What are melanocytes really doing all day long...?

Everyone knows and seems to agree that melanocytes are there to generate melanin - an intriguing, but underestimated multipurpose molecule that is capable of doing far more than providing pigment and UV protection to skin (1). What about the cell that generates melanin, then? Is this dendritic, neural crest-derived cell still serving useful (or even important) functions when no-one looks at the pigmentation of our skin and its appendages and when there is essentially no UV exposure? In other words, what do epidermal and hair follicle melanocytes do in their spare time - at night, under your bedcover? How much of the full portfolio of physiological melanocyte functions in mammalian skin has really been elucidated already? Does the presence or absence of melanocytes matter for normal epidermal and/or hair follicle functions (beyond pigmentation and UV protection), and for skin immune responses? Do melanocytes even deserve as much credit for UV protection as conventional wisdom attributes to them? In which interactions do these promiscuous cells engage with their immediate epithelial environment and who is controlling whom? What lessons might be distilled from looking at lower vertebrate melanophores and at extracutaneous melanocytes in the endeavour to reveal the 'secret identity' of melanocytes? The current Controversies feature explores these far too infrequently posed, biologically and clinically important questions. Complementing a companion viewpoint essay on malignant melanocytes (2), this critical re-examination of melanocyte biology provides a cornucopia of old, but under-appreciated concepts and novel ideas on the slowly emerging complexity of physiological melanocyte functions, and delineates important, thought-provoking questions that remain to be definitively answered by future research.

Identification of a cis-regulatory element and putative trans-acting factors responsible for 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of heme oxygenase expression in myelomonocytic cell lines.

The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with a variety of agents, including 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that during this process, the expression of heme oxygenase, a rate-limiting enzyme in heme catabolism, is induced. Treatment with TPA increases heme oxygenase mRNA in other myelomonocytic cell lines also, but not in cell lines of other lineages, such as HeLa cells. Increased heme oxygenase activity may represent one of the functions of activated macrophages, which sequestrate senescent erythrocytes and degrade heme derived from hemoglobin. This cell-type-specific induction by TPA treatment further investigated with respect to transcriptional regulation. We defined a cis-regulatory element, 5'-GTCATATGAC-3', located in the 5'-flanking region (positions -156 to -147) of the human heme oxygenase gene, which confers inducibility by TPA in THP-1 cells but not in HeLa cells. Nuclear proteins that bind to this element, which may be responsible for the cell specificity, were identified in THP-1 nuclear extracts. This element contains the consensus motif CANNTG, to which a large family of basic helix-loop-helix proteins binds. Our results suggest a novel mechanism of TPA-mediated transcriptional regulation in myelomonocytic cell lines.

Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.

Tyrosinase is a rate-limiting enzyme in melanin biosynthesis and is specifically expressed in differentiated melanocytes. We have identified the enhancer element in the 5'-flanking region of the human tyrosinase gene that is responsible for its pigment cell-specific transcription and have termed it tyrosinase distal element (TDE) (positions -1861 to -1842). Transient expression assays showed that TDE confers efficient expression of a firefly luciferase reporter gene linked to the tyrosinase gene promoter in MeWo pigmented melanoma cells but not in HeLa cells, which do not express tyrosinase. TDE was specifically bound by nuclear proteins of MeWo and HeLa cells, the binding properties of which were indistinguishable in gel mobility shift assays. TDE contains the CATGTG motif in its center, and mutation analysis indicates that the CA dinucleotides of this motif are crucial for protein binding and pigment cell-specific enhancer function. The CATGTG motif is consistent with the consensus sequence recognized by a large family of transcription factors with a basic helix-loop-helix structure, which prompted us to examine the possible involvement of a ubiquitous transcription factor, USF, and a novel factor, microphthalmia-associated transcription factor (MITF), recently cloned as the human homolog of the mouse microphthalmia (mi) gene product. The mi phenotype is associated with a mutant mi locus and characterized by small eyes and loss of melanin pigments. Both USF and MITF are predicted to contain a basic helix-loop-helix structure and a leucine zipper structure. We provide evidence that USF binds to TDE, whereas we were unable to detect the DNA-binding activity of MITF. Transient coexpression assays showed that MITF specifically transactivates the promoter activity of the tyrosinase gene through the CATGTG motif of TDE but not the promoter of the ubiquitously expressed heme oxygenase gene, while USF is able to activate both promoters. These results indicate that MITF is a cell-type-specific factor that is capable of activating transcription of the tyrosinase gene.

