DUSP5 and DUSP6 modulate corneal epithelial cell proliferation
DUSP5 and DUSP6 modulate corneal epithelial cell proliferation
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发表时间：
2010-08
期刊：
影响因子：
2.2
通讯作者：
Z. Wang;P. Reinach;Fan Zhang;Kati-Sisko Vellonen;A. Urtti;H. Turner;J. Wolosin
中科院分区：
文献类型：
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作者：
Z. Wang;P. Reinach;Fan Zhang;Kati-Sisko Vellonen;A. Urtti;H. Turner;J. Wolosin
目的双特异性磷酸酶(DUSPs)调节ERK1/2、p38和JNK1/2，丝裂原活化蛋白激酶(MAPK)的末端蛋白(TKS)的磷酸化持续时间和幅度。DUSP1、DUSP5和DUSP6三个DUSPs在眼表侧群干细胞(SPSCs)中高表达。我们的目的是确定这些酶对TK磷酸化和角膜上皮细胞增殖的影响。方法将SV40永生化和扩增的新鲜人角膜上皮细胞(EFHCECs)分别导入慢载体，诱导针对DUSP1、DUSP5和JNK1的shRNAmir表达，建立DUSP1i、DUSP5i和JNKi细胞亚系，或过表达DUSP6(以下简称DUSP6+)。免疫印迹法和3 H胸腺嘧啶核苷掺入法检测TK的磷酸化状态和细胞增殖率。结果在EF和svHCECs中，血清饥饿24 h后加入EGF后，3种类型TK的磷酸化水平均在5~15分钟迅速升高。然后在一小时内逐渐降低到低的磷酸化水平。这些下降与DUSP1和DUSP5蛋白表达的急剧增加相一致。在DUSP1i中，DUSP1的增加被取消。3种TKs均保持高磷酸化水平至少90min，细胞增殖率与未转导细胞无明显差异。在DUSP5i中，DUSP5蛋白的增加被阻止，峰后磷酸化水平仅在ERK1/2上发生下降，增殖率增加50%~60%。在JNK1i中，JNK1基本上被敲除，增殖率也显著升高。在稳定状态下，DUSP1i维持高水平的pJNK1/2表达。在DUSP6+ERK1/2中，磷酸化被阻止，增殖率降至50%以下。结论DUSP5和DUSP6选择性地调控ERK通路的活性和增殖。DUSP1基因敲除对细胞增殖的抑制作用的缺乏可以归因于其泛MAPK效应。由于pJNK1/2的抗增殖作用同时被释放，因此在DUSP1基因敲除后，由于ERK1/2的增强和持续的磷酸化，预期的增强的增殖反应不会发生。
Purpose Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation of Erk1/2, p38 and JNK1/2, the terminal kinases (TKs) of the mitogen activated protein kinase (MAPK) cascades. Three DUSPs, DUSP1, DUSP5, and DUSP6, are overexpressed in ocular surface side population stem cells (SPSCs). Our objective was to identify the impact of these enzymes on TK phosphorylation and proliferation of corneal epithelial cells. Methods SV40 immortalized (sv) and expanded fresh human corneal epithelial cells (efHCECs) were transduced with lentivectors to elicit expression of shRNAmir against DUSP1, DUSP5, and JNK1 to thereby create the DUSP1i, DUSP5i and JNKi cell sublines, or overexpress DUSP6 (henceforth DUSP6+), respectively. TK phosphorylation status and proliferation rates were determined by immunoblotting and 3H thymidine uptake. Results In both ef and svHCECs, EGF supplementation after a 24 h serum starvation caused a rapid 5–15 min spike in the phosphorylation of all three TK types. This was followed by gradual decreases to low phosphorylation levels within one h. These declines coincided with dramatic increases in DUSP1 and DUSP5 protein expression. In DUSP1i, the DUSP1 increase was abolished. All 3 TKs maintained high phosphorylation levels for at least 90 min and proliferation rates were unchanged from non-transduced cells. In DUSP5i, the DUSP5 protein increase was prevented, the post peak phosphorylation decrease occurred only on Erk1/2 and the proliferation rate increased by 50%–60%. In JNK1i, JNK1 was essentially knocked out and proliferation rates were also markedly elevated. At steady-state, DUSP1i maintained high levels of pJNK1/2 expression. In DUSP6+ Erk1/2 phosphorylation was prevented and proliferation rates decreased to less than 50%. Conclusions DUSP5 and DUSP6 selectively control ERK pathway activity and proliferation. The lack of an effect of DUSP1 knockdown on proliferation can be attributed to its pan-MAPK effect. The expected augmented proliferative response due to enhanced and prolonged phosphorylation of Erk1/2 following DUSP1 knockdown does not occur because a pJNK1/2 antiproliferative effect is simultaneously unleashed.