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1.
Clin Oral Investig ; 23(8): 3339-3349, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30506228

ABSTRACT

OBJECTIVES: The potential additive effect of an enamel matrix derivative (EMD) to a subepithelial connective tissue graft (CTG) for recession coverage is still controversially discussed. Therefore, the aim of this study was to histologically evaluate the healing of gingival recessions treated with coronally advanced flap (CAF) and CTG with or without EMD in dogs. MATERIALS AND METHODS: Gingival recession defects (5 mm wide and 7 mm deep) were surgically created on the labial side of bilateral maxillary canines in 7 dogs. After 8 weeks of plaque accumulation and subsequent 2 weeks of chemical plaque control, the 14 chronic defects were randomized to receive either CAF with CTG (CAF/CTG) or CAF with CTG and EMD (CAF/CTG/EMD). The animals were sacrificed 10 weeks after reconstructive surgery for histologic evaluation. RESULTS: Treatment with CAF/CTG/EMD demonstrated statistically significantly better results in terms of probing pocket depth reduction (P < 0.05) and clinical attachment level gain (P < 0.001). The length of the epithelium was statistically significantly shorter in the CAF/CTG/EMD group than in the CAF/CTG group (1.00 ± 0.75 mm vs. 2.38 ± 1.48 mm, respectively, P < 0.01). Cementum formation was statistically significantly greater in the CAF/CTG/EMD group than following treatment with the CAF/CTG group (3.20 ± 0.89 mm vs. 1.88 ± 1.58 mm, respectively, P < 0.01). The CAF/CTG/EMD group showed statistically significantly greater complete periodontal regeneration (i.e., new cementum, new periodontal ligament, and new bone) than treatment with CAF/CTG (0.54 ± 0.73 mm vs. 0.07 ± 0.27 mm, respectively, P < 0.05). CONCLUSION: Within their limits, the present findings indicate that the additional use of EMD in conjunction with CAF + CTG favors periodontal regeneration in gingival recession defects. CLINICAL RELEVANCE: The present findings support the use of EMD combined with CTG and CAF for promoting periodontal regeneration in isolated gingival recession defects.


Subject(s)
Connective Tissue , Dental Enamel Proteins , Gingival Recession , Animals , Connective Tissue/transplantation , Dogs , Gingiva , Gingival Recession/surgery , Gingivoplasty , Tooth Root , Treatment Outcome
2.
Clin Oral Investig ; 20(7): 1791-800, 2016 Sep.
Article in English | MEDLINE | ID: mdl-26612398

ABSTRACT

OBJECTIVE: This study aimed to evaluate the effects of a porcine acellular dermal matrix (PADM) with or without an enamel matrix derivative (EMD) on gingival recession defects treated with a coronally advanced flap (CAF) in dogs. MATERIALS AND METHODS: Miller class II gingival recession defects (5 mm wide and 7 mm deep) were surgically created on the labial side of bilateral maxillary canines in 12 dogs. After 8 weeks of plaque accumulation, the 24 chronic defects were randomly assigned to one of the following 4 treatments: CAF, CAF with PADM (CAF/PADM), CAF with EMD (CAF/EMD), and CAF with EMD and PADM (CAF/EMD/PADM). The animals were sacrificed 10 weeks after surgery for histologic evaluation. RESULTS: In all groups, root coverage was obtained to a varying degree. PADM was well incorporated in gingival connective tissue in the CAF/PADM and in the CAF/EMD/PADM groups. The height of newly formed bone was significantly greater in the CAF/EMD/PADM group than in the CAF and CAF/PADM groups. New cementum with periodontal ligament-like tissue was predominantly found in the CAF/EMD and CAF/EMD/PADM groups. The CAF/EMD/PADM group showed the greatest amount of new cementum among the groups examined, although the difference was not statistically significant. CONCLUSION: Within the limitations of the present study, it can be concluded that CAF/EMD/PADM treatment may promote periodontal regeneration in gingival recession defects. CLINICAL RELEVANCE: The present results suggest that the combination of EMD and PADM in conjunction with CAF may represent a promising approach for treating single Miller class II gingival recessions.


