Cell-specific expression of the transcriptional regulator RHAMM provides a timing mechanism that controls appropriate wound re-epithelialization

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

The efficient and robust repair of cutaneous injury is critically important for the health and survival of organisms. In adult mammals, cutaneous wounds repair quickly to restore barrier functions, which occur in distinct and timed phases with each phase preparing the wound for the subsequent steps necessary to restore function (1)(2)(3)(4)(5)(6). Key steps of repair are mesenchymal cell infiltration, deposition, and remodeling of a collagen-rich provisional extracellular matrix (ECM) 2 followed by resurfacing of the wound by keratinocytes, which migrate over and divide on the provisional matrix to re-establish a protective surface barrier. Temporal control of these repair steps is essential for restoration of cutaneous function and their dysregulation results in aberrant repair. For example, either inhibiting or stimulating keratinocyte migration (3,(7)(8)(9)(10)(11)(12) or proliferation (13) following excisional skin injury can modify wound inflammation and alter dermal cell differentiation, wound contraction, and remodeling (8,(14)(15)(16). However, the molecular nature of the factors that fine-tune migration speeds or proliferation rates in order to achieve appropriate timing of repair is not well-understood. As an example, dermal fibroblasts populate cutaneous wounds prior to the initiation of keratinocyte migration, yet fibroblast motility speed is severalfold slower than keratinocytes (17,18).
Because the creation of a wound gap is expected to release contact inhibition of motility and proliferation for both cell types (10,(19)(20)(21)(22)(23)(24), it is likely that factors, which restrain keratinocyte migration and proliferation, are produced to allow time for fibroblasts to repopulate the early wound site prior to re-epithelialization.
RHAMM (gene name HMMR) is a multifunctional intracellular and extracellular protein whose expression is normally restricted to repairing tissues (25)(26)(27)(28). Intracellular RHAMM regulates gene expression through association with transcriptional complexes (29) and centrosome/mitotic spindle stability through its association with proteins such as TPX2, AURKA, and BRCA1 (30,31). These intracellular functions are linked to regulation of cell proliferation. Extracellular (cell surface) RHAMM binds to hyaluronan and performs co-receptor signaling functions through CD44, other hyaluronan receptors, and growth factor receptors such as PDGF (26,32). RHAMM is transiently expressed during the inflammation (25,26,28,33,34) and granulation tissue stages of excisional injury (35,36). RHAMM signaling functions are required for macrophage and fibroblast migration in response to tissue injury, and genomic deletion of this gene reduces both innate immune cell influx/ function and fibroplasia during repair (33,35). In addition to its role in cell migration, RHAMM also regulates transition through the G 2 M phase of the cell cycle (30,37) predicting a role for this protein in controlling the cellular proliferation needed for tissue repair. RHAMM is expressed in macrophages, fibroblasts, and wound-edge keratinocytes during the early stages of excisional repair when macrophages and fibroblasts are migrating into the wound site but is unexpectedly strongly reduced when keratinocytes initiate migration over granulation tissue (35). These observations prompted us to assess whether the motogenic and mitogenic signaling functions of RHAMM are cell context-dependent. Here, we compare the consequences of RHAMM-loss on the migration and proliferation of dermal fibroblasts and keratinocytes and provide evidence for a role of RHAMM in orchestrating the temporal coordination of wound repair by stimulating fibroblast migration but restraining this function in keratinocytes. We further identify cell context-dependent mechanisms responsible for these opposing effects of RHAMM.

Rhamm-loss results in aberrant epidermal architecture and alters the timing of wound re-epithelialization
Granulation tissue is detected in excisional wounds of WT mice by day 3, and keratinocyte migration is initiated at the wound edge by day 7 (Fig. 1A). Re-epithelialization is complete by day 14 (Fig. 1B). In contrast, Rhamm Ϫ/Ϫ wounds produce very little granulation tissue by day 3 and have expanded a large depot of dermal adipocytes, which is lost by day 7 (Fig. 1A). . Rhamm ؊/؊ keratinocytes re-surface excisional skin wounds more rapidly than WT comparators. A, cross-sections of the center of excisional skin wounds were stained for pan-keratin (brown) and counterstained with hematoxylin. Red arrows indicate the leading edge of the migrating keratinocyte layer. Dotted black lines in day 3 images outline the underlying granulation tissue, and the black arrow indicates the clot. Subcutaneous fat (labeled dermal adipocytes) has uniquely expanded into the granulation tissue of Rhamm Ϫ/Ϫ mice. Rhamm Ϫ/Ϫ wounds are delayed in granulation tissue formation, but resurfacing of wounds by keratinocytes is accelerated relative to WT wounds. B, quantification of wound re-epithelialization shows that Rhamm Ϫ/Ϫ wounds have completed re-surfacing by day 7, and WT wounds complete this process by day 14. Values are the mean and S.E. n ϭ 3 mice. *, p Ͻ 0.01.

RHAMM expression is a timing mechanism in wound repair
Migration of keratinocytes is initiated at the wound edge by day 3 when the dermal microenvironment is aberrant. Rhamm Ϫ/Ϫ wound re-surfacing by keratinocytes is more rapid than in the WT comparator and is completed by day 7 (Fig. 1B). Although WT keratinocytes have organized into identifiable basal and supra-basal layers by day 14, Rhamm Ϫ/Ϫ keratinocytes are disorganized with no clear histological basal/supra-basal demarcation ( Fig. 2A). This lack of epidermal polarity resulting from Rhamm-loss is confirmed by aberrant expression of keratin 10 and the Partitioning defective protein 3 (PAR) polarity complex (38), detected by PAR staining (Fig. 2, B and C). The boundary between basal and suprabasal keratinocytes is clearly demarked in WT wounds by suprabasal keratin 10 staining (Fig. 2B) and strong PAR staining in basal keratinocytes (Fig. 2C). In contrast, all keratinocyte layers of the day 14 Rhamm Ϫ/Ϫ wound are keratin 10 -positive (Fig. 2B), and PAR is poorly expressed throughout the epidermis (Fig. 2C).
Collectively, these results confirm that Rhamm-loss alters mesenchymal differentiation and delays granulation tissue for-mation, as reported previously (35,39). It also identifies a novel effect on re-epithelialization and epidermal re-modeling.

