Jes K. Klarlund

Epithelial cell motility is triggered by activation of the EGF receptor through phosphatidic acid signaling.

Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid, and there is currently much interest in elucidating messenger functions for this molecule. We report here that wounding sheets of corneal epithelial and Madin Darby canine kidney cells induces strong activation of phospholipase D, and we provide evidence that activation is amplified through a positive feed-back loop. Short-chain analogues of phosphatidic acid induce motility robustly in corneal and other epithelial cell types. The effects of these analogues were not the result of their conversion to the corresponding diacylglycerol or lysophosphatidic acid, implying that phosphatidic acid acts directly on one or more cellular targets. Strikingly, phosphatidic acid signaling was found to stimulate the epidermal growth factor receptor (EGFR) through a transactivation process. Healing of wounds in sheets of corneal epithelial cells is absolutely dependent on epidermal growth factor receptor signaling, and the present data suggest that its activation is a result of wound-induced phospholipase D activation.

Wounding sheets of epithelial cells activates the epidermal growth factor receptor through distinct short- and long-range mechanisms.

Wounding epithelia induces activation of the epidermal growth factor receptor (EGFR), which is absolutely required for induction of motility. ATP is released from cells after wounding; it binds to purinergic receptors on the cell surface, and the EGFR is subsequently activated. Exogenous ATP activates phospholipase D, and we show here that ATP activates the EGFR through the phospholipase D2 isoform. The EGFR is activated in cells far (>0.3 cm) from wounds, which is mediated by diffusion of extracellular ATP because activation at a distance from wounds is abrogated by eliminating ATP in the medium with apyrase. In sharp contrast, activation of the EGFR near wounds is not sensitive to apyrase. Time-lapse microscopy revealed that cells exhibit increased motilities near edges of wounds; this increase in motility is not sensitive to apyrase, and apyrase does not detectably inhibit healing of wounds in epithelial sheets. This novel ATP/PLD2-independent pathway activates the EGFR by a transactivation process through ligand release, and it involves signaling by a member of the Src family of kinases. We conclude that wounding activates two distinct signaling pathways that induce EGFR activation and promote healing of wounds in epithelial cells. One pathway signals at a distance from wounds through release of ATP, and another pathway acts locally and is independent on ATP signaling.

Dual modes of motility at the leading edge of migrating epithelial cell sheets

Purse-string healing is driven by contraction of actin/myosin cables that span cells at wound edges, and it is the predominant mode of closing small round wounds in embryonic and some adult epithelia. Wounds can also heal by cell crawling, and my colleagues and I have shown previously that the presence of unconstrained, straight edges in sheets of epithelial cells is a sufficient signal to induce healing by crawling. Here, it is reported that the presence of highly concave edges, which are free or physically constrained by an inert material (agarose), is sufficient to induce formation of purse strings. It was determined that neither of the two types of healing required cell damage or other potential stimuli by using the particularly gentle procedure of introducing gaps by digesting agarose blocks imbedded in the cell sheets. Movement by crawling depends on signaling by the EGF receptor (EGFR); however, this was not required for purse-string contraction. A migrating epithelial cell sheet usually produces finger-like projections of crawling cells. The cells between fingers contain continuous actin cables, which were also determined to contain myosin IIA and exhibit additional characteristics of purse strings. When crawling was blocked by inhibition of EGFR signaling, the concave regions continued to move, suggesting that both mechanisms contribute to propel the sheets forward. Wounding epithelial cell sheets causes activation of the EGFR, which triggers movement by crawling. The EGFR was found to be activated only at straight and convex edges, which explains how both types of movement can coexist at leading epithelial edges.

Brief treatment with heparin-binding EGF-like growth factor, but not with EGF, is sufficient to accelerate epithelial wound healing

