Various effects of laminin-1 and laminin-2/4 on adhesion and migration of cultured human keratinocytes

The cell-matrix interaction is one of the factors defining the cell behavior in normal and wounded tissues. To determine the function of laminin-2/4, one of components of the skin basement membrane in the process of reepithelization, we studied its interaction with human keratinocytes. The adhesive properties of laminin-2/4 and its effect on keratinocytes migration in vitro were analysed. For comparison with our present investigation, we used the earlier studied laminin-1 from EHS mouse sarcoma. Laminin-2/4 appeared to be a good substrate for human keratinocytes, and this correlates with a greater number of cell surface receptors compared with laminin-1. Laminin-2/4 alone does not stimulate keratinocyte migration, but, in contrast to laminin-1, supports EGF-mediated migration. The obtained results give an insight into the function of laminin-2/4 in normal skin and during wound healing.     

Fibroblasts change spreading capability and mechanical properties in a direct interaction with keratinocytes in conditions mimicking wound healing

Keratinocytes are predominant in the uppermost layer of the skin, while fibroblasts dominate in the dermal layer. These cells interact with each other directly when fibroblasts migrate to a region of the wound where they induce keratinocytes proliferation through double paracrine signalling. Since a response from both keratinocytes and fibroblasts dominates during the inflammatory and proliferative phases, the exact knowledge how these two types of cells interact with each other is crucial for deeper understanding of mechanisms involved in the wound healing process. The aim of this study was to quantify alterations in mechanical properties of cells, i.e. fibroblasts and keratinocytes, in conditions mimicking direct cellular interactions observed in wound healing. Single cell elasticity was measured using atomic force microscope. To verify the influence of keratinocyte neighbors on fibroblasts elasticity (and vice versa), the effect of cellular confluency was studied in parallel. Our results enabled us to distinguish cellular density-related effects from intercellular interactions occurring between fibroblasts and keratinocytes. While the presence of keratinocytes affects fibroblasts spreading capability and mechanical properties, the keratinocytes remain unaffected by the fibroblasts. These results highlight the importance of the cellular deformability in understanding of the role of biomechanics in double paracrine signalling as fibroblast-keratinocyte interaction can change the potential of the wound healing.     

Exploring Hypotheses of the Actions of TGF-β1 in Epidermal Wound Healing Using a 3D Computational Multiscale Model of the Human Epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-β1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-β1 literature–derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units (keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-β1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged (by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-β1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-β1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing.     

A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair

Integrins are ubiquitous transmembrane receptors that play crucial roles in cell-cell and cell-matrix interactions. In this study, we have determined the effects of the loss of beta 1 integrins in keratinocytes in vitro and during cutaneous wound repair. Flow cytometry of cultured beta 1-deficient keratinocytes confirmed the absence of beta 1 integrins and showed downregulation of alpha 6 beta 4 but not of alpha v integrins. beta 1-null keratinocytes were characterised by poor adhesion to various substrates, by a reduced proliferation rate and by a strongly impaired migratory capacity. In vivo, the loss of beta 1 integrins in keratinocytes caused a severe defect in wound healing. beta 1-null keratinocytes showed impaired migration and were more densely packed in the hyperproliferative epithelium. Surprisingly, their proliferation rate was not reduced in early wounds and even increased in late wounds. The failure in re-epithelialisation resulted in a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes. Ultimately, beta 1-deficient epidermis did cover the wound bed, but the epithelial architecture was abnormal. These findings demonstrate a crucial role of beta 1 integrins in keratinocyte migration and wound re-epithelialisation. Movies available on-line     

Interaction of keratinocytes and fibroblasts modulates the expression of matrix metalloproteinases-2 and -9 and their inhibitors

Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process.     

