Glycogen synthase kinase-3 regulates formation of long lamellipodia in human keratinocytes
Glycogen synthase kinase-3 regulates formation of long lamellipodia in human keratinocytes
复制标题
DOI：
10.1242/jcs.00693
复制
发表时间：
2003-09-15
影响因子：
4
通讯作者：
Larjava, H
中科院分区：
文献类型：
--
作者：
Koivisto, L;Alavian, K;Larjava, H
在伤口愈合过程中，角质形成细胞通过延长板脂进入富含纤维粘连蛋白的临时基质而从伤口边缘开始迁移。虽然在表皮生长因子(EGF)作用下培养的角质形成细胞中也发现了片状脂膜样结构，但调节这些结构形成的信号通路尚不明确。在种植于纤维连接蛋白上的培养的人角质形成细胞中，我们发现包括星形孢子素在内的蛋白-丝氨酸/苏氨酸激酶抑制剂可以诱导浓度依赖的扩展片状脂膜(E-LAMS)的形成。蛋白酪氨酸激酶抑制剂赫比霉素A和金雀异黄素可抑制E-LAMS的形成，蛋白酪氨酸磷酸酶抑制剂原钒酸钠可促进E-LAMS的形成。星形孢子素治疗诱导酪氨酸磷酸化磷脂酶C-伽马1(PLC-Gamma1)重新定位到肌动蛋白组装开始的板脂末端。与PLC-Gamma1参与E-LAM形成相一致的是，在E-LAMS形成过程中，细胞内游离钙(Ca~(2+))升高，反之，E-LAM的形成可被BAPTA/AM的细胞内Ca~(2+)络合所阻断，但不能被EGTA降低细胞外Ca~(2+)所阻断。值得注意的是，糖原合成酶-3α/β(GSK-3α/β)被星形孢子素激活，Ser-21/9上的磷酸化程度降低。LiCl2或特定的化学抑制剂SB-415286抑制GSK-3的活性，阻止E-LAM的形成，但不改变细胞的扩散。此外，GSK-3抑制剂可阻断星状孢子素和表皮生长因子诱导的角质形成细胞在划痕伤培养中的迁移。我们认为GSK-3在人角质形成细胞的长板脂形成过程中起关键作用，并且可能是伤口愈合过程中上皮细胞迁移的中心调节分子。
During wound healing, keratinocytes initiate migration from the wound edge by extending lamellipodia into a fibronectin-rich provisional matrix. While lamellipodia-like structures are also found in cultured keratinocytes exposed to epidermal growth factor (EGF), the signaling pathway that regulates the formation of these structures is not defined. In cultured human keratinocytes seeded on fibronectin, we found that protein-serine/threonine kinase inhibitors including staurosporine, induced concentration-dependent formation of extended lamellipodia (E-lams). The formation of E-lams was inhibited by the protein-tyrosine kinase inhibitors herbimycin A and genistein and augmented by the protein-tyrosine phosphatase inhibitor sodium orthovanadate. Staurosporine treatment induced relocation of tyrosine phosphorylated phospholipase C-gamma1 (PLC-gamma1) to the tips of lamellipodia where actin assembly was initiated. Consistent with an involvement of PLC-gamma1 in E-lam formation, intracellular free calcium (Ca2+) was elevated during the formation of E-lams and conversely, E-lam formation was blocked by intracellular Ca2+ chelation with BAPTA/AM, but not by extracellular reduction of Ca2+ by EGTA. Notably, glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) was activated by staurosporine as evidenced by reduced phosphorylation on Ser-21/9. Suppression of GSK-3 activity by LiCl2 or by a specific chemical inhibitor, SB-415286, blocked E-lam formation but without altering cell spreading. Furthermore, GSK-3 inhibitors blocked both staurosporine- and EGF-induced keratinocyte migration in scratch-wounded cultures. We propose that GSK-3 plays a crucial role in the formation of long lamellipodia in human keratinocytes and is potentially a central regulatory molecule in epithelial cell migration during wound healing.