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1.
Cell ; 175(2): 488-501.e22, 2018 10 04.
Article in English | MEDLINE | ID: mdl-30270045

ABSTRACT

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.


Subject(s)
Capsid Proteins/immunology , Nuclear Matrix-Associated Proteins/immunology , Nuclear Matrix-Associated Proteins/physiology , Octamer Transcription Factors/immunology , Octamer Transcription Factors/physiology , RNA-Binding Proteins/immunology , RNA-Binding Proteins/physiology , Capsid/metabolism , Capsid Proteins/metabolism , Capsid Proteins/physiology , Cell Nucleus/metabolism , DNA, Viral/genetics , DNA, Viral/immunology , DNA-Binding Proteins , Dendritic Cells/immunology , HIV Infections/immunology , HIV-1/genetics , HIV-1/immunology , HIV-2/genetics , HIV-2/immunology , Host-Pathogen Interactions , Humans , Immunity, Innate/immunology , Macrophages/immunology , Membrane Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/physiology , RNA-Binding Proteins/metabolism , Signal Transduction/immunology
2.
Dev Biol ; 410(1): 36-44, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26708097

ABSTRACT

Reciprocal repression of inner cell mass specific factor OCT4 and trophectoderm specific factor CDX2 promotes mouse first lineage segregation. Studies in mouse embryonic stem (ES) cells revealed that they bind to each other's regulatory regions to reciprocally suppress transcription, additionally they form protein complex for mutual antagonism. However, so far the molecular interaction of Oct4 and Cdx2 in other mammal's early embryo is not yet investigated. Here, over-expression of Cdx2 in early porcine embryo showed CDX2 represses Oct4 through neither the transcriptional repression nor forming repressive complex, but promoting OCT4 nuclear export and proteasomal degradation. The results showed novel molecular regulation of CDX2 on Oct4, and provided important clues for clarifying the mechanism of interaction between CDX2 and Oct4 in embryo of mammals other than mouse.


Subject(s)
Homeodomain Proteins/physiology , Octamer Transcription Factors/physiology , Proteasome Endopeptidase Complex/physiology , Swine/embryology , Active Transport, Cell Nucleus , Animals , Cells, Cultured , Leupeptins/pharmacology , Octamer Transcription Factors/genetics , RNA, Messenger/analysis
3.
J Reprod Dev ; 61(6): 533-40, 2015.
Article in English | MEDLINE | ID: mdl-26255835

ABSTRACT

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.


Subject(s)
Blastocyst/physiology , Octamer Transcription Factors/physiology , Parthenogenesis/physiology , X Chromosome Inactivation/physiology , X Chromosome/genetics , Animals , Female , Gene Transfer Techniques , Genes, X-Linked , Humans , Lentivirus , Male , Mice , Real-Time Polymerase Chain Reaction , Swine
4.
Arch Gynecol Obstet ; 287(3): 477-85, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23073722

ABSTRACT

PURPOSE: Polycystic ovary syndrome (PCOS) is a reproductive endocrinopathy and is the most common cause of anovulatory infertility in women of reproductive age group. In PCOS research, there have been several reports which have shown the differential expression of genes from various tissue and cell types. However, little information can be gained about the regulation of these genes from the microarray data itself. Therefore, with the assumption that co-expression can also imply co-regulation in high-throughput gene expression studies, we investigated the role of various transcription factors by elucidating their binding sites (TFBSs) in these genes. METHODS: For this purpose, a group of 40 genes altered in a forskolin-treated PCOS gene expression study in theca cells were analyzed using in silico tools. 2,000 bp of the upstream region were extracted from all these genes, and the PAINT suite of programs was used to identify over-represented TFBSs in these genes. RESULTS: We identified three different TFBSs which were over-represented as compared with a human promoter background model. These three transcription factors (TFs) are Oct, HFH, and neuron-restrictive silencing factor. Each of these three TFs and their compatible members can be implicated in the pathogenesis of PCOS, as described in detail in this article. CONCLUSIONS: These factors might reveal a new insight into the complex pathogenesis of PCOS especially with respect to cAMP pathway.