Subject(s)
Acellular Dermis , Dental Enamel Proteins/pharmacology , Gingival Recession/drug therapy , Gingival Recession/surgery , Surgical Flaps , Wound Healing/physiology , Animals , Combined Modality Therapy , Dogs , Gingivoplasty/methods , Regeneration , Swine
3.
J Periodontal Res ; 49(1): 93-101, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23611485

ABSTRACT

BACKGROUND AND OBJECTIVE: Epidemiologic and clinical studies have indicated that diabetes is a risk factor for periodontal disease progression and healing. The aim of the present study was to evaluate short-term healing after enamel matrix derivative (EMD) application in combined supra/infrabony periodontal defects in diabetic rats. MATERIAL AND METHODS: Thirty male Wistar rats were initially divided into two groups, one with streptozotocin-induced diabetes and another one with healthy (non-diabetic) animals. Bony defects were surgically created on the mesial root of the first maxillary molars. After root surface planing and EDTA conditioning, EMD was applied to the roots at one side of the maxillae, while those on the contralateral sides were left untreated. Animals were killed 3 wk after surgery, and block sections were prepared for histologic and histomorphometric analysis. RESULTS: There was statistically significant more gingival recession in diabetic animals than in non-diabetic animals. The length of the junctional epithelium was significantly shorter in the EMD-treated sites in both diabetic and normoglycemic rats. Sulcus depth and length of supracrestal soft connective tissue showed no statistically significant differences between groups. In all animals, new bone formation was observed. Although new bone occurred more frequently in healthy animals, the extent of new bone was not significantly different between groups. In none of the teeth, a layer of new cementum was detectable. EMD had no influence on bone or cementum regeneration. Adverse reactions such as excessive inflammation due to bacterial root colonization, ankylosis and bone fractures were exclusively observed in diabetic animals, irrespective of EMD treatment. CONCLUSION: Within the limits of the present study, it can be concluded that periodontal healing was impaired in streptozotocin-induced diabetic rats. EMD had no beneficial effects on new bone and cementum formation during short-term healing in this defect model and could not ameliorate the adverse effects in the systemically compromised animals.


Subject(s)
Alveolar Bone Loss/surgery , Dental Enamel Proteins/therapeutic use , Diabetes Mellitus, Experimental/complications , Animals , Cementogenesis/drug effects , Connective Tissue/drug effects , Connective Tissue/pathology , Edetic Acid/therapeutic use , Epithelial Attachment/drug effects , Epithelial Attachment/pathology , Gingival Recession/etiology , Male , Maxillary Diseases/surgery , Molar/surgery , Osteogenesis/drug effects , Postoperative Complications , Rats, Wistar , Root Planing/methods , Streptozocin , Tooth Ankylosis/etiology , Tooth Fractures/etiology , Tooth Root/injuries , Tooth Root/surgery , Tooth Socket/drug effects , Tooth Socket/pathology , Wound Healing/physiology
4.
J Periodontal Res ; 48(1): 37-43, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22775137

