Alix Gazel

Pathway-specific Profiling Identifies the NF-κB-dependent Tumor Necrosis Factor α-regulated Genes in Epidermal Keratinocytes

Identification of tumor necrosis factor α (TNFα) as the key agent in inflammatory disorders led to new therapies specifically targeting TNFα and avoiding many side effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNFα, drugs targeting TNFα have a potential risk of delaying wound healing, secondary infections, and cancer. Indeed, increased risks of tuberculosis and carcinogenesis have been reported as side effects after anti-TNFα therapy. TNFα regulates many processes (e.g. immune response, cell cycle, and apoptosis) through several signal transduction pathways that convey the TNFα signals to the nucleus. Hypothesizing that specific TNFα-dependent pathways control specific processes and that inhibition of a specific pathway may yield even more precisely targeted therapies, we used oligonucleotide microarrays and parthenolide, an NF-κB-specific inhibitor, to identify the NF-κB-dependent set of the TNFα-regulated genes in human epidermal keratinocytes. Expression of ∼40% of all TNFα-regulated genes depends on NF-κB; 17% are regulated early (1–4 h post-treatment), and 23% are regulated late (24–48 h). Cytokines and apoptosis-related and cornification proteins belong to the “early” NF-κB-dependent group, and antigen presentation proteins belong to the “late” group, whereas most cell cycle, RNA-processing, and metabolic enzymes are not NF-κB-dependent. Therefore, inflammation, immunomodulation, apoptosis, and differentiation are on the NF-κB pathway, and cell cycle, metabolism, and RNA processing are not. Most early genes contain consensus NF-κB binding sites in their promoter DNA and are, presumably, directly regulated by NF-κB, except, curiously, the cornification markers. Using siRNA silencing, we identified cFLIP/CFLAR as an essential NF-κB-dependent antiapoptotic gene. The results confirm our hypothesis, suggesting that inhibiting a specific TNFα-dependent signaling pathway may inhibit a specific TNFα-regulated process, leaving others unaffected. This could lead to more specific anti-inflammatory agents that are both more effective and safer.

Inhibition of JNK Promotes Differentiation of Epidermal Keratinocytes

In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.

Transcriptional profiling defines the roles of ERK and p38 kinases in epidermal keratinocytes.

Epidermal keratinocytes respond to extracellular influences by activating cytoplasmic signal transduction pathways that change gene expression. Using pathway‐specific transcriptional profiling, we identified the genes regulated by two such pathways, p38 and ERK. These pathways are at the fulcrum of epidermal differentiation, proliferative and inflammatory skin diseases. We used SB203580 and PD98059 as specific inhibitors and Affymetrix Hu133Av2 microarrays, to identify the genes regulated after 1, 4, 24, and 48 h and compared them to genes regulated by JNK. Unexpectedly, inhibition of MAPK pathways is compensated by activation of the NFκB pathway and suppression of the DUSP enzymes. Both pathways promote epidermal differentiation; however, there is a surprising disconnect between the expression of steroid synthesis enzymes and differentiation markers. The p38 pathway induces the expression of extracellular matrix and proliferation‐associated genes, while suppressing microtubule‐associated genes. The ERK pathway induces nuclear envelope and mRNA splicing proteins, while suppressing steroid synthesis and mitochondrial energy production enzymes. Transcription factors SRY, c‐FOS, and N‐Myc are the principal targets of the p38 pathway, Elk‐1 SAP1 and HLH2 of ERK, while FREAC‐4, ARNT and USF are shared. The results suggest a list of targets potentially useful in therapeutic interventions in cutaneous diseases and wound healing. J. Cell. Physiol. 215: 292–308, 2008.

Specificity in Stress Response: Epidermal Keratinocytes Exhibit Specialized UV-Responsive Signal Transduction Pathways

UV light, a paradigmatic initiator of cell stress, invokes responses that include signal transduction, activation of transcription factors, and changes in gene expression. Consequently, in epidermal keratinocytes, its principal and frequent natural target, UV regulates transcription of a distinctive set of genes. Hypothesizing that UV activates distinctive epidermal signal transduction pathways, we compared the UV-responsive activation of the JNK and NFkappaB pathways in keratinocytes, with the activation of the same pathways by other agents and in other cell types. Using of inhibitors and antisense oligonucleotides, we found that in keratinocytes only UVB/UVC activate JNK, while in other cell types UVA, heat shock, and oxidative stress do as well. Keratinocytes express JNK-1 and JNK-3, which is unexpected because JNK-3 expression is considered brain-specific. In keratinocytes, ERK1, ERK2, and p38 are activated by growth factors, but not by UV. UVB/UVC in keratinocytes activates Elk1 and AP1 exclusively through the JNK pathway. JNKK1 is essential for UVB/UVC activation of JNK in keratinocytes in vitro and in human skin in vivo. In contrast, in HeLa cells, used as a control, crosstalk among signal transduction pathways allows considerable laxity. In parallel, UVB/UVC and TNFalpha activate the NFkappaB pathway via distinct mechanisms, as shown using antisense oligonucleotides targeted against IKKbeta, the active subunit of IKK. This implies a specific UVB/UVC responsive signal transduction pathway independent from other pathways. Our results suggest that in epidermal keratinocytes specific signal transduction pathways respond to UV light. Based on these findings, we propose that the UV light is not a genetic stress response inducer in these cells, but a specific agent to which epidermis developed highly specialized responses.

Transcriptional Profiling Defines the Effects of Nickel in Human Epidermal Keratinocytes

Nickel is a ubiquitous and virtually unavoidable environmental pollutant and occupational hazard, but its molecular and cellular effects are not well understood. Human epidermal keratinocytes are the sentinel and the primary target for nickel. We treated with nickel salts skin equivalents containing differentiating epidermal keratinocytes grown on air–liquid interface in standard cell culture conditions. We identified the transcriptional profiles affected by nickel in reconstructed human epidermis (RHE) using DNA microarrays. The Ni‐regulated genes were determined at two time points, immediate‐early, 30 min after treatment, and late, at 6 h. Using in silico data analysis, we determined that 134 genes are regulated by nickel; of these, 97 are induced and 37 suppressed. Functional categories of regulated genes suggest that Ni inhibits apoptosis, promotes cell cycle and induces synthesis of extracellular matrix proteins and extracellular proteases. Importantly, Ni also regulates a set of secreted signaling proteins, inducing VEGF, amphiregulin, PGF, GDF15, and BST2, while suppressing IL‐18, galectin‐3, and LITAF. These secreted proteins may be important in Ni‐caused allergic reactions. Ni induced inhibitors of the NFκB signaling pathway, and suppressed its activators. Correspondingly, NFκB binding sites were found to be overrepresented in the Ni‐suppressed genes, whereas cFOS/AP1 binding sites were common in the Ni‐induced genes. Significant parallels were found between the Ni‐regulated genes and the genes regulated by TGFβ, EGF, glucocorticoids, or Oncostatin‐M. The comprehensive identification of Ni‐regulated genes in human epidermal equivalents significantly advances our understanding of the molecular effects of nickel in skin. J. Cell. Physiol. 217: 686–692, 2008.