The epithelial barrier is critical to homeostasis. Because most epithelia are in continuous contact with a foreign and potentially harmful environment, a rapid and efficient mechanism to correct damage to this barrier must be in place. For small wounds, this takes the form of purse-string contraction4,7,9
rather than the slower lamellipodia-dependent cell migration typical of larger wounds.6,49
This purse-string mechanism depends on the assembly of a circumferential contractile actomyosin ring surrounding the wound7
and is used in a broad range of situations, including embryonic wound healing,11
apoptotic cell extrusion,4
and closure of plasma membrane wounds.12
Many of these processes occur in both healthy individuals and patients with intestinal disease. Thus, understanding the coordinated process of purse-string wound closure, which likely occurs far more frequently than migration-dependent closure of larger wounds, also has therapeutic implications, as new therapies that inhibit ROCK or MLCK have been proposed for a variety of intestinal and extraintestinal diseases, including cancer metastasis, acute lung injury, and inflammatory bowel disease.25,50–53
Therefore, definition of these mechanisms may allow early identification and intervention to minimize intestinal toxicity of these agents.
Because phosphorylation of the MLC is a primary regulator of actomyosin contraction, we posited that contraction of this actomyosin ring might depend on MLC phosphorylation. However, available data from disparate systems provide conflicting descriptions of the roles of rho, ROCK, MLCK, and MLC during purse-string actomyosin contraction.4,9,12,14,20,22,23
We sought to define the recruitment and activation patterns of these critical regulators in well-characterized in vitro systems of epithelial purse-string wound closure and in human biopsy specimens. We used live-cell imaging of EGFP-β-actin–expressing intestinal epithelial cells to follow actin dynamics in living cells during wound closure. This allowed us to define epithelial purse-string wound closure as a stepwise and precisely regulated process. Wound closure was reproducible and rapid, with the first recognizable event being actin concentration at the wound edge. This began within 2 minutes and was complete, with a continuous band of actin surrounding the wound edge, within 8 minutes after wounding. Initiation of this ring assembly phase coincided with recruitment of activated rho and its downstream effector ROCK to the wound edge. At the earliest time points activated rho and ROCK were present both in association with and separate from the initial sites of actin ring assembly. Thus, together with the observation that ROCK inhibition prevented ring assembly, these data suggest that ROCK may actually trigger ring assembly. This hypothesis is consistent with observations in 2 related models, healing of incisional wounds in the embryonic chick wing bud and puncture wounds in Xenopus
oocytes, showing that rho is required for actin ring assembly.12,23
Rho activity is also required for the assembly of actin bundles at wound edges in larger wounds, although neither rho activity nor formation of the bundles themselves is necessary for closure of these large wounds by lamellipodia-dependent cell migration.8
Thus, consistent with the roles of rho and rho family members in stress fiber assembly and actin polymerization in systems as diverse as Acanthamoeba
these data suggest that the roles of rho and ROCK in purse-string wound closure are to direct assembly of the actin ring. Because ROCK inhibition did not prevent contraction, our data also suggest that ROCK activity is not necessary for the second phase of purse-string wound closure. Our observation that transient filopodia were seen when ROCK was inhibited, but never in control wounds, suggest that, in the absence of ring assembly, alternative mechanisms of wound closure can be activated. This hypothesis is consistent with a recent study showing that small wounds in Drosophila
embryos, which normally heal by purse-string contraction, close using filopodial extensions in rho knockout flies.55
The absolute requirement for ROCK in purse-string closure of oligocellular epithelial wounds is at odds with work showing that barrier function recovers normally following the creation of single-cell wounds in intestinal epithelial monolayers treated with ROCK inhibitors9
but is consistent with studies in renal epithelia showing that ROCK inhibition blocks apoptotic cell extrusion, a process that also appears to occur via purse-string actomyosin contraction.4
While available data do not explain the differences between these disparate results in these unique experimental systems, it is important to note that the diameter of single-cell wounds induced with a current pulse (5–7 μm) is far less than the 20–40 μm diameter of the still quite small oligocellular wounds made by mechanical disruption. Thus, one possibility is that preexisting perijunctional actin rings that encircle each cell in an intact monolayer are sufficient to allow progression to the MLCK-dependent contraction phase in single-cell wounds, while ROCK-directed ring assembly is necessary in larger oligocellular wounds.
In normal wound healing, actin ring assembly is followed by contraction. We have shown that contraction begins within 8 minutes after wounding, as ring assembly is completed, and can initially be identified as wound edge rounding, signaling the development of tension within the actin ring. This process continues until wound closure is complete. The critical role for MLCK in wound closure is emphasized by the coincidence of the recruitment and activation of MLCK with MLC phosphorylation and the initiation of wound closure. The effects of the specific peptide inhibitor of MLCK, PIK, also support the essential role of MLCK in late phases of wound closure. Thus, the data suggest that MLCK, and not ROCK, mediates the phosphorylation of MLC that is necessary for purse-string contraction.
The use of the peptide inhibitor of MLCK, PIK, directed against the substrate-binding site is a significant advance over previous studies using other inhibitors that target the adenosine triphosphate–binding site of MLCK. For example, the most commonly used MLCK inhibitor, ML-7, inhibits adenosine 3′,5′-cyclic monophosphate–dependent protein kinase with a Ki
close to that for MLCK, while PIK does not inhibit adenosine 3′,5′-cyclic monophosphate–dependent protein kinase appreciably at any concentration.46
Moreover, the observation that biotinylated PIK can be used as a specific probe for activated MLCK in fixed cells will likely make this an important tool in future work.
While these studies are not possible in intact tissues, we have asked whether components of this wound closure pathway can be identified in vivo. We analyzed oligocellular wounds in colonic mucosa from patients with active inflammatory bowel disease. MLC phosphorylation was markedly enhanced at the edges of oligocellular wounds in a pattern strikingly similar to that observed in the in vitro model. Thus, these data suggest that this mechanism of wound closure is active in vivo as well as in vitro.
Together the data suggest a model of epithelial purse-string wound closure where the rho-ROCK and MLCK pathways of actomyosin regulation serve spatially and temporally distinct functions (). In this model, rho is rapidly recruited to and activated at the wound edge where it activates ROCK. ROCK then directs local actin polymerization, resulting in the coordinated assembly of a circumferential actin ring at the wound edge. MLCK recruitment and activation and the resulting MLC phosphorylation then drive actomyosin contraction. This culminates with restoration of epithelial barrier function. Thus, we have shown epithelial purse-string wound closure to be a precisely orchestrated process that can be separated into 2 distinct phases regulated by separate kinases: ROCK and MLCK.
Figure 10 Model describing the roles of ROCK and MLCK during epithelial purse-string wound closure. (A) Schematic of initial wound within a previously intact epithelial sheet. A continuous multicellular actin ring is not present at the wound edge, and the profile (more ...)