Role of NF-kappaB in the apoptotic-resistant phenotype of keratinocytes

Several studies point to a role for NF-kappaB in modulating epidermal thickness and apoptotic susceptibility of keratinocytes. When phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) are topically applied, prominent epidermal thickening occurs, and exposure to interferon (IFN)-gamma promotes increased epidermal thickness producing psoriatic lesions. While keratinocytes derived from psoriatic plaque resist apoptosis, and combination of TPA and IFN-gamma activates NF-kappaB, the molecular mechanism linking NF-kappaB activation and keratinocyte apoptosis resistance was unknown. Therefore, we examined the ability of IFN-gamma plus TPA to influence NF-kappaB activity, gene expression, and response to UV light-induced apoptosis. These responses in normal keratinocytes were compared with immortalized keratinocytes (HaCaT cells). Exposure of normal keratinocytes to IFN-gamma plus TPA produced a synergistic activation of NF-kappaB, compared with when each reagent was used individually. Normal keratinocytes when exposed to IFN-gamma plus TPA acquired a resistance to UV light-induced apoptosis, which was dependent on NF-kappaB because expression of a dominant negative form of IkappaBalpha overcame the resistance. Compared with normal keratinocytes, HaCaT cells have a dysfunctional constitutive NF-kappaB signaling pathway not induced by IFN-gamma and TPA, rendering HaCaT cells highly susceptible to UV-induced apoptosis. Thus, immortalized HaCaT cells have an abnormal constitutive and dysfunctional NF-kappaB signaling system. These results provide evidence that activation and proper regulation of NF-kappaB is essential for acquisition of an apoptotic-resistant phenotype for epidermal-derived keratinocytes.     

STAT-1 alpha and IFN-gamma as modulators of TNF-alpha signaling in macrophages: regulation and functional implications of the TNF receptor 1:STAT-1 alpha complex

TNF-alpha and IFN-gamma cooperate in the activation of macrophages. TNF-alpha-dependent activation of NF-kappaB is stronger in the presence of IFN-gamma. STAT-1alpha associates with TNFR1 in TNF-alpha-treated cells, and this association attenuates TNF-alpha-mediated NF-kappaB activation. We hypothesized that nuclear localization of STAT-1alpha due to IFN-gamma signaling would preclude it from being recruited to the TNFR1 and therefore enhance TNF-alpha-induced NF-kappaB activation. In the RAW264.7 macrophage cell line, TNF-alpha treatment indeed recruits STAT-1alpha to the TNFR1, and this association is abrogated when cells are exposed to IFN-gamma. TNF-alpha treatment induces a more robust activation of NF-kappaB in STAT-1alpha-deficient cells, and restoration of STAT-1alpha inhibits TNF-alpha-dependent NF-kappaB activation. Our results suggest that a receptor-proximal level of cross-talk exists between these two cytokine pathways: IFN-gamma limits STAT-1alpha availability to the TNFR1 by depleting STAT-1alpha from the cytoplasm, thus allowing for optimal NF-kappaB activation upon TNF-alpha ligation.     

IL-4 and IL-13 negatively regulate TNF-alpha- and IFN-gamma-induced beta-defensin expression through STAT-6, suppressor of cytokine signaling (SOCS)-1, and SOCS-3

Human beta-defensins (HBDs) are a major class of antimicrobial peptides that play an important role in the innate immune response, however, the induction and regulation of these antimicrobial peptides is not well understood. We demonstrate here that stimulation of keratinocytes with TNF-alpha/IFN-gamma induces HBD-2 and HBD-3 by activating STAT-1 and NF-kappaB signaling. We further demonstrate that IL-4 and IL-13 activate STAT-6 and induce the suppressors of cytokine signaling (SOCS)-1 and -3. This interferes with STAT-1 and NF-kappaB signaling, thereby inhibiting TNF-alpha/IFN-gamma-mediated induction of HBD-2 and HBD-3. These data suggest that targeting the STAT-1-signaling pathway or suppressor of cytokine signaling expression enhances beta-defensin expression and represents a new therapeutic strategy for reduction of infection in human diseases associated with beta-defensin deficiency.     

