Effects of Tumor Necrosis Factor-α (TNFα) in Epidermal Keratinocytes Revealed Using Global Transcriptional Profiling

Abstract

Identification of tumor necrosis factor-α (TNFα) as the key agent in inflammatory disorders, e.g. rheumatoid arthritis, Crohn's disease, and psoriasis, led to TNFα-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, TNFα orchestrates the cutaneous responses to environmental damage and inflammation. To define TNFα action in epidermis, we compared the transcriptional profiles of normal human keratinocytes untreated and treated with TNFα for 1, 4, 24, and 48 h by using oligonucleotide microarrays. We found that TNFα regulates not only immune and inflammatory responses but also tissue remodeling, cell motility, cell cycle, and apoptosis. Specifically, TNFα regulates innate immunity and inflammation by inducing a characteristic large set of chemokines, including newly identified TNFα targets, that attract neutrophils, macrophages, and skin-specific memory T-cells. This implicates TNFα in the pathogenesis of psoriasis, fixed drug eruption, atopic and allergic contact dermatitis. TNFα promotes tissue repair by inducing basement membrane components and collagen-degrading proteases. Unexpectedly, TNFα induces actin cytoskeleton regulators and integrins, enhancing keratinocyte motility and attachment, effects not previously associated with TNFα. Also unanticipated was the influence of TNFα upon keratinocyte cell fate by regulating cell-cycle and apoptosis-associated genes. Therefore, TNFα 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 TNFα effects and define the mechanisms and genes that achieve these outcomes, both of which are important for TNFα-targeted therapies. Identification of tumor necrosis factor-α (TNFα) as the key agent in inflammatory disorders, e.g. rheumatoid arthritis, Crohn's disease, and psoriasis, led to TNFα-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, TNFα orchestrates the cutaneous responses to environmental damage and inflammation. To define TNFα action in epidermis, we compared the transcriptional profiles of normal human keratinocytes untreated and treated with TNFα for 1, 4, 24, and 48 h by using oligonucleotide microarrays. We found that TNFα regulates not only immune and inflammatory responses but also tissue remodeling, cell motility, cell cycle, and apoptosis. Specifically, TNFα regulates innate immunity and inflammation by inducing a characteristic large set of chemokines, including newly identified TNFα targets, that attract neutrophils, macrophages, and skin-specific memory T-cells. This implicates TNFα in the pathogenesis of psoriasis, fixed drug eruption, atopic and allergic contact dermatitis. TNFα promotes tissue repair by inducing basement membrane components and collagen-degrading proteases. Unexpectedly, TNFα induces actin cytoskeleton regulators and integrins, enhancing keratinocyte motility and attachment, effects not previously associated with TNFα. Also unanticipated was the influence of TNFα upon keratinocyte cell fate by regulating cell-cycle and apoptosis-associated genes. Therefore, TNFα 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 TNFα effects and define the mechanisms and genes that achieve these outcomes, both of which are important for TNFα-targeted therapies. Tumor necrosis factor-α (TNFα) 1The abbreviations used are: TNFα, tumor necrosis factor-α; PBS, phosphate-buffered saline; MMP, matrix metalloproteinase; FACS, fluorescence-activated cell sorter. is a multifunctional cytokine that mediates inflammation, immune response, and apoptosis (1Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Google Scholar). Inappropriate production or persistent activation of TNFα participates in a wide spectrum of diseases, including septic shock, diabetes, cancer, graft rejection, rheumatoid arthritis, and Crohn's disease (2McDermott M.F. Cell. Mol. Biol. Res. 2001; 47: 619-635Google Scholar). Accumulating evidence indicates that TNFα also has a significant role in normal development and homeostasis of several organs. Mice deficient in TNFα lack germinal centers and show increased susceptibility to microbial pathogens due to incomplete inflammatory responses (3Pasparakis M. Alexopoulou L. Episkopou V. Kollias G. J. Exp. Med. 1996; 184: 1397-1411Google Scholar). TNFR1-mutant mice show similar abnormalities, in addition to defective formation of Peyer's patches (4Fu Y.X. Chaplin D.D. Annu. Rev. Immunol. 1999; 17: 399-433Google Scholar). In skin, TNFα is the master cytokine regulator in inflammatory diseases, such as psoriasis, contact dermatitis, drug eruptions, cutaneous T-cell lymphoma, etc. (5Ettehadi P. Greaves M.W. Wallach D. Aderka D. Camp R.D. Clin. Exp. Immunol. 