Expression of neutrophil gelatinase-associated lipocalin in skin epidermis.
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Acute-Phase Proteins↗
Biomedical subjects
Jeung-Hoon Lee
Publications and source records attributed to Jeung-Hoon Lee.
Expression of vanilloid receptor 1 in cultured fibroblast.
Although the vanilloid receptor 1 (VR1) was originally discovered on primary sensory neurons, its broad tissue expression in non-neuronal cells has been reported on. Recently, VR1 expression was clearly demonstrated in a variety of cutaneous components, such as keratinocytes, glandular epithelium, mast cells and sebocytes, except for melanocytes and fibroblasts. However, we demonstrated the VR1 expression in the cultured human skin fibroblasts as follows. Previously cloned human VR1 primers that corresponded to the expected size of 680 bp by reverse transcriptase polymerase chain reaction were identified on the fibroblasts, the same as was noted for the positive control, the HaCaT cells. A positive immunoreactivity of the VR1 was observed both on fibroblasts and on HaCaT cells by Western blotting analysis. Fibroblasts treated with capsaicin, an agonist to the VR1, induced significant changes of the membrane current and the intracellular calcium level, and these changes were antagonized by capsazepin. Capsaicin treatment also showed a positive immunocytochemistry result. Our results suggest the existence of VR1 on fibroblasts; this receptor is likely to be influenced by ligand-dependent activation.
Blotting, Western↗
Central trichoptilosis with onycholysis.
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Adult↗
Altered immune cell proportions in the radiodermatitis induced hairless mice-1 (HR-1).
Accidental radiation exposures or radiation therapy can cause internal and external damage including radiodermatitis. Even though radiodermatitis is one of the dose limiting factors in radiotherapy, the immunological nature of it is not yet been clearly understood. In this study, we have examined the alteration in immune cell population during the radiodermatitis process. A radiodermatitis model was established in HR-1 mice by locally exposing a posterior dorsal region to 10 Gy X-ray/day for 4 consecutive days. Collagen accumulation, redness, erythema, and dry desquamation of the skin were detected after X-irradiation. The size and total cell number of the spleen decreased immediately after X-irradiation, compared to those of the sham-irradiated mice, and recovered to the normal levels two weeks later. Reduction and recovery of the bone marrow cell population preceded a similar change of the spleen cell population. The proportion of CD4+ T cell increased, while the proportion of CD8+ T cell decreased ahead of the obvious skin damage, in both lymph node and spleen of the irradiated mice. Interestingly, the proportion of splenic monocytes/macrophages was expanded gradually at a similar kinetics with the aggravation of the radiodermatitis. The infiltration of the CD11b+ monocyte/macrophage to the X-irradiated skin also coincided with the development of radiodermatitis. These altered proportions of immune cells may play important roles in radiodermatitis.
Adaptation, Physiological↗
Identification of genes responding to ultraviolet B irradiation in HaCaT keratinocytes cultured in vitro.
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Cell Line, Transformed↗
CpG-oligodeoxynucleotide protects immune cells from gamma-irradiation-induced cell death.
Synthetic oligodeoxynucleotides (CpG-ODNs) and bacterial DNA containing unmethylated CpG dinucleotides in the context of particular base sequences (CpG motifs) are known to inhibit anti-IgM-induced growth arrest and apoptosis of WHEI 231 B lymphocytes, and spontaneous apoptosis of mature spleen B cells in a sequence-specific fashion of the CpG-ODN. Here we report that CpG-ODN protects from the cell death induced by gamma-irradiation of primary mouse spleen cells as well as mouse RAW 264.7 macrophage cells and human RPMI 8226 B cells. Experimental results showed that CpG-ODN promotes growth of the cells, and protects the cells from gamma-irradiation-induced cell death accompanying Bcl-xS/L and Bcl-2 upregulation. Furthermore, survival of macrophages was enhanced when splenocytes were pretreated with CpG-ODN. Our results suggest the potential application of CpG-ODNs for more efficient cancer radiotherapy by enhancing survival of normal immune cells after radiation damage.
Animals↗
Analysis of calcium-inducible genes in keratinocytes using suppression subtractive hybridization and cDNA microarray.