Identification of novel interaction between annexin A2 and keratin 17: evidence for reciprocal regulation

Keratins are cytoplasmic intermediate filament proteins providing crucial structural support in epithelial cells. Keratin expression has diagnostic and even prognostic value in disease settings, and recent studies have uncovered modulatory roles for select keratin proteins in signaling pathways regulating cell growth and cell death. Elevated keratin expression in select cancers is correlated with higher expression of EGF receptor (EGFR), whose overexpression and/or mutation give rise to cancer. To explore the role of keratins in oncogenic signaling pathways, we examined the regulation of epithelial growth-associated keratin 17 (K17) in response to EGFR activation. K17 is specifically up-regulated in detergent-soluble fraction upon EGFR activation, and immunofluorescence analysis revealed alterations in K17-containing filaments. Interestingly, we identified AnxA2 as a novel interacting partner of K17, and this interaction is antagonized by EGFR activation. K17 and AnxA2 proteins show reciprocal regulation. Modulating expression of AnxA2 altered K17 stability, and AnxA2 overexpression delays EGFR-mediated change in K17 detergent solubility. Down-regulation of K17 expression, in turn, results in decreased AnxA2 phosphorylation at Tyr-23. These findings uncover a novel interaction involving K17 and AnxA2 and identify AnxA2 as a potential regulator of keratin filaments.     

Keratin gene mutations in disorders of human skin and its appendages

Keratins, the major structural protein of all epithelia are a diverse group of cytoskeletal scaffolding proteins that form intermediate filament networks, providing structural support to keratinocytes that maintain the integrity of the skin. Expression of keratin genes is usually regulated by differentiation of the epidermal cells within the stratifying squamous epithelium. Amongst the 54 known functional keratin genes in humans, about 22 different genes including, the cornea, hair and hair follicle-specific keratins have been implicated in a wide range of hereditary diseases. The exact phenotype of each disease usually reflects the spatial expression level and the types of mutated keratin genes, the location of the mutations and their consequences at sub-cellular levels as well as other epigenetic and/or environmental factors. The identification of specific pathogenic mutations in keratin disorders formed the basis of our understanding that led to re-classification, improved diagnosis with prognostic implications, prenatal testing and genetic counseling in severe keratin genodermatoses. Molecular defects in cutaneous keratin genes encoding for keratin intermediate filaments (KIFs) causes keratinocytes and tissue-specific fragility, accounting for a large number of genetic disorders in human skin and its appendages. These diseases are characterized by keratinocytes fragility (cytolysis), intra-epidermal blistering, hyperkeratosis, and keratin filament aggregation in severely affected tissues. Examples include epidermolysis bullosa simplex (EBS; K5, K14), keratinopathic ichthyosis (KPI; K1, K2, K10) i.e. epidermolytic ichthyosis (EI; K1, K10) and ichthyosis bullosa of Siemens (IBS; K2), pachyonychia congenita (PC; K6a, K6b, K16, K17), epidermolytic palmo-plantar keratoderma (EPPK; K9, (K1)), monilethrix (K81, K83, K86), ectodermal dysplasia (ED; K85) and steatocystoma multiplex. These keratins also have been identified to have roles in apoptosis, cell proliferation, wound healing, tissue polarity and remodeling. This review summarizes and discusses the clinical, ultrastructural, molecular genetics and biochemical characteristics of a broad spectrum of keratin-related genodermatoses, with special clinical emphasis on EBS, EI and PC. We also highlight current and emerging model tools for prognostic future therapies. Hopefully, disease modeling and in-depth understanding of the molecular pathogenesis of the diseases may lead to the development of novel therapies for several hereditary cutaneous diseases.     

Suppressor of cytokine signaling 1 inhibits cytokine induction of CD40 expression in macrophages

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer's disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-gamma induces CD40 expression in macrophages and microglia. IFN-gamma leads to STAT-1alpha activation directly and up-regulation of NF-kappaB activity due to the secretion and subsequent autocrine signaling of TNF-alpha. However, TNF-alpha alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1alpha activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-gamma-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-gamma-induced TNF-alpha secretion, as well as STAT-1alpha and NF-kappaB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-gamma-mediated STAT-1alpha activation, which also then results in suppression of IFN-gamma-induced TNF-alpha secretion and subsequent NF-kappaB activation.