Interaction of keratinocytes and fibroblasts modulates the expression of matrix metalloproteinases-2 and -9 and their inhibitors

Disruption of epidermal-mesenchymal communication due to a delay in epithelialization, increases the frequency of developing fibrotic conditions in skin. As matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) are two key enzymes involved in wound healing and tissue remodeling, here we examined the efficacy of keratinocyte-fibroblast interaction on modulation of these enzymes and their inhibitors. The conditioned media derived from keratinocytes and fibroblasts grown in upper and lower chambers of a co-culture system, respectively, were analyzed for MMP-2 and -9. Keratinocyte or fibroblast conditioned medium (FCM) was used as a control. Gelatinolytic activity analyzed by zymography showed that keratinocytes mainly express MMP-9 and to a lesser extent MMP-2; while fibroblasts express only MMP-2. In a co-culture system, the activities of both MMP-2 and MMP-9 markedly increased in conditioned media collected from bottom chambers. These findings were consistent with the level of MMP-2 and MMP-9 measured by Western blot. Using the same experimental setting, the levels of tissue inhibitors of MMPs (TIMPs) secreted by keratinocytes and fibroblasts grown in the same co-culture system were also evaluated. Western blot showed that fibroblasts secrete only TIMP-1 and TIMP-2 whose levels were increased by co-culturing fibroblasts with keratinocytes. In contrary the level of TIMP-3, which was mainly expressed by keratinocytes, increased by co-culturing these cells with fibroblasts. In conclusion, interaction of fibroblast-keratinocyte modulates the levels of MMP-2 and -9 and their inhibitors produced by these cells and this interaction may be critical for a better healing quality at a late stage of the wound healing process. (Mol Cell Biochem 269: 209–216, 2005)     

Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin

As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication.     

Direct evidence for spatial and temporal regulation of transforming growth factor beta 1 expression during cutaneous wound healing

The expression of transforming growth factor (TGF beta 1) protein in human and porcine skin has been analyzed by immunohistochemistry with two polyclonal antibodies (anti-CC and anti-LC) following cutaneous injury. The anti-LC antibody binds intracellular TGF beta 1 constitutively expressed in the nonproliferating, differentiated suprabasal keratinocytes in the epidermis of normal human skin, while the anti-CC antibody does not react with the form of TGF beta 1 present in normal skin as previously shown. TGF beta 1 may play a role in wound healing as suggested by its effect on multiple cell types in vitro and its acceleration of wound repair in animals. We have evaluated the natural expression and localization of TGF beta 1 protein in situ during initiation, progression, and resolution of the wound healing response in two models of cutaneous injury: the human suction blister and the dermatome excision of partial thickness procine skin. Anti-CC reactive TGF beta 1 in the epidermis is rapidly induced within 5 minutes following injury and progresses outward from the site of injury. The induction reflects a structural or conformational change in TGF beta 1 protein and can be blocked by the protease inhibitor leupeptin or by EDTA, suggesting a change in TGF beta 1 activity. One day post-injury anti-CC reactive TGF beta 1 is present in all epidermal keratinocytes adjacent to the wound including the basal cells. This corresponds temporally to the transient block of the basal keratinocyte mitotic burst following epithelial injury. Three to 4 days post-injury anti-CC reactive TGF beta 1 is localized around the suprabasal keratinocytes, in blood vessels, and in the papillary dermis in cellular infiltrates. The exclusion of TGF beta 1 from the rapidly proliferating basal cells and its extracellular association with suprabasal keratinocytes may represent physiological compartmentation of TGF beta 1 activity. Anti-CC staining is strong in the leading edge of the migrating epithelial sheet. The constitutive anti-LC reactivity with suprabasal keratinocytes seen in normal epidermis is neither relocalized nor abolished adjacent to the injury, but anti-LC staining is absent in the keratinocytes migrating within the wound. As the wound healing response resolves and the skin returns to normal, anti-CC reactive TGF beta 1 disappears while constitutive anti-LC reactive TGF beta 1 persists. Thus, changes in the structure or conformation of TGF beta 1, its localization, and perhaps its activity vary in a spatial and temporal manner following cutaneous injury and correlate with physiological changes during wound healing.     

