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M Blumenberg

Publications and source records attributed to M Blumenberg.

At least 19 recordsLinked to original sources
PubMed2001

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.

Chemokines↗
PubMed2001

Interleukin-1 induces transcription of keratin K6 in human epidermal keratinocytes.

Keratinocytes respond to injury by releasing the proinflammatory cytokine interleukin-1, which serves as the initial "alarm signal" to surrounding cells. Among the consequences of interleukin-1 release is the production of additional cytokines and their receptors by keratinocytes and other cells in the skin. Here we describe an additional effect of interleukin-1 on keratinocytes, namely the alteration in the keratinocyte cytoskeleton in the form of the induction of keratin 6 expression. Keratin 6 is a marker of hyperproliferative, activated keratinocytes, found in wound healing, psoriasis, and other inflammatory disorders. Skin biopsies in organ culture treated with interleukin-1 express keratin 6 in all suprabasal layers of the epidermis, throughout the tissue. In cultured epidermal keratinocytes, the induction of keratin 6 is time and concentration dependent. Importantly, only confluent keratinocytes respond to interleukin-1, subconfluent cultures do not. In the cells starved of growth factors, epidermal growth factor or tumor necrosis factor-alpha, if added simultaneously with interleukin-1, they synergistically augment the effects of interleukin-1. Using DNA-mediated cell transfection, we analyzed the molecular mechanisms regulating the keratin 6 induction by interleukin-1, and found that the induction occurs at the transcriptional level. We used a series of deletions and point mutations to identify the interleukin-1 responsive DNA element in the keratin 6 promoter, and determined that it contains a complex of C/EBP binding sites. The transcription factor C/EBPbeta binds this element in vitro, and the binding is augmented by pretreatment of the cells with interleukin-1. The interleukin-1 responsive element is clearly distinct from the epidermal growth factor responsive one, which means that the proinflammatory and proliferative signals independently regulate the expression of keratin 6. Thus, interleukin-1 initiates keratinocyte activation not only by triggering additional signaling events, but also by inducing directly the synthesis of keratin 6 in epidermal keratinocytes, and thus changing the composition of their cytoskeleton.

Base Sequence↗
PubMed2001

Keratins and the keratinocyte activation cycle.

In wound healing and many pathologic conditions, keratinocytes become activated: they turn into migratory, hyperproliferative cells that produce and secrete extracellular matrix components and signaling polypeptides. At the same time, their cytoskeleton is also altered by the production of specific keratin proteins. These changes are orchestrated by growth factors, chemokines, and cytokines produced by keratinocytes and other cutaneous cell types. The responding intracellular signaling pathways activate transcription factors that regulate expression of keratin genes. Analysis of these processes led us to propose the existence of a keratinocyte activation cycle, in which the cells first become activated by the release of IL-1. Subsequently, they maintain the activated state by autocrine production of proinflammatory and proliferative signals. Keratins K6 and K16 are markers of the active state. Signals from the lymphocytes, in the form of Interferon-gamma, induce the expression of K17 and make keratinocytes contractile. This enables the keratinocytes to shrink the provisional fibronectin-rich basement membrane. Signals from the fibroblasts, in the form of TGF-beta, induce the expression of K5 and K14, revert the keratinocytes to the healthy basal phenotype, and thus complete the activation cycle.

Humans↗
PubMed2000

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 alpha (TNFalpha) 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 TNFalpha, a proinflammatory cytokine. Specifically, TNFalpha induces the transcription of the K6b gene promoter. By using co-transfection, specific inhibitors, and antisense oligonucleotides, we have identified NFkappaB and C/EBPbeta as the transcription factors that convey the TNFalpha signal. Both transcription factors are necessary for the induction of K6b by TNFalpha and act as a complex, although only C/EBPbeta binds the K6b promoter DNA. By using transfection, site-directed mutagenesis, and footprinting, we have mapped the site that responds to TNFalpha, NFkappaB, and C/EBPbeta. This site is separate from the one responsive to EGF and AP1. Our results show that the proinflammatory (TNFalpha) 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.

Base Sequence↗
PubMed2000

Novel mechanism of steroid action in skin through glucocorticoid receptor monomers.

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.

Gene Expression Regulation↗
PubMed1998

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.

Gene Expression Regulation↗
PubMed1997

Specific organization of the negative response elements for retinoic acid and thyroid hormone receptors in keratin gene family.

