Miroslav Blumenberg

Novel Genomic Effects of Glucocorticoids in Epidermal Keratinocytes INHIBITION OF APOPTOSIS, INTERFERON-γ PATHWAY, AND WOUND HEALING ALONG WITH PROMOTION OF TERMINAL DIFFERENTIATION

Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48, and 72 h using large scale microarray analyses. The majority of genes were found to be regulated only after 24 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFNγ-regulated genes, including IFNγ receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UV-induced keratinocyte apoptosis. Consequently, treatment with GCs blocked UV-induced apoptosis of keratinocytes. GCs have profound effect on wound healing by inhibiting cell motility and the expression of the proangiogenic factor, vascular endothelial growth factor. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGFβ1 and -2 and MMP1, -2, -9, and -10 and inducing TIMP-2. Finally, GCs promote terminal epidermal differentiation while simultaneously inhibiting early stage differentiation. These results provide new insights into the beneficial and adverse effects of GCs in the epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies.

Transforming Growth Factor-β and microRNA:mRNA Regulatory Networks in Epithelial Plasticity

Noncoding microRNAs act as posttranscriptional repressors of gene function and are often deregulated in cancers and other diseases. Here we review recent findings on microRNA roles in tumorigenesis and report a microRNA profiling screen in transforming growth factor-β1 (TGF-β)-induced epithelial-mesenchymal transition (EMT) in human keratinocytes, a model of epithelial cell plasticity underlying epidermal injury and skin carcinogenesis. We describe a novel EMT-specific microRNA signature that includes induction of miR-21, a candidate oncogenic microRNA associated with carcinogenesis. By integrating the microRNA screen results with target prediction algorithms and gene expression profiling data, we outline a framework for TGF-β-directed microRNA:messenger RNA (mRNA) regulatory circuitry and discuss its biological relevance for tumor progression.

Rays and arrays: the transcriptional program in the response of human epidermal keratinocytes to UVB illumination

The epidermis, our first line of defense from ultraviolet (UV) light, bears the majority of photodamage, which results in skin thinning, wrinkling, keratosis, and malignancy. Hypothesizing that skin has specific mechanisms to protect itself and the organism from UV damage, we used DNA arrays to follow UV‐caused gene expression changes in epidermal keratinocytes. Of the 6,800 genes examined, UV regulates the expression of at least 198. Three waves of changes in gene expression can be distinguished, 0.5–2, 4–8, and 16–24 h after illumination. The first contains transcription factors, signal transducing, and cytoskeletal proteins that change cell phenotype from a normal, fast‐growing cell to an activated, paused cell. The second contains secreted growth factors, cytokines, and chemokines; keratinocytes, having changed their own physiology, alert the surrounding tissues to the UV damage. The third wave contains components of the cornified envelope, as keratinocytes enhance the epidermal protective covering and, simultaneously, terminally differentiate and die, removing a carcinogenic threat. UV also induces the expression of mitochondrial proteins that provide additional energy, and the enzymes that synthesize raw materials for DNA repair. Using a novel skin organ culture model, we demonstrated that the UV‐induced changes detected in keratinocyte cultures also occur in human epidermis in vivo.

Epidermal signal transduction and transcription factor activation in activated keratinocytes

