Lampasso J D, Marzec N, Margarone J, Dziak R
Department of Oral Biology, University at Buffalo, New York 14214, USA.
J Bone Miner Res. 2002 Nov;17(11):1968-76. doi: 10.1359/jbmr.2002.17.11.1968.
Protein kinase C (PKC) isoforms have been shown to have specific expression profiles and individual isoforms are believed to play distinct roles in the cells in which they are found. The goal here was to determine which specific isoform(s) is involved in proliferation of primary human osteoblasts. In primary human osteoblasts, 10 microM of acute sphingosine-1-phosphate (S1P) treatment induced an increase in proliferation that correlated with an increase in PKCalpha and PKCiota expression. To further delineate which isoforms are involved in osteoblastic cell proliferation, the effect of low versus high serum culture conditions on PKC isoform expression was determined. Likewise, the effect of antisense oligodeoxynucleotides (ODNs) to specific PKC isoforms on proliferation and MAPK activation was studied. The effect of S1P on intracellular translocation of activated PKC isoforms was also evaluated. The results indicated that in primary human osteoblasts, PKCalpha was not expressed under conditions of low proliferative rate while PKCdelta and PKCiota expression was not affected. The specific inhibition of PKCalpha by antisense ODNs resulted in inhibition of MAPK activity leading to a significant decrease in proliferation. S1P up-regulated antisense ODN inhibited PKCalpha expression and MAPK activity and led to an increase in proliferation. Subsequent experiments using platelet-derived growth factor (PDGF) as an additional mitogen generated similar data. PDGF stimulation resulted in a significant increase in proliferation that correlated with an up-regulation of inhibited PKCalpha expression in antisense ODN-treated cells. Immunofluorescence methods showed that mitogenic stimulation of PKCa resulted in nuclear translocation. Our findings present original data that PKCalpha is the isoform specifically involved in the proliferation of primary human osteoblasts.
蛋白激酶C（PKC）亚型已显示出具有特定的表达谱，并且据信各个亚型在其所在的细胞中发挥不同的作用。此处的目标是确定哪种特定亚型参与原代人成骨细胞的增殖。在原代人成骨细胞中，10微摩尔的急性鞘氨醇-1-磷酸（S1P）处理诱导增殖增加，这与PKCα和PKCι表达的增加相关。为了进一步阐明哪些亚型参与成骨细胞增殖，确定了低血清与高血清培养条件对PKC亚型表达的影响。同样，研究了针对特定PKC亚型的反义寡脱氧核苷酸（ODN）对增殖和丝裂原活化蛋白激酶（MAPK）激活的影响。还评估了S1P对活化的PKC亚型细胞内转位的影响。结果表明，在原代人成骨细胞中，在低增殖率条件下不表达PKCα，而PKCδ和PKCι的表达不受影响。反义ODN对PKCα的特异性抑制导致MAPK活性受到抑制，从而导致增殖显著降低。S1P上调反义ODN抑制PKCα表达和MAPK活性，并导致增殖增加。随后使用血小板衍生生长因子（PDGF）作为额外的促有丝分裂原进行的实验产生了类似的数据。PDGF刺激导致增殖显著增加，这与反义ODN处理的细胞中受抑制的PKCα表达上调相关。免疫荧光方法显示，PKCα的促有丝分裂刺激导致核转位。我们的研究结果提供了原始数据，表明PKCα是特异性参与原代人成骨细胞增殖的亚型。