Subject(s)
Forkhead Transcription Factors/physiology , Octamer Transcription Factors/physiology , Polycystic Ovary Syndrome/genetics , Repressor Proteins/physiology , Theca Cells/metabolism , Binding Sites/genetics , Female , Gene Expression Regulation , Humans , Models, Genetic , Promoter Regions, Genetic
5.
Biochem J ; 411(1): 201-9, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18042045

ABSTRACT

We report that a DBHS (Drosophila behaviour, human splicing) family protein, p54(nrb), binds both BRG1 (Brahma-related gene 1) and Brm (Brahma), catalytic subunits of the SWI/SNF (switch/sucrose non-fermentable) chromatin remodelling complex, and also another core subunit of this complex, BAF60a. The N-terminal region of p54(nrb) is sufficient to pull-down other core subunits of the SWI/SNF complex, suggesting that p54(nrb) binds SWI/SNF-like complexes. PSF (polypyrimidine tract-binding protein-associated splicing factor), another DBHS family protein known to directly bind p54(nrb), was also found to associate with the SWI/SNF-like complex. When sh (short hairpin) RNAs targeting Brm were retrovirally expressed in a BRG1-deficient human cell line (NCI-H1299), the resulting clones showed down-regulation of the TERT (telomerase reverse transcriptase) gene and an enhancement of ratios of exon-7-and-8-excluded TERT mRNA that encodes a beta-site-deleted inactive protein. All of these clones display growth arrest within 2 months of the Brm-knockdown. In NCI-H1299 cells, Brm, p54(nrb), PSF and RNA polymerase II phosphorylated on CTD (C-terminal domain) Ser(2) specifically co-localize at a region incorporating an alternative splicing acceptor site of TERT exon 7. These findings suggest that, at the TERT gene locus in human tumour cells containing a functional SWI/SNF complex, Brm, and possibly BRG1, in concert with p54(nrb), would initiate efficient transcription and could be involved in the subsequent splicing of TERT transcripts by accelerating exon-inclusion, which partly contributes to the maintenance of active telomerase.


Subject(s)
DNA Helicases/physiology , Nuclear Matrix-Associated Proteins/physiology , Nuclear Proteins/physiology , Octamer Transcription Factors/physiology , RNA Splicing , RNA-Binding Proteins/physiology , Telomerase/genetics , Transcription Factors/physiology , Transcriptional Activation , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/metabolism , DNA Helicases/metabolism , DNA-Binding Proteins , Humans , Nuclear Proteins/metabolism , RNA, Messenger/genetics , Transcription Factors/metabolism , Transcription, Genetic
6.
Brain Res ; 1179: 16-27, 2007 Nov 07.
Article in English | MEDLINE | ID: mdl-17936731

ABSTRACT

Induction of nitric oxide synthase-2 (iNOS) by cytokines and bacterial products is associated with protein binding at the proximal promoter and in an upstream enhancer region of the Nos2 gene. To clarify how ethanol suppresses rat iNOS activity, we constructed several deletion mutants of the Nos2 promoter fused to the luciferase gene and transfected the constructs into C6 glial cells. Acute ethanol exposure of stably transfected cells for 24 h inhibits induced activity of Nos2 promoter constructs containing deletions in the 5' flanking region, including a 94 bp promoter that lacks any known NF-kappaB site but which carries a C/EBPbeta and overlapping gamma-IRE, GAS and Oct motifs. Ethanol failed to inhibit the endogenous activity of a smaller, 78 bp promoter that lacks the C/EBPbeta and overlapping, gamma-IRE and GAS motifs and showed no inducible activity. As another approach, in vivo DNA footprinting was used and identified protein protections at five regions of the proximal Nos2 promoter in induced cells. Exposure to acute ethanol diminished protein occupation in the five promoter regions including the gamma-IRE/NF-kappaB and the overlapping gamma-IRE/GAS/Oct sites. Site-directed mutagenesis in the octamer domain of the gamma-IRE/GAS/Oct motifs was studied in a 1002 bp promoter to examine its role in ethanol inhibition of cytokine and lipopolysaccharide induced activity. The data indicate that ethanol failed to inhibit promoter activity when the Oct motif is missing. Electrophoretic mobility shift assays performed using a 22-mer probe containing the overlapping gamma-IRE/GAS/Oct sites showed three complexes with one of the complexes being competed by an octamer-1 antibody. These observations demonstrate the role of protein-DNA binding at the core promoter, and the likely involvement of the octamer motif, in ethanol modulation of cytokine and lipopolysaccharide induced iNOS expression.