Association of susceptibility to the development of pneumonia in the older Japanese population with haem oxygenase-1 gene promoter polymorphism.

BACKGROUND: Oxidative stresses including cigarette smoking are implicated in the pathogenesis of cerebrovascular diseases, which are associated with pneumonia because of frequent aspiration. Haem oxygenase-1 (HO-1) acts in cytoprotection against oxidants, provides anti-inflammatory effects, and inhibits atherogenesis. A (GT)(n) dinucleotide repeat in the human HO-1 promoter modulates HO-1 gene expression and shows length polymorphism, which is grouped into three classes: class S (<27 repeats), class M (> or = 27, <33 repeats), and class L (> or = 33 repeats) alleles. OBJECTIVE: To investigate the correlation between the HO-1 gene polymorphism and development of pneumonia in elderly Japanese. METHODS: The length of the (GT)n repeats was analysed in 200 elderly patients with pneumonia and 200 control subjects. The association of the HO-1 gene polymorphism with risk of pneumonia was estimated by logistic regression. RESULTS: The proportion of allele frequencies in class L, and the proportion of genotypic frequencies in the L-allele carriers (L/L, L/M, and L/S), was significantly higher in patients with pneumonia than in controls (20% v 10% in class L, and 34% v 18% in L-allele carriers). After adjustment for potentially confounding factors, both cerebrovascular disorders and HO-1 gene L-allele carriers were significant and independent risk factors for pneumonia. The adjusted odds ratio for L-allele carriers v non-L-allele carrier was 2.1 (95% confidence interval, 1.2 to 3.6). CONCLUSIONS: The large size of a (GT)n repeat in the HO-1 gene promoter may be associated with susceptibility to pneumonia in the older Japanese population.

Molecular cloning of cDNA encoding a human TFEC isoform, a newly identified transcriptional regulator.

TFEC is a transcriptional repressor originally identified in rat chondrosarcoma and contains a basic helix-loop-helix and leucine zipper (bHLH/LZ) structure. TFEC shares a closely related bHLH/LZ structure with microphthalmia-associated transcription factor (MITF) and TFE3. In the course of cDNA cloning for a factor structurally related to MITF which is also a regulator for cell differentiation, we have isolated cDNA clones from a THP-1 human monocytic leukemia cell line. These cDNAs encode a protein of 347 amino acids, termed TFECL, a human homolog of a putative rat TFEC isoform. TFECL contains an acidic domain that corresponds to a transcriptional activation domain of TFE3 but its equivalent region is deleted in rat TFEC. We explored a function of TFECL using a melanocyte-specific tyrosinase gene and a ubiquitously expressed heme oxygenase-1 gene, each promoter containing the cis-acting CANNTG motifs. By transient coexpression assays, we showed that TFECL is able to activate or inhibit transcription of a reporter gene linked to either the tyrosinase or the heme oxygenase-1 gene promoter, depending on cell types. These results suggest that TFECL may function as a transcriptional activator under certain conditions.

Molecular cloning and functional analysis of a cDNA coding for human DOPAchrome tautomerase/tyrosinase-related protein-2.

We have cloned the cDNAs encoding tyrosinase-related protein-2 (TRP-2) from a human melanoma cDNA library. Transient expression of the isolated cDNA in HeLa cells established that TRP-2 is DOPAchrome tautomerase. Human TRP-2/DOPAchrome tautomerase is composed of 519 amino acids with a molecular weight of 59,000 and has about 84% identity with the mouse counterpart.

Identification of a composite enhancer of the human tyrosinase-related protein 2/DOPAchrome tautomerase gene.

The human tyrosinase-related protein 2 (TRP-2) gene promoter contains a cis-regulatory element (positions -447 to -416), termed DOPAchrome tautomerase distal enhancer 1 (DDE1). DDE1 functions as an enhancer in cultured melanoma cells and its core element includes a potential binding site for transcription factors containing a high-mobility-group domain. This core element is bound in vitro by multiple nuclear proteins, which are preferentially expressed in melanoma cells. DDE1 represents a composite enhancer that may be involved in melanocyte-specific transcription of the human TRP-2 gene.

Positive and negative regulation of the human heme oxygenase-1 gene expression in cultured cells.

