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Regulation of dopachrome tautomerase expression and use in the study of melanocyte development
Journal of the American Academy of Dermatology Volume 39, Number 2, Part 1
anabolic steroid stanozolol and that, for the most part, this effect may be the result of enhanced TGF-~l synthesis. Our results point to a novel mechanism of action of anabolic steroids. REFERENCES 1. Falanga V, Kirsner RS, Eaglstein WH, et al. Stanozolol in treatment of leg ulcers due to cryofibrinogenemia. Lancet 1991;338:347-8. 2. Helfman T, Falanga V. Stanozolol as a novel therapeutic agent in dermatology. J Am Acad Dermatol 1995;32: 254-8. 3. Gribbin HR, Flavell Matts SG. Mode of action and use of anabolic steroids. Br J Clin Pract 1976;30:3-9.
Regulation of dopachrome tautomerase expression and use in the study of melanocyte development Thomas J. Hornyak, MD, PhD: New York University, New York, N.Y.
Sponsored by Irwin M. Freedberg, MD, George Miller Mackee Professor and Chairman, Ronald O. Perelman Department of Dermatology, New York University Medical Center
The dopachrome tautomerase (dct) gene encodes the melanogenic enzyme dopachrome tautomerase (dct), also known as tyrosinase-related protein-2 (TRP-2), and is expressed early in melanocyte development. 1 To begin to characterize factors important for the activation of dct in the mouse melanocyte cell line melan-a, reporter gene constructs were made with a 3.5 kb region of upstream genomic DNA from the TRP-2 gene and the reporter genes luciferase and ~-galactosidase (lacZ). A transgenic mouse line containing a transgene expressing the lacZ gene from this regulatory region also was generated. Transfection of cells at low cell density with the luciferase reporter vector revealed no significant dct promoter activity, whereas cells stably transfected with the lacZ construct showed differential expression of dct promoter activity, highest in the center of the colonies at high cell density. To test directly whether dct gene expression was induced by cell density, total RNA harvested from melan-a cells grown to increasing density was subjected to 'Ronald O. Perelman Department of Dermatology, New York University Medical Center, 550 First Ave, Room H-100, New York, NY 10016.
AAD 273 Northern blot analysis. Induction of dct gene expression at increased cell density was observed, with nearly a 20-fold difference between relative levels of dct expression at low and high density. Western blotting using an anti-dopachrome tautomerase antibody (aPEP8)2 confirmed that dct protein was induced in a similar pattern. Densitydependent dct activation was independent of both the time of cell growth in culture and the presence of phorbol ester. These results demonstrate that cell-cell interactions are important for the transcriptional activation of dct gene expression and for dct protein product formation in cultured melanocytes. Analysis of transgenic mouse embryos shows early transgene expression in the eye, in individual cells surmounting and adjacent to the dorsal neural tube in the caudal aspect of the embryo, in the wall of the dorsal aorta, and in the dorsal telencephalon, with telencephalic expression corresponding to proliferative cell populations in embryonic and early postnatal mice. To utilize the early expression of the trans gene in presumptive melanoblasts to study early events in melanocyte development, matings were arranged between transgenic mice and mice of the coat color mutant allele MITFmi, containing a mutation of the MITF gene, a gene encoding a basic-helix-Ioop-helixleucine zipper transcription factor.3 Homozygous MITFmilMITFmi mice lack pigment caused by the failure of melanocytes to migrate to the hair follicle epithelium.4 Matings between transgenic MITFmi/+ heterozygotes generated litters of transgenic embryos with variations in melanocyte development apparent at the earliest stages of neural crest migration from the neural tube. The results of these studies utilizing transgenic animals suggest that dct expression in vivo may be regulated by a variety of tissue-specific mechanisms and that the transcription factor MITF acts early in the development of the neural crest to promote either the specification of or early differentiation of melanoblasts. REFERENCES 1. Steel KP, Davidson DR, Jackson n. TRP-2IDT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 1992; 115:1111-9. 2. Tsukamoto K, Jackson n, Kazunori U, Montague PM, Hearing VJ. A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J 1992;1l:519-26.