RHAMM protein expression is transient but ubiquitous in excisional wounds
We have previously shown that RHAMM mRNA expression is undetectable in homeostatic skin but is transiently expressed between days 1 and 7 in excisional wounds (35). To facilitate identification of mechanism(s) for increased re-epithelialization of Rhamm Ϫ/Ϫ wounds, the localization of RHAMM protein in excisional wounds was assessed using immunohistochemistry (Fig. 3A). This analysis confirmed the transient expression of RHAMM and showed that protein expression is maximum on day 1 after excisional wounding and is ubiquitously expressed at the wound edge in all skin layers. Staining is gradually reduced between days 3 and 14, raising the possibility that RHAMM may suppress keratinocyte functions involved in re-epithelialization, and loss of expression releases this inhibition. Positive staining for RHAMM in the epidermis was con- Figure 2. Rhamm-loss causes aberrant epidermal differentiation. A, cross-sections of WT and Rhamm Ϫ/Ϫ excisional skin wounds at day 14 post-wounding were stained for pan-keratin and show that WT epidermis has re-organized into discernable basal, suprabasal, and cornified layers by day 14; however, these layers are not distinguishable in Rhamm Ϫ/Ϫ epidermis. B, cytokeratin 10 (K10) staining of day 14 Rhamm Ϫ/Ϫ and WT wounds was used to identify the suprabasal keratinocyte layer. C, Par3 expression was detected by immunohistochemistry and used as an epidermal polarization marker. Loss of RHAMM reduces expression of this protein at day 14 wound centers and edges.

RHAMM expression is a timing mechanism in wound repair
firmed by identifying keratinocytes with the markers keratin 10 and 14 (Fig. 3B). Notably, RHAMM expression is highest when keratin 10 and 14 expression is low. As RHAMM expression disappears, keratin 10 and 14 staining becomes stronger (Fig.  3B). We have previously shown that RHAMM is also expressed in fibroblasts and macrophages of excisional skin wounds (35).
Aberrant re-epithelialization and dermal fibroplasia of excisional wounds can result from de-regulated cell migration and/or proliferation (22,40). Because intracellular RHAMM affects centrosome/mitotic spindle stability and cell-surface RHAMM regulates progression through the cell cycle (30), we first assessed the consequences of its expression-loss on fibroblast and keratinocyte proliferation.

Rhamm-loss has no detectable effect on keratinocyte and fibroblast proliferation
Quantification of Ki67 staining in the epidermis (Fig. 4A) and dermis (Fig. 4B), when active proliferation occurs in both compartments, revealed no significant difference between Rhamm Ϫ/Ϫ fibroblasts or keratinocytes compared with WT counterparts (p Ͼ 0.05). To further assess this, Rhamm Ϫ/Ϫ and Rhamm-rescued dermal fibroblasts were cultured. Re-expressed RHAMM was confirmed by immunoblot analysis (Fig.   4C). No difference in staining for the proliferation marker Ki67 was observed (Fig. 4D). To address the effect of cell-surface RHAMM function-loss in keratinocytes, in particular that of cell-surface RHAMM, a human keratinocyte line, HaCaT, was used. These cells express RHAMM protein (Fig. 4C). A function blocking RHAMM antibody has no effect on the proliferation of these keratinocytes (Fig. 4E), mirroring the lack of effect of Rhamm-loss on keratinocyte proliferation observed in vivo. Because differences in proliferation do not account for the effect of Rhamm-loss on excisional repair, we next investigated the consequence of this loss on cellular migration.

Rhamm-loss suppresses fibroblast motility but promotes this function in keratinocytes
Rhamm-loss alters the migration properties of both primary keratinocyte and fibroblast migration in culture (Figs. 5-8). As we have reported previously (35), deletion of Rhamm in fibroblasts reduces their migration (Fig. 5). Both the migration speed and net translocation of immortalized Rhamm-rescued fibroblasts are significantly greater than Rhamm Ϫ/Ϫ counterparts (Fig. 5, A and B). However, migration persistence of these cells is not affected by Rhamm-loss (Fig. 5C). The migration velocity of primary WT dermal fibroblasts is also more rapid than

RHAMM expression is a timing mechanism in wound repair
Rhamm Ϫ/Ϫ counterparts (Fig. 5D). The function-blocking antibody, which was used to assess cell proliferation in keratinocytes, significantly inhibits motility speed of WT fibroblasts but does not significantly alter Rhamm Ϫ/Ϫ motility speed. Collectively, these results indicate that dermal fibroblasts are dependent on RHAMM expression for motility speed and net translocation.
Unexpectedly, Rhamm-loss increases rather than decreases keratinocyte migration (Figs. 6 -8). We first compared the migration properties of primary WT and Rhamm Ϫ/Ϫ keratinocytes responding to EGF. EGF is the major motogenic cytokine that promotes re-epithelialization in excisional wounds. Both the migration velocity (Fig. 6, A and B) and net translocation of keratinocytes (Fig. 6, C and D) are increased by Rhamm-loss. Directional persistence is unaffected by Rhamm-loss (Fig. 7, B and C). Blocking RHAMM cell-surface signaling with the RHAMM antibody also increases migration speed of WT keratinocytes (Fig. 8, A and B). The specificity of this antibody for RHAMM protein is demonstrated by its lack of effect on Rhamm Ϫ/Ϫ keratinocyte migration speed (Fig. 8, C and D). Collectively, these results show that the motogenic effect of RHAMM expression is cell-type-dependent because it promotes fibroblast motility but inhibits this function in keratinocytes.
Temporal regulation of motogenic and mitogenic signaling cascades such as the MAP kinases controls appropriate migration necessary for normal wound repair (41)(42)(43). We have previously shown that RHAMM-induced fibroblast migration requires ERK1/2 activity (31), and we confirm this finding here (Fig. 5D). We therefore next investigated whether RHAMM also regulates activity of these kinases in keratinocytes. To facilitate these analyses, we utilized a keratinocyte cell line (HaCaT). These cells express RHAMM (Fig. 4C), and the RHAMM antibody significantly increases their migration velocity in scratchwound assays (Fig. 9, A and B) and random motility (Fig. 9C), confirming a role for cell-surface RHAMM signaling in regulating migration of this immortalized keratinocyte line.

