The Role of PLC and PKC in Collective Migration of Keratinocytes

Introduction::
In the US nearly 7 million people are affected by chronic wounds, and this number is expected to increase in the next decade [1]. The first step in wound healing is re-epithelization, where the keratinocytes respond to the injury by migrating as a collective group to reform the outer barrier. Re-epithelization is regulated by a variety of growth factors, including epidermal growth factor (EGF), which is found in the wound in both a soluble and immobilized form. Our prior work suggests that the intracellular signal PLC is activated in cells exposed to immobilized EGF, and this activation leads to increased persistence in collective migration in HaCaTs, an immortalized human keratinocyte cell line [2]. However, the mechanism linking PLC activity to persistence in collective migration is unknown.




Materials and Methods::
To determine the mechanism responsible for activation of PLC and its effectors in collective migration in response to delivery of immobilized EGF, we first covalently immobilized EGF to the 24-well tissue culture polystyrene plates (20 ng/well) via heterobifunctional cross-linker Sulfo-SANPAH. Briefly, 2.5mM EGF in HEPES buffered saline solution was reacted with Sulfo-SANPAH in a 1:50 M ratio for 5 hr, and the resulting solution was pipetted into each well (250 μL/well) and dried at 40 °C for 6 hr. After, Sulfo-SANPAH-EGF mixture was immobilized by UV light (365 nm) for 4 mins, and unreacted EGF was removed by four rinses with diH2O on a shaker. Control plates were treated with HEPES buffer without Sulfo-SANPAH-EGF mixture. Next, RFP-labeled HaCaTs were cultured to confluence inside a removable, silicone fence (100,000 cells/ml). Following 12 hr cell attachment, the fence was lifted, and cells were washed with 1x PBS and 0.5% serum media containing treatments of interest (5mM U73122 (PKC inhibitor), 2mM Go6976 (PKC inhibitor), 5mM m-3M3FBS (PLC activator), and 10 mM OAG (PKC activator)). In a subset of experiments, to identify the isoforms of PLC and PKC responsible for cell migration outcomes, knockdown by siRNA was used prior to initiating the wound assay. Finally, using time-lapse microscopy, cell migration was evaluated by quantifying individual cell speed and persistence, as well as distance the leading edge had migrated over 8 h.




Results, Conclusions, and Discussions::
HaCaTs on immobilized EGF exhibited increased migration persistence compared to control plates without EGF. This increase in persistence was reversed by PLC inhibition with U73122. Additionally, activation of PLC by m-3M3FBS in the absence of immobilized EGF led to increased persistence. Together, these two findings suggest that PLC is necessary and sufficient for increased collective migration. We next sought to identify the PLC isoform(s) responsible for these outcomes. siRNA knockdown of PLCγ1 revealed that this is the responsible isoform since we saw a decrease in collective migration as well as persistence and speed in knockdown compared to scrambled condition. PKC is a downstream effector of PLC, and both have been shown to be involved in keratinocyte migration, therefore we examined if improved collective migration occurred through PKC activation downstream of PLC. Inhibition of PKC with Go6976 resulted in decreased persistence and overall migration. To confirm that PKC pathway is the one enacting the PLC-based response, we inhibited PLC with U73122 while activating PKC with OAG and confirmed that PKC activation was able to recover migration in the absence of PLC activation (Figure 1). As there are multiple PKC isoforms, we used siRNA to confirm that there is a PKC-β specific effect.




Our future work will identify the role of PLCγ1 in actin cytoskeletal remodeling that leads to improved collective migration. Understanding the mechanism by which PLC activation and its effectors lead to increased persistence will help develop therapies capable of modulating cell migration and enhancing treatment of chronic wounds.




Acknowledgements (Optional): :
This study was supported by the National Institute for Health research grant R01GM143795-01A1.




References (Optional): : [1]Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt TK, et al. Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Regen. 2009

[2]Kim CS, Mitchell IP, Desotell AW, Kreeger PK, Masters KS. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1. FASEB J. 2016 Jul;30(7):2580-90. doi: 10.1096/fj.201600252. Epub 2016 Mar 29. PMID: 27025961; PMCID: PMC4904288.