Williams J P, Dong S S, Whitaker C H, Jordan S E, Blair H C
Department of Pathology, University of Alabama at Birmingham, USA.
J Cell Physiol. 1996 Dec;169(3):411-9. doi: 10.1002/(SICI)1097-4652(199612)169:3<411::AID-JCP1>3.0.CO;2-R.
Osteoclasts mediate bone resorption by secretion at the site of bone attachment. This process depends on calmodulin concentrated at a specialized acid-secreting membrane. We hypothesized that increased calmodulin and bone attachment were required for acid secretion. We tested this by studying calmodulin, bone attachment, and membrane acid transport in osteoclasts and their precursor mononuclear cells. Osteoclasts and macrophages were isolated from medullary bone of hens; cell fractions were prepared after culturing cells with or without bone. Calmodulin was visualized by Western analysis; calmodulin mRNA was determined by Northern hybridization, and ATP-dependent membrane acid transport was assayed by acridine orange uptake. Calmodulin decreased in osteoclasts cultured without bone. Calmodulin in isolated macrophages was approximately 25% of osteoclast levels, but increased several fold by 5 days. Bone had no effect. Calmodulin mRNA was similar in osteoclasts with or without bone. However, only osteoclasts cultured with bone retained acid transport capacity. Macrophage calmodulin mRNA was not affected by bone, but increased three fold by day 5, paralleling protein production. Macrophages developed acid transport capacity at 3-5 days, but at lower levels than osteoclasts, and bone had no measurable effect. Chicken cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated. We conclude that, following isolation, calmodulin levels decline in osteoclasts via a post-transcriptional mechanism. In cultured macrophages, by contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentiation in vitro. Increased calmodulin correlated with membrane acid transport capacity in both cell types. The macrophage findings indicate that stimuli other than bone influence acid transport capacity in this family of cells.
破骨细胞通过在骨附着部位分泌来介导骨吸收。这一过程依赖于集中在专门的酸分泌膜上的钙调蛋白。我们推测酸分泌需要增加钙调蛋白和骨附着。我们通过研究破骨细胞及其前体单核细胞中的钙调蛋白、骨附着和膜酸转运来验证这一推测。从母鸡的骨髓骨中分离出破骨细胞和巨噬细胞；在有或没有骨的情况下培养细胞后制备细胞组分。通过蛋白质免疫印迹分析可视化钙调蛋白；通过Northern杂交确定钙调蛋白mRNA，并通过吖啶橙摄取测定ATP依赖性膜酸转运。在无骨培养的破骨细胞中钙调蛋白减少。分离的巨噬细胞中的钙调蛋白约为破骨细胞水平的25%，但在5天时增加了几倍。骨对此没有影响。有骨或无骨的破骨细胞中钙调蛋白mRNA相似。然而，只有与骨一起培养的破骨细胞保留了酸转运能力。巨噬细胞钙调蛋白mRNA不受骨的影响，但在第5天时增加了三倍，与蛋白质产生平行。巨噬细胞在3 - 5天时发展出酸转运能力，但水平低于破骨细胞，且骨没有可测量的影响。鸡细胞表达1.6 kb和可诱导的1.9 kb钙调蛋白转录本；在巨噬细胞和破骨细胞中，1.9 kb转录本占主导。我们得出结论，分离后，破骨细胞中的钙调蛋白水平通过转录后机制下降。相比之下，在培养的巨噬细胞中，钙调蛋白mRNA、蛋白质和酸分泌随时间增加，与骨基质无关，这可能反映了体外分化。两种细胞类型中钙调蛋白增加都与膜酸转运能力相关。巨噬细胞的研究结果表明，除骨之外的其他刺激影响该细胞家族的酸转运能力。