Rhamm-loss alters temporal regulation of wound ERK1/2 activity
Immunohistochemical analyses of excisional wounds in vivo show that the levels of active ERK1/2, detected by nuclear phospho-ERK1/2 (p-ERK1/2), are higher in keratinocytes of Rhamm Ϫ/Ϫ versus WT wounds between days 3 and 14 postexcisional injury (Fig. 10, A and B). By day 14, p-ERK1/2 staining is largely restricted to basal keratinocytes in WT wounds ( Fig. 10A). At this time point, strongly p-ERK1/2-positive Rhamm Ϫ/Ϫ keratinocytes occur throughout a multilayered, disorganized epidermis that does not have a clear basal layer. These results predict that RHAMM expression suppresses ERK1/2 activity, which loss of Rhamm releases, and that this may impact both keratinocyte migration and differentiation. Cell cultures were therefore utilized to directly assess a role of ERK1/2 activation in RHAMM-regulated keratinocyte migration.

Cultured keratinocytes and fibroblasts are dependent on ERK1/2 activity for motility and RHAMM regulates this activity
We have previously reported that RHAMM expression promotes growth factor-regulated ERK1/2 activity in fibroblasts and that these MAP kinases are required for RHAMM-dependent fibroblast motility (35). These previous results are con-firmed here. RHAMM expression does not affect basal levels of ERK1/2 activity but increase and prolong activation of these MAP kinases in response to PDGF (Fig. S1). Inhibition of the upstream kinase MEK suppresses migration of fibroblasts expressing RHAMM but has no significant effect on Rhamm Ϫ/Ϫ comparators (Fig. 5D). As observed in vivo, p-ERK1/2 levels are significantly higher in cultured primary Rhamm Ϫ/Ϫ versus WT keratinocytes (Fig. 10C). Furthermore, blocking ERK1/2 activity with a MEK inhibitor strongly decreases migration velocity of Rhamm Ϫ/Ϫ keratinocytes (Fig. 10D). Because keratinocytes are migrating in response to EGF, we next investigated the relationship among EGFR, RHAMM, and ERK1/2 in RHAMM-regulated keratinocyte migration using the HaCaT cell line.

Extracellular RHAMM blocks EGFR motogenic signaling through ERK1/2
We first confirmed that RHAMM antibody-stimulated HaCaT migration in response to EGF is inhibited by blocking EGFR signaling (Fig. 11A). Because EGFR-stimulated ERK1/2 activity is required for keratinocyte migration (44), we next confirmed that blocking cell-surface RHAMM alters ERK1/2 activity. As shown in immunoblot assays, the RHAMM antibody increases phospho-ERK1/2 in HaCaT keratinocytes ( Fig.   Figure 5. Rhamm-loss reduces fibroblast migration. Immortalized Rhamm Ϫ/Ϫ fibroblasts were transfected with a full-length mouse Rhamm cDNA, and motility of the null and rescued cells was quantified. Rescue of Rhamm Ϫ/Ϫ fibroblasts significantly increases cell motility velocity (A) and net translocation (B) but migration persistence is not affected (C). D, speed of WT fibroblasts that express RHAMM is significantly greater than Rhamm Ϫ/Ϫ counterparts in defined medium. WT fibroblast migration is increased by fetal calf serum (FCS) and is strongly reduced by either a RHAMM function-blocking antibody or MEK inhibitor. Rhamm Ϫ/Ϫ fibroblasts are unresponsive to these stimuli and inhibitors. Results are shown as scatter plots. *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001. 11B). Furthermore, RHAMM antibody-stimulated HaCaT migration requires ERK1/2 activity because inhibition of MEK1 blocks this increase (Fig. 11C). These results confirm a role for EGFR and ERK1/2 activity in the migration of HaCaT keratinocytes stimulated by blocking cell-surface RHAMM. We therefore next assessed the association of RHAMM with EGFR and the consequences of blocking cell-surface RHAMM on EGFR activation. Immunostaining shows that EGFR and RHAMM are co-localized in a subset of HaCaT keratinocytes and that the RHAMM antibody significantly reduces this association (Fig. 11D). However, the RHAMM antibody did not result in detectable changes in EGFR activity as detected by phosphorylation status (Fig. 11E). These results suggest that RHAMM dissociation from EGFR affects ERK1/ 2-dependent motility downstream of EGFR activation. We therefore next analyzed ERK1/2-dependent motogenic gene expression in keratinocytes and fibroblasts.

MMP-9 activity is required for migration of keratinocytes and fibroblasts
To identify motogenic genes that are differentially regulated by RHAMM in keratinocytes and fibroblasts, focused PCR arrays for motility-associated genes were performed ( Fig. 12) for primary mouse keratinocytes. Results of these arrays were compared with results from unbiased Affymetrix microarrays of serum-stimulated fibroblasts. Significant gene-expression changes result from Rhamm-loss in keratinocytes (Fig. 12A) and fibroblasts (Fig. 12B), which pathway analyses (Ingenuity Pathway Analysis) confirmed are linked to RHAMM in motility signaling (p Ͻ 6.9E-07). Only two genes Mmp-9 and Ptk2, which are known ERK1/2 targets (45)(46)(47), are differentially affected by Rhamm-loss in keratinocytes versus fibroblasts. Rhamm-loss in keratinocytes increases expression of these genes in keratinocytes but decreases their expression in fibroblasts. Because MMP-9 is well-characterized to promote the motility of keratinocytes (48) and fibroblasts (49) during wound Figure 6. Rhamm-loss promotes keratinocyte migration. A-D, Rhamm Ϫ/Ϫ primary keratinocytes migrate more rapidly than WT counterparts in random migration assays. Primary keratinocytes were isolated from Rhamm Ϫ/Ϫ or WT 1-2-day-old mouse pups, and migration was stimulated by EGF. A, velocity range distribution of WT or Rhamm Ϫ/Ϫ keratinocytes: n ϭ 64 cells/assay, n ϭ 2 assays, B, velocity of WT and Rhamm Ϫ/Ϫ keratinocytes. Box and whisker plots of n ϭ 64 cells. C, displacement plot of individual WT and Rhamm Ϫ/Ϫ keratinocytes. D, Box and whisker plot showing net displacement of WT and Rhamm Ϫ/Ϫ keratinocytes. n ϭ 50 cells. **, p Ͻ 0.01; ****, p Ͻ 0.001.

