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 NFkappaB 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.

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 NFkappaB signaling pathway, and suppressed its activators. Correspondingly, NFkappaB 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 TGFbeta, 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.

Transcriptional profiling of epidermal differentiation.

In epidermal differentiation basal keratinocytes detach from the basement membrane, stop proliferating, and express a new set of structural proteins and enzymes, which results in an impermeable protein/lipid barrier that protects us. To define the transcriptional changes essential for this process, we purified large quantities of basal and suprabasal cells from human epidermis, using the expression of beta4 integrin as the discriminating factor. The expected expression differences in cytoskeletal, cell cycle, and adhesion genes confirmed the effective separation of the cell populations. Using DNA microarray chips, we comprehensively identify the differences in genes expressed in basal and differentiating layers of the epidermis, including the ECM components produced by the basal cells, the proteases in both the basal and suprabasal cells, and the lipid and steroid metabolism enzymes in suprabasal cells responsible for the permeability barrier. We identified the signaling pathways specific for the two populations and found two previously unknown paracrine and one juxtacrine signaling pathway operating between the basal and suprabasal cells. Furthermore, using specific expression signatures, we identified a new set of late differentiation markers and mapped their chromosomal loci, as well as a new set of melanocyte-specific markers. The data represent a quantum jump in understanding the mechanisms of epidermal differentiation.

Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays.

Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large "transcriptome." To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis.

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.

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.

A characteristic subset of psoriasis-associated genes is induced by oncostatin-M in reconstituted epidermis.

The pathological manifestations of psoriasis are orchestrated by many secreted proteins, but only a handful, tumor necrosis factor-alpha, IFN-gamma and IL-1, have been studied in great detail. Oncostatin-M (OsM) has also been found in psoriatic skin and we hypothesized that it makes a unique and characteristic contribution to the psoriatic processes. To define in-depth the molecular effects of OsM in epidermis, we used high-density DNA microarrays for transcriptional profiling of OsM-treated human skin equivalents. We identified 374 unambiguously OsM-regulated genes, out of 22,000 probed. OsM suppressed the expression of the "classical" epidermal differentiation markers, but strongly and specifically induced the S100A proteins. Cytoskeletal and complement proteins, proteases, and their inhibitors were also induced by OsM. Interestingly, a large set of genes was induced by OsM at early time points but suppressed later; these genes are known regulatory targets of IFN and thus provide a nexus between the OsM and IFN pathways. OsM induces IL-4 and suppresses the T-helper 1-type and IL-1-responsive signals, potentially attenuating the psoriatic pathology. The data suggest that OsM plays a unique role in psoriasis, different from all other, more thoroughly studied cytokines.

Pathway-specific profiling identifies the NF-kappa B-dependent tumor necrosis factor alpha-regulated genes in epidermal keratinocytes.

Identification of tumor necrosis factor alpha (TNF alpha) as the key agent in inflammatory disorders led to new therapies specifically targeting TNF alpha and avoiding many side effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNF alpha, drugs targeting TNF alpha 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-TNF alpha therapy. TNF alpha regulates many processes (e.g. immune response, cell cycle, and apoptosis) through several signal transduction pathways that convey the TNF alpha signals to the nucleus. Hypothesizing that specific TNF alpha-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-kappa B-specific inhibitor, to identify the NF-kappa B-dependent set of the TNF alpha-regulated genes in human epidermal keratinocytes. Expression of approximately 40% of all TNF alpha-regulated genes depends on NF-kappa B; 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-kappa B-dependent group, and antigen presentation proteins belong to the "late" group, whereas most cell cycle, RNA-processing, and metabolic enzymes are not NF-kappa B-dependent. Therefore, inflammation, immunomodulation, apoptosis, and differentiation are on the NF-kappa B pathway, and cell cycle, metabolism, and RNA processing are not. Most early genes contain consensus NF-kappaB binding sites in their promoter DNA and are, presumably, directly regulated by NF-kappa B, except, curiously, the cornification markers. Using siRNA silencing, we identified cFLIP/CFLAR as an essential NF-kappa B-dependent antiapoptotic gene. The results confirm our hypothesis, suggesting that inhibiting a specific TNF alpha-dependent signaling pathway may inhibit a specific TNF alpha-regulated process, leaving others unaffected. This could lead to more specific anti-inflammatory agents that are both more effective and safer.

Transcriptional responses of human epidermal keratinocytes to Oncostatin-M.

Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes.

Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling.

Identification of tumor necrosis factor-alpha (TNF alpha) as the key agent in inflammatory disorders, e.g. rheumatoid arthritis, Crohn's disease, and psoriasis, led to TNF alpha-targeting therapies, which, although avoiding many of the side-effects of previous drugs, nonetheless causes other side-effects, including secondary infections and cancer. By controlling gene expression, TNF alpha orchestrates the cutaneous responses to environmental damage and inflammation. To define TNF alpha action in epidermis, we compared the transcriptional profiles of normal human keratinocytes untreated and treated with TNF alpha for 1, 4, 24, and 48 h by using oligonucleotide microarrays. We found that TNF alpha regulates not only immune and inflammatory responses but also tissue remodeling, cell motility, cell cycle, and apoptosis. Specifically, TNF alpha regulates innate immunity and inflammation by inducing a characteristic large set of chemokines, including newly identified TNF alpha targets, that attract neutrophils, macrophages, and skin-specific memory T-cells. This implicates TNF alpha in the pathogenesis of psoriasis, fixed drug eruption, atopic and allergic contact dermatitis. TNF alpha promotes tissue repair by inducing basement membrane components and collagen-degrading proteases. Unexpectedly, TNF alpha induces actin cytoskeleton regulators and integrins, enhancing keratinocyte motility and attachment, effects not previously associated with TNF alpha. Also unanticipated was the influence of TNF alpha upon keratinocyte cell fate by regulating cell-cycle and apoptosis-associated genes. Therefore, TNF alpha initiates not only the initiation of inflammation and responses to injury, but also the subsequent epidermal repair. The results provide new insights into the harmful and beneficial TNF alpha effects and define the mechanisms and genes that achieve these outcomes, both of which are important for TNF alpha-targeted therapies.