ABSTRACT

BACKGROUND AND OBJECTIVE: Although the application of EMD is a widely accepted periodontal-regenerative therapy, its effects on noncontained intrabony defects are unpredictable because of the lack of a space-making property. The combined use of EMD and autogenous bone grafts reportedly stimulates significant periodontal regeneration in intrabony defects. The aim of the present study was to evaluate the effects of EMD in combination with bone swaging (BS) and injectable calcium phosphate bone cement (CPC), which was placed into the spaces between the grafted swaged bone and the proximal host bone, on periodontal healing in one-wall intrabony defects in dogs. MATERIAL AND METHODS: One-wall intrabony defects (3 mm wide and 5 mm deep) were surgically created on the mesial and distal sides of the bilateral mandibular premolars in four dogs. The 16 defects were assigned to one of the following treatments: EMD only, BS only, EMD with BS (EMD + BS), or EMD with BS and CPC (EMD + BS + CPC). The animals were killed 8 wk after surgery for histologic evaluation. RESULTS: The height of newly formed bone was significantly greater in the EMD + BS + CPC group (3.73 ± 0.30 mm) than in the BS-only (2.74 ± 0.33 mm; p < 0.05) and EMD + BS (2.88 ± 0.98 mm; p < 0.05) groups. The area of newly formed bone was significantly larger in the EMD + BS + CPC group (5.68 ± 1.66 mm(2)) than in the EMD-only (3.68 ± 0.33 mm(2); p < 0.05), BS-only (3.48 ± 1.26 mm(2); p < 0.05) and EMD + BS (3.38 ± 1.37 mm(2); p < 0.05) groups. The EMD-only (4.63 ± 0.42 mm), EMD + BS (4.67 ± 0.30 mm) and EMD + BS + CPC (4.78 ± 0.54 mm) groups showed significantly greater cementum formation than did the BS-only group (3.93 ± 0.56 mm; p < 0.05). CONCLUSION: These results indicate that treatment with EMD + BS + CPC promotes favorable periodontal healing in one-wall intrabony defects in dogs.


Subject(s)
Alveolar Bone Loss/surgery , Bone Cements/therapeutic use , Bone Transplantation/methods , Calcium Phosphates/therapeutic use , Dental Enamel Proteins/therapeutic use , Alveolar Process/drug effects , Alveolar Process/pathology , Animals , Bone Regeneration/drug effects , Cementogenesis/drug effects , Collagen/drug effects , Connective Tissue/drug effects , Connective Tissue/pathology , Dental Cementum/drug effects , Dental Cementum/pathology , Dogs , Epithelial Attachment/drug effects , Epithelial Attachment/pathology , Male , Mandible/surgery , Osteogenesis/drug effects , Periodontal Ligament/drug effects , Periodontal Ligament/pathology , Tooth Cervix/drug effects , Tooth Cervix/pathology , Tooth Root/drug effects , Tooth Root/pathology , Wound Healing/physiology
5.
Br J Dermatol ; 166(2): 322-30, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21936856

ABSTRACT

BACKGROUND: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are characterized by keratinocyte apoptosis and necrosis, resulting in epidermal detachment. Although monocytes abundantly infiltrate the epidermis in SJS/TEN skin lesions, the properties and functions of these cells have not been fully examined. OBJECTIVES: To determine the properties of monocytes infiltrating into the epidermis in SJS/TEN. METHODS: Immunostaining of skin sections was performed to examine the membrane markers of monocytes infiltrating into skin lesions. RESULTS: Immunostaining of cryosections from 11 SJS/TEN skin lesions revealed numerous CD14+ monocytes located along the dermoepidermal junction and throughout the epidermis. The cells coexpressed CD16, CD11c and HLA-DR. CD14+ CD16+ cells were identified in very early lesions without epidermal damage, suggesting that their infiltration is a cause, rather than a result, of epidermal damage. Moreover, these cells expressed CD80, CD86 and CD137 ligand, indicative of their ability to facilitate the proliferation and cytotoxicity of CD8+ T cells. CD16+ cells infiltrating the epidermis and detected at the dermoepidermal junction were immunostained and counted in paraffin-embedded skin sections obtained from 47 patients with drug rash manifested as TEN, SJS, maculopapular-type rash or erythema multiform-type rash. The number of CD16+ monocytes infiltrating the epidermis increased significantly, depending on the grade of epidermal damage. CONCLUSIONS: These findings suggest that the appearance of CD14+ CD16+ cells of monocyte lineage plays an important role in the epidermal damage associated with SJS/TEN, most probably by enhancing the cytotoxicity of CD8+ T cells.