RHAMM expression is a timing mechanism in wound repair
repair, the role of this metalloproteinase in RHAMM-regulated migration was further investigated.
The changes in Mmp-9 mRNA expression in Rhamm Ϫ/Ϫ keratinocytes and fibroblasts detected by PCR arrays were first confirmed with qRT-PCR (Fig. 13, A and B), and the dependence of Mmp-9 expression on ERK1/2 activity in these two cell types was confirmed (Fig. 13, A and B). The dependence of fibroblasts and keratinocytes migration on Mmp-9 activity was then assessed.
An inhibitor specific for MMP-9 significantly reduces migration of HaCaT keratinocytes in response to the RHAMM antibody in the scratch-wound assays (Fig. 13C). The MMP-9 inhibitor also strongly blunts migration of Rhamm-rescued null fibroblasts (Fig. 13D). To confirm that RHAMM expression/ function affects MMP-9 activity and that the inhibitor is specific for this metalloproteinase, zymograms using conditioned medium and in situ degradation assays using live cells were performed. Results show that the RHAMM antibody selectively increases MMP-9 activity (Fig. 14, A and B). Furthermore, the MMP-9 inhibitor used for migration assays is specific for this metalloproteinase (Fig. 14B). In situ degradation assays show that Rhamm-rescued fibroblasts significantly degrade more denatured rhodamine B-isothiocyanate (RITC)-collagen than Rhamm Ϫ/Ϫ fibroblasts and that this is blocked by the MMP-9 inhibitor (Fig. 14, C and D). Collectively, these results show that inhibiting RHAMM function or expression releases an EGFR/ MMP-9 mechanism that supports keratinocyte migration. In contrast, gain of RHAMM function or expression activates a serum/MMP-9 mechanism that supports fibroblast migration.

RHAMM-blocking antibody promotes MMP-9-dependent CD44 shedding
To assess a role for CD44 in RHAMM-regulated keratinocyte migration, HaCaT cells were scratch-wounded and treated with a function-blocking CD44 antibody Ϯ RHAMM antibody or nonimmune IgG (Fig. 15A). Blocking CD44 function stimulated HaCaT keratinocyte migration to a similar level as blocking RHAMM function (Fig. 15A). Simultaneous addition of both anti-CD44 and anti-RHAMM antibodies did not further increase motility predicting that CD44 and RHAMM coordinate suppression of migration.
CD44 suppression of migration can be released by ectodomain shedding of this protein (67,68). A number of enzymes (68), notably MMP-9 (69,70), proteolyze CD44 prompting us to assess whether exposure of HaCaT keratinocytes to RHAMM antibodies releases CD44 and whether this requires

RHAMM expression is a timing mechanism in wound repair
expression and activity of MMP-9. RHAMM-blocking antibodies significantly promoted ectodomain shedding of CD44 into supernatant medium collected from HaCaT cells, as detected by an ELISA (Fig. 15B). Furthermore, shedding was blocked by inhibiting MMP-9 activity. Results also show that control cultures exhibit a lower level of CD44 shedding that can also be blocked by inhibiting MMP-9 activity (Fig. 15B). MMP-9 activity is a critical factor in releasing RHAMM-and CD44-mediated migration inhibition because addition of recombinant-active MMP-9 by itself is sufficient to significantly stimulate keratinocyte motility (Fig. 15C). These results show that MMP-9 expression is required to release CD44 and promote keratinocyte migration and that this is mediated by blocking extracellular RHAMM function (e.g. Fig. 16A).

Discussion
Here, we show that RHAMM expression is required for the re-formation of a polarized epidermal architecture and function following wounding. Its absence may therefore be related to wound-healing abnormalities such as keloids, diabetic ulcers, and hypertrophic scarring. RHAMM inhibits keratinocyte migration in vivo and in culture, while conversely, it stimulates dermal fibroblast migration. RHAMM is transiently-expressed by both cell types in excisional wounds when mesenchymal cells are migrating into the wound to produce a pro-visional matrix, and keratinocyte migration has not yet been initiated. Therefore, we propose that this opposing effect of RHAMM on cell migration is a critical factor in coordinating the timing of excisional wound re-surfacing by keratinocytes relative to in-migration of fibroblasts/mesenchymal cells so that keratinocytes migrate on the provisional matrix (Fig. 16B). We predict that alteration of normal coordination has longterm consequences for re-establishing appropriate epidermal architecture. Thus, Rhamm genomic-loss impairs the development of the basal and supra-basal layers of the epidermis, and analyses of repaired tissue show that the polarity of basal keratinocytes is not appropriately re-established. Paracrine communication among keratinocytes and dermal cells, such as fibroblasts and adipocytes, is known to be required for establishing appropriate mesenchymal differentiation and skin tissue architecture (71,72) predicting that Rhamm-loss modifies this interaction. Either Rhamm genomic-loss or blocking extracellular RHAMM function (39) promotes premature dermal adipogenesis, and therefore Rhamm Ϫ/Ϫ keratinocytes migrate over an inappropriately predominant adipocyte microenvironment, which likely alters key paracrine/cytokine signals required for establishing epidermal architecture. Intracellular RHAMM functions have previously been linked to establishing epithelial polarity (73,74), and loss of both extracellular and

RHAMM expression is a timing mechanism in wound repair
intracellular functions therefore likely contributed to this aberrant effect of Rhamm-loss.
The processes of cell migration and proliferation are uncoupled in both fibroblasts and keratinocytes during fibrotic wound repair (6,16,(75)(76)(77). For example, keratinocytes migrating under the clot to initiate wound re-covering do not proliferate, whereas keratinocytes in back of the wound edge proliferate to ensure the supply of migrating cells is sufficient to cover the wound (75). Nonproliferating fibroblasts contribute to wound closure not only by producing the ECM for keratinocytes to migrate on but also by differentiating into myofibroblasts to promote wound-site contraction (7,75,78). Deregulation of this temporal control is associated with abnormal skin repair.
Our results show that the key factors in the cell context-dependent regulation of migration by RHAMM are CD44 and MMP-9. Both keratinocytes and fibroblasts exhibit co-distribution as well as a functional interdependence of RHAMM, CD44, and MMP-9 activity for migration. However, RHAMM and CD44 suppress an EGFR-ERK1/2:MMP-9 pathway signaling required to promote keratinocyte migration. When this suppression is relieved, EGFR signaling is activated, and MMP-9 is expressed, which cleaves CD44 to promote migration. In contrast, dermal fibroblasts require RHAMM and CD44 to activate a PDGF receptor, TGF␤1, and/or a serum:ERK1/2 pathway that promotes MMP-9 expression, which is able to activate fibro-blast motogens such as TGF␤1 (79) and which promotes focal adhesion formation (80) necessary for fibroblast migration. These results are summarized in the model shown in Fig. 16A.
CD44 is a hyaluronan and growth factor co-receptor that is subject to extensive alternative splicing. Its resulting biology is complex because although this receptor has most frequently been shown to promote cell adhesion and motility, for example (81,82), an increasing number of reports are identifying context-dependent conditions where CD44 shedding or its genomic-loss is required for stimulation of motility. For example, blocking cleavage of CD44 prevents migration of glioma cells on HA substrates (83), and CD44 shedding or genetic-loss promotes migration of fibroblasts and other cells across small wound gaps (67). Conversely, fibroblasts require the surface display of CD44 for migrating across larger wound gaps that mimic excisional wounds (35). These few examples and the data presented in this study illustrate a mechanistic complexity and contextual dependence underlying regulation of cell migration during wound repair that is understudied and that needs to be addressed for the development of effective wound control.