274 AAD 3. Hodgkinson CA, Moore KJ, Nakayama A, Steingrimsson E, Copeland NG, Jenkins NA, Arnheiter H. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helixloop-helix-zipper protein. Cell 1993;74:395-404. 4. Silvers WK. The coat colors of mice. In: A model for mammalian gene action and interaction. New York: Springer-Verlag; 1979. p. 269.
Regulation of E-cadherin function in skinderived dendritic cells by proinflammatory epidermal cytokines: Implications for the mobilization of Langerhans cells in vivo Thilo Jakob, MD,* Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. Sponsored by Mark C. Udey, MD, PhD, Senior Investigator, Dermatology Branch, National Cancer Institute, National Institutes of Health
Migration of antigen-bearing Langerhans cells (LCs) from epidermis to regional lymph nodes is a crucial step in the initiation of primary immune responses to antigens encountered in the epidermis. Although various mediators that induce LC mobilization have been identified, the exact mechanisms that allow LCs to leave the keratinocyte environment are poorly understood. In earlier studies, our laboratory demonstrated that LCs express E-cadherin,l an adhesion molecule previously identified on epithelial cells (eg, keratinocytes). Cadherins comprise a family of molecules that maintain tissue homeostasis via mediating homophilic adhesion, that is, binding the same adhesion molecule on an adjacent celI.2 In vitro adhesion studies identified E-cadherin as the crucial adhesion molecule mediating the leukocyteepithelial adhesion of LCs and keratinocytes. 1 Attenuation of this E-cadherin-mediated adhesion to keratinocytes thus appears to be the initial step in LC migration; however, direct evidence for this is lacking. Further studies of LC E-cadherin biology have been hampered by difficulties associated with isolating large numbers of LCs. We developed an in vitro culture system that allowed the generation of Langerhans cell-like dendritic cells from fetal murine skin (fetal skin-derived dendritic cells [FSDDCsD in large 'Dermatology Branch, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bldg 10, 12N 238, Bethesda, MD 20892.
Journal of the American Academy of Dermatology August 1998
numbers and that enabled us to study E-cadherin biology in vitro.3 Characterization of FSDDCs demonstrated a striking resemblance to LCs in terms of morphology, surface phenotype, cytokine profile, and functional activity. 3 In the presence of calcium, FSDDCs were isolated as trypsin-resistant aggregates of tightly clustered cells, suggesting that the intercellular adhesion is a cadherin-dependent process (in the absence of calcium, cadherins are susceptible to cleavage by trypsin). Flow cytometry showed that FSDDCs expressed high surface levels of E-cadherin. Inhibition studies with monoclonal antibodies directed against various adhesion molecules expressed on FSDDCs (E-cadherin, CD54, CDlla, CDllb) confirmed that the intercellular adhesion within the cell aggregates was an E-cadherin-dependent process. 3 Because FSDDCs proved to be a suitable system to study the E-cadherin biology of LC-like dendritic cells, we utilized them to analyze the effects of mediators that are known to mobilize LCs in vivo. 4 -7 FSDDC aggregates were subcultured in the presence or absence of various cytokines or bacterial products (eg, lipopolysaccharide), and the loss of cell adhesion (ie, the dissociation of cell aggregates into single cells) was monitored visually and quantified in a newly developed Coulter counter-based disaggregation assay. Epidermal proinflammatory cytokines interleukin-l (IL-l) and tumor necrosis factor-a (TNFex), as well as LPS, induced a pronounced decrease in homotypic cell adhesion that began within 4 to 8 hours and was complete by 20 hours, whereas other cytokines did not. 8 Inhibition studies with receptor-blocking or neutralizing monoclonal antibodies demonstrated that IL-l and TNF-a induced loss of cell adhesion directly and independently of each other and that the effect of LPS was TNF-dependent, but not IL-l dependent. 8 To determine whether the loss of E-cadherin-mediated adhesion was accompanied by a change in E-cadherin surface expression or mRNA levels, we performed flow cytometry and Northern blot analysis after different times of stimulation. IL-l, TNF-a, and LPS induced a reduction of E-cadherin surface expression that was paralleled by the loss of Ecadherin-mediated adhesion. In addition, IL-l and TNF-a caused a rapid reduction in FSDDC E-cadherin mRNA levels, which preceded the reduction of E-cadherin surface expression. 8 In summary, mediators that induce LC emigra-
anabolic steroid stanozolol and that, for the most part, this effect may be the result of enhanced TGF-~l synthesis. Our results point to a novel mechanism of action of anabolic steroids. REFERENCES 1. Falanga V, Kirsner RS, Eaglstein WH, et al. Stanozolol in treatment of leg ulcers due to cryofibrinogenemia. Lancet 1991;338:347-8. 2. Helfman T, Falanga V. Stanozolol as a novel therapeutic agent in dermatology. J Am Acad Dermatol 1995;32: 254-8. 3. Gribbin HR, Flavell Matts SG. Mode of action and use of anabolic steroids. Br J Clin Pract 1976;30:3-9.