1994; 96: 146-151Google Scholar, 6Becke F.M. Hehlgans T. Brockhoff G. Mannel D.N. Eur. Cytokine Network. 2001; 12: 45-50Google Scholar, 7Teraki Y. Moriya N. Shiohara T. Am. J. Pathol. 1994; 145: 550-560Google Scholar). TNFα is found in skin after injury (8Kondo T. Ohshima T. Int. J. Legal Med. 1996; 108: 231-236Google Scholar, 9Wakefield P.E. James W.D. Samlaska C.P. Meltzer M.S. J. Am. Acad. Dermatol. 1991; 24: 675-685Google Scholar, 10Kock A. Schwarz T. Kirnbauer R. Urbanski A. Perry P. Ansel J.C. Luger T.A. J. Exp. Med. 1990; 172: 1609-1614Google Scholar) and is considered essential for angiogenesis during wound healing (11Leibovich S.J. Polverini P.J. Shepard H.M. Wiseman D.M. Shively V. Nuseir N. Nature. 1987; 329: 630-632Google Scholar). Agents targeting TNFα are now in clinical trials for treatment of inflammatory diseases, including psoriasis (12Wolf R. Matz H. Orion E. Ruocco V. Clin. Dermatol. 2002; 20: 522-530Google Scholar). However, our understanding of the TNFα action has not been sufficient to predict the full effects and side effects of such therapies because TNFα has a remarkable variety of functions and the anti-TNFα treatments can simultaneously affect many cellular processes in both abnormal and normal tissues (13Antoni C. Braun J. Clin. Exp. Rheumatol. 2002; 20: S152-S157Google Scholar). TNFα is a homodimer of 157 amino acid subunits produced primarily by activated macrophages but also by other cell types, including epidermal keratinocytes (14Chen G. Goeddel D.V. Science. 2002; 296: 1634-1635Google Scholar). A low level of TNFα is present in the upper layer of the healthy epidermis, but its synthesis and release from keratinocytes are greatly augmented by injury, infection, UV irradiation, and contact sensitizers (8Kondo T. Ohshima T. Int. J. Legal Med. 1996; 108: 231-236Google Scholar, 9Wakefield P.E. James W.D. Samlaska C.P. Meltzer M.S. J. Am. Acad. Dermatol. 1991; 24: 675-685Google Scholar, 10Kock A. Schwarz T. Kirnbauer R. Urbanski A. Perry P. Ansel J.C. Luger T.A. J. Exp. Med. 1990; 172: 1609-1614Google Scholar, 15Griffiths C.E. Barker J.N. Kunkel S. Nickoloff B.J. Br. J. Dermatol. 1991; 124: 519-526Google Scholar). Of the two distinct cell-surface receptors for TNFα, TNFR1 and TNFR2, keratinocytes mainly express TNFR1 (16Kondo S. Sauder D.N. Eur. J. Immunol. 1997; 27: 1713-1718Google Scholar). The binding of TNFα to TNFR1 triggers a series of intracellular events resulting in the activation of transcription factors, including NFκB, AP-1, CCAAT enhancer-binding protein β, and others (17Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Google Scholar), which are responsible for the induction of genes important for diverse biological processes, including cell growth and death, oncogenesis, and immune, inflammatory, and stress responses (14Chen G. Goeddel D.V. Science. 2002; 296: 1634-1635Google Scholar). TNFα activates the immune responses through inducing the production of additional signals, such as interleukin 1 (IL-1) and IL-8, transforming growth factor type β (TGF-β), intercellular adhesion molecule 1 (ICAM-1), etc. (18Barker J.N. Mitra R.S. Griffiths C.E. Dixit V.M. Nickoloff B.J. Lancet. 1991; 337: 211-214Google Scholar). TNFα affects epidermis in cutaneous inflammatory diseases, and therefore we set out to identify and comprehensively analyze the TNFα-regulated genes in epidermal keratinocytes by using DNA microarrays. Several reports of microarray analyses of TNFα-regulated genes focused on cell lines or pathological conditions (19Murakami T. Mataki C. Nagao C. Umetani M. Wada Y. Ishii M. Tsutsumi S. Kohro T. Saiura A. Aburatani H. Hamakubo T. Kodama T. J. Atheroscler. Thromb. 2000; 7: 39-44Google Scholar, 20Shakhov A.N. Lyakhov I.G. Tumanov A.V. Rubtsov A.V. Drutskaya L.N. Marino M.W. Nedospasov S.A. J. Leukocyte Biol. 2000; 68: 151-157Google Scholar, 21Zhou A. Scoggin S. Gaynor R.B. Williams N.S. Oncogene. 2003; 22: 2054-2064Google Scholar). However, we decided to use primary cultures of healthy human epidermal keratinocytes and profile the transcriptional changes 1, 4, 24, and 48 h after TNFα treatment. The gene expression patterns were compared with the corresponding untreated control at each time point, which allowed us to describe novel responses not previously associated with TNFα action or cutaneous inflammation. We found that TNFα works fast, with many genes regulated already after 1 h. Among the regulated genes, we found those involved in immune and inflammatory responses, and although TNFα was known to regulate some of many are newly identified Unexpectedly, TNFα regulates genes that affect the cell motility and which activates keratinocyte TNFα also