Subject(s)
Lipopolysaccharide Receptors/metabolism , Monocytes/metabolism , Receptors, IgG/metabolism , Stevens-Johnson Syndrome/pathology , 4-1BB Ligand/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis/physiology , B7-1 Antigen/metabolism , B7-2 Antigen/metabolism , Cell Lineage , Cell Proliferation , Epidermis/pathology , Female , Humans , Immunohistochemistry , Keratinocytes/pathology , Male , Middle Aged , Stevens-Johnson Syndrome/immunology
7.
Oncogene ; 26(3): 339-48, 2007 Jan 18.
Article in English | MEDLINE | ID: mdl-16862184

ABSTRACT

Promyelocytic leukemia zinc-finger (PLZF) is a transcriptional repressor and tumor suppressor. PLZF is expressed in melanocytes but not in melanoma cells, and recovery of PLZF expression markedly suppresses melanoma cell growth. Several target genes regulated by PLZF have been identified, but the precise function of PLZF remains uncertain. Here, we searched for candidate target genes of PLZF by DNA microarray analysis. Pre-B-cell leukemia transcription factor 1 (Pbx1) was one of the prominently suppressed genes. Pbx1 was highly expressed in melanoma cells, and its expression was reduced by transduction with the PLZF gene. Moreover, the growth suppression mediated by PLZF was reversed by enforced expression of Pbx1. Knockdown of Pbx1 by specific small interfering RNAs suppressed melanoma cell growth. We also found that Pbx1 binds HoxB7. Reverse transcription-polymerase chain reaction analysis demonstrated that repression of Pbx1 by PLZF reduces the expression of HoxB7 target genes, including tumor-associated neoangiogenesis factors such as basic fibroblast growth factor, angiopoietin-2 and matrix metalloprotease 9. These findings suggest that deregulation of Pbx1 expression owing to loss of PLZF expression contributes to the progression and/or pathogenesis of melanoma.


Subject(s)
DNA-Binding Proteins/genetics , Melanoma/pathology , Proto-Oncogene Proteins/genetics , Transcription Factors/genetics , Angiopoietin-2/genetics , Angiopoietin-2/metabolism , Blotting, Western , Cell Proliferation , DNA-Binding Proteins/metabolism , Down-Regulation , Electrophoretic Mobility Shift Assay , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/metabolism , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Immunoprecipitation , Kruppel-Like Transcription Factors , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Melanocytes/metabolism , Melanoma/genetics , Oligonucleotide Array Sequence Analysis , Pre-B-Cell Leukemia Transcription Factor 1 , Promoter Regions, Genetic/genetics , Promyelocytic Leukemia Zinc Finger Protein , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolism , Transcription, Genetic , Tumor Cells, Cultured , Zinc Fingers
9.
Br J Dermatol ; 149(1): 185-8, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12890216

ABSTRACT

Darier's disease is an autosomal dominant skin disorder that is characterized by multiple keratotic papules, focal loss of adhesion and abnormal keratinization. Mutations in the ATP2A2 gene encoding sarco/endoplasmic reticulum calcium pumping ATPase type 2 have been identified as the molecular basis of Darier's disease. Segmental Darier's disease is a rare type of Darier's disease in which there is characteristic localization of the keratotic papules in a linear pattern following Blaschko's lines. In this study we examined ATP2A2 mutations in a Japanese patient with segmental Darier's disease. The samples from affected skin, unaffected skin and peripheral leucocytes were subjected to polymerase chain reaction (PCR). Direct sequencing of the PCR products was performed. Sequence analysis revealed that the patient had 160A-->G substitution mutation which predicts I54V. This novel mutation was present in the affected skin, but not in the unaffected skin or peripheral leucocytes. This is the first report of segmental Darier's disease caused by mosaicism for an ATP2A2 mutation in Japan.