Subject(s)
Brain Neoplasms/metabolism , Central Nervous System Depressants/pharmacology , DNA/physiology , Ethanol/pharmacology , Glioma/metabolism , Nitric Oxide Synthase Type II/genetics , Octamer Transcription Factors/physiology , Promoter Regions, Genetic/drug effects , Animals , Cell Line, Tumor , Cytokines/biosynthesis , DNA Footprinting , Electrophoretic Mobility Shift Assay , Gene Expression Regulation/physiology , Lipopolysaccharides/toxicity , Luciferases/genetics , Neuroglia/drug effects , Neuroglia/enzymology , Phenotype , Rats
7.
PLoS One ; 12(12): e0189340, 2017.
Article in English | MEDLINE | ID: mdl-29216297

ABSTRACT

Transient receptor potential channel M5 (Trpm5)-expressing cells, such as sweet, umami, and bitter taste cells in the oropharyngeal epithelium, solitary chemosensory cells in the nasal respiratory epithelium, and tuft cells in the small intestine, that express taste-related genes function as chemosensory cells. Previous studies demonstrated that Skn-1a/Pou2f3, a POU homeodomain transcription factor is expressed in these Trpm5-expressing chemosensory cells, and is necessary for their generation. Trpm5-expressing cells have recently been found in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. They are considered to be involved in protective responses to potential hazardous compounds as Skn-1a-dependent bitter taste cells, respiratory solitary chemosensory cells, and intestinal tuft cells are. In this study, we examined the expression and function of Skn-1a/Pou2f3 in Trpm5-expressing cells in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. Skn-1a/Pou2f3 is expressed in a majority of Trpm5-expressing cells in all tissues examined. In Skn-1a/Pou2f3-deficient mice, the expression of Trpm5 as well as marker genes for Trpm5-expressing cells were absent in all tested tissues. Immunohistochemical analyses demonstrated that two types of microvillous cells exist in trachea, urethra, and thymus, Trpm5-positive and Trpm5-negative cells. In Skn-1a/Pou2f3-deficient mice, a considerable proportion of Trpm5-negative and villin-positive microvillous cells remained present in these tissues. Thus, we propose that Skn-1a/Pou2f3 is the master regulator for the generation of the Trpm5-expressing microvillous cells in multiple tissues.


Subject(s)
Octamer Transcription Factors/physiology , TRPM Cation Channels/physiology , Animals , Digestive System/cytology , Digestive System/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Trachea/cytology , Trachea/metabolism
8.
Neurosci Lett ; 648: 53-58, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28359935

ABSTRACT

The connections between taste receptor cells (TRCs) and innervating gustatory neurons are formed in a mutually dependent manner during development. To investigate whether a change in the ratio of cell types that compose taste buds influences the number of innervating gustatory neurons, we analyzed the proportion of gustatory neurons that transmit sour taste signals in adult Skn-1a-/- mice in which the number of sour TRCs is greatly increased. We generated polycystic kidney disease 1 like 3-wheat germ agglutinin (pkd1l3-WGA)/Skn-1a+/+ and pkd1l3-WGA/Skn-1a-/- mice by crossing Skn-1a-/- mice and pkd1l3-WGA transgenic mice, in which neural pathways of sour taste signals can be visualized. The number of WGA-positive cells in the circumvallate papillae is 3-fold higher in taste buds of pkd1l3-WGA/Skn-1a-/- mice relative to pkd1l3-WGA/Skn-1a+/+ mice. Intriguingly, the ratio of WGA-positive neurons to P2X2-expressing gustatory neurons in nodose/petrosal ganglia was similar between pkd1l3-WGA/Skn-1a+/+ and pkd1l3-WGA/Skn-1a-/- mice. In conclusion, an alteration in the ratio of cell types that compose taste buds does not influence the number of gustatory neurons that transmit sour taste signals.


Subject(s)
Neurons/cytology , Octamer Transcription Factors/physiology , Taste Buds/cytology , Taste , Animals , Mice , Mice, Knockout , Neurons/metabolism , Octamer Transcription Factors/genetics , Signal Transduction , Taste Buds/metabolism , Wheat Germ Agglutinins/metabolism
9.
Oncogene ; 35(5): 567-76, 2016 Feb 04.
Article in English | MEDLINE | ID: mdl-25893301

