Schwartz Z, Sylvia V L, Del Toro F, Hardin R R, Dean D D, Boyan B D
Department of Orthopaedics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA.
J Cell Physiol. 2000 Mar;182(3):390-401. doi: 10.1002/(SICI)1097-4652(200003)182:3<390::AID-JCP10>3.0.CO;2-T.
Recent studies have shown that 24R,25-(OH)(2)D(3) mediates its effects on growth plate chondrocytes via membrane receptors. This study examined the roles of phospholipase A(2) (PLA(2)) and cyclooxygenase (Cox) in the mechanism of action of 24R, 25-(OH)(2)D(3) in resting zone chondrocytes in order to determine whether the activity of one or both enzymes provides a regulatory checkpoint in the signaling pathway resulting in increased protein kinase C (PKC) activity. We also determined whether constitutive or inducible Cox is involved. Cultures were incubated with 24R, 25-(OH)(2)D(3) for 90 min to measure PKC or for 24 h to measure physiological responses ([(3)H]-thymidine incorporation, alkaline phosphatase-specific activity, [(35)S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, resting zone chondrocytes express mRNAs for both Cox-1 and Cox-2. Levels of mRNA for both proteins were unchanged from control levels after a 24-h incubation with 24R,25-(OH)(2)D(3). To examine the role of Cox, the cultures were also treated with resveratrol (a specific inhibitor of Cox-1), NS-398 (a specific inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). Cox-1 inhibition resulted in effects on proliferation, differentiation, and matrix production typical of 24R, 25-(OH)(2)D(3). In contrast, inhibition of Cox-2 had no effect, indicating that 24R,25-(OH)(2)D(3) exerts its effects via Cox-1. Inhibition of Cox-1 also blocked 24R,25-(OH)(2)D(3)-dependent increases in PKC. Activation of PLA(2) with melittin inhibited 24R, 25-(OH)(2)D(3)-dependent stimulation of PKC, and inhibition of PLA(2) with quinacrine stimulated PKC in response to 24R, 25-(OH)(2)D(3). Inclusion of resveratrol reduced the melittin-dependent inhibition of PLA(2) and caused an increase in quinacrine-stimulated PLA(2) activity. Metabolism of arachidonic acid to leukotrienes is not involved in the response to 24R, 25-(OH)(2)D(3) because inhibition of lipoxygenase had no effect. The effect of 24R,25-(OH)(2)D(3) was specific because 24S,25-(OH)(2)D(3), the biologically inactive stereoisomer, failed to elicit a response from the cells. These results support the hypothesis that 24R, 25-(OH)(2)D(3) exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA(2). PKC regulation may occur at multiple stages in the signal transduction cascade.
最近的研究表明，24R,25-(OH)₂D₃通过膜受体介导其对生长板软骨细胞的作用。本研究检测了磷脂酶A₂（PLA₂）和环氧化酶（Cox）在24R,25-(OH)₂D₃作用于静止区软骨细胞机制中的作用，以确定一种或两种酶的活性是否在导致蛋白激酶C（PKC）活性增加的信号通路中提供了一个调节检查点。我们还确定了组成型或诱导型Cox是否参与其中。将培养物与24R,25-(OH)₂D₃孵育90分钟以测量PKC，或孵育24小时以测量生理反应（[³H]-胸腺嘧啶掺入、碱性磷酸酶特异性活性、[³⁵S]-硫酸盐掺入）。基于逆转录聚合酶链反应（RT-PCR）和Northern印迹分析，静止区软骨细胞表达Cox-1和Cox-2的信使核糖核酸（mRNA）。与24R,25-(OH)₂D₃孵育24小时后，两种蛋白的mRNA水平与对照水平相比没有变化。为了检测Cox的作用，还对培养物用白藜芦醇（Cox-1的特异性抑制剂）、NS-398（Cox-2的特异性抑制剂）或吲哚美辛（一种通用的Cox抑制剂）进行处理。抑制Cox-1导致了24R,25-(OH)₂D₃典型的对增殖、分化和基质产生的影响。相反，抑制Cox-2没有效果，表明24R,25-(OH)₂D₃通过Cox-1发挥其作用。抑制Cox-1也阻断了24R,25-(OH)₂D₃依赖性的PKC增加。用蜂毒肽激活PLA₂抑制了24R,25-(OH)₂D₃依赖性的PKC刺激，而用喹吖因抑制PLA₂则刺激了PKC对24R,25-(OH)₂D₃的反应。加入白藜芦醇减少了蜂毒肽依赖性的PLA₂抑制，并导致喹吖因刺激的PLA₂活性增加。花生四烯酸代谢为白三烯不参与对24R,25-(OH)₂D₃的反应，因为抑制脂氧合酶没有效果。24R,25-(OH)₂D₃的作用是特异性的，因为生物学上无活性的立体异构体24S,25-(OH)₂D₃未能引起细胞反应。这些结果支持这样的假说，即24R,25-(OH)₂D₃通过多种信号通路发挥其作用且这些通路通过PLA₂的调节相互关联。PKC调节可能发生在信号转导级联反应的多个阶段。