Retinoic acid and thyroid hormone are important regulators of epidermal growth, differentiation, and homeostasis. Retinoic acid is extensively used in the treatment of many epidermal disorders ranging from wrinkles to skin cancers. Retinoic acid and thyroid hormone directly control the transcription of differentiation-specific genes including keratins. Their effect is mediated through nuclear receptors RAR and T3R. We have previously identified the response element in the K14 gene, K14RARE/TRE, to which these receptors bind, and found that it consists of a cluster of five half-sites with variable spacing and orientation. To determine whether this specific structure is found in other keratin genes, we have mapped and analyzed the RARE/TRE elements in three additional epidermal keratin genes: K5, K6, and K17. We used three different approaches to identify these elements: co-transfection of promoter deletion constructs, gel-shift assays, and site-specific mutagenesis. We localized the RARE/TRE elements relatively close to the TATA box in all three promoters. All three RARE/TRE elements have a similar structural organization: they consist of clusters of 3-6 half-sites with variable spacing and orientation. This means that the clustered structure of the RARE/TREs is a common characteristic for keratin genes. RARE and TRE in the K5 promoter are adjacent to each other whereas in the K17 promoter they overlap. All three keratin REs bind specifically both RAR and T3R in gel-shift assays. Interestingly, addition of ligand to the receptor changes the binding pattern ofthe T3R from homodimer to monomer, reflecting the change in regulation from induction to inhibition.

Base Sequence↗
PubMed1997

Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NF-kappaB family members.

The expression of keratins K5 and K14 is restricted to the basal layers of the healthy epidermis, whereas the expression of K6 and K17 is induced in response to proliferative and inflammatory signals, respectively. The control of keratin expression occurs primarily at the transcriptional level. We studied the effects of transcription factors of the AP-1 and NF-kappaB families on the expression of those four keratin genes. We chose AP-1 and NF-kappaB proteins because they are activated by many extracellular signals, including those in hyperproliferative and inflammatory processes. DNA constructs expressing the transcription factors were, in various combinations, cotransfected with constructs containing keratin gene promoters and the CAT reporter gene into HeLa cells or keratinocytes. We found that the K5 and K14 promoters, which are coexpressed in vivo, are regulated in parallel by the cotransfected genes. Both were activated by the c-Fos and c-Jun components of AP-1, but not by Fra1. On the other hand, the NF-kappaB proteins, especially p65, suppressed these two promoters. The K17 promoter was specifically activated by c-Jun, whereas the other transcription factors tested had no significant effect. In contrast, the K6 promoter was very strongly activated by all AP-1 proteins, especially by the c-Fos + c-Jun and Fra1 + c-Jun combinations. It was also strongly activated by the p65 NF-kappaB protein. AP-1 and NF-kappaB acted synergistically in activating the K6 promoter, although the AP-1 and the NF-kappaB responsive sites could be separated physically. These results suggest that the interplay of AP-1 and NF-kappaB proteins regulates epidermal gene expression and that the activation of these transcription factors by extracellular signaling molecules brings about the differential expression of keratin genes in epidermal differentiation, cutaneous diseases, and wound healing.

Base Sequence↗
PubMed1996

Close linkage of the two keratin gene clusters in the human genome.

Mapping studies of functional keratin genes in the human genome have localized most of the acidic keratin genes to chromosome 17q12-q21 and the basic keratin genes to chromosome 12q11-q13. Within the acidic keratin locus two clusters were identified, one containing the genes for K15 and K19, the other the genes for K14, K16, and K17. The relative positions and the distance between the two clusters have not been determined previously. In this paper we describe our analysis of P1 clones containing multiple acidic keratin genes, which were studied using restriction analysis and Southern blot hybridization with PCR-amplified probes specific for functional human keratin genes 15, 17, and 19. Our results show that the two clusters are very closely linked to each other, within a 55-kb region in the human genome. The genes are organized 5' to 3' in the following order: 5'-K19-K15-K17-K16-K14. Between K15 and K17 at least one additional, unidentified keratin gene is present.

Base Sequence↗
PubMed1996

Codominant regulation of keratin gene expression by cell surface receptors and nuclear receptors.

Epidermal keratinocytes are subject to a large variety of signals that modulate their differentiation in health and their activation in disease. Hormones and vitamins, which act via nuclear receptors, affect the differentiation process, whereas growth factors and cytokines, which act via cell surface receptors, affect keratinocyte activation and related events. Using expression of keratin genes as markers for keratinocyte phenotype, we examined the interaction between the nuclear receptor and cell surface receptor pathways. We expected to find dominance of one of the pathways. Surprisingly, we found that the two pathways are codominant. Specifically, while EGF induces expression of K6 and K16 keratin genes, retinoic acid suppresses their expression, and when both mediators are present simultaneously, the level of expression is intermediate, a product of both signals. Similar codominant effects were found on other keratin genes using interferon gamma, TGF beta, and thyroid hormone signaling molecules. These codominant effects are specific only for genes that are regulated by both pathways. Our results suggest that a judicious combination of hormones, vitamins, growth factors, and cytokines may be used to target specific expression of appropriate genes in the treatment of human epidermal diseases.

Epidermal Growth Factor↗
PubMed1996

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 NH2-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.

Base Sequence↗
PubMed1996

Characterization of nuclear protein binding sites in the promoter of keratin K17 gene.