Growth-regulated peptide-alpha (GRO-alpha) production by oral keratinocytes: a comparison with skin keratinocytes

Growth regulated peptide (GRO-alpha) is chemotactic for neutrophils. It also stimulates keratinocyte proliferation and migration, and angiogenesis in cutaneous wound healing. We compared GRO-alpha production by normal human skin and oral keratinocytes, and the effects of cytokine stimulation. Resting keratinocytes produced little, if any, GRO-alpha. TNF-alpha induced a large increase in GRO-alpha mRNA and protein production in both cell types (P<0.001). However, the response of oral keratinocytes was significantly higher (P<0.01). Oral, but not skin, keratinocytes also produced significant amounts of GRO-alpha in response to IL-1 alpha (P<0.005) and IL-4 (P<0.01) stimulation. Indeed, there was an additive effect on GRO-alpha production when oral keratinocytes were stimulated with combinations of TNF-alpha and IL-1 alpha or TNF-alpha and IL-4. Neither cell type responded to interferon gamma. Keratinocyte GRO-alpha production may help selectively recruit neutrophils in mucocutaneous inflammatory diseases, and differences in production by skin and oral keratinocyte could explain the different presentation of these diseases at the two sites. The increased GRO-alpha responsiveness of oral keratinocytes may also help explain the excellent wound healing properties of oral mucosa.     

miR-96-5p regulates wound healing by targeting BNIP3/FAK pathway

Cutaneous wound healing is a highly orchestrated basic biological process and one of the key processes in restoring skin integrity. The role of microRNAs (miRNAs) during this process has raised numerous attention and is poorly explored. The aim of this study is to investigate the potential function of BCL2 interacting protein (BNIP3) and its target miRNA, miR-96-5p, in cutaneous wound healing. The results demonstrated that BNIP3 was significantly increased and miR-96-5p was obviously decreased during wound healing. Overexpression of BNIP3 significantly increased, while inhibition of BNIP3 decreased cell proliferation and migration of human primary keratinocytes. miR-96-5p was predicted to be a target miRNA for BNIP3 and luciferase reporter assay confirmed that miR-96-5p directly targeted the 3′-untranslated region of BNIP3. Moreover, miR-96-5p overexpression significantly decreased, while miR-96-5p inhibition dramatically increased BNIP3 protein expression and focal adhesion kinase (FAK) pathway activation. Furthermore, miR-96-5p inhibited cell proliferation and migration of human primary keratinocytes. Overall, our findings suggest that miR-96-5p might be critical in the regulation of wound healing by mediating BNIP3 and FAK pathway.     

Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.     

Specific stimulation of migration of human keratinocytes by mu-opiate receptor agonists

There are several indications that neuropeptides, especially the opiate receptor agonists, modulate the immune response by stimulating the formation of granulation tissue and enhancing the reepithelialization. We observed that the mu-opiate receptor ligand beta-endorphin stimulates the migration of cultured human foreskin keratinocytes. After 1 hour exposure to 1 microM beta-endorphin, the keratinocytes experienced an increase of cell diameter by cellular elongation and stimulation of migration. Dynorphin had a lesser effect under the same condition. The opiate receptor antagonist naltrexone significantly reduced the effect of beta-endorphin on keratinocyte migration. This migratory effect of mu-opiate receptor agonists in vitro indicates that the opioid peptides, released in wounds, could play a key role in the final reepithelialization and tissue regeneration in wound healing. This new knowledge will help us not only to understand the mechanism of wound healing but also to improve the therapeutic strategy in the healing of painful chronic wounds.     

E2F-1 is essential for normal epidermal wound repair.

E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1(-/-) keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins alpha(5), alpha(6), beta(1), and beta(4) in E2F-1(-/-) keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1(-/-) keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-gamma1. E2F-1(-/-) keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G(1) and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1(-/-) mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1(-/-) mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of re-epithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo.     

Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.     

14-3-3ζ/τ heterodimers regulate Slingshot activity in migrating keratinocytes

Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an α6β4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3ζ, we demonstrate that in keratinocytes 14-3-3ζ/τ heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3ζ acts as a dominant negative and inhibits the interaction between 14-3-3τ and SSH1. These results implicate 14-3-3ζ/τ heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.