Subject(s)
Calcium-Transporting ATPases/genetics , Darier Disease/genetics , Mosaicism , Adult , Amino Acid Substitution , Base Sequence , DNA Mutational Analysis , Darier Disease/pathology , Female , Humans , Molecular Sequence Data , Sarcoplasmic Reticulum Calcium-Transporting ATPases
10.
J Biol Chem ; 276(2): 999-1004, 2001 Jan 12.
Article in English | MEDLINE | ID: mdl-11029458

ABSTRACT

Cells differentiate in response to various extracellular stimuli. This cellular response requires intracellular signaling pathways. The mitogen-activated protein (MAP) kinase cascade is a core signal transduction pathway that determines the fate of many kinds of cell. MAP kinase kinase kinase activates MAP kinase kinase, which in turn activates MAP kinase. Apoptosis signal-regulating kinase (ASK1) was identified as a MAP kinase kinase kinase involved in the stress-induced apoptosis-signaling cascade that activates the SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades. Expression of the constitutively active form of ASK1 (ASK1-DeltaN) in keratinocytes induced significant morphological changes and differentiation markers, transglutaminase-1, loricrin, and involucrin. A transient increase in p21(Cip1/WAF1) reduced DNA synthesis, and cell cycle analysis verified the differentiation. p38 MAP kinase inhibitors, SB202190 and SB203580, abolished the induction of differentiation markers, transglutaminase-1, loricrin, and involucrin. In turn, the induction of differentiation with ceramide in keratinocytes caused an increase in ASK1 expression and activity. Furthermore, normal human skin expresses ASK1 protein in the upper epidermis, implicating ASK1 in in vivo keratinocyte differentiation. We propose that the ASK1-p38 MAP kinase cascade is a new intracellular regulator of keratinocyte differentiation.


Subject(s)
Cell Differentiation/physiology , Keratinocytes/cytology , Keratinocytes/enzymology , MAP Kinase Kinase 4 , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System/physiology , Cell Differentiation/drug effects , Cells, Cultured , Enzyme Inhibitors/pharmacology , Genes, Reporter , Humans , Imidazoles/pharmacology , JNK Mitogen-Activated Protein Kinases , Keratinocytes/drug effects , Kinetics , MAP Kinase Kinase Kinase 5 , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Pyridines/pharmacology , Transfection , p38 Mitogen-Activated Protein Kinases
11.
J Cell Biol ; 151(2): 209-20, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11038170

ABSTRACT

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.


Subject(s)
Cell Movement , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Keratinocytes/physiology , Skin/injuries , Wound Healing/physiology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Cell Movement/drug effects , Cells, Cultured , Female , Heparin-binding EGF-like Growth Factor , Humans , Hydroxamic Acids/pharmacology , Intercellular Signaling Peptides and Proteins , Keratinocytes/cytology , Ligands , Matrix Metalloproteinase Inhibitors , Mice , Mice, Inbred BALB C , Protease Inhibitors/pharmacology
12.
J Exp Med ; 192(2): 171-82, 2000 Jul 17.
Article in English | MEDLINE | ID: mdl-10899904

ABSTRACT

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.


Subject(s)
B-Lymphocytes/physiology , Hematopoietic Stem Cells/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Animals , Bone Marrow Cells/physiology , Cell Survival , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/physiology , Proto-Oncogene Proteins c-bcl-2/analysis , Proto-Oncogene Proteins c-bcl-2/physiology , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Transgenes , bcl-X Protein
13.
J Cell Sci ; 113 ( Pt 10): 1803-11, 2000 May.
Article in English | MEDLINE | ID: mdl-10769211

ABSTRACT

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.


Subject(s)
Cell Adhesion/physiology , Cytoskeletal Proteins/genetics , Desmosomes/genetics , Desmosomes/metabolism , Keratinocytes/metabolism , Mutation/genetics , Animals , Cadherins/genetics , Cadherins/metabolism , Calcium/metabolism , Cells, Cultured , Cytoskeletal Proteins/metabolism , DNA, Complementary/genetics , Desmocollins , Desmoglein 3 , Desmogleins , Desmoplakins , Genetic Vectors , Humans , Mice , Precipitin Tests , Protein Structure, Tertiary/genetics , Tight Junctions/genetics , Tight Junctions/metabolism , gamma Catenin
14.
J Biol Chem ; 275(8): 5748-53, 2000 Feb 25.
Article in English | MEDLINE | ID: mdl-10681561

