Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.     

Activities of the matrix metalloproteinase stromelysin-2 (MMP-10) in matrix degradation and keratinocyte organization in wounded skin

The matrix metalloproteinase stromelysin-2 is expressed in keratinocytes of the epithelial tongue of skin wounds, suggesting a role in keratinocyte migration. Here, we show that stromelysin-2 enhances migration of cultured keratinocytes. To gain insight into the in vivo activities of stromelysin-2 in epithelial repair, we generated transgenic mice expressing a constitutively active stromelysin-2 mutant in keratinocytes. These animals had no alterations in skin architecture, and the healing rate of skin wounds was normal. Histologically, however, we found abnormalities in the organization of the wound epithelium. Keratinocytes at the migrating epidermal tip were scattered in most sections of mice with high expression level, and there was a reduced deposition of new matrix. In particular, the staining pattern of laminin-5 at the wound site was altered. This may be due to proteolytic processing of laminin-5 by stromelysin-2, because degradation of laminin-5 by this enzyme was observed in vitro. The inappropriate matrix contact of keratinocytes was accompanied by aberrant localization of beta1-integrins and phosphorylated focal adhesion kinase, as well as by increased apoptosis of wound keratinocytes. These results suggest that a tightly regulated expression level of stromelysin-2 is required for limited matrix degradation at the wound site, thereby controlling keratinocyte migration.     

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.     

The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration

After an injury, keratinocytes acquire the plasticity necessary for the reepithelialization of the wound. Here, we identify a novel pathway by which a nuclear hormone receptor, until now better known for its metabolic functions, potentiates cell migration. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARbeta/delta activity amplifies the response of keratinocytes to a chemotactic signal, promotes integrin recycling and remodeling of the actin cytoskeleton, and thereby favors cell migration. Using three-dimensional wound reconstructions, we demonstrate that these defects have a strong impact on in vivo skin healing, since PPARbeta/delta-/- mice show an unexpected and rare epithelialization phenotype. Our findings demonstrate that nuclear hormone receptors not only regulate intercellular communication at the organism level but also participate in cell responses to a chemotactic signal. The implications of our findings may be far-reaching, considering that the mechanisms described here are important in many physiological and pathological situations.     

Normal development, wound healing, and adenovirus susceptibility in beta5-deficient mice

Integrins have been shown to play important roles in embryonic development, wound healing, metastasis, and other biological processes. alphavbeta5 is a receptor for RGD-containing extracellular matrix proteins that has been suggested to be important in cutaneous wound healing and adenovirus infection. To examine the in vivo function of this receptor, we have generated mice lacking beta5 expression, using homologous recombination in embryonic stem cells. Mice homozygous for a null mutation of the beta5 subunit gene develop, grow, and reproduce normally. Keratinocytes harvested from beta5(-/-) mice demonstrate impaired migration on and adhesion to the alphavbeta5 ligand, vitronectin. However, the rate of healing of cutaneous wounds is not different in beta5(-/-) and beta5(+/+) mice. Furthermore, keratinocytes and airway epithelial cells obtained from null mice show adenovirus infection efficiency equal to that from wild-type mice. These data suggest that alphavbeta5 is not essential for normal development, reproduction, adenovirus infection, or the healing of cutaneous wounds.     

Impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis

Recently, we demonstrated a strong upregulation of activin expression after skin injury. Furthermore, overexpression of this transforming growth factor beta family member in the skin of transgenic mice caused dermal fibrosis, epidermal hyperthickening and enhanced wound repair. However, the role of endogenous activin in wound healing has not been determined. To address this question we overexpressed the soluble activin antagonist follistatin in the epidermis of transgenic mice. These animals were born with open eyes, and the adult mice had larger ears, longer tails and reduced body weight compared with non-transgenic littermates. Their skin was characterized by a mild dermal and epidermal atrophy. After injury, a severe delay in wound healing was observed. In particular, granulation tissue formation was significantly reduced, leading to a major reduction in wound breaking strength. The wounds, however, finally healed, and the resulting scar area was smaller than in control animals. These results implicate an important function of endogenous activin in the control of wound repair and scar formation.     

Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing

The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin. We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization. Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells. Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia.     