ABSTRACT

The main risk factor for skin cancer is ultraviolet (UV) exposure, which causes DNA damage. Cells respond to UV-induced DNA damage by activating the intra-S-phase checkpoint, which prevents replication fork collapse, late origin firing and stabilizes fragile sites. Recently, the 54-kDa multifunctional protein NONO was found to be involved in the non-homologous end-joining DNA repair process and in poly ADP-ribose polymerase 1 activation. Interestingly, NONO is mutated in several tumour types and emerged as a crucial factor underlying both melanoma development and progression. Therefore, we set out to evaluate whether NONO could be involved in the DNA-damage response to UV radiations. We generated NONO-silenced HeLa cell clones and found that lack of NONO decreased cell growth rate. Then, we challenged NONO-silenced cells with exposure to UV radiations and found that NONO-silenced cells, compared with control cells, continued to synthesize DNA, failed to block new origin firing and impaired CHK1S345 phosphorylation showing a defective checkpoint activation. Consistently, NONO is present at the sites of UV-induced DNA damage where it localizes to RAD9 foci. To position NONO in the DNA-damage response cascade, we analysed the loading onto chromatin of various intra-S-phase checkpoint mediators and found that NONO favours the loading of topoisomerase II-binding protein 1 acting upstream of the ATM and Rad3-related kinase activity. Strikingly, re-expression of NONO, through an sh-resistant mRNA, rescued CHK1S345 phosphorylation in NONO-silenced cells. Interestingly, NONO silencing affected cell response to UV radiations also in a melanoma cell line. Overall, our data uncover a new role for NONO in mediating the cellular response to UV-induced DNA damage.


Subject(s)
DNA Damage , Nuclear Matrix-Associated Proteins/physiology , Octamer Transcription Factors/physiology , RNA-Binding Proteins/physiology , S Phase Cell Cycle Checkpoints/physiology , S Phase Cell Cycle Checkpoints/radiation effects , DNA/metabolism , DNA Repair , DNA-Binding Proteins , HeLa Cells , Humans , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , Octamer Transcription Factors/genetics , Octamer Transcription Factors/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , S Phase Cell Cycle Checkpoints/genetics , Transfection , Ultraviolet Rays
10.
J Invest Dermatol ; 135(2): 471-480, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25178105

ABSTRACT

Neuropeptides and their receptors are present in human skin, and their importance for cutaneous homeostasis and during wound healing is increasingly appreciated. However, there is currently a lack of understanding of the molecular mechanisms by which their signaling modulates keratinocyte function. Here, we show that δ-opioid receptor (DOPr) activation inhibits proliferation of human keratinocytes, resulting in decreased epidermal thickness in an organotypic skin model. DOPr signaling markedly delayed induction of keratin intermediate filament (KRT10) during in vitro differentiation and abolished its induction in the organotypic skin model. This was accompanied by deregulation of involucrin (IVL), loricrin, and filaggrin. Analysis of the transcription factor POU2F3, which is involved in regulation of KRT10, IVL, and profilaggrin expression, revealed a DOPr-mediated extracellular signal-regulated kinase (ERK)-dependent downregulation of this factor. We propose that DOPr signaling specifically activates the ERK 1/2 mitogen-activated protein kinase pathway to regulate keratinocyte functions. Complementing our earlier studies in DOPr-deficient mice, these data suggest that DOPr activation in human keratinocytes profoundly influences epidermal morphogenesis and homeostasis.


Subject(s)
Epidermis/physiology , Extracellular Signal-Regulated MAP Kinases/physiology , Homeostasis/physiology , Octamer Transcription Factors/antagonists & inhibitors , Receptors, Opioid, delta/physiology , Calcium/metabolism , Cell Differentiation , Cell Proliferation , Cells, Cultured , Filaggrin Proteins , Humans , Keratinocytes/cytology , Octamer Transcription Factors/physiology
12.
J Pharm Sci ; 100(9): 4006-12, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21437911

ABSTRACT

The present study was conducted to assess the functional characteristics of human organic cation transporter 1 (hOCT1) for the transport of 4',6-diamidino-2-phenylindol (DAPI), a fluorescent compound that may be used as a probe substrate for rapid assays of its functionality. The specific uptake of DAPI by hOCT1 heterologously introduced into Madin-Darby canine kidney II cells by stable transfection was found to be, when assessed by DAPI-derived fluorescence intensity, rapid and saturable with a Michaelis constant of 8.94 µM, indicating that DAPI is a good substrate of hOCT1. The specific uptake of DAPI was insensitive to the membrane potential and extracellular pH, indicating a mode of operation different from that for typical cationic substrates such as tetraethylammonium (TEA), for which hOCT1 has been suggested to be driven by an inside-negative membrane potential and favor higher pH for optimal operation. However, many organic cations were found to inhibit the specific DAPI uptake with extents well correlated with those of inhibition of the specific uptake of [(14) C]TEA, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of hOCT1.


