Glycogen synthase kinase-3 regulates formation of long lamellipodia in human keratinocytes

Abstract

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 proteintyrosine 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-γ1 (PLC-γ1) to the tips of lamellipodia where actin assembly was initiated. Consistent with an involvement of PLC-γ1 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-3α/β (GSK-3α/β) 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.

Expression of junctional adhesion molecule-A prevents spontaneous and random motility

“…3B) and number of tracks (Fig. 3C) Inactivation of GSK-3β mimics JAM-A-dependent inhibition of motility Similarly to the expression of JAM-A in our system, inactivation of GSK-3β was shown to reduce long protrusions in keratinocytes (Koivisto et al, 2003). Thus, we surmised that JAM-A expression might inhibit protrusions (and possibly motility) by inactivating GSK-3β.…”

“…First, GSK-3β inhibitors prevent the formation of long lamellipodia in keratinocytes (Koivisto et al, 2003) and filopodia in neurons (Owen and Gordon-Weeks, 1999). In addition, inactive GSK-3β localizes to the leading edge of growth cones (Eickholt et al, 2002).…”

The Pivotal Roles of GSK3β in Glioma Biology

“…Arf6 is also involved in vesicle trafficking, membrane ruffling, as well as lamellipodia formation (Turner & Brown, 2001). It has been shown that GSK3 activity is required for keratinocytes to form lamellipodia and migrate directionally in response to wound signaling (Koivisto et al, 2003). Similarly, GSK3 activates Rac in response to wound signaling in intestinal epithelial cells.…”

Glycogen Synthase Kinase 3 and h-prune Regulate Cell Migration by Modulating Focal Adhesions

“…GSK-3 has also been shown to be involved in signaling activated by cell adhesion in nonneuronal cells (23,37). The formation of extending lamellipodia in migrating keratinocytes is blocked by GSK-3 inhibitors (29). The initiation and stimulation of sperm motility are accompanied by the inactivation of GSK-3 (41).…”