Teti A, Colucci S, Grano M, Argentino L, Zambonin Zallone A
Institute of Human Anatomy, School of Pharmacy, University of Bari, Italy.
Am J Physiol. 1992 Jul;263(1 Pt 1):C130-9. doi: 10.1152/ajpcell.1992.263.1.C130.
The effects of protein kinase C (PKC) in the control of osteoclast activity are still unknown. We investigated the role of the enzyme in the control of microfilament organization, podosome assembly, bone resorption, and extracellular Ca2+ sensing in chicken and rabbit osteoclasts treated with agents known to affect PKC activity. Cells were treated for 20 min with a PKC activator [phorbol 12-myristate 13-acetate (PMA)], a PKC inhibitor (staurosporine), a protein kinase A (PKA) inhibitor (H-9), a guanosine 3',5'-cyclic monophosphate-dependent protein kinase-PKA-PKC inhibitor (H-7), or with the inactive phorbol, 4 alpha-phorbol, to examine microfilaments by decoration with rhodamine-phalloidin. In PMA-treated osteoclasts, the number of microfilament-containing adhesion structures (podosomes) per cell decreased. However, enlarged microfilamentous cores in podosomes and stress fiber-like filaments, otherwise absent in controls, appeared. Whereas H-7 induced increase of the number of podosomes, staurosporine, H-9, and 4 alpha-phorbol failed to change microfilament organization. Chicken osteoclasts received also long-term treatment with the agents in the presence of [3H]proline-prelabeled chicken or rat bone particles to measure bone resorption. PMA, as well as staurosporine and H-7, stimulated the resorbing activity, whereas cells were insensitive to H-9 and 4 alpha-phorbol. Measurement of cytosolic free calcium concentration in PMA-treated fura-2-loaded single osteoclasts demonstrated a synergistic effect of PKC activation on the inhibitory extracellular calcium concentration-sensing mechanism, which was, by contrast, blocked by H-7, staurosporine, and H-9 and was insensitive to 4 alpha-phorbol. These results indicate that PKC regulates osteoclast activity inducing both morphological and functional modifications.
蛋白激酶C（PKC）在破骨细胞活性控制中的作用仍不清楚。我们在用已知影响PKC活性的试剂处理的鸡和兔破骨细胞中，研究了该酶在微丝组织、足体组装、骨吸收和细胞外Ca2+感知控制中的作用。用PKC激活剂[佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）]、PKC抑制剂（星形孢菌素）、蛋白激酶A（PKA）抑制剂（H - 9）、环磷酸鸟苷依赖性蛋白激酶 - PKA - PKC抑制剂（H - 7）或无活性的佛波醇4α - 佛波醇处理细胞20分钟，通过用罗丹明 - 鬼笔环肽染色来检测微丝。在PMA处理的破骨细胞中，每个细胞中含微丝的粘附结构（足体）数量减少。然而，足体中扩大的微丝核心和对照中不存在的应力纤维样细丝出现了。而H - 7诱导足体数量增加，星形孢菌素、H - 9和4α - 佛波醇未能改变微丝组织。在存在[3H]脯氨酸预标记的鸡或大鼠骨颗粒的情况下，鸡破骨细胞也接受了这些试剂的长期处理以测量骨吸收。PMA以及星形孢菌素和H - 7刺激了吸收活性，而细胞对H - 9和4α - 佛波醇不敏感。对用PMA处理的、加载fura - 2的单个破骨细胞的胞质游离钙浓度测量表明，PKC激活对抑制性细胞外钙浓度感知机制有协同作用，相比之下，该作用被H - 7、星形孢菌素和H - 9阻断，且对4α - 佛波醇不敏感。这些结果表明PKC通过诱导形态和功能修饰来调节破骨细胞活性。