ABSTRACT

Epiregulin is a new member of the epidermal growth factor (EGF) family purified from conditioned medium of NIH-3T3 clone T7. Some EGF family growth factors play essential roles in human keratinocytes in an autocrine manner. We show here that epiregulin is another autocrine growth factor for human keratinocytes. Epiregulin stimulated human keratinocyte proliferation under both subconfluent and confluent culture conditions in the absence of exogenous EGF family growth factors. Immunoprecipitation of [(35)S]methionine-labeled conditioned medium revealed a 5-kDa band corresponding to epiregulin. Northern blot analysis detected a 4. 8-kilobase transcript of epiregulin, and the addition of epiregulin up-regulated epiregulin mRNA synthesis. Furthermore, an anti-epiregulin blocking antibody reduced DNA synthesis by 25%. Epiregulin up-regulated the mRNA levels of heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and TGF-alpha. In turn, the addition of EGF, HB-EGF, amphiregulin, and TGF-alpha increased epiregulin mRNA levels. These results demonstrate that epiregulin acts as an autocrine growth factor in human epidermal keratinocytes and is part of auto- and cross-induction mechanisms involving HB-EGF, amphiregulin, and TGF-alpha. The mRNA expression profile resulting from induction of differentiation with high calcium and fetal calf serum revealed the differential expression of epiregulin, HB-EGF, amphiregulin, and TGF-alpha in keratinocytes. This indicates that these four growth factors have distinct, non-redundant biological functions.


Subject(s)
Epidermal Growth Factor/physiology , Intercellular Signaling Peptides and Proteins , Keratinocytes/chemistry , Amphiregulin , Antimetabolites/pharmacokinetics , Autocrine Communication , Blotting, Northern , Bromodeoxyuridine/pharmacokinetics , Cell Division , Dose-Response Relationship, Drug , EGF Family of Proteins , Epidermal Growth Factor/biosynthesis , Epidermal Growth Factor/pharmacology , Epiregulin , ErbB Receptors/metabolism , Glycoproteins/pharmacology , Growth Substances/pharmacology , Heparin-binding EGF-like Growth Factor , Humans , Phosphorylation , Protein Binding , RNA, Messenger/metabolism , Recombinant Proteins , Reverse Transcriptase Polymerase Chain Reaction , Skin/metabolism , Time Factors , Transforming Growth Factor alpha/biosynthesis
15.
Oncogene ; 18(50): 6965-73, 1999 Nov 25.
Article in English | MEDLINE | ID: mdl-10597295

ABSTRACT

Hepatitis B virus (HBV) X protein activates many viral and cellular genes in trans and functional disruption of the p53 tumor suppressor gene product occurs when X protein is transiently expressed in the cytoplasm of cultured cells. We have carried out investigations to determine the exact location of X protein in X gene transfected cells by using a fluorescent staining technique as well as by biochemical analyses. Aggregation of mitochondrial structures became evident at the periphery of nucleus in the cytoplasm of X transfected cells. X protein was found associated with the aggregated mitochondrial structures. Furthermore, transiently expressed p53 protein co-localized with X protein in X transfected cells. However, the appearance of aggregated mitochondrial structures at the nuclear periphery was independent of the presence of p53 protein in X transfected cells. X protein expression also caused an appearance of TUNEL positive nucleus, cytochrome c release from mitochondrial, the decrease of mitochondrial membrane potential and the membrane blebbing of X transfected cells, which are characteristic of cell death. Our data suggest that X protein causes an abnormal aggregation of mitochondrial structures in the cell, which may be eventually connected with cell death.