To elucidate the regulation of the human heme oxygenase-1 (hHO-1) gene expression, we assessed approximately 4 kb of the 5'-flanking region of the hHO-1 gene for basal promoter activity and sequenced approximately 2 kb of the 5'-flanking region. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in HepG2 human hepatoma cells and HeLa cervical cancer cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found a positive regulatory region at position -1976 to -1655 bp. This region functions in HepG2 cells but not in HeLa cells. A negative regulatory region was also found at position -981 to -412 bp that functions in both HepG2 cells and HeLa cells.

Sequential activation of genes for heme pathway enzymes during erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells.

Changes in the level of transcripts encoding enzymes of the heme biosynthetic pathway as well as those encoding ubiquitous proteins were examined in murine Friend virus-transformed erythroleukemia cells during erythroid cell differentiation induced by chemicals including dimethyl sulfoxide (DMSO). Early changes following DMSO treatment were marked decreases in mRNAs for three ubiquitous proteins, i.e., a 70 kDa heat shock protein (less than 6 h), heme oxygenase and nonspecific delta-aminolevulinate synthase (ALAS) (less than 12 h). These changes were followed by sequential increases in mRNAs for enzymes in the heme biosynthetic pathway. Namely, mRNAs for the erythroid-specific ALAS, delta-aminolevulinate dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase started to increase at 12, 18, 18-24 and 24 h, respectively. Nuclear runoff studies revealed that these changes are largely transcriptional. Treatments with other inducers of erythroid differentiation, e.g., hexamethylene bisacetamide, n-butyric acid and N'-methylnicotinamide, also showed similar effects on mRNAs as those following DMSO. These findings suggest that both suppression of ubiquitous genes and activation of heme pathway enzyme genes are associated with erythroid differentiation, and the former occurs preceding changes in the latter.

Structural organization of the human microphthalmia-associated transcription factor gene containing four alternative promoters.

Microphthalmia-associated transcription factor (MITF) affects the development of many types of cells, including melanocytes and retinal pigment epithelium (RPE). MITF consists of at least three isoforms, MITF-A, MITF-H and MITF-M, differing at their amino-termini and expression patterns. Here, we characterize the structural organization of the human MITF gene. The gene contains at least four isoform-specific first exons, exons 1A, 1H, 1B and 1M in the 5' to 3' direction, each of which encodes the unique amino-terminus of a given isoform, including newly identified MITF-B. The 5'-flanking regions of these isoform-specific exons are termed promoters A, H, B and M, respectively, which showed different promoter activities, as judged by transient transfection assay. Promoter A directs the expression of a reporter gene in RPE, cervical cancer and melanoma cells, whereas promoter M is functional only in melanoma cells. Promoter H showed the significant activity in RPE and cervical cancer cells but not in melanoma cells. In contrast, the 1.7 kb 5'-flanking region of exon 1B showed no noticeable promoter activity in these cell lines. Therefore, alternative promoters provide the MITF gene with the diversity in transcriptional regulation and the capability of generating structurally different protein isoforms.

Increased expression of heme oxygenase-1 and bilirubin accumulation in foam cells of rabbit atherosclerotic lesions.

Heme oxygenase-1 (HO-1) catalyzes the regiospecific oxidative degradation of heme to biliverdin IXalpha, iron, and carbon monoxide. Biliverdin IXalpha is subsequently reduced to bilirubin IXalpha by biliverdin reductase. HO-1 expression is induced under various disease conditions, including atherosclerosis, but it is unknown whether HO-1 catalyzes heme breakdown in the regions at risk. Using hypercholesterolemic rabbits fed a cholesterol-enriched diet, we attempted to demonstrate the involvement of HO-1 induction and bilirubin IXalpha production in atherosclerotic regions. Expression levels of HO-1 mRNA were elevated in the aortas of hypercholesterolemic rabbits. In situ hybridization and immunohistochemistry revealed that mRNA and protein of HO-1 are induced in endothelial cells and foam cells (lipid-filled macrophages) in atherosclerotic lesions. Furthermore, immunohistochemistry with the use of an anti-bilirubin-IXalpha monoclonal antibody, 24G7, demonstrated accumulation of bilirubin IXalpha in foam cells, indicating that heme is actually degraded in atherosclerotic lesions. Remarkably, bilirubin IXalpha, like HO-1 protein, is predominantly accumulated in the perinuclear regions of foam cells. These results provide the first in vivo evidence of the colocalization of HO-1 and bilirubin IXalpha in foam cells, suggesting a role of HO-1 induction in the modulation of macrophage activation in atherosclerosis.