RHAMM expression is a timing mechanism in wound repair
and MMP-9 loss delays re-epithelialization (92). This metalloproteinase can also promote motility by proteolytic-independent, hemopexin domain-dependent mechanisms (80, 93, 94). CD44 is a key MMP-9 -binding partner for both of these motogenic mechanisms. Our data suggest that Rhamm-loss is also a critical partner in these mechanisms since we show that blocking this protein promotes the proteolytic selectivity of MMP-9 for CD44, which results in increased keratinocyte migration.
In addition to its role in cell migration, RHAMM has also been reported to regulate cell proliferation. For example, RHAMM promotes progression of cultured cells through G 2 M, and it contributes to mitotic spindle dynamics and orientation during development and disease (25,26,81,95). However, our results support a dominant effect of RHAMM on migration rather than proliferation per se, as we were unable to observe a strong effect of Rhamm-loss on keratinocyte or fibroblast proliferation during excisional repair or in 2D culture. Keratinocyte migration and proliferation during cutaneous repair are uncoupled, which we also observed in WT excisional wounds of this study. Rhamm-loss appears to extend this period of uncoupling even after re-epithelialization of the wound has occurred. This subtle consequence of Rhamm-loss could alter stem cell re-population dynamics and contribute to the observed aberrant epidermal architecture. These results are consistent with a previous report documenting a role for RHAMM in coupling migration to the cell cycle (96).
In conclusion, a number of diseases (97,98) result from aberrantly regulated keratinocyte migration, including skin disorders (e.g. ulcers), burns, diseases resulting from chronic inflammation, and the spread of keratinocyte cancers. The worldwide increase in patients with aberrant wound repair related to aging, diabetes, malnutrition, chemotherapy, and hereditary diseases provides an imperative for identifying the mechanisms of the healing steps in order to rationally manipulate and improve skin wound repair. Our results suggest that blocking RHAMM function could be used to accelerate keratinocyte migration in some of these skin disorders.
C57BL/6 mice were obtained from Charles River Laboratories; the preparation of C57BL/6 Rhamm Ϫ/Ϫ mice has previously been described and characterized (100), and deletion of exons 8 -15 of the Rhamm gene is described in detail elsewhere (100). In brief, to delete exons 8 -15 of the murine Rhamm gene, a construct was generated that consisted of the hypoxanthineguanine phosphoribosyltransferase selection marker flanked by genomic Rhamm sequence upstream of exon 8 and downstream of exon 15. This construct was transfected by electroporation into Hprt-negative HM-1 mouse embryonic stem cells. Hprt-positive clones were isolated by selection in HAT medium. Clones with homologous recombination events were identified by Southern blotting analyses. Rhamm ϩ/Ϫ ES cells were injected into C57BL/6 blastocysts that were implanted into pseudopregnant females. To generate Rhamm ϩ/Ϫ mice, chimeric males were bred to C57BL/6 females. Breeding of Rhamm ϩ/Ϫ males with Rhamm ϩ/Ϫ females produced the expected ratio of WT, Rhamm Ϫ/Ϫ , and Rhamm ϩ/Ϫ offspring. WT and Rhamm Ϫ/Ϫ mice, which were maintained as homozygotes on a C57BL/6 background, were cared for in keeping with the Guide to the Care and use of Experimental Animals from the Canadian Council on Animal Care, and our use of mice was reviewed and approved (protocol 2009-060) by the Animal Care Committees at the London Regional Health Centre and University of Western Ontario. Food and water access were ad libitum. For breeding, adult C57Bl/6 males were housed with two C57Bl/6 females until pregnancy was obvious, at which time pregnant females were housed individually. For keratinocyte isolation, newborn pups were removed and euthanized by 30 min of CO 2 inhalation.

Methods
Genotyping-Frozen tail samples were digested overnight at 55°C in 350 l of lysis buffer containing 50 mM Tris, 0.1 M EDTA, pH 8, 1% SDS, 100 mM NaCl, 800 g/ml proteinase K. Samples were continuously mixed at 800 rpm during the digest. Digested samples were centrifuged at 15,500 ϫ g for 10 min to remove debris. The supernatant was retrieved and mixed with 500 l of isopropyl alcohol. Samples were centrifuged again at 15,500 ϫ g for 15 min to pellet the DNA. The supernatant was decanted, and the DNA pellet was washed twice with 500 l ice cold 70% ETOH, dried at room temperature for several hrs and the pellet was dissolved in 100 l ddH 2 O. DNA concentration was measured using a NANODROP instrument and adjusted to a concentration of 50 ng/l. The following PCR conditions were used: 1. incubate at 94°C for 15 min; 2. incubate at 94°C for 30 s; incubate at 50°C for 1 min (WT allele) or 55°C for 1 min (Rhamm knock out allele); incubate at 72°C for 1 min. Return to step 2 for 39 more times. Incubate at 72°C for 10 min and hold at 4°C. Add 2 l of 6ϫ DNA loading buffer to 10 l of PCR sample and run sample on a 1% agarose gel.
Excisional wounding-All animal experiments followed policies and guidelines established by the Canadian Council for Animal Care and were previously approved by the Animal Use Committee of Western University (protocol no. 2009-060). Excisional wounds were placed as described previously (35). In brief, 8 -12-month-old female Rhamm Ϫ/Ϫ and WT mice were anesthetized using isoflurane inhalation. Dorsal hair was removed using an electric razor. Using a metal punch with a 4-mm diameter, one full-thickness punch biopsy was taken from the middle of the dorsum, resulting in two full-thickness skin wounds. Using an 8-mm metal punch, the wound area was harvested at different times after wounding. Wound samples were fixed in 4% paraformaldehyde/phosphate buffered saline (PBS) at pH 7.4, and paraffin was processed for histology tissue sections.