regulates genes involved in cell and as as basement membrane components and matrix these genes and processes important of we predict that as a of TNFα-targeted therapies. The results that TNFα affects a wide of processes to the epidermal responses to injury and that TNFα is responsible not only for the initiation of inflammation, but also for the subsequent repair and This has significant for the development of TNFα-targeted and Cytokine used the D. A. G. M. J. 2001; Scholar, T. M. L. M. 2003; Scholar). normal human epidermal keratinocytes were from M. The cultures were using and in the keratinocytes were in a keratinocyte growth with epidermal growth factor and at in We using because can of keratinocyte The was and the were through for the were with which was with of keratinocytes were used at primary cultures provide a cell and by using a large of we due to growth We the from to keratinocyte h treatment to the effects of the in growth were treated with human TNFα and by at 1, 4, 24, and 48 h after treatment. 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Biol. 2000; Scholar), the activation of to its 1, A and The transcriptional induction of the genes results in the of untreated keratinocytes is by and 48 h after the TNFα treatment 1, and cell a of the transcriptional changes by TNFα, we primary cultures of normal human epidermal keratinocytes and treated with of TNFα, which was by for microarray Of the genes present on the some were found in of some genes were regulated by TNFα, to our Of the genes, were were and were both and at time We used several to analyze the microarray The A and expression of keratinocytes untreated corresponding show the of genes during the and that the of regulated genes increased with the time TNFα works genes were at 1 h. of TNFα is that the of genes is that of the In TNFα is from e.g. or UV which and of genes D. A. G. M. J. 2001; Scholar, T. M. L. M. 2003; Scholar). a we compared the regulated genes at each time after TNFα and found that the and 48 h time were similar to each a of regulated genes and a and of TNFα We of regulated genes at each time and the that these genes. The matrix of indicates a significant of the genes at time of the genes similar are the of genes regulated at the and time the of the To the we of genes We both and genes and both and To our of the genes in the two of the TNFα-regulated the cellular processes in keratinocytes by TNFα we the regulated genes in a of cellular functions The level of indicates the or of each the these functions into cellular or and the to a by TNFα. we on the of TNFα-regulated genes, those that are involved in the innate inflammation, tissue repair and remodeling, epidermal cell-cycle and apoptosis. and TNFα is known to of innate immunity and inflammation in skin P.E. James W.D. Samlaska C.P. Meltzer M.S. J. Am. Acad. Dermatol. 1991; 24: 675-685Google Scholar, S. Sauder D.N. Eur. J. Immunol. 1997; 27: 1713-1718Google Scholar), those effects not in TNFα induces of its the and others The and a for the neutrophils, and attract and macrophages, the memory T-cells. TNFα interleukin receptors and and receptors and TNFα also a and which on components and of acid and were also these results that TNFα induces chemokines, growth factors, and cell-surface receptors that in neutrophils, memory and macrophages, of which to the innate immunity and inflammation. upon and Cell effects of TNFα upon tissue remodeling, and cell not been The of the matrix important factor for tissue repair during and after inflammation. The induction of matrix was is the to and is involved in tissue wound and tumor C.E. Rev. Mol. Cell. Biol. 2002; Scholar). that was by TNFα, which in a of tissue by the in In the induction of and as as a of basement with and growth in role for TNFα in the production of basement membrane R.S. J. Biol. Scholar). and membrane protein were also by TNFα. TNFα regulates the through the induction of and its and A TNFα both adhesion and in a characteristic of cell and Specifically, and and genes and were regulated by TNFα. are receptors that the the cytoskeleton and the as as to and from the cell Cell. Scholar). TNFα several adhesion and This is the of induction by induction at h after TNFα a in TNFα many to keratinocytes from inflammation. such as a role in epidermal S. C. A. D. M. J. A. Y. Y. A. 2000; Scholar) and is protein microbial infections James R.M. G. C. 2003; Scholar). these changes in expression of integrins, adhesion and genes changes of the of the actin we found in the actin in the treated actin into and into and In to the of control the from and out We that the changes by TNFα affect cell in cell and To the of TNFα on keratinocyte we of cell