Gender specific effects on the actin-remodelling protein Flightless I and TGF-beta1 contribute to impaired wound healing in aged skin

Impaired wound healing in the elderly presents a major clinical challenge. Understanding the cellular mechanisms behind age-related impaired healing is vital for developing new wound therapies. Here we show that the actin-remodelling protein, Flightless I (FliI) is a contributing factor to the poor healing observed in elderly skin and that gender plays a major role in this process. Using young and aged, wild-type and FliI overexpressing mice we found that aging significantly elevated FliI expression in the epidermis and wound matrix. Aging exacerbated the negative effect of FliI on wound repair and wounds in aged FliI transgenic mice were larger with delayed reepithelialisation. When the effect of gender was further analysed, despite increased FliI expression in young and aged male and female mice, female FliI transgenic mice had the most severe wound healing phenotype suggesting that male mice were refractory to FliI gene expression. Of potential importance, males, but not females, up-regulated transforming growth factor-β1 and this was most pronounced in aged male FliI overexpressing wounds. As FliI also functions as a co-activator of the estrogen nuclear receptor, increasing concentrations of β-estradiol were added to skin fibroblasts and keratinocytes and significantly enhanced FliI expression and translocation of FliI from the cytoplasm to the nucleus was observed. FliI further inhibited estrogen-mediated collagen I secretion suggesting a mechanism via which FliI may directly affect provisional matrix synthesis. In summary, FliI is a contributing factor to impaired healing and strategies aimed at decreasing FliI levels in elderly skin may improve wound repair.     

Peroxisome proliferator-activated receptors and skin development

PPARs are nuclear hormone receptors. PPAR subtypes (alpha, gamma, delta, the latter a xPPARbeta homologue) were initially investigated in skin because of their known role in regulating lipid metabolism. Studies adding specific PPAR ligand activators to cultured skin or skin cells are compatible with the concepts that PPARalpha activation mediates early lipogenic steps common to the function of both skin epidermal cells (keratinocytes) and sebaceous cells (sebocytes), PPARgamma activation plays a unique role in stimulating sebocyte lipogenesis, and PPARdelta activation may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances. Epidermal keratinocytes appear to express small amounts of PPARalpha and PPARdelta mRNA and a trace of PPARgamma mRNA which is up-regulated with differentiation. Sebocytes express all subtypes; PPARgamma gene expression excedes that in epidermis. The emerging data on PPAR protein expression suggests that epidermis normally expresses predominantly PPARalpha, while sebocytes express more PPARgamma than PPARalpha. These expression patterns may change during hyperplasia, differentiation and inflammation. Gene disruption studies in mice are compatible with a contribution of PPARalpha to skin barrier function, suggest that PPARgamma is necessary for sebocyte differentiation, and indicate that PPARdelta can ameliorate inflammatory responses in skin. PPARs appear to play a role in keratinocyte synthesis of the lipids that they export to the intercellular space to form the skin permeability barrier. They also appear to be important for sebocyte formation of the intracellular fused lipid droplets that constitute the holocrine secretion of the sebaceous gland. In addition, they may play roles in keratinocyte growth and differentiation and the inhibition of skin inflammation by diverse mechanisms not necessarily related to fat metabolism.     

Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis.

To elucidate the biological role of Stat3 in the skin, conditional gene targeting using the Cre-loxP system was performed as germline Stat3 ablation leads to embryonic lethality. K5-Cre;Stat3(flox/-) transgenic mice, whose epidermal and follicular keratinocytes lack functional Stat3, were viable and the development of epidermis and hair follicles appeared normal. However, hair cycle and wound healing processes were severely compromised. Furthermore, mutant mice expressed sparse hair and developed spontaneously occurring ulcers with age. Growth factor-dependent in vitro migration of Stat3-disrupted keratinocytes was impaired despite normal proliferative responses. We therefore conclude that Stat3 plays a crucial role in transducing a signal required for migration but not for proliferation of keratinocytes, and that Stat3 is essential for skin remodeling, including hair cycle and wound healing.     

8S-lipoxygenase products activate peroxisome proliferator-activated receptor alpha and induce differentiation in murine keratinocytes.