Figure 13. MMP-9 expression is regulated by ERK1/2 in keratinocytes and fibroblasts and is required for migration of both cell types.
A, RHAMMblocking antibody significantly increases the expression of MMP-9 mRNA in HaCaT keratinocytes as detected by qRT-PCR using GAPDH as a loading control. Expression is reduced by inhibiting ERK1/2 activity with the MEK inhibitor PD98059. Box and whisker plot of n ϭ 9 replicates. B, Rhamm Ϫ/Ϫ fibroblasts express little MMP-9 mRNA and expression is increased by Rhamm-rescue. The increased in MMP-9 expression stimulated by Rhamm-rescue is blocked by inhibiting ERK1/2 activity using PD98059. Scatter plot of n ϭ 4 replicates. C, keratinocyte migration stimulated by the RHAMM antibody is blocked by inhibiting MMP-9 activity. Box and whisker plot of n ϭ 25. D, fibroblast migration stimulated by Rhamm-rescued is blocked by inhibiting MMP-9 activity. Values are the mean and S.E. n ϭ 60 cells/condition. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001

RHAMM expression is a timing mechanism in wound repair
Immunohistochemistry-Paraffin-processed tissue sections were obtained as described previously (35). In brief, sections were de-paraffinized by passage through xylene and an ethanol series. Antigen retrieval was performed by boiling slides in 0.01 M sodium citrate, pH 6, for 20 min using a microwave. Endogenous peroxidase was blocked by incubating slides for 10 min in 3% H 2 O 2 /PBS at room temperature. Nonspecific antibody binding was blocked by incubating slides in 3% BSA/PBS for 1 h at room temperature. Primary antibodies were diluted 1:250 in 1% BSA/PBS and added to the slides. IgG was used as negative control. Slides were incubated overnight with antibodies (pankeratin, p-ERK1/2) at 4°C in a humidified chamber at dilutions recommended by the manufacturer. Slides were then washed in PBS and incubated with a biotinylated secondary antibody (1:500 in 1% BSA/PBS) for 1 h at room temperature. Slides were washed to remove unbound secondary antibody, and washed slides were incubated with streptavidin-HRP (1:500 in PBS) for 1 h at room temperature to visualize bound streptavidin secondary antibodies. Slides were washed to remove unbound streptavidin-HRP, which was then visualized using DAB Plus according to the manufacturer's instructions. Slides were counterstained with hematoxylin, washed, passed through an increasing ethanol series followed by xylene, and mounted using Cytoseal 60.
Image analysis of immunohistochemistry-Slides were scanned using an Aperio Scan Scope or photographed using an AX70 Provis Olympus microscope equipped with air objectives (ϫ4, NA 0.16; ϫ20, NA 0.7; Olympus, Tokyo, Japan) and a color camera (Cooke SensiCam CCD imaging; PCO-TECH, Romulus, MI) controlled by Image-Pro Plus 4.5.1.2.9 (Media Cybernetics, Rockville, MD) software. Images were saved as tiff files which were then analyzed by ImageJ software (RRID: SCR_003070). Color deconvolution was performed using the DAB/hematoxylin setting of the color deconvolution plugin. Using the threshold function and by comparing images of antibody versus control nonimmune IgG, brown pixels were Figure 14. RHAMM regulates MMP-9 activity. A and B, scratch wounds of HaCaT keratinocyte cultures were treated with either RHAMM antibodies or control IgG. MMP-9 activity in conditioned medium was analyzed by zymogram gels. RHAMM antibodies increased MMP-9 activity. MMP-9 activity was inhibited by an MMP-9 inhibitor, demonstrating its specificity. A, scatter plot of n ϭ 3-4. *, p Ͻ 0.05; ***, p Ͻ 0.001. B, zymogram images. C and D, MMP-9 activity in fibroblasts is rescued by RHAMM expression. C, images of collagen degradation. D, scatter plot of n ϭ 10 cells. **, p Ͻ 0.01.

RHAMM expression is a timing mechanism in wound repair
selected. The number of selected pixels in a constant region of interest was quantified using the analysis function.
Fibroblast cultures-Primary WT and Rhamm Ϫ/Ϫ dermal fibroblasts were isolated from explanted skin. Immortalized Rhamm Ϫ/Ϫ fibroblasts were obtained as described previously and rescued by transfection with a full-length mouse Rhamm cDNA. Cells were plated onto fibronectin-coated culture flasks (Falcon) for 12 h and then placed on a heated stage (Tokai Hit/Nikon, Mississauga, Ontario, Canada) at 37°C, and the displacement of cells was recorded at 5-min intervals for 12 h by video microscopy using a Nikon Eclipse TE300 microscope (Mississauga, Ontario, Canada) with NSI Elements AR software (Nikon), and a Hewlett Packard computer (Mississauga, Ontario, Canada).
HaCaT keratinocyte cell line-This human keratinocyte cell line (HaCaT) was a kind gift from Dr. Trevor Shepherd (London Regional Cancer Program). Cells were grown in growth medium (DMEM with 10% FBS and 1ϫ antibiotic/antimycotic) at 37°C at 5% CO 2 . Cells were passaged at 80% confluence by incubating the monolayer in 10 ml of D-PBS containing 0.2 mM EDTA for 15 min at 37°C. The D-PBS/EDTA solution was removed, and the monolayers were then incubated with 2 ml of 0.25% trypsin for 10 min, which was stopped by adding fresh DMEM ϩ 10% FBS. Dispersed cells were recovered by centrifugation (100 ϫ g) and subcultured at a 1:5 dilution into DMEM ϩ 10% FBS.
Cell motility of primary keratinocytes, HaCaT keratinocytes, and fibroblasts-Cells were washed with DMEM, and fresh growth medium was added to the flasks. RHAMM antibody (1: 50 dilution) or nonimmune IgG was added to the media. Inhibitors PD98059 (50 M) and UO126 (50 M) were added to the media. Control media contained the same concentration of Figure 15. Loss of CD44 promotes keratinocyte migration and RHAMM regulates CD44 shedding. A, CD44 function-blocking antibody increases HaCaT motility to a similar extent as the RHAMM antibody. Addition of both antibodies together does not further increase motility predicting that CD44 and RHAMM are acting on the same motogenic pathway. Box and whisker plots of n ϭ 65 cells/condition. B, RHAMM antibody increases CD44 shedding, which requires MMP-9 activity. Shedding was quantified using a CD44 ELISA that detects all CD44 isoforms. Box and whisker plots of n ϭ 6 replicates. a-MMP-9, activated recombinant MMP-9 protein; MMP-9I, MMP-9 inhibitor. C, a-MMP-9 stimulates HaCaT keratinocytes migration, which was quantified with a scratch-wound assay. Left panel shows migration vectors of individual cells, and right panel shows the averaged motility of HaCaT keratinocytes exposed to active MMP-9 protein or buffer alone. Values are the mean and S.E. n ϭ 120. *, p Ͻ 0.05; ***, p Ͻ 0.001.