In the area of biology, many laboratories around the world are dissecting and characterizing signal transduction mechanisms and transcription factors responsive to various growth factors and cytokines, in various cell types. However, because of the differences in systems used, it is not clear whether these systems coexist, whether they interact meaningfully, and what their relative roles are. Epidermal keratinocytes are the perfect cell type in which to integrate this knowledge, because in these cells these mechanisms are known to be relevant. Keratinocytes both produce and respond to growth factors and cytokines, especially in pathological conditions and during wound healing, when the physiology of keratinocytes is altered in a way specified by the presence of a subset growth factors and cytokines. In fact, growth factors and cytokines cause the major changes in gene expression and keratinocyte behavior in various cutaneous diseases. In some cases, such as in wound healing, these responses are highly beneficial; in others, such as in psoriasis, they are pathological. It is not clear at present which are operating in which conditions, which are important for the healing process and which are harmful. Growth factors and cytokines affect keratinocytes sometimes simultaneously, at other times individually. In this manuscript we describe the signal transduction pathways responsible for the effects of interferons, the EGF/TGF alpha family and the TNF alpha/IL-1 family of signaling molecules. We also describe the important transcription factors known to be functional in epidermis, with particular emphasis on those factors that are activated by growth factors and cytokines. Finally, we describe what is known about transcriptional regulation of keratin genes, especially those specifically expressed in pathological processes in the epidermis. We expect that the enhanced understanding of the pathways regulating gene expression in keratinocytes will identify the pharmacological targets, the signal transducing proteins and the corresponding transcription factors, used by growth factors and cytokines. This research will led to development of compounds precisely aimed at those targets, allowing us to isolate and inhibit the harmful side effects of growth factors and cytokines. Such compounds should lead to highly specific and therefore more effective treatments of the cutaneous disorders in which these pathways play significant roles.

Novel Mechanism of Steroid Action in Skin through Glucocorticoid Receptor Monomers

ABSTRACT Glucocorticoids (GCs), important regulators of epidermal growth, differentiation, and homeostasis, are used extensively in the treatment of skin diseases. Using keratin gene expression as a paradigm of epidermal physiology and pathology, we have developed a model system to study the molecular mechanism of GCs action in skin. Here we describe a novel mechanism of suppression of transcription by the glucocorticoid receptor (GR) that represents an example of customizing a device for transcriptional regulation to target a specific group of genes within the target tissue, in our case, epidermis. We have shown that GCs repress the expression of the basal-cell-specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17 but not the differentiation-specific keratins K3 and K10 or the simple epithelium-specific keratins K8, K18, and K19. We have identified the negative recognition elements (nGREs) in all five regulated keratin gene promoters. Detailed footprinting revealed that the function of nGREs is to instruct the GR to bind as four monomers. Furthermore, using cotransfection and antisense technology we have found that, unlike SRC-1 and GRIP-1, which are not involved in the GR complex that suppresses keratin genes, histone acetyltransferase and CBP are. In addition, we have found that GR, independently from GREs, blocks the induction of keratin gene expression by AP1. We conclude that GR suppresses keratin gene expression through two independent mechanisms: directly, through interactions of keratin nGREs with four GR monomers, as well as indirectly, by blocking the AP1 induction of keratin gene expression.

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.

Transcriptional responses of human epidermal keratinocytes to cytokine interleukin-1

Interleukin‐1 is a proinflammatory and immunomodulatory cytokine that plays a crucial role in inflammatory diseases of the skin, including bacterial infections, bullous diseases, UV damage, and especially psoriasis. To characterize the molecular effects of IL‐1 in epidermis, we defined the transcriptional changes in human epidermal keratinocytes 1, 4, 24, and 48 h after treatment with IL‐1α. IL‐1 significantly regulated 388 genes, including genes associated with proteolysis, adhesion, signal transduction, proliferation, and epidermal differentiation. IL‐1 induces many genes that have antimicrobial function. Secreted cytokines, chemokines, growth factors, and their receptors are the prominent targets of IL‐1 regulation, including IL‐8, IL‐19, elafin, C3, and S100A proteins, which implicate IL‐1 in the pathogenesis of inflammatory diseases. IL‐1 induced not only proliferation‐associated genes but also differentiation marker genes such as transglutaminase‐1 and involucrin, which suggests that IL‐1 plays an important role in the aberrant proliferation and differentiation seen in psoriasis. Correlation of IL‐1 regulated genes with the TNFα and IFNγ regulated ones showed more similarities between IL‐1 and TNFα than IL‐1 and IFNγ, whereas Oncostatin‐M (OsM) affected a largely unrelated set of genes. IL‐1 regulates many genes previously shown to be specifically over‐expressed in psoriasis. In summary, IL‐1 regulates a characteristic set of genes that define its specific contribution to inflammation and aberrant differentiation in skin diseases. J. Cell. Physiol. 214:1–13, 2008.

