了环氧化酶（COX）-1和COX-2以及PGE2受体亚型EP1、EP2、EP3和EP4在生长板原位和体外的表达及空间分布。通过质谱法测定PGE2合成，通过DNA[3H] - 胸腺嘧啶核苷掺入法测定细胞增殖，通过RT-PCR测定培养细胞和匀浆生长板中环氧化酶和EP受体的mRNA表达。为确定细胞表达情况，使用针对COX-1、COX-2、EP1、EP2、EP3和EP4的多克隆抗体进行免疫组织化学染色，对大鼠胫骨生长板冰冻切片和原代软骨细胞培养物进行检测。培养的生长板软骨细胞将PGE2短暂分泌到培养基中。虽然两种酶在体外和体内的软骨细胞中均有表达，但似乎主要是COX-2促成了PGE2依赖性增殖。外源性添加的PGE2以剂量依赖性方式刺激DNA合成，并呈现钟形曲线，在10-8 M时达到最大值。EP1/EP3特异性激动剂舒前列素和EP1选择性激动剂ONO-D1-004增加了DNA合成。PGE2的作用被ONO-8711抑制。观察到EP1、EP2、EP3和EP4受体在原位和体外的表达；EP2在生长板原位的所有区域均呈均匀表达，而EP1表达不均匀，储备区有未受累细胞。在培养细胞中，这四种受体仅在一部分细胞中表达。在被基质包围的多边形细胞中发现EP1受体染色最强。在大鼠生长板中也观察到了EP3和EP4受体蛋白的表达。然而，在培养的软骨细胞中，仅检测到EP3和EP4受体的弱表达。我们认为，在生长板软骨细胞中，COX-2负责PGE2的释放，PGE2通过EP1受体刺激细胞增殖。