RHAMM expression is a timing mechanism in wound repair
DMSO (0.1%) as the inhibitor-containing media, and cells were incubated for 1 h at 37°C with 5% CO 2 prior to filming. Sealed flask(s) were placed on a heated stage (Tokai Hit/Nikon, Mississauga, Ontario, Canada) at 37°C, and the displacement of cells was recorded at 5-min intervals for 12 h by video microscopy using a Nikon Eclipse TE300 microscope (Mississauga, Ontario, Canada) with NSI Elements AR software (Nikon), and a Hewlett Packard computer (Mississauga, Ontario, Canada). The NSI Elements AR analysis program (Nikon) was used to track the path length of each migrating cell and to calculate the total distance traveled by each cell at the corresponding time, which was used to calculate the migration velocity of each cell. Individual migration rates as well as the recorded population average were calculated and graphed on GraphPad software. Directional persistence was defined as the total distance from origin (in micrometers) divided by the net path length (in micrometers) (103).
HaCaT scratch-wound assay-HaCaT cells were plated onto fibronectin-coated IBIDI 8-well chambers using a cell density that achieved confluence after overnight incubation in growth medium. The next day, the monolayer was scratch-wounded with a 1000-l (blue) pipette tip. Detached cells were removed by gentle washing, and injured monolayers were incubated with blocking RHAMM antibody or control IgG in low-calcium keratinocyte medium containing 10% calcium-free FBS overnight Ϯ 10 nM MMP-9 inhibitor I, 10 ng/ml active MMP-9 recombinant protein, or 1:100 blocking CD44 antibody or blocking EGFR antibody (10 M). DMSO was included in controls where relevant. Cell migration was analyzed by time-lapse video microscopy with images taken at 5-min intervals for 24 h using the above Nikon microscope equipped with NSI Elements AR software. The NSI Elements AR analysis program was then used to measure the distance between the cell front at time 0 and individual cells after 12 h. Results were copied to EXCEL and then sorted from lowest to highest value and displayed in a bar graph.
Immunofluorescent staining and co-localization analysis-Scratch-wounded, subconfluent HaCaT keratinocytes were exposed to the blocking RHAMM antibody or nonimmune IgG as above for 24 h and then fixed in 4% paraformaldehyde/PBS, pH 7.5, for 10 min at room temperature. Fixed cells were washed to remove fixative and then incubated for 10 min at room temperature with 0.01% Triton X-100/PBS. Nonspecific antibody-binding sites were blocked with 3% BSA/PBS for 1 h. Coverslips were then incubated with primary antibodies (EGFR, IQGAP1 1:500; RHAMM 1:250; CD44 1:250; Ki67 1:200, Alexa 488 ERK 1:100 or nonimmune IgG) overnight at 4°C, washed, and then incubated with fluorescent secondary Figure 16. Models for RHAMM-regulated motogenic signaling in keratinocytes and temporal-spatial coordination of keratinocyte and fibroblast migration in excisional wounds. A, signaling model for regulation of keratinocyte migration by RHAMM. RHAMM/CD44 interactions block EGFR-regulated ERK1/2 activity and downstream expression of the motogenic target gene, MMP-9. Blocking extracellular RHAMM function releases the pathway and stimulates the expression and release of MMP-9, which promotes CD44 shedding, resulting in increased keratinocyte migration. B, RHAMM expression is ubiquitous in wounds by 24 h after excisional injury and then decreases over time. Maximal expression corresponds to influx of innate immune cells and initiation of fibroblast migration to form granulation tissue. As RHAMM protein levels decrease, keratinocyte migration is initiated. Aberrant RHAMM expression alters the timing of keratinocyte and fibroblast migration resulting in altered rate of wound closure and dysregulated differentiation within the wound site.