Subject(s)
Apoptosis/genetics , Cell Nucleus/metabolism , Mitochondria/metabolism , Trans-Activators/metabolism , Cell Line , Humans , In Situ Nick-End Labeling , Trans-Activators/genetics , Transfection , Tumor Suppressor Protein p53/metabolism , Viral Regulatory and Accessory Proteins
16.
J Immunol ; 163(12): 6589-97, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10586053

ABSTRACT

We and others previously observed that IgM and CD40 stimulation in murine B cells resulted in activation of extracellular signal-regulated kinase (ERK), a subfamily of mitogen-activated protein kinase. The present study demonstrated that ERK was rapidly phosphorylated and translocated to the nucleus in murine B cells upon stimulation with CD40, whereas it was preferentially localized within the cytosol after stimulation with IgM, suggesting that signaling through CD40 and IgM differentially regulates ERK subcellular localization. Costimulation with CD40 and IgM (CD40/IgM) resulted in subcellular localization of ERK within the cytosol, supporting the notion that stimulation with IgM delivers the signal responsible for inhibition of ERK nuclear transport. Consistent with these observations, IgM and CD40/IgM stimulation resulted in activation of ribosomal S6 kinase, which is a cytoplasmic substrate for ERK, whereas CD40 stimulation had little effect on its activity. Disruption of the microtubule by colchicine in WEHI231 cells resulted in reduction of ERK activity in IgM signaling, but not in CD40 signaling, compatible with the notion that the microtubule network may hold cytoplasmic ERK activity mediated by IgM stimulation. These results support the notion that ERK could mediate different effector functions in B cells upon stimulation with IgM and CD40.


Subject(s)
B-Lymphocytes/enzymology , B-Lymphocytes/immunology , CD40 Antigens/pharmacology , Immunoglobulin M/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Animals , CD40 Antigens/physiology , Cells, Cultured , Colchicine/toxicity , Cytoplasm/enzymology , Enzyme Activation/drug effects , Enzyme Activation/immunology , Immunoglobulin M/physiology , Mice , Mice, Inbred C57BL , Microtubules/drug effects , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction/immunology , Subcellular Fractions/enzymology , Subcellular Fractions/immunology , Substrate Specificity/immunology
17.
J Dermatol Sci ; 21(1): 8-12, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10468186

ABSTRACT

Topical vitamin D3 therapy is one of the mainstays of psoriasis treatment. However, the effectiveness of combination therapy with topical vitamin D3 and corticosteroids is still controversial. It has been reported that topical vitamin D3 treatment following topical corticosteroids is less effective than that without preceding corticosteroid treatment. We hypothesized that vitamin D receptor (VDR) in the skin is down-regulated by topical corticosteroids. To obtain support for this hypothesis, we determined VDR protein levels in cultured keratinocytes and fibroblasts after corticosteroid treatment. VDR levels were quantified by Western blot analysis with a Fluorolmager. Keratinocytes and fibroblasts were obtained from four psoriasis patients and four normal controls. VDR levels were altered in neither normal nor psoriatic keratinocytes by 2-day incubation with dexamethasone (1x10(-9)-1x10(-6) M) or clobetasol propionate (1x10(-9)-1x10(-6) M). Similarly, VDR levels in normal and psoriatic fibroblasts were not affected by 2-day incubation with dexamethasone (1x10(-6) M). These findings suggest that down-regulation of VDR by topical corticosteroids in keratinocytes and fibroblasts of psoriasis is unlikely.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Clobetasol/analogs & derivatives , Dexamethasone/pharmacology , Fibroblasts/drug effects , Keratinocytes/drug effects , Receptors, Calcitriol/biosynthesis , Administration, Topical , Adult , Aged , Blotting, Western , Cells, Cultured , Clobetasol/pharmacology , Down-Regulation/drug effects , Female , Fibroblasts/metabolism , Glucocorticoids , Humans , Keratinocytes/metabolism , Male , Middle Aged , Psoriasis/metabolism , Psoriasis/pathology , Skin/cytology , Skin/drug effects , Skin/metabolism
18.
Br J Dermatol ; 140(2): 351-7, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10233237

