Schwartz Z, Graham E J, Wang L, Lossdörfer S, Gay I, Johnson-Pais T L, Carnes D L, Sylvia V L, Boyan B D
Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
J Cell Physiol. 2005 Apr;203(1):54-70. doi: 10.1002/jcp.20212.
Phospholipase A2 (PLA2) is pivotal in the rapid membrane-mediated actions of 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. Microarray analysis indicated that PLA2 activating protein (PLAA) mRNA is upregulated 6-fold before rat growth plate cells exhibit 1alpha,25(OH)2D3-dependent protein kinase C (PKC) increases, suggesting that it plays an important role in 1alpha,25(OH)2D3's mechanism of action. PLAA mRNA was confirmed in 1alpha,25(OH)2D3-responsive growth zone (prehypertrophic and upper hypertrophic cell zones) chondrocytes by RT-PCR and Northern blot in vitro and by in situ hybridization in vivo. PLAA protein was shown by Western blot and immunohistochemistry. PLAAs role in 1alpha,25(OH)2D3 signaling was evaluated in growth zone cell cultures using PLAA peptide. Arachidonic acid release was increased as was PLA2-specific activity in plasma membranes and matrix vesicles. PKCalpha, but not PKCbeta, PKCepsilon, or PKCzeta, was increased. PLAAs effect was comparable to that of 1alpha,25(OH)2D3 and was additive with 1alpha,25(OH)2D3. PLA2 inhibitors quinacrine and AACOCF3, and cyclooxygenase inhibitor indomethacin blocked the effect of PLAA peptide on PKC, indicating arachidonic acid and its metabolites were involved. This was confirmed using exogenous arachidonic acid. Prostaglandin acted via EP1 based on inhibition by SC19220 and not via EP2 since AH6809 had no effect. Like 1alpha,25(OH)2D3, PLAA peptide also increased activity of phospholipase C-specific activity via beta-1 and beta-3 isoforms, but not delta-1 or gamma-1; the effect of PLAA was via lysophospholipid but not via arachidonic acid. PLAA peptide decreased [3H]-thymidine incorporation to 50% of the decrease caused by 1alpha,25(OH)2D3. In contrast, PLAA peptide increased alkaline phosphatase-specific activity and proteoglycan production in a manner similar to 1alpha,25(OH)2D3. This indicates that PLAA is a specific activator of PLA2 in growth plate chondrocytes, and suggests that it mediates the membrane effect of 1alpha,25(OH)2D3, thereby modulating physiological response.
磷脂酶A2（PLA2）在1,25 - 二羟基维生素D3[1α,25(OH)2D3]快速的膜介导作用中起关键作用。微阵列分析表明，在大鼠生长板细胞出现1α,25(OH)2D3依赖性蛋白激酶C（PKC）增加之前，磷脂酶A2激活蛋白（PLAA）的mRNA上调了6倍，这表明它在1α,25(OH)2D3的作用机制中发挥重要作用。通过体外逆转录聚合酶链反应（RT-PCR）和Northern印迹以及体内原位杂交，在1α,25(OH)2D3反应性生长区（前肥大细胞区和上肥大细胞区）软骨细胞中证实了PLAA mRNA的存在。通过蛋白质免疫印迹和免疫组织化学显示了PLAA蛋白。使用PLAA肽在生长区细胞培养物中评估了PLAA在1α,25(OH)2D3信号传导中的作用。花生四烯酸释放增加，同时质膜和基质小泡中的PLA2特异性活性也增加。PKCα增加，而PKCβ、PKCε或PKCζ没有增加。PLAA的作用与1α,25(OH)2D3相当，并且与1α,25(OH)2D3具有相加作用。PLA2抑制剂奎纳克林和AACOCF3以及环氧化酶抑制剂吲哚美辛阻断了PLAA肽对PKC的作用，表明花生四烯酸及其代谢产物参与其中。使用外源性花生四烯酸证实了这一点。基于SC19220的抑制作用，前列腺素通过EP1起作用，而AH6809没有作用，因此不是通过EP2起作用。与1α,25(OH)2D3一样，PLAA肽也通过β-1和β-3亚型增加磷脂酶C的特异性活性，但不通过δ-1或γ-1；PLAA的作用是通过溶血磷脂而不是花生四烯酸实现的。PLAA肽使[3H] - 胸腺嘧啶掺入量降低至1α,25(OH)2D3引起降低量的50%。相反，PLAA肽以类似于1α,25(OH)2D3的方式增加碱性磷酸酶特异性活性和蛋白聚糖的产生。这表明PLAA是生长板软骨细胞中PLA2的特异性激活剂，并表明它介导了1α,25(OH)2D3的膜效应，从而调节生理反应。
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