Functional analysis of the tyrosinase gene and brown-locus protein gene promoters.

Tyrosinase is a rate-limiting enzyme of melanin biosynthesis and the brown (b)-locus protein is responsible for the formation of black melanin rather than brown. To identify the cis-acting element(s) required for pigment cell-specific gene transcription, we analyzed the promoter function of two pigment cell-specific genes encoding mouse tyrosinase and b-locus protein using a cell-free transcription system prepared from mouse melanoma cells. Functional and structural analysis of both gene promoters reveals that three elements are conserved in both genes at equivalent positions, suggesting that these elements may be responsible for pigment cell-specific transcription. We discuss possible mechanisms for pigment cell-specific expression of the tyrosinase and b-locus protein genes.

MAT-1, a monoclonal antibody that specifically recognizes human tyrosinase.

There is no available monoclonal antibody which reacts specifically recognizes human tyrosinase. Employing a synthetic peptide, MEKEDYHSLYQSHL, corresponding to the carboxyl terminus of human tyrosinase as an immunogen, we produced a mouse monoclonal antibody MAT-1 of the IgG1 isotype. The epitope for MAT-1 was determined to be EDYH, the sequence of which is not present in human tyrosinase-related protein-1 (TRP-1) or tyrosinase-related protein-2 (TRP-2). By transient expression assays and immunofluorescence technique, we show that MAT-1 reacts specifically with cells expressing human tyrosinase cDNA but not with cells expressing TRP-1 or TRP-2 cDNA. The results of immunohistochemical staining also confirmed that MAT-1 reacts specifically with epidermal melanocytes in human skin sections. MAT-1 should be invaluable for studying the interaction between tyrosinase and TRPs and for detecting the changes in the levels of tyrosinase expression. In addition, MAT-1 should be useful as a sensitive immunohistochemical tool for investigation of various pigmentary disorders and possibly for the diagnosis of melanoma.

The monoclonal antibodies TMH-1 and TMH-2 specifically bind to a protein encoded at the murine b-locus, not to the authentic tyrosinase encoded at the c-locus.

Three hybridomas, TMH-1, TMH-2, and TMH-3, were previously reported by Tomita et al to produce monoclonal antibodies against murine and human T4-tyrosinase localized in melanosome for the formation of melanin pigment. However, TMH antibodies were unable to react with K1735 cells transfected with the authentic tyrosinase-cDNA construct, but did react with those transfected with the pMT4-cDNA construct. The cDNA pMT4 was initially cloned as a putative tyrosinase cDNA by Shibahara et al, but it is now known to encode mouse brown (b) locus protein, which was named "tyrosinase-related protein" by Jackson or "b protein" by Hearing and Jimenez. Furthermore, TMH antibodies recognize hair bulbs of C57BL/6J-c2J/c2J mouse (B/B, c/c) lacking tyrosinase activity, but do not recognize hair bulbs of b-locus mutated DBA/2 mouse (b/b, C/C), which have authentic tyrosinase. Considering these observations, we conclude that TMH antibodies specifically recognize the protein encoded at b-locus.

Expression of melanin-concentrating hormone receptor messenger ribonucleic acid in tumor tissues of pheochromocytoma, ganglioneuroblastoma, and neuroblastoma.

Expression of melanin-concentrating hormone (MCH) receptor messenger ribonucleic acid (mRNA) was studied by RT-PCR and Northern blot analysis in human brain; pituitary; adrenal glands; tumor tissues of adrenal tumors, ganglioneuroblastomas, and neuroblastomas; and various cultured tumor cell lines. RT-PCR analysis showed that MCH receptor mRNA was widely expressed in brain tissues, pituitary, normal portions of adrenal glands (cortex and medulla), tumor tissues of adrenocortical tumors (12 of 13 cases), pheochromocytoma (all 7 cases), ganglioneuroblastoma (1 case), neuroblastoma (all 5 cases), and various cultured tumor cell lines (6 of 7 cell lines), including 2 neuroblastoma cell lines. Northern blot analysis showed the expression of MCH receptor mRNA ( approximately 2.4 kb) only in the tumor tissues of 5 pheochromocytomas, 1 ganglioneuroblastoma, and 4 neuroblastomas, indicating that the expression levels of MCH receptor mRNA are much higher in these tumors than in the other tissues. These findings raised the possibility that MCH or MCH-like peptides may be related to the pathophysiology of these neural crest-derived tumors.

Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor.

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin-releasing factor precursor (prepro-CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro-CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro-CRF than is the cryptic portion.

Simple beta-lactam compounds derived from 6-aminopenicillanic acid.

As part of a general program of structural modification in beta-lactam antibiotics, we have synthesized several simple penicillins from 6-aminopenicillanic acid where the C-3 carboxyl group has been replaced by a hydroxy or an acetoxy group and the C-6 side chain has been substituted by bromine or hydrogen. Some of the compounds exhibit mild activity against the Gram-positive strain Bacillus subtilis.

Synthesis and structure-activity relationships of peptidyl alpha-keto heterocycles as novel inhibitors of prolyl endopeptidase.

The preparation and in vitro prolyl endopeptidase (PEP) inhibitory activity of a series of alpha-keto heterocyclic compounds is described. The design is based on the introduction of alpha-keto heterocycles at the C-terminal end of substrate-like peptides. Many of the compounds including those substituted with thiazole, benzothiazole, benzoxazole, imidazole, and pyridine groups exhibit IC50 potencies of PEP inhibition at nanomolar levels. Structure-activity studies of the C-terminal heterocyclic groups indicate the importance of an sp2 nitrogen atom at a beta-position from the adjoining ketone carbonyl group. This heterocyclic nitrogen atom would provide a critical hydrogen-bond interaction with the histidine residue of the catalytic triad in PEP. Our inhibitors would extend the generality of the alpha-keto heterocycle design to another serine protease.

Coordinated expression of messenger RNAs for nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in the rat hippocampus following transient forebrain ischemia.

Changes in nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 messenger RNA expression in the rat hippocampus following 20 min of transient forebrain ischemia were evaluated using Northern blot analysis and in situ hybridization histochemistry. Twelve hours after the insult, the level of nerve growth factor messenger RNA increased markedly in the granular cell layer of the dentate gyrus and by day 2 returned to control levels. The level of brain-derived neurotrophic factor messenger RNA showed a persistent and moderate increase. The highest expression of brain-derived neurotrophic factor messenger RNA was seen in the dentate granule cells on day 2 after the insult, and then the expression returned to the control levels. At 2 days post-ischemia, contents of messenger RNAs for nerve growth factor and brain-derived neurotrophic factor were reduced in the CA1 region, which may represent delayed loss of vulnerable CA1 pyramidal neurons. In contrast to brain-derived neurotrophic factor and nerve growth factor messenger RNA expression, the level of neurotrophin-3 messenger RNA declined in the CA1, the CA2 and the dentate granular layer immediately after ischemic insult. In the CA1 region, the reduced expression persisted for at least seven days, but in the dentate gyrus, neurotrophin-3 messenger RNA expression returned to the control levels after two days of post-ischemic recovery. These results suggest that nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 genes are differentially regulated and that each of their gene products may play different roles in the central nervous system under pathophysiological conditions.

Induction of heparin-binding growth-associated molecule expression in reactive astrocytes following hippocampal neuronal injury.

Heparin-binding growth-associated molecule is a potent neurotrophic factor. To obtain a better understanding of its role in the central nervous system, we studied the changes of its expression in adult rat brain after two types of neuronal injury. In the control hippocampus, expression of heparin-binding growth-associated molecule messenger RNA was confined to CA1 pyramidal neurons and some hilar cells. Following transient forebrain ischaemia, the messenger RNA expression decreased within the first two days. On day 4, however, both the messenger RNA level and the number of expression-positive cells markedly increased in the CA1 subfield, where the selective neuronal losses were seen following ischaemia. Double-staining with a heparin-binding growth-associated molecule complementary RNA probe and an anti-glial fibrillary acidic protein antibody revealed that most of the expressing cells were reactive astrocytes. Moreover, the protein induction of heparin-binding growth-associated molecule after neuronal injury was demonstrated by immunohistochemistry using the affinity-purified antibodies. This molecule was also induced after intraventricular kainate injection, which is known to cause selective pyramidal cell necrosis in the CA3 region. Four days after the insult, the number of cells expressing the messenger RNA prominently increased in the CA3 subfield ipsilateral to the injection. As observed after the ischaemic insult, most of the expression-positive cells were identified as astrocytes. The data presented here suggest that heparin-binding growth-associated molecule, produced by the reactive astrocytes, may play important roles in the repair process after neuronal injury.