Regulation of dopachrome tautomerase expression and use in the study of melanocyte development Thomas J. Hornyak, MD, PhD: New York University, New York, N.Y.
Sponsored by Irwin M. Freedberg, MD, George Miller Mackee Professor and Chairman, Ronald O. Perelman Department of Dermatology, New York University Medical Center
The dopachrome tautomerase (dct) gene encodes the melanogenic enzyme dopachrome tautomerase (dct), also known as tyrosinase-related protein-2 (TRP-2), and is expressed early in melanocyte development. 1 To begin to characterize factors important for the activation of dct in the mouse melanocyte cell line melan-a, reporter gene constructs were made with a 3.5 kb region of upstream genomic DNA from the TRP-2 gene and the reporter genes luciferase and ~-galactosidase (lacZ). A transgenic mouse line containing a transgene expressing the lacZ gene from this regulatory region also was generated. Transfection of cells at low cell density with the luciferase reporter vector revealed no significant dct promoter activity, whereas cells stably transfected with the lacZ construct showed differential expression of dct promoter activity, highest in the center of the colonies at high cell density. To test directly whether dct gene expression was induced by cell density, total RNA harvested from melan-a cells grown to increasing density was subjected to 'Ronald O. Perelman Department of Dermatology, New York University Medical Center, 550 First Ave, Room H-100, New York, NY 10016.
AAD 273 Northern blot analysis. Induction of dct gene expression at increased cell density was observed, with nearly a 20-fold difference between relative levels of dct expression at low and high density. Western blotting using an anti-dopachrome tautomerase antibody (aPEP8)2 confirmed that dct protein was induced in a similar pattern. Densitydependent dct activation was independent of both the time of cell growth in culture and the presence of phorbol ester. These results demonstrate that cell-cell interactions are important for the transcriptional activation of dct gene expression and for dct protein product formation in cultured melanocytes. Analysis of transgenic mouse embryos shows early transgene expression in the eye, in individual cells surmounting and adjacent to the dorsal neural tube in the caudal aspect of the embryo, in the wall of the dorsal aorta, and in the dorsal telencephalon, with telencephalic expression corresponding to proliferative cell populations in embryonic and early postnatal mice. To utilize the early expression of the trans gene in presumptive melanoblasts to study early events in melanocyte development, matings were arranged between transgenic mice and mice of the coat color mutant allele MITFmi, containing a mutation of the MITF gene, a gene encoding a basic-helix-Ioop-helixleucine zipper transcription factor.3 Homozygous MITFmilMITFmi mice lack pigment caused by the failure of melanocytes to migrate to the hair follicle epithelium.4 Matings between transgenic MITFmi/+ heterozygotes generated litters of transgenic embryos with variations in melanocyte development apparent at the earliest stages of neural crest migration from the neural tube. The results of these studies utilizing transgenic animals suggest that dct expression in vivo may be regulated by a variety of tissue-specific mechanisms and that the transcription factor MITF acts early in the development of the neural crest to promote either the specification of or early differentiation of melanoblasts. REFERENCES 1. Steel KP, Davidson DR, Jackson n. TRP-2IDT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 1992; 115:1111-9. 2. Tsukamoto K, Jackson n, Kazunori U, Montague PM, Hearing VJ. A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO J 1992;1l:519-26.
