Susanne Braun

Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound

ABSTRACT Keratinocyte growth factor (KGF) is a potent mitogen for epithelial cells, and it promotes survival of these cells under stress conditions. In a search for KGF-regulated genes in keratinocytes, we identified the gene encoding the transcription factor NF-E2-related factor 2 (Nrf2). Nrf2 is a key player in the cellular stress response. This might be of particular importance during wound healing, where large amounts of reactive oxygen species are produced as a defense against invading bacteria. Therefore, we studied the wound repair process in Nrf2 knockout mice. Interestingly, the expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf2 knockout animals, and the late phase of repair was characterized by prolonged inflammation. However, these differences in gene expression were not reflected by obvious histological abnormalities. The normal healing rate appears to be at least partially due to an up-regulation of the related transcription factor Nrf3, which was also identified as a target of KGF and which was coexpressed with Nrf2 in the healing skin wound. Taken together, our results reveal novel roles of the KGF-regulated transcription factors Nrf2 and possibly Nrf3 in the control of gene expression and inflammation during cutaneous wound repair.

Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing.

ABSTRACT The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.

Keratinocyte growth factor: effects on keratinocytes and mechanisms of action.

Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, and it can protect these cells from various insults. Due to these properties, it is of particular importance for the repair of injured epithelial tissues, and it is currently therapeutically explored for the treatment of radiation- and chemotherapy-induced mucosal epithelial damage in cancer patients. In this review we summarize the current knowledge on the role of KGF in tissue repair and cytoprotection, and we report on its mechanisms of action in keratinocytes.

Roles and mechanisms of action of the Nrf2 transcription factor in skin morphogenesis, wound repair and skin cancer

Roles and mechanisms of action of the Nrf2 transcription factor in skin morphogenesis, wound repair and skin cancer

Keratinocyte growth factor protects epidermis and hair follicles from cell death induced by UV irradiation, chemotherapeutic or cytotoxic agents

Owing to its potent cytoprotective properties for epithelial cells, keratinocyte growth factor (KGF) is successfully used for the treatment of chemotherapy- and radiotherapy-induced oral mucositis in cancer patients. It is therefore of major interest to determine possible clinical applications of KGF in other organs and in different stress situations and to unravel common and organ-specific mechanisms of KGF action. Here we show that KGF protects human keratinocytes from the toxicity of xenobiotics with electrophilic and oxidative properties and reduces the cell death induced by UV irradiation. In contrast to other cell types, cytoprotection of keratinocytes by KGF is not a direct anti-apoptotic effect but requires de novo protein synthesis. The in vitro findings are clinically relevant because KGF protected keratinocytes in organ-cultured human scalp hair follicles from the toxicity of the xenobiotic menadione. Moreover, injection of KGF into murine back skin markedly reduced cell death in the epidermis after UVB irradiation. This activity is dependent on FGF receptor signaling because it was abrogated in transgenic mice expressing a dominant-negative FGF receptor mutant in keratinocytes. Taken together, our results encourage the use of KGF for skin protection from chemical and physical insults.

Id proteins: novel targets of activin action, which regulate epidermal homeostasis.

Activin is a member of the transforming growth factor β (TGF-β) family, which plays a crucial role in skin morphogenesis and wound healing. To gain insight into the underlying mechanisms of action, we searched for activin-regulated genes in cultured keratinocytes. One of the identified target genes encodes Id1, a negative regulator of helix–loop–helix transcription factors. We show that Id1, Id2, and Id3 are strongly downregulated by activin in keratinocytes in vitro and in vivo. To determine the role of Id1 in keratinocyte biology, we generated stable HaCaT keratinocyte cell lines overexpressing this protein. Our results revealed that enhanced levels of Id1 do not affect proliferation of keratinocytes in monoculture under exponential culture conditions or in response to activin or TGF-β1. However, in three-dimensional organotypic cultures, Id1-overexpressing HaCaT cells formed a hyperthickened and disorganized epithelium that was characterized by enhanced keratinocyte proliferation, abnormal differentiation, and an increased rate of apoptosis. These results identify an important function of Id1 in the regulation of epidermal homeostasis.

