Human IP-9: A keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3).
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Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses. (+info)
UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.
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We have previously shown that hydrogen peroxide is an important mediator of ultraviolet B induced phosphorylation of the epidermal growth factor receptor in human keratinocytes. Here we demonstrate that physiologic doses of ultraviolet B and hydrogen peroxide stimulate activation of two related but distinct mitogen-activated protein kinase pathways: extracellular regulated kinase 1 and 2 (ERK1/2), as well as p38, the mammalian homolog of HOG1 in yeast which is a major kinase for a recently identified stress-induced signaling pathway. The time-dependent activation of ERK1/2 and p38 are distinct, and ultraviolet B-induced ERK1/2 activation is downregulated more rapidly than p38. Using dihydrorhodamine or Amplex as specific fluorescent dye probes, we show that ultraviolet B-induced peroxides can be inhibited by ascorbic acid. Ascorbic acid strongly blocks ERK1/2 and p38 activation by ultraviolet B and hydrogen peroxide whereas pyrrolidine dithiocarbamate and butyl hydroxyanisole are less effective. Pyrrolidine dithiocarbamate was unable to inhibit ultraviolet B-induced p38 activation. Cell death was increased after ultraviolet B when ERK1/2 activation was attenuated by the specific inhibitor PD098059. The distinct time courses and extents of activation and inhibition of ERK1/2 and p38 indicate that these pathways are separate and regulated independently in keratinocytes. Specific types of reactive oxygen species induced by ultraviolet B as well as selective activation or inhibition of specific phosphatases may mediate these responses in keratinocytes. These findings demonstrate that reactive oxygen species are important multifunctional mediators of ultraviolet B-induced ERK1/2 and p38 signaling transduction pathways and suggest that ERK1/2 may play an important part in protecting keratinocytes from cell death following oxidative stress. (+info)
Activation of p38 mitogen-activated protein kinase and caspases in UVB-induced apoptosis of human keratinocyte HaCaT cells.
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Exposure of human keratinocyte HaCaT cells to ultraviolet B-irradiation induced apoptotic morphologic changes. In this study, we found that the ultraviolet B irradiation (0.25 J per cm2) induced phosphorylation of p38 mitogen-activated protein kinase and c-jun N-terminal protein kinase, and also significant activation of caspase-3 (CPP32-like protease) and a small increase of caspase-1 (ICE-like protease) activity in the early stages of ultraviolet B-induced apoptosis. Pretreatments of the cells with a p38 mitogen-activated protein kinase inhibitor, SB203580, and a caspase-3 inhibitor, Ac-Asp-Met-Gln-Asp-1-aldehyde, suppressed the ultraviolet B irradiation-induced apoptosis by approximately 60% as estimated by nuclear staining and DNA laddering. Pretreatment with caspase-1 inhibitor, Ac-Tyr-Val-Lys-Asp-aldehyde was without effect. Ultraviolet B-induced caspase-3 activation resulted in cleavage of poly(ADP) ribose polymerase, which was abolished by the caspase-3 inhibitor. SB203580 pretreatment prevented activation of caspase-3 and caspase-1, and also suppressed the cleavage of poly(ADP) ribose polymerase. Neither ceramide generation nor sphingomyelinase activation (neutral and acid) was observed in the ultraviolet B-irradiated HaCaT cells. Also various antioxidants did not affect the caspase activation induced by ultraviolet B irradiation. These results indicated that activation of p38 mitogen-activated protein kinase upstream of caspases may play an important part in the apoptotic process of keratinocytes exposed to ultraviolet B irradiation. (+info)
Spatial and temporal expression of parathyroid hormone-related protein during wound healing.
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Parathyroid hormone-related protein is produced by many normal tissues including the skin, where it regulates growth and differentiation of keratinocytes. To define better the role of parathyroid hormone-related protein in the skin, we investigated the spatial and temporal expression of parathyroid hormone-related protein and mRNA by immunohistochemistry and in situ hybridization during the healing of skin wounds, and the effects of topical administration of a parathyroid hormone-related protein agonist [parathyroid hormone-related protein (1-36)] and a parathyroid hormone-related protein antagonist [parathyroid hormone (7-34)] on the healing rate and morphology of the wounds. Wounds were produced on the back of guinea pigs with a 4 mm punch, and wound sites were collected at different time points during the healing process. Parathyroid hormone-related protein was expressed in normal skin by all viable keratinocyte layers, hair follicles, and adnexae. Following injury, migratory keratinocytes at wound margins and the newly restored epidermis expressed increased levels of parathyroid hormone-related protein. The remodeling phase was associated with progressive restoration of the pattern of parathyroid hormone-related protein expression in normal epidermis. Granulation tissue myofibroblasts and infiltrating macrophages also expressed parathyroid hormone-related protein. In vitro studies using THP-1 cells (a promonocytic cell line) confirmed that macrophages expressed parathyroid hormone-related protein, especially after activation. Topical application of parathyroid hormone related protein (1-36) or parathyroid hormone (7-34) did not result in significant changes in the healing rate and morphology of the wounds. These findings demonstrated that, in addition to keratinocytes, myofibroblasts and macrophages also represent sources of parathyroid hormone-related protein during the healing of skin wounds. Although the data suggest a role for parathyroid hormone-related protein in the healing of skin and in the restoration of epidermal homeostasis, parathyroid hormone-related protein does not appear to be required for proper re-epithelialization in response to injury, potentially because of redundancy in epidermal growth and wound healing, as has been shown for other paracrine and autocrine growth factors of the epidermis. (+info)
Expression of interferon-beta is associated with growth arrest of murine and human epidermal cells.