Inflammatory versus proliferative processes in epidermis. Tumor necrosis factor alpha induces K6b keratin synthesis through a transcriptional complex containing NFkappa B and C/EBPbeta

Epidermal keratinocytes respond to injury by becoming activated, i.e. hyperproliferative, migratory, and proinflammatory. These processes are regulated by growth factors and cytokines. One of the markers of activated keratinocytes is keratin K6. We used a novel organ culture system to show that tumor necrosis factor α (TNFα) induces the expression of K6 protein and mRNA in human skin. Multiple isoforms of K6 are encoded by distinct genes and have distinct patterns of expression. By having shown previously that proliferative signals, such as epidermal growth factor (EGF), induce expression of the cytoskeletal protein keratin K6b, we here demonstrate that the same isoform, K6b, is also induced by TNFα, a proinflammatory cytokine. Specifically, TNFα induces the transcription of the K6b gene promoter. By using co-transfection, specific inhibitors, and antisense oligonucleotides, we have identified NFκB and C/EBPβ as the transcription factors that convey the TNFα signal. Both transcription factors are necessary for the induction of K6b by TNFα and act as a complex, although only C/EBPβ binds the K6b promoter DNA. By using transfection, site-directed mutagenesis, and footprinting, we have mapped the site that responds to TNFα, NFκB, and C/EBPβ. This site is separate from the one responsive to EGF and AP1. Our results show that the proinflammatory (TNFα) and the proliferative (EGF) signals in epidermis separately and independently regulate the expression of the same K6b keratin isoform. Thus, the cytoskeletal responses in epidermal cells can be precisely tuned by separate proliferative and inflammatory signals to fit the nature of the injuries that caused them.

Attenuation of the transforming growth factor beta-signaling pathway in chronic venous ulcers.

Transforming growth factor β (TGFβ) is important in inflammation, angiogenesis, reepithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFβ signaling pathway in biopsies from 10 patients with nonhealing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFβ-treated keratinocytes, we found deregulation of TGFβ target genes in VUs. Using quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, we found suppression of TGFβRI, TGFβRII and TGFβRIII, and complete absence of phosphorylated Smad2 (pSmad2) in VU epidermis. In contrast, pSmad2 was induced in the cells of the migrating epithelial tongue of acute wounds. TGFβ-inducible transcription factors (GADD45β, ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFβ (FABP5, CSTA and S100A8) were induced in nonhealing VUs. An inhibitor of Smad signaling, Smad7 was also downregulated in VUs. We conclude that TGFβ signaling is functionally blocked in VUs by downregulation of TGFβ receptors and attenuation of Smad signaling resulting in deregulation of TGFβ target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFβ may not be a beneficial treatment for VUs.

Novel Regulation of Keratin Gene Expression by Thyroid Hormone and Retinoid Receptors

Expression of keratin proteins, markers of epidermal differentiation and pathology, is uniquely regulated by the nuclear receptors for retinoic acid (RAR) and thyroid hormone (T3R) and their ligands: it is constitutively activated by unliganded T3R, but it is suppressed by ligand-occupied T3R or RAR. This regulation was studied using gel mobility shift assays with purified receptors and transient transfection assays with vectors expressing various receptor mutants. Regulation of keratin gene expression by RAR and T3R occurs through direct binding of these receptors to receptor response elements of the keratin gene promoters. The DNA binding “C” domain of these receptors is essential for both ligand-dependent and -independent regulation. However, the NH-terminal “A/B” domain of T3R is not required for either mode of regulation of keratin gene expression. Furthermore, v-ErbA, an oncogenic derivative of cT3R, also activates keratin gene expression. In contrast to the previously described mechanism of gene regulation by T3R, heterodimerization with the retinoid X receptor is not essential for activation of keratin gene expression by unliganded T3R. These findings indicate that the mechanism of regulation of keratin genes by RAR and T3R differs significantly from the mechanisms described for other genes modulated by these receptors.