Drissi H, Hott M, Marie P J, Lasmoles F
INSERM U 349, Biologie cellulaire et moléculaire de l'os et du cartilage Hospital Lariboisière, Paris, France.
J Bone Miner Res. 1997 Nov;12(11):1805-14. doi: 10.1359/jbmr.1997.12.11.1805.
There is general agreement that calcitonin (CT) inhibits bone resorption by its effects on osteoclast function. CT was also found to have direct effects on osteoblast-like cells. In this study, we investigated the expression of CT and calcitonin gene-related peptide (CGRP), the two peptides encoded by the CT/CGRP gene, in human osteosarcoma cell lines and in normal human trabecular osteoblastic cells (HOB), and we studied the modulation of CT/CGRP gene expression by dibutyryl cyclic adenosine monophosphate ((Bu)2, cAMP), a cAMP analog. We first detected by Northern blot hybridization the presence of CT and CGRP mRNAs in different osteosarcoma cell lines (OHS-4, MG-63, Saos-2, HOS-TE85) and HOB cells. In the steady state, OHS-4 cells express slightly more CT and CGRP mRNAs than other cell lines or normal human osteoblasts, in parallel with messengers of differentiated osteoblasts, such as osteocalcin (OC) and alkaline phosphatase (ALP). OHS-4 cells also express CT and CGRP proteins, as demonstrated by immunocytochemistry. Stimulation of OHS-4 cells with 1 mM (Bu)2 cAMP induced a significant increase in mRNA levels for CT (x 2.5) and CGRP (x 3), as determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure. The involvement of a transcriptional mechanism in this effect was evidenced by nuclear run-off transcription assay. In addition, (Bu)2 cAMP increased OC (x 4) and ALP (x 3) mRNA levels in OHS-4 cells. These effects were observed at 24 h and were maximal at 48 h, indicating that (Bu)2, cAMP induced cell differentiation and increased the transcription of the CT/CGRP gene in OHS-4 osteoblast-like cells. The results indicate that human osteosarcoma cells and primary human osteoblastic cells express CT and CGRP mRNA and proteins, and that (Bu)2 cAMP, an activator of protein kinase A, induces up-regulation of osteoblastic phenotypic genes and enhances CT and CGRP gene transcription, indicating that induction of osteoblastic differentiation by (Bu)2 cAMP is associated with enhanced expression of CT and CGRP in human osteoblastic cells.
人们普遍认为，降钙素（CT）通过对破骨细胞功能的影响来抑制骨吸收。还发现CT对成骨样细胞有直接作用。在本研究中，我们调查了CT/CGRP基因编码的两种肽，即CT和降钙素基因相关肽（CGRP），在人骨肉瘤细胞系和正常人小梁成骨细胞（HOB）中的表达，并研究了环磷酸腺苷（cAMP）类似物二丁酰环磷腺苷酸（(Bu)2cAMP）对CT/CGRP基因表达的调节作用。我们首先通过Northern印迹杂交检测了不同骨肉瘤细胞系（OHS-4、MG-63、Saos-2、HOS-TE85）和HOB细胞中CT和CGRP mRNA的存在情况。在稳定状态下，OHS-4细胞比其他细胞系或正常人成骨细胞表达略多的CT和CGRP mRNA，与分化成骨细胞的信使分子，如骨钙素（OC）和碱性磷酸酶（ALP）平行。免疫细胞化学证明，OHS-4细胞也表达CT和CGRP蛋白。用1 mM (Bu)2cAMP刺激OHS-4细胞，通过半定量逆转录-聚合酶链反应（RT-PCR）程序测定，CT（x 2.5）和CGRP（x 3）的mRNA水平显著增加。核转录分析证明了转录机制参与了这种效应。此外，(Bu)2cAMP增加了OHS-4细胞中OC（x 4）和ALP（x 3）的mRNA水平。这些效应在24小时时观察到，48小时时达到最大值，表明(Bu)2cAMP诱导细胞分化并增加了OHS-4成骨样细胞中CT/CGRP基因的转录。结果表明，人骨肉瘤细胞和原代人成骨细胞表达CT和CGRP mRNA及蛋白，蛋白激酶A的激活剂(Bu)2cAMP诱导成骨细胞表型基因上调并增强CT和CGRP基因转录，表明(Bu)2cAMP诱导的成骨细胞分化与人成骨细胞中CT和CGRP表达增强有关。