274 AAD 3. Hodgkinson CA, Moore KJ, Nakayama A, Steingrimsson E, Copeland NG, Jenkins NA, Arnheiter H. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helixloop-helix-zipper protein. Cell 1993;74:395-404. 4. Silvers WK. The coat colors of mice. In: A model for mammalian gene action and interaction. New York: Springer-Verlag; 1979. p. 269.
Regulation of E-cadherin function in skinderived dendritic cells by proinflammatory epidermal cytokines: Implications for the mobilization of Langerhans cells in vivo Thilo Jakob, MD,* Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. Sponsored by Mark C. Udey, MD, PhD, Senior Investigator, Dermatology Branch, National Cancer Institute, National Institutes of Health
Migration of antigen-bearing Langerhans cells (LCs) from epidermis to regional lymph nodes is a crucial step in the initiation of primary immune responses to antigens encountered in the epidermis. Although various mediators that induce LC mobilization have been identified, the exact mechanisms that allow LCs to leave the keratinocyte environment are poorly understood. In earlier studies, our laboratory demonstrated that LCs express E-cadherin,l an adhesion molecule previously identified on epithelial cells (eg, keratinocytes). Cadherins comprise a family of molecules that maintain tissue homeostasis via mediating homophilic adhesion, that is, binding the same adhesion molecule on an adjacent celI.2 In vitro adhesion studies identified E-cadherin as the crucial adhesion molecule mediating the leukocyteepithelial adhesion of LCs and keratinocytes. 1 Attenuation of this E-cadherin-mediated adhesion to keratinocytes thus appears to be the initial step in LC migration; however, direct evidence for this is lacking. Further studies of LC E-cadherin biology have been hampered by difficulties associated with isolating large numbers of LCs. We developed an in vitro culture system that allowed the generation of Langerhans cell-like dendritic cells from fetal murine skin (fetal skin-derived dendritic cells [FSDDCsD in large 'Dermatology Branch, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bldg 10, 12N 238, Bethesda, MD 20892.
Journal of the American Academy of Dermatology August 1998
numbers and that enabled us to study E-cadherin biology in vitro.3 Characterization of FSDDCs demonstrated a striking resemblance to LCs in terms of morphology, surface phenotype, cytokine profile, and functional activity. 3 In the presence of calcium, FSDDCs were isolated as trypsin-resistant aggregates of tightly clustered cells, suggesting that the intercellular adhesion is a cadherin-dependent process (in the absence of calcium, cadherins are susceptible to cleavage by trypsin). Flow cytometry showed that FSDDCs expressed high surface levels of E-cadherin. Inhibition studies with monoclonal antibodies directed against various adhesion molecules expressed on FSDDCs (E-cadherin, CD54, CDlla, CDllb) confirmed that the intercellular adhesion within the cell aggregates was an E-cadherin-dependent process. 3 Because FSDDCs proved to be a suitable system to study the E-cadherin biology of LC-like dendritic cells, we utilized them to analyze the effects of mediators that are known to mobilize LCs in vivo. 4 -7 FSDDC aggregates were subcultured in the presence or absence of various cytokines or bacterial products (eg, lipopolysaccharide), and the loss of cell adhesion (ie, the dissociation of cell aggregates into single cells) was monitored visually and quantified in a newly developed Coulter counter-based disaggregation assay. Epidermal proinflammatory cytokines interleukin-l (IL-l) and tumor necrosis factor-a (TNFex), as well as LPS, induced a pronounced decrease in homotypic cell adhesion that began within 4 to 8 hours and was complete by 20 hours, whereas other cytokines did not. 8 Inhibition studies with receptor-blocking or neutralizing monoclonal antibodies demonstrated that IL-l and TNF-a induced loss of cell adhesion directly and independently of each other and that the effect of LPS was TNF-dependent, but not IL-l dependent. 8 To determine whether the loss of E-cadherin-mediated adhesion was accompanied by a change in E-cadherin surface expression or mRNA levels, we performed flow cytometry and Northern blot analysis after different times of stimulation. IL-l, TNF-a, and LPS induced a reduction of E-cadherin surface expression that was paralleled by the loss of Ecadherin-mediated adhesion. In addition, IL-l and TNF-a caused a rapid reduction in FSDDC E-cadherin mRNA levels, which preceded the reduction of E-cadherin surface expression. 8 In summary, mediators that induce LC emigra-