Novel roles of NM23 proteins in skin homeostasis, repair and disease

Keratinocyte growth factor (KGF) is an important regulator of epidermal homeostasis and repair. Therefore, the identification of KGF target genes in keratinocytes should contribute to our understanding of the molecular mechanisms underlying these processes. In a search for KGF-regulated genes, we identified the gene encoding the nucleoside diphosphate kinase NM23-H1. Apart from a housekeeping function, NM23 proteins are involved in the regulation of many cellular processes as well as in tumor metastasis, but their functions in epidermal homeostasis and repair are largely unknown. Here, we show a high expression of NM23-H1 and NM23-H2 in the KGF-responsive keratinocytes of the hyperproliferative epidermis of mouse skin wounds and of patients suffering from the skin disease psoriasis. To determine if this overexpression is functionally important, we generated HaCaT keratinocyte cell lines overexpressing NM23-H1 and/or -H2. Whereas the enhanced levels of NM23 did not affect cell proliferation in monoculture, NM23-H2 and double transfectants but not NM23-H1 transfectants formed a strongly hyperthickened epithelium in three-dimensional organotypic cultures. The abnormal epithelial morphology resulted from enhanced proliferation, reduced apoptosis and alterations in the differentiation pattern. These findings suggest that epidermal homeostasis depends on a tight regulation of the levels of NM23 isoforms.

Upregulation and activation of the Nrf-1 transcription factor in the lesioned hippocampus as a novel neuroprotective mechanism against reactive oxygen species

The Nrf‐1 and Nrf‐2 transcription factors play a pivotal role in the cellular defence against the toxic effects of reactive oxygen species (ROS). Although ROS are key effectors of neuronal death after ischaemic and traumatic brain injury, it is not known whether Nrf‐1 and Nrf‐2 are involved in neuroprotective signalling. Here, we analysed the temporal and spatial expression pattern of Nrf‐1 and Nrf‐2 after unilateral excitotoxic lesion of mouse hippocampus. In marked contrast to previous in vitro studies, where upregulation of these transcription factors on the mRNA level was never detected, we found a strong induction of Nrf‐1 mRNA and protein expression in neurons of the lesioned hippocampus, accompanied by a weak elevation of Nrf‐2 mRNA levels. Nrf‐1 predominantly localized to the nucleus in the injured hippocampus. Furthermore, expression of the cytoprotective enzyme, heme oxygenase‐1, a major target of Nrf‐1 and Nrf‐2 action, was coregulated with Nrf‐1 in the same hippocampal neurons, suggesting that Nrf‐1 is functionally active. Because Nrf‐1 and Nrf‐2 are potent inducers of various cytoprotective proteins, our data suggest a role of Nrf‐1 and Nrf‐2 in neuronal survival after acute brain injury.

Upregulation and activation of the Nrf‐1 transcription factor in the lesioned hippocampus

The Nrf‐1 and Nrf‐2 transcription factors play a pivotal role in the cellular defence against the toxic effects of reactive oxygen species (ROS). Although ROS are key effectors of neuronal death after ischaemic and traumatic brain injury, it is not known whether Nrf‐1 and Nrf‐2 are involved in neuroprotective signalling. Here, we analysed the temporal and spatial expression pattern of Nrf‐1 and Nrf‐2 after unilateral excitotoxic lesion of mouse hippocampus. In marked contrast to previous in vitro studies, where upregulation of these transcription factors on the mRNA level was never detected, we found a strong induction of Nrf‐1 mRNA and protein expression in neurons of the lesioned hippocampus, accompanied by a weak elevation of Nrf‐2 mRNA levels. Nrf‐1 predominantly localized to the nucleus in the injured hippocampus. Furthermore, expression of the cytoprotective enzyme, heme oxygenase‐1, a major target of Nrf‐1 and Nrf‐2 action, was coregulated with Nrf‐1 in the same hippocampal neurons, suggesting that Nrf‐1 is functionally active. Because Nrf‐1 and Nrf‐2 are potent inducers of various cytoprotective proteins, our data suggest a role of Nrf‐1 and Nrf‐2 in neuronal survival after acute brain injury.