Terminal differentiation of skin keratinocytes is a vertically directed multistep process that is tightly controlled by the sequential expression of a variety of genes. To gain further insight into the molecular events involved in this process, we used suppression subtraction hybridization (SSH) and cDNA microarray analysis. Messenger RNAs were isolated from primary skin keratinocytes cultured in vitro after treatment with calcium and then SSH was performed. A total of 840 cDNA clones were obtained from subtracted libraries, and these cDNA clones were used to make the microarray slides. Time-course cDNA microarray analysis (1, 3, 7, and 14 days after calcium treatment) revealed the global gene expression profile during keratinocyte differentiation. Of the 840 genes tested, 290 showed a greater than twofold change in expression level at least once over four time points. The genes were clustered into six groups according to their expression pattern using self-organizing map analysis and showed the global feature of function-related regulation. The genes related to keratinocyte differentiation were markedly up-regulated by calcium treatment. In addition, a unique pattern of increase was seen in the expression of genes related to ribosomal proteins. On the other hand, transcripts involved in metabolism, DNA repair, transcription, and translation were generally down-regulated. These results demonstrate the complexity of the gene expression profile that contributes to the spatiotemporal regulation of keratinocyte differentiation.
Calcium↗
IFN-gamma regulates the expression of B7-H1 in dermal fibroblast cells.
BACKGROUND: Programmed cell death ligand 1 (B7-H1) was recently cloned in antigen presenting cells (APCs) and represents a third member of the B7 family. Thus, B7-H1 may be a novel target for clinical intervention in human inflammatory disease. OBJECTIVE: The aim of this study is to investigate the signal transduction mechanism and transcriptional regulation of B7-H1 expression in human dermal fibroblasts. METHODS: We performed reverse transcription PCR (RT-PCR) for the detection of mRNA expression, luciferase reporter assays with B7-H1 promoter constructs, and Western blot analysis. RESULTS: From RT-PCR analysis, IFN-gamma can induce the expression of B7-H1 mRNA in dermal fibroblast. This expression is similar to the results of luciferase reporter assay with B7-H1 promoter. Western blot analysis and EMSA revealed that NF-kappaB transcription factors mediate the induction of B7-H1 expression via the transient phosphorylation of ERK1/2 and PI3K when cells are stimulated by IFN-gamma. Also, Specific destruction of the NF-kappaB binding site abolished the induction of the promoter activity by IFN-gamma. CONCLUSION: Our data not only provides the first evidence to demonstrate that dermal fibroblast express the B7-H1 mRNA in the process of skin inflammation, but also suggests the involvement of NF-kappaB and MAPK and PI3K, that may play some important roles in inflammation process in human skin diseases.
Antigens, CD↗
Upregulation of class II beta-tubulin expression in differentiating keratinocytes.
The diverse functions of microtubules (MT) in different cells and tissues may be facilitated by compositional changes in tubulin isotypes. We obtained partial cDNA clones of class II beta-tubulin from a library of differentiating normal human epidermal keratinocytes (NHEK) cells, whereas screening via subtractive hybridization for genes involved in calcium-induced keratinocyte differentiation. Analysis of the isotypic composition of beta-tubulin from NHEK cells revealed elevations in class II beta-tubulin concentrations at both protein and message levels during cell differentiation, resulting in increased rates of incorporation of class II beta-tubulin into MT. Immunohistochemistry of normal and pathologic skin tissues showed that class II beta-tubulin occurred in the granular layer of the epidermis and in differentiated areas of carcinomas. Class II beta-tubulin was, however, not observed in the uppermost granular and cornified layers of normal epidermis. Further experiments showed that MT were likely to decay in the final stage of terminal differentiation during formation of the cornified envelope. Our results suggest that there is differential modulation of MT composition and stability during keratinocyte differentiation.
Acetylation↗
Molecular cloning and expression of human keratinocyte proline-rich protein (hKPRP), an epidermal marker isolated from calcium-induced differentiating keratinocytes.
We isolated a human gene encoding keratinocyte proline-rich protein (hKPRP). hKPRP gene is located in the region of epidermal differentiation complex on chromosome 1q21, and its approximately 2.5 kb mRNA encodes 579 amino acid protein with high proline content (18%). The mRNA level of hKPRP was markedly increased at both 7 and 14 d after treatment with 1.2 mM calcium in cultured normal human epidermal keratinocytes. In situ hybridization demonstrated that hKPRP was expressed in upper granular layer of normal epidermis with characteristic intermittent pattern. In psoriatic lesion, hKPRP expression was increased as compared with normal skin and showed continuous pattern. Immunohistochemical analysis also confirmed the expression of hKPRP at the protein level. Western blot analysis showed that hKPRP protein of approximately 70 kDa size was significantly increased by calcium in a time-dependent manner. In mouse tissue blot assays, the expression of KPRP was detected in stomach and skin tissues, and began at 17.5 embryonic days. Additionally, hKPRP expression was detected in the periderm of human fetal skin from 16 wk estimated gestational age. Together, these results suggest that hKPRP is an epidermal marker expressed in stratified squamous epithelia and has a potential role in keratinocytes differentiation.