the of the of keratinocyte cultures with or TNFα. the untreated keratinocytes out the the keratinocytes and the of the by in and out of This that TNFα the motility of We the of keratinocytes using from of keratinocytes during the h after TNFα treatment. We found that the keratinocytes the untreated in and many Therefore, the changes of expression of keratinocyte motility, essential of the not previously associated with TNFα. of Cell and Cell TNFα only affects the cell in its effects upon growth and apoptosis However, our show that TNFα affects these TNFα affects the cell of The gene genes and were the were This that TNFα the keratinocyte cell-cycle in the We the of TNFα on cell by and fluorescence-activated cell which a significant of the in the of the cell TNFα induces cell in some tumor cell lines B.J. P.E. Shepard H.M. Science. our cultures not show cell after the TNFα treatment. that TNFα both and genes a for the of or TNFα two and which the the to apoptosis Mol. Cell. Biol. 2002; 22: Scholar), influence of these genes on the Tumor also affect the cell and regulate apoptosis. TNFα the P. J. Biol. 2003; Scholar). these changes of gene expression effects upon the cell cell and cell death, by inducing the expression of that keratinocytes in the and and induces the expression of many transcription TNFα to both the subunits and the factor 1 and effects upon NFκB, and are the components of factor and two were a multifunctional transcriptional was The induction of and were because regulates cell growth and apoptosis G. G. S. E. J. Cytokine Res. 2002; 22: Scholar), is for the development of and M.W. H. Immunol. Scholar). transcription a variety of important in is known in We that the induction of and by TNFα has important role in epidermal tissue repair and transcription and transcription regulators and is a transcription H. M. Res. 1997; Scholar). and affects transcription factor to DNA S. J. N. D. Res. 2002; Scholar). many transcription and regulators are for the immune response, and The of the TNFα time of gene expression the of the biological events to TNFα We the and of for each of regulated genes from these the effects of TNFα to the at the of the and the immune and inflammatory TNFα motility, and which are by the induction of matrix that in TNFα the cell or cell death, and the of the TNFα is known as the regulator of diverse inflammatory and immune processes in human skin diseases, but the of its action is not We the gene expression changes in normal human epidermal keratinocytes treated with TNFα. We identified comprehensively the set of TNFα-regulated genes that significant in skin innate immunity and inflammation. we novel functions of TNFα and a wide spectrum of genes many additional cellular processes that not been associated previously with TNFα action or cutaneous inflammation. The with keratinocytes not in inflammatory skin diseases, although similar processes are TNFα control in as processes tissue repair and remodeling, cell keratinocyte and results describe of the epidermal transcriptional responses by TNFα in to skin injury and inflammation. of the TNFα responses can and others are and TNFα-targeting therapies both into Specifically, TNFα, as the regulator of innate immunity and inflammation, induces to attract and macrophages to the skin in the of inflammation. results the pathological in infection, the increased production of TNFα and of can TNFα also a skin-specific memory T-cell the of TNFα in fixed drug and other skin inflammation processes, such as psoriasis and atopic and allergic contact T. Y. Y. Clin. Immunol. 2002; Scholar, H. A. H. L. T. E. M. H. D. H. To J. T. P. A. Med. 2002; Scholar). We show that TNFα also induces the expression of in In addition to innate immunity and inflammation processes, TNFα also to the repair of angiogenesis is considered essential for wound and has important role in wound is after skin injury (11Leibovich S.J. Polverini P.J. Shepard H.M. Wiseman D.M. Shively V. Nuseir N. Nature. 1987; 329: 630-632Google Scholar, 9Wakefield P.E. James W.D. Samlaska C.P. Meltzer M.S. J. Am. Acad. Dermatol. 