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Jang-Kyu Park

Publications and source records attributed to Jang-Kyu Park.

14 recordsLinked to original sources
PubMed2005

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↗
PubMed2005

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↗
PubMed2005

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↗
PubMed2005

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↗
PubMed2004

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↗
PubMed2004

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↗
PubMed2004

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↗
PubMed2002

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↗
PubMed2002

Identification of calcium-induced genes in HaCaT keratinocytes by polymerase chain reaction-based subtractive hybridization.

Suppression subtractive hybridization, a PCR-based method for cDNA subtraction, was used to identify differentially expressed genes in keratinocytes. Differentiation was induced by elevating the calcium level in the cell culture medium. Using HaCaT immortalized keratinocytes cultured in the presence of a high calcium concentration, we isolated 60 clones representing 48 different genes. By reverse Northern analysis, 13 genes were scored as overexpressed in these HaCaT cells. Northern blot analysis was used to confirm differential gene expression. Six genes, keratin 1, plasminogen activator inhibitor type 2 (PAI-2), ferritin H, peroxiredoxin 5 (PRDX5), insulin-like growth factor binding protein-3 (IGFBP-3), and one EST gene, were differentially expressed in HaCaT cells cultured in the presence of a high calcium concentration. Two of these genes, keratin 1 and PAI-2, are differentially expressed during keratinocyte terminal differentiation. IGFBP-3, which has reduced expression during epidermal differentiation, was increased after culture in a high-calcium medium for 2 or 5 days. Overexpression of the ferritin H and PRDX5 genes due to elevated calcium has not been reported in keratinocytes. We demonstrated the expression of IGFBP-3, ferritin H, PRDX5, and one gene of a matching sequence from the EST database during differentiation in primary cultured normal human keratinocytes. The EST gene expressed two transcripts of 1.8 kb and 2.5 kb in HaCaT cells, and the transcripts were confirmed to increase in keratinocytes cultured in a high-calcium medium.

Calcium↗