ABSTRACT

Pemphigus is an autoimmune blistering disease with two major subtypes, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Patients with pemphigus have circulating antidesmoglein (Dsg)1 and/or anti-Dsg3 IgG autoantibodies. We have previously developed enzyme-linked immunosorbent assays (ELISAs) using recombinant Dsg1 and Dsg3 expressed by baculovirus as a diagnostic tool for pemphigus. The purpose of this study was to evaluate the practical application of these ELISAs for clinical use with a large number of serum samples. We used 81 PV sera, 48 PF sera, 114 bullous pemphigoid (BP) sera, 124 collagen disease sera, nine sera of other non-pemphigus bullous diseases and 179 normal control sera. A cut-off value was determined by receiver-operating-characteristic plots. Forty-seven of 48 PF sera (97.9%) were positive in the Dsg1 ELISA and 79 of 81 PV sera (97.5%) were positive in the Dsg3 ELISA, while only two (1. 1%) and four (2.2%) of 179 normal sera were positive in Dsg1 and Dsg3 ELISAs, respectively. However, some disease control sera of BP and collagen diseases exceeded the cut-off value. Introduction of a grey zone helped to decrease the number of these false-positive sera. Furthermore, in three patients studied, the respective Dsg1 and Dsg3 ELISA scores showed parallel fluctuation with the disease activity along the time course. We conclude that Dsg1 and Dsg3 ELISAs provide a simple, sensitive and highly specific assay for the diagnosis of patients with PV and PF and that these ELISAs may be a valuable tool to monitor the disease activity. We also propose diagnostic criteria for pemphigus based on ELISA reactivity: if a serum is positive against Dsg3 it indicates a diagnosis of PV, regardless of reactivity against Dsg1; if a serum is negative for Dsg3 and positive for Dsg1, it indicates a diagnosis of PF.


Subject(s)
Autoantigens/immunology , Autoimmune Diseases/diagnosis , Cadherins/immunology , Cytoskeletal Proteins/immunology , Pemphigus/diagnosis , Aged , Autoantigens/analysis , Cell Adhesion Molecules/immunology , Desmoglein 1 , Desmoglein 3 , Desmogleins , Desmoplakins , Desmosomes/immunology , Diagnosis, Differential , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Male , Middle Aged , ROC Curve , Recombinant Proteins/immunology , Sensitivity and Specificity
19.
J Cell Physiol ; 179(1): 40-4, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10082130

ABSTRACT

It has been reported that p21, p53, and p16 affect the cell cycle and cell senescence. However, their roles in keratinocyte senescence are not clear. We established primary keratinocyte strains from 15 donors and maintained them until replicative senescence; their population doublings ranged from 5.7-45.2. These strains were classified based on their population doublings as short (5.7-10.4), intermediate (13.9-17.4), and long (21.5-45.2). To investigate the roles of p21, p53, and p16 in the cellular senescence of the cultured keratinocytes, we quantitatively analyzed p21, p53, and p16 levels of keratinocyte strains with different life spans by Western blot with Fluorol mager. p21 levels increased in the senescent phase but not in the nonsenescent phase in all of the short, intermediate, and long life-span strains. Northern blot analysis also revealed induction of p21 mRNA was similar to that of p21 protein levels. There were no apparent differences in p53 levels between senescent and nonsenescent cells. The short life-span strains exhibited a significant increase in p16 levels in the senescent phase (eighth or tenth passage). However, in two long life-span strains, p16 levels were increased in the nonsenescent phase (eighth passage) but then declined as the cells reached senescence (twenty-seventh passage). Therefore, induction of p16 appeared not to be associated with senescence in long life-span strains. In conclusion, p21 but not p16 or p53 may play roles in keratinocyte senescence.


Subject(s)
Cellular Senescence/physiology , Cyclins/physiology , Keratinocytes/cytology , Adolescent , Adult , Cell Division , Cells, Cultured , Child , Child, Preschool , Culture Media, Serum-Free , Cyclin-Dependent Kinase Inhibitor p16/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Cyclins/genetics , Female , Gene Expression Regulation , Humans , Infant , Keratinocytes/metabolism , Male , Middle Aged , RNA, Messenger/biosynthesis , Tumor Suppressor Protein p53/physiology
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