1991; 24: 675-685Google Scholar, J. Annu. Rev. Cell Biol. 1990; Scholar, D.N. J. Exp. Med. Scholar). TNFα induces which is for the normal repair of skin and a role in and adhesion at injury which initiates and J. C. M.J. Med. 1996; Scholar, S. G. M. Y. 2002; Scholar, A. M. M. T. E. S. S. J. 2003; 17: Scholar). This on the TNFα triggers the initiation of inflammation, but on the other for the of inflammation and its as was the by TNFα, tissue is associated with M. P. E. M. R. J. Cell. 1996; Scholar), and deficient of were found in is A.N. C.E. J. 1999; 7: Scholar). responsible for the keratinocytes from the basement to the tissue T. M. M. H. H. 1994; Scholar). the TNFα-regulated a role both in tissue and in repair from macrophages and keratinocytes TNFα and TNFα induces production of secondary However, of these inflammatory the inflammation and to such as septic we that TNFα also mechanisms for TNFα regulators of and including and many We that some of the TNFα-regulated is its are important in inflammatory skin diseases, such as psoriasis J. T. T. Dermatol. Res. 1987; Scholar), but also been involved in the of the epidermal R.M. H. M. Nature. Scholar). TNFα regulates two processes, adhesion and by inducing and that affect both actin and The is important because cell both a formation of at the and at the of the cell A. J. Cell Scholar). In addition to integrins, TNFα protein and that affect the Scholar). TNFα regulates both and which are essential for the keratinocyte and TNFα induces which was identified as adhesion molecule by injury T. J. 1996; 17: Scholar). initiates and cell adhesion and through its with Trends Cell Biol. 2001; 11: Scholar). is a with a significant role in inflammatory angiogenesis and in actin through its and as as a role in the inflammatory angiogenesis by TNFα N. T. H. T. Cell Biol. 2002; Scholar, A. H. R.M. Polverini P.J. Dixit V.M. Science. Scholar). is a of the protein and regulates the of the actin cytoskeleton through to cell changes D.M. J. Biol. 2001; Scholar). has been to in the of the actin E. S. P. A. T. Mol. Cell. Biol. 2003; Scholar). actin similar to C. J. H. J. Cell. 2002; Scholar). is known the of a protein that and actin 2002; 24: Scholar). The of some of the with to keratinocyte is TNFα induces both and We not apoptosis in the We that TNFα keratinocytes to to additional these the apoptosis was that TNFα the growth of human keratinocytes S. D.D. J. Clin. Scholar, T. T. M. N. S. Ishii M. T. J. Dermatol. 1990; 17: Scholar). We found that TNFα which the transcription of to of the cell at the N. C. R. C. C. P. P. R. J. M. C. A. 1996; Scholar), and which has also been in the N.S. 2001; Scholar). our microarray that the keratinocytes in the of the cell We that the is for the cell fate of for a to by or apoptosis. is a in the effects of TNFα upon apoptosis and cell TNFα induces that keratinocytes in the and the cell cycle, or effects full cell-cycle or TNFα in the to additional that the or from these We that TNFα the keratinocytes to and to such additional The additional which growth factors, or are in psoriasis, for in atopic or in wound and the fate of keratinocytes or apoptosis. This to in our of TNFα are a of many genes and a of genes the In the responses of keratinocytes to UV or were with a of genes regulated in the D. A. G. M. J. 2001; Scholar, T. M. L. M. 2003; Scholar). keratinocytes to TNFα, because TNFα a and e.g. from infection, UV which or which indicates that activated are The to TNFα is the induction of and that the to the as as of transcription that subsequent transcriptional the cell fate changes are The mechanisms to TNFα NFκB, and other transcription factors, of which can both and transcription of genes J. Clin. Exp. 22: is many genes are by TNFα. UV and TNFα activates set of transcription factors, works primarily through and of transcription 1 which as a a transcriptional and not a A. Cytokine Rev. 2001; 12: Scholar). Therefore, additional to describe the of the and transcription factor activation in to TNFα and other has been for the treatment of many inflammatory R. 2001; Scholar, R. 2001; Scholar). However, the TNFα has a of wound secondary and due to of the immune and inflammatory processes and of tissue repair. In and been as side effects after anti-TNFα 2003; Scholar). for each We that a the spectrum of gene expression regulated by TNFα, which has been to these treatments both and In a of the transcriptional changes by TNFα in epidermal keratinocytes and we two significant and novel TNFα is essential not for the inflammation and to injury, but also for the subsequent repair and and TNFα the keratinocytes to additional and to and through or apoptosis. TNFα activates a series of immune responses and triggers inflammation, which is by tissue and repair to repair epidermis, with and from each other and the basement keratinocytes by apoptosis as a of the inflammatory response, some on the newly basement membrane and upon additional also to a epidermal tissue results a of TNFα as the key of responses to skin injury, regulating a wide of biological processes from immune and inflammation to cell epidermal and tissue repair. We that the of gene expression for of these processes can by a TNFα, in a cell the epidermal We M. for the of of our and Y. for and and for the