BACKGROUND Heparin-binding EGF-like growth factor (HB-EGF) contains, in contrast to EGF, a domain that binds to negatively charged glycans on cell surfaces and in extracellular matrix. We speculated that a short exposure to HB-EGF induces prolonged biological effects such as healing of wounds after immobilization in tissues. METHODS Epithelial cell sheets in tissue and corneas in organ culture were treated briefly with HB-EGF or EGF and binding of the growth factors, time course of activation of the EGF receptor, and healing of wounds were compared. RESULTS Treating human corneal epithelial cells for 2 min with HB-EGF resulted in 8h of detectable activation of the EGF receptor, but activation was much shorter after EGF treatment. A brief treatment with HB-EGF, but not with EGF, induced significant acceleration of healing in wounds in epithelial sheets in tissue and organ culture. Bound HB-EGF was detectable up to 16 h after brief treatments. Neutralizing antibodies added after HB-EGF treatment blocked acceleration of healing, demonstrating the role of bound HB-EGF in accelerating healing. CONCLUSIONS A brief exposure to HB-EGF, but not to EGF, is sufficient to induce prolonged activation of the EGF receptor and to enhance healing. GENERAL SIGNIFICANCE Bound HB-EGF can serve as a pool that induces prolonged activation of the EGF receptor. EGF has been used experimentally to treat poorly healing wounds, but the frequent applications that are necessary have hampered its use clinically. The findings imply that HB-EGF may be a useful long-acting alternative to EGF.

Free edges in epithelial cell sheets stimulate epidermal growth factor receptor signaling.

Epithelia tend to migrate when edges are present, for instance, after wounding or during development. Using a new tissue culture model, we found that the existence of free edges is in itself a signal that causes activation of the epidermal growth factor and cell motility.

Putting on the brakes: Bacterial impediment of wound healing.

The epithelium provides a crucial barrier to infection, and its integrity requires efficient wound healing. Bacterial cells and secretomes from a subset of tested species of bacteria inhibited human and porcine corneal epithelial cell migration in vitro and ex vivo. Secretomes from 95% of Serratia marcescens, 71% of Pseudomonas aeruginosa, 29% of Staphylococcus aureus strains, and other bacterial species inhibited epithelial cell migration. Migration of human foreskin fibroblasts was also inhibited by S. marcescens secretomes indicating that the effect is not cornea specific. Transposon mutagenesis implicated lipopolysaccharide (LPS) core biosynthetic genes as being required to inhibit corneal epithelial cell migration. LPS depletion of S. marcescens secretomes with polymyxin B agarose rendered secretomes unable to inhibit epithelial cell migration. Purified LPS from S. marcescens, but not from Escherichia coli or S. marcescens strains with mutations in the waaG and waaC genes, inhibited epithelial cell migration in vitro and wound healing ex vivo. Together these data suggest that S. marcescens LPS is sufficient for inhibition of epithelial wound healing. This study presents a novel host-pathogen interaction with implications for infections where bacteria impact wound healing and provides evidence that secreted LPS is a key factor in the inhibitory mechanism.

Activation of the epidermal growth factor receptor by hydrogels in artificial tears.

Most formulations of artificial tears include high-molecular weight hydrophilic polymers (hydrogels) that are usually thought to serve to enhance viscosity and to act as demulcents. A few reports have indicated that application of some of the polymers accelerates healing of wounds in epithelia. Since activation of the epidermal growth factor (EGF) receptor is critical for spontaneous corneal epithelial wound healing, we tested commonly used hydrogels for their ability to activate the EGF receptor and enhance closure of wounds. Five structurally unrelated hydrogels used in artificial tears were found to activate the EGF receptor. Importantly, two of the hydrogels enhanced wound healing in an organ culture model. We propose that the efficacy of hydrogels in treating dry eye may be related to their ability to activate the EGF receptor, and that hydrogels are inexpensive, safe agents to promote healing of wounds in the cornea and possibly in other tissues.

Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor

Wounding usually causes considerable cell damage, and released ATP promotes migration of nearby epithelium. ATP binds to purinergic receptors on the cell surface and induces transactivation of the EGF receptor through signaling by the Src family kinases (SFKs). Here we tested whether ATP activates these kinases through Pyk2, a member of the focal adhesion kinase family. Pyk2 was rapidly and potently activated by treating corneal epithelial cells with ATP, and physical interaction of Pyk2 with the SFKs was enhanced. Disruption of Pyk2 signaling either by siRNA or by expression of a dominant-negative mutant led to inhibition of ATP-induced activation of the SFKs and the EGF receptor. Inhibiting Pyk2 activity also blocked ATP stimulation of healing of wounds in epithelial cell sheets. These data suggest that ATP stimulates sequential activation of Pyk2, SFKs, and the EGF receptor to induce cell migration.