Gap-junctional communication is required for the maturation process of osteoblastic cells in culture
Gap-junctional communication is required for the maturation process of osteoblastic cells in culture
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DOI：
10.1016/s8756-3282(00)00458-0
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发表时间：
2001-04-01
期刊：
影响因子：
4.1
通讯作者：
Howard, GA
中科院分区：
文献类型：
--
作者：
Schiller, PC;D'ippolito, G;Howard, GA
成骨细胞在长期培养中经历表型成熟过程，导致细胞外基质（ECM）的产生和骨结节（BN）的形成。通过间隙连接（GJC）的细胞与细胞之间的通信可以检测到成骨细胞在24小时内的平板。我们在成骨细胞的长期培养中评估了抑制GJC对表型的影响：成熟过程和与该过程相关的特异性基因的表达，将MC 3 T3-E1细胞接种，24小时（第0天）后，将细胞暴露于18-α-大黄次酸（阿加），一种GJC、GJC、碱性磷酸酶（AP）活性、BN形成，骨钙素（OC）、骨涎蛋白（bSP）、骨桥蛋白（OP）转录本的相对水平。在第0天评价胶原α 1 I型（α 1 ICol）和延伸因子-1a（EF 1a），此后每4-7天评价一次，直到第30天，通过荧光染料转移评价GJC。通过北方印迹和半定量逆转录聚合酶链反应分析基因表达。GJC在第0天可检测到，并随培养时间增加，阿加（100 μ mol/L）强烈抑制GJC在所有测试的时间点。此外，AGA暴露告诉显示出剂量依赖性的AP活性降低和BN的出现延迟。这种延迟的表型表达与对成骨细胞特异性基因OC和bSP表达的抑制作用相一致，α 1 ICol mRNA的表达也受到影响，但程度较小，而OP和EF 1a不受影响。用油酰胺（一种额外的GJC可逆抑制剂）获得了类似的结果。相反，暴露于载体或100 μ mol/L大黄酸（18-β-大黄次酸的非抑制性糖苷）的细胞与未处理的细胞在所有评价的参数上都无法区分。我们的结论是，GJC抑制干扰培养中成骨细胞的成熟过程，可能是通过影响调节成骨细胞表型成熟/分化中所涉及的基因表达的信号，（Bone 28：362-369; 2001）（C）2001，Elsevier Science Inc. All rights reserved.
Osteoblastic cells in long-term culture undergo a phenotypic maturation process leading to extracellular matrix (ECM) production and hone nodule (BN) formation. Cell-to-cell communication via gap junctions (GJC) can be detected between osteoblastic cells within 24 h of plating. We evaluated, in long-term cultures of osteoblastic cells, the effect of inhibiting GJC on the phenotypic: maturation process and the expression of specific genes associated with this process, MC3T3-E1 cells were plated, and, after 24 h (day 0) cells were exposed to 18-alpha -glycyrrhetinic acid (AGA), a nontoxic reversible inhibitor of GJC, GJC, alkaline phosphatase (AP) activity, BN formation, and the relative level of transcripts encoding osteocalcin (OC), bone sialoprotein (bSP), osteopontin (OP). collagen alpha1 type I(alpha 1ICol), and elongation factor-1a (EF1a) were evaluated on day 0 and every 4-7 days thereafter through day 30, GJC was assessed by fluorescent dye transfer. Gene expression was analyzed by northern blot and semiquantitative reverse transcription-polymerase chain reaction. GJC was detectable at day 0 and increased with time in culture, AGA (100 mu mol/L) strongly inhibited GJC at all timepoints tested. Moreover, AGA-exposed tells showed a dose-dependent decrease in AP activity and a delay in the appearance of BN. This delayed phenotypic expression coincided with an inhibitory effect on the expression of the osteoblast-specific genes OC and bSP, Expression of alpha 1ICol mRNA was also affected, but to a lesser extent, whereas OP and EF1a were not affected. Similar results were obtained with oleamide, an additional reversible inhibitor of GJC, In contrast, cells exposed to either vehicle or 100 mu mol/L glycyrrhizic acid (a noninhibitory glycoside of 18-beta -glycyrrhetinic acid) were indistinguishable from untreated cells for ail parameters evaluated. We conclude that GJC inhibition interferes with the maturation process of osteoblastic cells in culture, possibly by affecting signals regulating the expression of genes involved in the maturation/differentiation of the osteoblastic phenotype, (Bone 28:362-369; 2001) (C) 2001 by Elsevier Science Inc. All rights reserved.