Amino Acid Sequence↗
Involvement of urokinase-type plasminogen activator in sphingosylphosphorylcholine-induced angiogenesis.
Sphingosylphosphorylcholine (SPC) has been shown to accelerate wound healing. As angiogenesis is fundamental to proper wound healing, we examined the effect of SPC on angiogenesis using a well-established rat aortic ring assay. SPC significantly stimulated the sprouting of endothelial cells from rat aortic ring. Recognizing its potential effect on angiogenesis, we further investigated the action of SPC using human umbilical vein endothelial cells (HUVECs) cultured in vitro. SPC significantly accelerated the closure of in vitro wound. In addition, SPC markedly enhanced the chemotactic migration and capillary-like tube formation. Subsequently, we examined whether SPC affected the production of urokinase-type plasminogen activator (uPA), an important regulator of angiogenesis, and found that SPC stimulated the expression of uPA at both the transcriptional and translational levels. Consistent with these results, SPC increased the activity of cell-surface-associated plasminogen activator. Pretreatment with antiuPA antibody significantly diminished both the chemotactic migration and capillary-like tube formation, indicating the potential importance of uPA in SPC-induced angiogenesis. Together, these results suggest that SPC may affect angiogenesis in the wound-healing process via regulation of uPA production.
Animals↗
Induction of connective tissue growth factor expression by sphingosylphosphorylcholine in cultured human skin fibroblasts.
Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In an effort to find downstream effectors of SPC, we performed microarray analysis and found that the expression of the gene for connective tissue growth factor (CTGF) was significantly affected in human skin fibroblasts cultured in vitro. Northern blot analysis showed that SPC markedly induced CTGF mRNA expression in a dose- and time-dependent manner. Consistent with this result, Western blot analysis also showed that SPC significantly induced the CTGF production. Pretreatment with cycloheximide did not prevent the CTGF induction by SPC, indicating that SPC stimulates CTGF mRNA expression without the increased synthesis of a regulatory protein. Inhibition by pretreatment with Y27632, but not by PD98059 (a mitogen-activated protein kinase 1/2 inhibitor) and LY294002 (a phosphatidylinositol 3-kinase inhibitor), indicated that rho-kinase pathway was involved in SPC-induced CTGF expression. Together, these results reveal the potential importance of CTGF induction as a downstream event in SPC-induced cellular responses.
Base Sequence↗
Daxx-mediated transcriptional repression of MMP1 gene is reversed by SPOP.
Daxx-mediated transcriptional repression was modulated by a speckled POZ domain protein SPOP which was first identified as an autoantigen from the serum of a scleroderma patient. This is the first report on the biochemical and functional interactions between Daxx and SPOP. The COOH-terminal region of Daxx interacts with the NH2-terminal region of SPOP. SPOP reversed the transcriptional repression mediated by Daxx which binds with ETS1 transcription factor to repress ETS1-responsive gene expression. Mutagenesis study suggests that the ability of SPOP to self-associate as well as its ability to bind with Daxx was important for the modulation of Daxx-mediated transcriptional repression.
Animals↗
Identification of PUVA-inducible genes in primary cultured dermal fibroblasts using suppression subtractive hybridization.
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Cells, Cultured↗
Signaling events during induction of plasminogen activator inhibitor-1 expression by sphingosylphosphorylcholine in cultured human dermal fibroblasts.
Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In a search for effectors downstream of SPC in the wound-healing process, we found that the expression of the gene for plasminogen activator inhibitor-1 (PAI-1) was significantly affected. ELISA and western blot analyses showed that SPC markedly induced PAI-1 production in human dermal fibroblasts cultured in vitro. Inhibition by pre-treatment with pertussis toxin (PTx), but not by tyrphostin A47 (a receptor tyrosine kinase inhibitor), indicated that PTx-sensitive G proteins were involved in SPC-induced PAI-1 expression. SPC elicited a rapid and transient increase in intracellular calcium levels ([Ca2+]i), measured using laser scanning confocal microscopy, which was partly mediated through PTx-sensitive G proteins. Pre-treatment with thapsigargin, but not with EGTA, abolished SPC-induced PAI-1 expression, indicating the importance of Ca2+ release from internal stores. Phorbol-12-myristate-13-acetate (PMA) induced the expression of PAI-1, and pre-treatment with Ro 31-8220 (a PKC inhibitor) markedly suppressed SPC-induced PAI-1 expression. SPC-induced PAI-1 expression was also significantly suppressed by PD98059 (a specific MAPK kinase 1/2 inhibitor). Consistent with this result, SPC stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase (ERK). Together, these results suggest that SPC induces PAI-1 production through a G protein-coupled calcium increase and downstream kinase signaling events in cultured human dermal fibroblasts.