RHAMM expression is a timing mechanism in wound repair
antibodies (1:200) for 1 h at room temperature, with the exception of the fluorescently-labeled p-ERK antibody, which was immediately mounted in DAPI-containing ProLong Gold antifade mounting reagent. Coverslips were washed in PBS and then mounted with DAPI-containing ProLong Gold anti-fade mounting reagent as above. Cells were imaged using an IX81 Olympus confocal microscope equipped with FV10-ASW 4.2 software, and images were saved as tiff files. Co-localization and fluorescent intensity were quantified using ImageJ. Fluorescent channels were separated (Image-Color-Split Channels), and the co-localization of red and green channel was determined using the co-localization plugin (Analyze-Co-localization-Colocalization Threshold). Fluorescence intensity was quantified using the "Measure" function.
The protocol for immunofluorescent staining of paraffinembedded sections was similar to immunohistochemistry described above, with the endogenous peroxidase quenching step omitted. Nonspecific antibody binding was blocked using 3% BSA/PBS at room temperature for 1 h, and intermediate washing steps were performed using PBS. Primary antibodies (RHAMM, keratin 10, keratin 14, and PAR) were added to slides at dilutions recommended by the manufacturer, with nonimmune IgG used as the negative control. All slides were incubated overnight in humidifying chambers at 4°C. Following three washes in PBS, fluorescent secondary antibody (Alexa 488, 1:100) was added to slides incubated at room temperature for 2 h and then washed in PBS. All slides were mounted with DAPI-containing ProLong Gold anti-fade mounting reagents and imaged using an IX81 Olympus confocal microscope. Quantification of staining was performed with ImageJ.
Cell proliferation analysis-Subconfluent HaCaT cell cultures were stained with Ki67 antibody as described above. Cell proliferation of HaCaT keratinocytes and dermal fibroblasts were quantified by counting Ki67-positive and -negative nuclei and calculating the percentage of Ki67-positive nuclei.
CD44 ELISA-Sufficient HaCaT cells were seeded onto fibronectin-coated 6-well plates to generate confluent monolayers after 24 h. Four scratch wounds/well were placed using a 1000-l blue pipette tip, and cultures were gently washed with keratinocyte growth medium to remove detached cells. Keratinocyte growth medium Ϯ active MMP-9 recombinant protein (10 ng/ml), blocking RHAMM Ab, and/or nonimmune IgG were then added for 20 h at 37°C, and culture medium was collected and centrifuged at 1000 ϫ g for 4 min. Supernatant was added to the CD44 ELISA plate undiluted (200 l/well), and the plate was incubated overnight at 4°C. Detection of CD44 was performed as described in the manufacturer's instructions.
RT-PCR analysis-Total RNA was isolated from cultures of migrating cells using TRIzol reagent (1 ml/plate) according to the manufacturer's instructions. Cells were scraped off using rubber cell scrapers, and detached cells were mixed with 200 l of chloroform and centrifuged at 15,500 ϫ g for 10 min at 4°C. The top phase was pipetted and mixed with 500 l of isopropyl alcohol to precipitate RNA. Samples were stored at Ϫ20°C overnight and then centrifuged at 15,500 ϫ g at 4°C for 30 min. Isopropyl alcohol was removed, and the RNA pellet was washed with 70% ice-cold ethanol, and centrifuged at 15,500 ϫ g at 4°C for 15 min. Ethanol was removed from the precipitated RNA pellet, which was dried at room temperature, dissolved in 100 l of ultrapure ddH 2 O, and measured using a NANODROP instrument. 2 g of RNA was used to synthesize cDNA with the RT 2 Profiler reverse transcription kit used according to the manufacturer's instructions. RT 2 Profiler PCR arrays were used and analyzed following manufacturer's instructions.
MMP-9 qRT-PCR-Subconfluent HaCaT cells were treated with RHAMM antibody or IgG as described above. RNA was isolated using TRIzol reagent and following the manufacturer's instructions. RNA concentration was determined using a NANODROP instrument. 2 g of RNA was used to synthesize cDNA with SuperScript VILO cDNA synthesis kit and following the manufacturer's instructions. cDNA was diluted 1:10 with ddH 2 O, and 2 l of diluted cDNA was used per qPCR. SYBR Green mastermix, MMP-9-specific primers, and 2 l of diluted cDNA were used for each qRT-PCR. GAPDH was used as a housekeeping gene. Fold-expression was calculated using the ⌬CT method. Mouse fibroblast MMP-9 mRNA expression was performed as described previously (93).
PCR conditions: 1. incubate at 94°C for 10 min; 2. incubate at 94°C for 30 s; incubate at 55°C for 45 s; and incubate at 72°C for 45 s. Return to step 2 for 39 more times. Incubate at 72°C for 10 min.
Western blotting assays-Subconfluent HaCaT cell cultures were treated with RHAMM antibody or IgG as described above. Cells were washed once with cold PBS and then lysed in RIPA buffer containing proteinase and phosphatase inhibitors. Protein concentration was determined using BCA protein assay kit following the manufacturer's instructions. Laemmli sample buffer was added to the lysate, and samples were heated to 95°C for 5 min. 30 g of protein per sample were loaded onto Bolt 4 -12% polyacrylamide gels. Following electrophoresis, gels were transferred on Immobilon-P transfer membrane using NuPAGE transfer buffer containing 10% methanol. Membranes were blocked with TBS-T (TBS plus 0.25% Tween 20) containing 5% skim milk overnight at 4°C. Primary antibodies (RHAMM, p-EGFR, and p-ERK1/2, AbCAM) were diluted 1:500 to 1:1000 in TBS-T containing 1% milk. Membranes were incubated with primary antibodies for 2 h at room temperature, washed with TBS-T three times for 10 min, then incubated with 1:2000 diluted secondary HRP-coupled antibody for 1 h at room temperature, and washed with TBS-T three times for 10 min. Membranes were incubated with HRP substrate and imaged using Image Lab software (Bio-Rad). Protein bands were quantified using Image Lab software.
Zymograms-HaCaT cells were cultured on fibronectincoated 6-cm cell culture plates using DMEM plus 10% FBS and antibiotics/antimycotics. Confluent cultures were scratchwounded using a blue pipette tip. Cultures were washed once with serum-free Keratinocyte Growth Medium containing EGF. Cells were cultured overnight in serum-free Keratinocyte Growth Medium plus EGF, containing either RHAMM Ab or IgG. Conditioned medium was mixed with 2ϫ Tris-glycine SDS Sample buffer (Novex/Thermo Fisher Scientific), and 30 ml/well were loaded onto a zymogram gel containing gelatin (Novex/Thermo Fisher Scientific). 1ϫ Tris-glycine SDS running buffer (Novex/Thermo Fisher Scientific) was used during gel electrophoresis. Following gel electrophoresis, gels were incubated two times for 30 min at room temperature in 1ϫ zymogram Renaturation buffer (Novex/Thermo Fisher Scientific). Gels were then incubated for 30 min at room temperature in 1ϫ Zymogram Developing buffer (Novex/Thermo Fisher Scientific) followed by 36 h of incubation at 37°C in 1ϫ Zymogram Developing buffer. Where indicated, MMP-9 inhibitor was added to the Developing buffer. Zymograms were stained with Coomassie Blue (2.5 of Coomassie Brilliant Blue in 450 ml of methanol, 100 ml of acetic acid, 450 ml of H 2 O) for 2 h at room temperature followed by de-staining in de-staining solution (450 ml of methanol, 100 ml of acetic acid, 450 ml of H 2 O). Stained gels were imaged with a digital camera. Degraded areas were quantified using ImageJ software.
For cell zymograms, fibroblasts were plated onto RITC-fibronectin (Cytoskeleton)-coated tissue coverslips and grown for 12 h. Cultures were fixed in 3% paraformaldehyde, washed, and mounted in ProLong Gold anti-fade mounting reagents and imaged using an IX81 Olympus confocal microscope. Areas cleared of RITC-fibronectin were quantified using ImageJ.
Statistical analyses-A two-way Student's t test was used to determine statistical significance between two experimental groups. A p value of Ͻ0.05 was considered to be statistically significant.