Subject(s)
Fluorescent Dyes , Indoles/pharmacokinetics , Octamer Transcription Factors/physiology , Animals , Cells, Cultured , Dogs , Humans , Hydrogen-Ion Concentration , Tetraethylammonium/metabolism
13.
Oncogene ; 27(9): 1263-72, 2008 Feb 21.
Article in English | MEDLINE | ID: mdl-17724474

ABSTRACT

The GADD45gamma protein is a potential tumor suppressor whose expression is reduced in several tumors. However, very little is known about the regulation of its expression. We have determined that the most relevant region of its promoter lies between nucleotides -112 and -54, relative to the transcription start site. Putative Oct and NF-Y elements were found in this region and factors belonging to these families interacted with these elements in vitro and with the promoter in vivo. Mutation of these elements reduced the basal activity of the promoter, suggesting that both sites are essential for basal expression. These factors interact with chromatin modifying proteins and we found that histone deacetylase 1 or silencing mediator for retinoid and thyroid hormone receptor overexpression reduced the basal activity of the promoter. In contrast, forced expression of the histone acetylase protein PCAF or cell treatment with the HDAC inhibitor trichostatin A increased GADD45gamma mRNA levels and induced GADD45gamma promoter activity through its Oct and NF-Y elements. Moreover, ectopic expression of a dominant-negative version of NF-YA strongly inhibited trichostatin A-induced activation of the promoter. Our data strongly suggest that inhibition of deacetylase activity could potentially be used for treatment of tumors where GADD45gamma expression is reduced.


Subject(s)
CCAAT-Binding Factor/metabolism , Gene Expression Regulation/drug effects , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Octamer Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites/physiology , CCAAT-Binding Factor/physiology , Cell Line , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mutagenesis, Site-Directed , NIH 3T3 Cells , Octamer Transcription Factors/physiology , Point Mutation , Promoter Regions, Genetic , Protein Binding/physiology , Sequence Deletion
14.
Biochem Biophys Res Commun ; 353(3): 644-9, 2007 Feb 16.
Article in English | MEDLINE | ID: mdl-17194450

ABSTRACT

We have previously indicated that Xenopus Suppressor of Hairless 2, XSu(H)2, is involved in the morphogenesis of gastrula embryos in a different manner from the XESR-1-mediated Notch signaling pathway. To address the downstream factors of XSu(H)2, we investigated the effect of XSu(H)2 on XenopusPOU-V genes, Xoct25 and Xoct91. Knockdown of XSu(H)2 caused the downregulation of Xoct25, Xoct91 and Xbrachyury. Dominant-negative Xoct25 caused the delay of blastopore closure with a decrease of Xbrachyury expression. Overexpression of Xoct25 or Xoct91 could rescue the decrease of Xbrachyury expression caused by XSu(H)2 depletion. XSu(H)2EnR inhibited Xoct25 and Xoct91 expressions in CHX-treated animal caps. Promoter analysis of the Xoct25 showed that the upstream region of Xoct25 contains the prospective Su(H) binding motif, which is essential for the transcription of Xoct25. These results suggest that XSu(H)2 is engaged in the cell fate decision during gastrulation through the gene expression of the Xoct25/91-mediated pathway.


Subject(s)
Gastrula/physiology , Gene Expression Regulation, Developmental , Octamer Transcription Factors/physiology , Transcription Factors/physiology , Xenopus Proteins/physiology , Xenopus laevis/embryology , Animals , POU Domain Factors/biosynthesis
15.
J Invest Dermatol ; 127(5): 1107-14, 2007 May.
Article in English | MEDLINE | ID: mdl-17195013

ABSTRACT

Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.


Subject(s)
Cell Differentiation/physiology , Keratinocytes/enzymology , Metalloendopeptidases/physiology , Octamer Transcription Factor-6/physiology , Octamer Transcription Factors/physiology , Animals , COS Cells , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Differentiation/genetics , Cell Line , Cell Movement/genetics , Cell Movement/physiology , Chlorocebus aethiops , Gene Expression Regulation , Humans , Keratin-14/genetics , Keratin-14/physiology , Keratin-5/genetics , Keratin-5/physiology , Keratinocytes/cytology , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/physiology , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/physiology , Matrix Metalloproteinases, Secreted , Membrane Proteins/genetics , Membrane Proteins/physiology , Metalloendopeptidases/genetics , Octamer Transcription Factor-6/genetics , Octamer Transcription Factors/genetics , Protein Precursors/genetics , Protein Precursors/physiology
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