Keratin K17, while not present in healthy skin, is expressed under various pathological conditions, including psoriasis and cutaneous allergic reactions. The regulatory circuits involved in transcription of the human keratin K17 gene are poorly understood. To begin an analysis of the molecular mechanisms that regulate K17 gene transcription, we have studied the interactions between the nuclear proteins and the promoter region of the human K17 gene. That promoter region comprised 450 bp upstream from the translation initiation site. For these studies, we used electrophoretic mobility-shift assays, computer analysis, site-directed mutagenesis, and DNA-mediated cell transfection. In addition to the previously characterized interferon-gamma-responsive elements, we identified eight protein binding sites in the promoter. Five of them bind the known transcription factors NF1, AP2, and Sp1 and three others bind still unidentified proteins. Using site-directed mutagenesis, we have demonstrated the importance of the protein binding sites for the promoter function involved in both constitutive and interferon-induced expression of the K17 keratin gene.

Antineoplastic Agents↗
PubMed1996

Regulation of epidermal expression of keratin K17 in inflammatory skin diseases.

Keratin K17, the myoepithelial keratin, is expressed in psoriasis but is not present in healthy skin. Psoriasis is associated with production of gamma interferon (IFN gamma), which induces the expression of keratin K17 by activating transcription factor STAT1. Our hypothesis states that the induction of K17 is specific for the inflammatory reactions associated with high levels of IFN gamma and activation of STAT1. One of the corollaries of the hypothesis is that the STAT1-activating cytokines should induce the expression of keratin K17, whereas those cytokines that work through other mechanisms should not. Furthermore, because the STAT activation pathway is dependent upon protein phosphorylation events, phosphorylation inhibitors should attenuate the induction of keratin K17, whereas protein phosphatase inhibitors should augment it. To test this hypothesis, we analyzed lesional samples of inflammatory diseases using immunofluorescence, transfected keratinocytes with K17 gene promoter DNAs in the presence of various cytokines, and followed nuclear translocation of STAT1 in keratinocytes using specific antibodies. Confirming the hypothesis, we found that K17 is induced in psoriasis and dermatitis caused by delayed type hypersensitivity, which are associated with high levels of IFN gamma, but not in samples of atopic dermatitis, which is not. Two cytokines, interleukin-6 and leukemia inhibitory factor, which can induce phosphorylation of STAT1, can also induce K17 expression, whereas interleukin-3, interleukin-4, interleukin-10, and granulocyte macrophage colony stimulating factor have no effect on K17 expression. As expected, staurosporine and genistein inhibited, whereas okadaic acid augmented, the induction of K17 by IFN gamma. Our data indicate that in inflammatory skin diseases, lymphocytes, through the cytokines they produce, differently regulate not only each other, but also keratin gene expression in epidermis one of their target tissues.

Cytokines↗
PubMed1995

Keratinocyte growth factor and keratin gene regulation.

Keratinocyte growth factor (KGF) is a stromally derived paracrine mitogen that belongs to the fibroblast growth factor (FGF) family. It is secreted by dermal fibroblasts and specifically promotes keratinocyte proliferation. We have recently shown that epidermal growth factor (EGF) and transforming growth factor beta (TGF beta), modulators of keratinocyte proliferation, regulate expression of specific keratin genes. However KGF, unlike EGF and TGF beta, allows keratinocytes to differentiate normally. With this in mind, we sought to determine whether KGF may be involved in keratinocyte differentiation through a mechanism that does not involve regulation of keratin gene expression. We transfected human epidermal keratinocytes with ten different keratin gene promoters linked to a reporter gene, and grew the transfected cells in the presence or absence of KGF. Interestingly, no significant change in keratin gene regulation was observed in the presence of KGF relative to control. The possibility that KGF influences the induction of keratin gene expression by other keratinocyte modulators, such as EGF, TGF beta and gamma interferon (IFN gamma), was also explored. In these experiments, the transformed keratinocytes were exposed simultaneously to KGF and another modulator. KGF did not significantly change the effects of EGF, TGF beta or IFN gamma on keratin gene expression. KGF's lack of ability to directly regulate keratin gene expression suggests that KGF affects keratinocyte growth and differentiation through a pathway independent of keratin gene regulation. These results illustrate that regulation of keratinocyte proliferation can be separated from the regulation of keratin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1995

Retinoid receptors and keratinocytes.

In 1987, a tremendous boost in our understanding of the action of dietary vitamin A occurred with the discovery and characterization of nuclear receptors for retinoic acid, the active form of the vitamin, in the laboratories of P. Chambon and R. Evans. They have shown that the nuclear receptors are ligand-activated transcription factors capable of specific gene regulation. Since that discovery, it has been determined that there are at least six retinoic acid receptors belonging to two families, RARs and RXRs, that they are differentially expressed in various mammalian tissues, and that they act as homo- and heterodimers interacting with other ligand-activated nuclear receptors. The domain structure of the receptors has been described, and their DNA-binding, ligand-binding, dimerization, and transcriptional activation regions characterized. Among the most important retinoid-regulated genes are the homeobox proteins, regulatory transcription factors which are responsible for body axis formation, patterning, limb formation, and other crucial processes during development. Retinoic acid and its receptors also regulate many differentiation markers which are particularly important in stratified epithelia, such as skin and oral epithelia. Our increased understanding led to improved therapy of a large number of skin disorders, ranging from acne to wrinkles and including epidermal and oral carcinomas.

Animals↗