Animals↗
Characterization of Ets-binding sequence of human transglutaminase 3 gene promoter.
The Ets- and Sp1-transcription factors have been implicated in the epithelial specific expression of transglutaminase 3 gene (TGM3) in vitro. By electrophoretic mobility shift assay (EMSA), the core motif of Ets-binding sequence of TGM3 was determined as ACAGGAAT (-118 to -111 bp from transcriptional start site). However, a sequence extending from -120 to +10 of TGM3 proximal promoter region failed to induce the expression of CAT reporter in transfected normal human epidermal keratinocytes (NHEKs). In contrast, a construct extending from -126 to +10 bp showed the highest expression of CAT gene, indicating the presence of an important element(s) between -126 and -120 bp that affects TGM3 expression in NHEKs. To find the critical sequences in this promoter region, we performed EMSA analysis with competitor oligonucleotides, in which the upstream nucleotides of Ets core motif were mutated. The results showed that the mutation of A at -125 dramatically reduced the binding of Ets to its recognition sequence. Transient transfection assay revealed that the mutation in this nucleotide greatly diminished the expression of CAT reporter gene in NHEKs. Together, these results suggest that the upstream sequence of Ets core motif is critical for the expression of TGM3 in NHEKs cultured in vitro.
Binding Sites↗
Identification of calcium-inducible genes in primary keratinocytes using suppression-subtractive hybridization.
Terminal differentiation in epidermal keratinocytes involves major biochemical changes including the expression of many new differentiation-specific genes. To further understand this process, we performed suppression-subtractive hybridization of keratinocytes cultured under high-calcium condition, known to induce differentiation in vitro. We randomly isolated 300 clones representing 90 different genes. By reverse Northern blot analyses, 20 different genes were found to be overexpressed, of which 13 were confirmed as differentially expressed genes during keratinocyte differentiation by Northern blot analysis. Of those, five genes, transglutaminase 1, keratin 6, interleukin-1 receptor antagonist, kallikrein 7, and heat shock protein 27, are known to be up-regulated during epidermal differentiation. Six genes, ferritin-L chain, ribosomal protein S6, tumor-associated calcium signal transducer 2, neuroendocrine secretory protein 55, phosphoserine aminotransferase, and neutrophil gelatinase-associated lipocalin, heretofore were not known to be up-regulated during keratinocyte differentiation. We also identified two novel genes. One of these maps to chromosome 1q21 of the epidermal differentiation complex, and its expression level was strongly increased in differentiating keratinocytes. These differentially expressed genes may provide significant opportunities for further understanding of the epidermal keratinocyte differentiation.
Blotting, Northern↗
Sphingosylphosphorylcholine stimulates cellular fibronectin expression through upregulation of IL-6 in cultured human dermal fibroblasts.
Sphingosylphosphorylcholine (SPC) has been reported to stimulate wound healing by its potent mitogenic effect. Fibronectin (FN) is a cell-adhesion protein that plays an important role in cell migration and collagen deposition during wound healing. In order to elucidate further the mechanism involved in the accelerated wound healing stimulated by SPC, we studied the role of SPC in FN production in cultured human dermal fibroblasts. We demonstrated that SPC dose- and time-dependently enhanced the expression of FN in human dermal fibroblast at the protein and mRNA levels. IL-6 is known to stimulate the production of FN in fibroblasts. SPC also markedly induced IL-6 production in cultured human dermal fibroblasts in a dose- and time-dependent manner. We also demonstrated that FN mRNA expression in human dermal fibroblasts was upregulated 4 h after IL-6 treatment. Moreover, pretreatment with neutralizing anti-IL-6 antibodies partially blocked the upregulation of FN mRNA expression induced by SPC in human dermal fibroblasts. These results indicate that SPC may stimulate FN synthesis through IL-6 production in cultured human dermal fibroblasts.
Antibodies↗