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The cytokine interferon-beta is a regulator of cell replication and function, including invasion and induction of angiogenesis. The goal of this study was to determine whether the expression of interferon-beta by cells in the epidermis correlated with terminal differentiation. In situ hybridization analysis and immunohistochemical staining of formalin-fixed, paraffin-embedded specimens of normal human and murine epidermis and human and murine skin tumors of epithelial origin revealed that only differentiated, nondividing cells of the epidermis expressed interferon-beta protein. Keratinocyte cultures established from the epidermis of 3 d old mice were maintained under conditions permitting continuous cell division or induction of differentiation. Continuously dividing cells did not produce interferon-beta whereas nondividing differentiated cells expressing keratin 1 did. Growth-arrested, undifferentiated keratinocytes also expressed interferon-beta protein. Neutralizing interferon-beta in the culture medium inhibited differentiation, but the addition of exogenous interferon-beta did not stimulate differentiation. These data indicate that interferon-beta is produced by growth-arrested, terminally differentiated keratinocytes. (+info)
A partial catalog of proteins secreted by epidermal keratinocytes in culture.
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Proteins secreted by epidermal keratinocytes are known to engage in functions other than those directly associated with barrier formation. We have used a previously published culture model to collect proteins secreted by adult human epidermal keratinocytes. Electrophoresis and microsequencing allowed us to identify 20 proteins. The list of proteins includes those known to be produced by keratinocytes (beta-2 microglobulin, betaIG-H3, calgranulin A, cathepsin B and D, E-cadherin, gelatinase B, gelsolin, interstitial collagenase, laminin B2t, plasminogen activator inhibitor-1, protein 14-3-3epsilon, SCC antigen, stratifin, and translationally controlled tumor protein) as well as those not previously known to be secreted by keratinocytes (epididymis secretory protein, maspin, and anti-neoplastic urinary protein). In addition, two proteins were identified that are not known to be secreted (glutathione-S-transferase and heat shock protein 27/28 kDa). The varied nature of the proteins identified suggests that epidermal keratinocytes have physiologic functions that have yet to be identified. (+info)
Trichostatin A up-regulates human papillomavirus type 11 upstream regulatory region-E6 promoter activity in undifferentiated primary human keratinocytes.
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Human papillomavirus (HPV) gene expression in squamous epithelia is differentiation dependent in benign patient lesions and in organotypic raft cultures of primary human keratinocytes (PHKs). Using the lacZ reporter in raft cultures, we previously showed that this transcriptional regulation of the HPV type 11 (HPV-11) enhancer-promoter located in the upstream regulatory region (URR) appears to have resulted from coordination between the transcription transactivators AP1, Oct1, and Sp1 in differentiated upper strata and the repressor C/EBP in proliferating basal cells. We report here that trichostatin A, a specific inhibitor of histone deacetylase, dramatically stimulated reporter gene activity from the wild-type HPV-11 URR or the C/EBP mutation in PHKs grown in undifferentiated submerged cultures. In epithelial raft cultures, up-regulation occurred predominantly in basal and parabasal strata; this effect was promoter specific, as expression of the lacZ reporter gene driven by the murine leukemia virus long terminal repeat (LTR), the keratin 14 promoter, or the involucrin promoter was not altered, nor was expression of endogenous keratin 10 and profilaggrin affected. However, the responses were not cell type or species specific, as identical results were observed for both HPV-11 URR-lacZ and LTR-lacZ in murine retrovirus producer cell lines of fibroblast origin. (+info)
Cell type and gene-specific activity of the retinoid inverse agonist AGN 193109: divergent effects from agonist at retinoic acid receptor gamma in human keratinocytes.
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Retinoids are important regulators of epithelial differentiation. AGN 193109 is a high-affinity antagonist and inverse agonist for the nuclear retinoic acid receptors (RARs). Paradoxically, both AGN 193109 and retinoid agonists inhibit the expression of the differentiation marker MRP-8 in normal human keratinocytes (NHKs). TTNPB, an RAR agonist, and AGN 193109 mutually antagonize MRP-8 inhibition at both mRNA and protein levels. We find that this antagonism, which is greatest at an AGN 193109:TTNPB ratio of about 10:1, is absent when either compound is in significant excess. The potent RARalpha-specific agonist, AGN 193836, has no effect on MRP-8 regulation. These data indicate that inverse agonists and agonists suppress MRP-8 in NHKs through RARgamma using distinct and mutually inhibitory mechanisms. The activity of AGN 193109 on MRP-8 is cell type specific. In differentiating ECE16-1 cervical cells, TTNPB inhibits while AGN 193109 induces MRP-8 mRNA levels. The effect of AGN 193109 on genes inhibited by retinoid agonists in NHKs is also selective; expression of the differentiation markers transglutaminase 1 and keratin 6 is not down-regulated by AGN 193109 whereas stromelysin-1 expression is suppressed. These results show a complex gene and cell context-specific interplay between agonist and inverse agonist for the regulation of gene expression. (+info)