To determine the function and mechanism of action of the 8S-lipoxygenase (8-LOX) product of arachidonic acid, 8S-hydroxyeicosatetraenoic acid (8S-HETE), which is normally synthesized only after irritation of the epidermis, transgenic mice with 8-LOX targeted to keratinocytes through the use of a loricrin promoter were generated. Histological analyses showed that the skin, tongue, and stomach of transgenic mice are highly differentiated, and immunoblotting and immunohistochemistries of skin showed higher levels of keratin-1 expression compared with wild-type mice. The labeling index, however, of the transgenic epidermis was twice that of the wild-type epidermis. Furthermore, 8S-HETE treatment of wild-type primary keratinocytes induced keratin-1 expression. Peroxisome proliferator activated receptor alpha (PPARalpha) was identified as a crucial component of keratin-1 induction through transient transfection with expression vectors for PPARalpha, PPARgamma, and a dominant-negative PPAR, as well as through the use of known PPAR agonists. From these studies, it is concluded that 8S-HETE plays an important role in keratinocyte differentiation and that at least some of its effects are mediated by PPARalpha.     

Regulation of eNOS in normal and diabetes-impaired skin repair: implications for tissue regeneration.

An important role of inducible nitric oxide (NO) synthase for epithelial action during skin repair has been well established. Although a delayed healing of skin wounds has been recently described for eNOS-deficient mice, a participation of endothelial-type NO synthase (eNOS) in skin repair largely remains unclear. In this study we determined the expression pattern of eNOS during wound healing in healthy and in diabetic mice. Remarkably, normal repair in healthy animals was characterized by a moderate induction of eNOS at the mRNA and protein level, whereas diabetes-impaired healing was associated with a clearly reduced eNOS protein expression. Immunohistochemistry revealed the endothelial lining of blood vessels within the granulation tissue, and also keratinocytes of the wound margins, the developing neo-epithelium, and the hair follicles to express eNOS protein. Keratinocyte-derived expression of eNOS could be confirmed at the mRNA level in vitro for human primary keratinocytes and the keratinocyte cell line HaCaT. Furthermore, eNOS enzymatic activity most likely contributes to epithelial regeneration, as eNOS-deficient (eNOS -/-) animals exhibited reduced wound margin epithelia associated with reduced keratinocyte proliferation.     

Comparison of proliferation and motile activity between human keratinocytes isolated from skin and oral mucosa

OBJECTIVE: Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. For healing of skin wounds the skin keratinocytes can be replaced by oral mucosa epithelial cells grown in vitro. The presented experiments were carried out in order to compare the proliferation, morphology, and migration between human keratinocytes isolated from human skin and oral mucosa. MATERIALS AND METHODS: Human epidermal and oral mucosa keratinocytes from primary culture were used in all experiments. Cell motility and shape were determined using computer-aided methods. RESULTS AND CONCLUSIONS: It was demonstrated that although both cell types exhibit the same typical epithelial morphology, oral mucosa keratinocytes locomote significantly faster than skin keratinocytes. They also differ in proliferation activity. Oral mucosa keratinocytes exhibited faster growth and different actin cytoskeleton organisation than skin keratinocytes under in vitro conditions. Autologous oral mucosa keratinocytes may be expanded in vitro and used for skin wound healing in vivo.     

Cyclophilin C-associated protein is up-regulated during wound healing

Cyclophilin C-associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon-gamma increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNgamma and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing.     

Overexpression of TIMP-1 under the MMP-9 promoter interferes with wound healing in transgenic mice

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.The expert technical assistance of M. Jarva, L. Ollitervo, S. Kangas, and R. Jokisalo is gratefully acknowledged. This work was supported in part by grants from the Finnish Academy of Science, the Swedish Cancer Foundation, the Novo Nordisk Foundation and EC contract QLG1-CT-2000-01131 (K.T.), the Finnish Dental Society Apollonia and the Northern Finland Cancer Foundation (M.P.), as well as the K. Albin Johansson Foundation and the Einar and Karin Stroems Foundation (E.P.)     

Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair

The ability to heal wounds is vital to all organisms. In mammalian tissues, alterations in intermediate filament (IF) gene expression represent an early reaction of cells surviving injury. We investigated the role of keratin IFs during the epithelialization of skin wounds using a keratin 6alpha and 6beta (K6alpha/K6beta)-null mouse model. In skin explant culture, null keratinocytes exhibit an enhanced epithelialization potential due to increased migration. The extent of the phenotype is strain dependent, and is accompanied by alterations in keratin IF and F-actin organization. However, in wounded skin in vivo, null keratinocytes rupture as they attempt to migrate under the blood clot. Fragility of the K6alpha/K6beta-null epidermis is confirmed when applying trauma to chemically treated skin. We propose that the alterations in IF gene expression after tissue injury foster a compromise between the need to display the cellular pliability necessary for timely migration and the requirement for resilience sufficient to withstand the rigors of a wound site.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.