Murray E J, Grisanti M S, Bentley G V, Murray S S
Geriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Sepulveda, CA 91343, USA.
Metabolism. 1997 Sep;46(9):1090-4. doi: 10.1016/s0026-0495(97)90284-5.
Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.
甲状旁腺激素（PTH）可激活钙蛋白酶I和II（钙激活的木瓜蛋白酶样蛋白酶），并刺激成骨细胞中组织蛋白酶B（一种溶酶体半胱氨酸蛋白酶）的合成与分泌。合成代谢剂量的PTH还可刺激骨祖细胞增殖并分化为能够合成骨基质的成熟、功能完备的成骨细胞，而分解代谢剂量的PTH则刺激钙动员和基质更新。此前在其他细胞类型中的研究表明，钙激活的钙蛋白酶通过催化核蛋白、转录因子和酶的有限调节性蛋白水解，在调节增殖和分化中起主要作用。我们检验了这样一个假说，即抑制细胞内半胱氨酸蛋白酶（如钙蛋白酶）将消除PTH介导的成骨细胞增殖和分化，这是骨合成代谢的两个基本指标。在短期PTH处理前，用膜通透性、不可逆的半胱氨酸蛋白酶抑制剂E64d（10微克/毫升）进行短暂预孵育，可减弱PTH诱导的亚汇合培养物中的细胞增殖，并在长期汇合培养物中减弱增殖并抑制分化。这证实了这样一个假说，即半胱氨酸蛋白酶（如钙蛋白酶）在介导PTH对培养的MC3T3-E1细胞的增殖、促分化或合成代谢作用中很重要。免疫荧光定位显示，钙蛋白酶I、钙蛋白酶II和钙蛋白酶抑制蛋白（内源性钙蛋白酶抑制剂）在活跃增殖的MC3T3-E1前成骨细胞中大量存在且分布广泛。由于钙蛋白酶在成骨细胞的中性细胞内pH水平下具有活性且稳定，而组织蛋白酶则不然，我们的结果支持这些钙激活的调节蛋白酶在介导PTH在骨中的合成代谢作用方面发挥作用。
