MacPherson H, Noble B S, Ralston S H
Department of Medicine and Therapeutics, University of Aberdeen Medical School, Foresterhill, UK.
Bone. 1999 Mar;24(3):179-85. doi: 10.1016/s8756-3282(98)00173-2.
Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regulating osteoblast function is less clear and the isoforms of nitric oxide synthase (NOS) that are expressed by human osteoblasts have not been determined. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on osteoblast growth and differentiation. Low levels of NO were produced constitutively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was found in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produced only by hOB cells after stimulation with the cytokines IL-1beta, TNF-alpha, and IFN-gamma. The NOS inhibitor, L-NMMA, did not affect growth or alkaline phosphatase activity in unstimulated osteoblasts. Incubation of hOB cells with cytokines inhibited growth and stimulated alkaline phosphatase activity and these effects were abrogated by L-NMMA. Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO independent because they were not influenced by L-NMMA. Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect on osteoblast growth or differentiation under basal conditions. In contrast, IL-1beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting and differentiation-inducing effects on osteoblasts that were partly NO dependent, indicating that NO may act predominantly as a modulator of cytokine-induced effects on osteoblast function.
先前的研究已显示，来自不同物种的培养成骨细胞样细胞中存在组成型和细胞因子诱导型一氧化氮（NO）合酶活性的证据。尽管已发现细胞因子诱导的NO产生会抑制成骨细胞生长，但组成型NO产生在调节成骨细胞功能中的作用尚不清楚，并且人类成骨细胞表达的一氧化氮合酶（NOS）同工型尚未确定。在此，我们研究了培养的人类成骨细胞样细胞中NOS的表达，并研究了组成型和细胞因子诱导型NO对成骨细胞生长和分化的影响。通过分析培养基中亚硝酸盐浓度反映出，成骨细胞样细胞组成型产生低水平的NO，并且在原代成骨细胞（hOBs）、TE85和MG63骨肉瘤细胞中发现了内皮型一氧化氮合酶（ecNOS）mRNA、蛋白质和生物活性的证据。然而，没有一个成骨细胞样细胞表达神经元型一氧化氮合酶（nNOS），并且只有在用细胞因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）刺激后，hOB细胞才产生诱导型一氧化氮合酶（iNOS）。NOS抑制剂L-NMMA对未刺激的成骨细胞的生长或碱性磷酸酶活性没有影响。用细胞因子孵育hOB细胞会抑制生长并刺激碱性磷酸酶活性，而这些作用会被L-NMMA消除。细胞因子也会抑制TE85细胞和MG63细胞的生长，但这些作用似乎不依赖于NO，因为它们不受L-NMMA的影响。我们的实验表明，人类成骨细胞通过ecNOS途径组成型产生NO，但证明在基础条件下，这似乎对成骨细胞生长或分化没有明显影响。相比之下，IL-1β、TNF-α和IFN-γ对成骨细胞发挥生长抑制和分化诱导作用，部分依赖于NO，这表明NO可能主要作为细胞因子诱导的成骨细胞功能效应的调节剂发挥作用。
