Using confocal laser scanning microscopy we investigated the Ca(2+) distribution in single corticotropin releasing factor- and urocortin-stimulated human skin cells. The models tested included melanoma cells, neonatal melanocytes and keratinocytes, and immortalized HaCaT keratinocytes. The changes in intracellular Ca(2+) signal intensities observed after stimulation of different cell types with corticotropin releasing factor and urocortin showed that: (1) the increase of intracellular Ca(2+) concentration was caused by a Ca(2+) influx (inhibition by EGTA); (2) this Ca(2+) influx took place through voltage-activated Ca(2+) ion channels (inhibition by d-cis-diltiazem, verapamil) and (3) cyclic nucleotide-gated ion channels were not involved in this process (no effect of Mg(2+)). The effects were also observed at very low peptide concentrations (10(-13) M) with no apparent linear correlation between peptide dosage and increase of fluorescence intensity, which implied co-expression of different corticotropin releasing factor receptor forms in the same cell. Immortalized (HaCaT) keratinocytes exhibited the strongest differential increases of a Ca(2+) fluorescence after peptide-stimulation. Corticotropin releasing factor induced Ca(2+) flux into the cytoplasm, while urocortin Ca(2+) flux into the nucleus with a remarkable oscillatory effect. The latter indicated the presence of an intracellular urocortin-induced signal transduction pathway that is unique to keratinocytes.

HGF/SF and its receptor (Met) are principal mediators of mesenchymal‐epithelial interactions in several different systems and have recently been implicated in the control of hair follicle (HF) growth. We have studied their expression patterns during HF morphogenesis and cycling in C57BL/6 mice, whereas functional hair growth effects of HGF/SF were assessed in vivo by analysis of transgenic mice and in skin organ culture. In normal mouse skin, follicular expression of HGF/SF and Met was strikingly localized: HGF/SF was found only in the HF mesenchyme (dermal papilla fibroblasts) and Met in the neighboring hair bulb keratinocytes. Both HGF/SF and Met expression peaked during the initial phases of HF morphogenesis, the stage of active hair growth (early and mid anagen), and during the apoptosis‐driven HF regression (cata‐gen). Met+ cells in the regressing epithelial strand appeared to be protected from undergoing apoptosis. Compared to wild‐type controls, transgenic mice overexpressing HGF/SF under the control of the MT‐1 promoter had twice as many developing HF and displayed accelerated HF development on postnatal day 3. They also showed significant catagen retardation on P17. In organ culture and in vivo, HGF/SF i.c. resulted in a significant catagen retardation. These results demonstrate an important role of HGF/SF and Met in murine hair growth control and suggest that Met‐mediated signaling might be exploited for therapeutic manipulation of human hair growth disorders.—Lindner, G., Menrad, A., Gherardi, E., Merlino, G., Welker, P., Handjiski, B., Roloff, B., Paus, R. Involvement of hepatocyte growth factor/scatter factor and Met receptor signaling in hair follicle morphogenesis and cycling. FASEB J. 14, 319–332 (2000)

Since the skin produces POMC peptides, in the present work we investigated local production of urocortin, a peptide related to CRH, the normal endogenous stimulant for POMC. Urocortin immunoreactivity was detected by direct RIA in extracts of human skin, mouse skin (C57BL-6 strain), cultured cells from established lines of human melanoma and squamous cell carcinoma, human keratinocytes (HaCaT), and hamster melanomas. Addition of a reverse phase high performance liquid chromatography step before the RIA confirmed the presence of urocortin, as the immunoreactivity eluted at the same retention time as urocortin standard in extracts from HaCaT keratinocytes and mouse skin. Using the tandem technique of liquid chromatography-mass spectrometry, we identified a peptide with the same mass and retention time as the urocortin standard in human skin extracts. The urocortin antigen could be immunolocalized to normal keratinocytes of the epidermis and hair follicle, epithelium of sweat and sebaceous glands, dermal skeletal muscle, and nevocytes; it was also detected in melanoma and basal cell carcinoma cells. RT-PCR amplification of ribonucleic acid from human skin, cultured keratinocytes, and melanoma cells showed a 145-kb fragment from the coding region of exon 2 of the urocortin gene in all of the tested sources. Lastly, sequencing of the amplified fragment confirmed 100% homology with the known sequence of the urocortin gene. In conclusion, we now demonstrate that human skin and mouse skin as well as cultured keratinocytes and melanoma cells exhibit functional expression of the urocortin gene with actual production of urocortin peptide.

Annals of the New York Academy of SciencesVolume 885, Issue 1 p. 433-439 Developmentally Regulated Expression of α-MSH and MC-1 Receptor in C57BL/6 Mouse Skin Suggests Functions Beyond Pigmentationa V. A. BOTCHKAREV, V. A. BOTCHKAREV Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, GermanySearch for more papers by this authorN. V. BOTCHKAREVA, N. V. BOTCHKAREVA Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, GermanySearch for more papers by this authorA. SLOMINSKI, A. SLOMINSKI Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, GermanySearch for more papers by this authorB. ROLOFF, B. ROLOFF Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, Germany Department of Pathology, Loyola University, Maywood, Illinois 60153, USASearch for more papers by this authorT. LUGER, T. LUGER Department of Dermatology, University of Münster, D-48149, Münster, GermanySearch for more papers by this authorR. PAUS, Corresponding Author R. PAUS Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, Germany Address for correspondence: Dr. R. Paus, Department of Dermatology, University Hospital Eppendorf, University of Hamburg, D-20246 Hamburg, Germany, paus@uke.uni-hamburg.de (e-mail).Search for more papers by this author V. A. BOTCHKAREV, V. A. BOTCHKAREV Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, GermanySearch for more papers by this authorN. V. BOTCHKAREVA, N. V. BOTCHKAREVA Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, GermanySearch for more papers by this authorA. SLOMINSKI, A. SLOMINSKI Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, GermanySearch for more papers by this authorB. ROLOFF, B. ROLOFF Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, Germany Department of Pathology, Loyola University, Maywood, Illinois 60153, USASearch for more papers by this authorT. LUGER, T. LUGER Department of Dermatology, University of Münster, D-48149, Münster, GermanySearch for more papers by this authorR. PAUS, Corresponding Author R. PAUS Department of Dermatology, Charité, Humboldt Universität zu Berlin, D-10117, Berlin, Germany Address for correspondence: Dr. R. Paus, Department of Dermatology, University Hospital Eppendorf, University of Hamburg, D-20246 Hamburg, Germany, paus@uke.uni-hamburg.de (e-mail).Search for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb08706.xCitations: 25 a Note: The results reported here represent preliminary results. An extended manuscript containing specific data in preparation. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume885, Issue1CUTANEOUS NEUROIMMUNOMODULATION THE PROOPIOMELANOCORTIN SYSTEMOctober 1999Pages 433-439 RelatedInformation

ABSTRACT: The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH‐R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic‐pituitary‐adrenal axis functions in mammalian skin, in response to local stress (see Reference 1 ). To further define such system we used immunocytochemistry, RP‐HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH‐R1 was documented in mouse and human skin using RT‐PCR and Northern blot techniques; CRH binding sites and CRH‐R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose‐dependent increases in intracellular Ca 2+ . The latter were inhibited by the CRH antagonist α‐helical‐CRH(9–41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH‐R1, supports the existence of a local CRH/CRH‐R neuroendocrine pathway that may be activated within the context of a skin stress response system .

We demonstrate the presence and hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors (CRF-R) in C57BL/6 mouse skin. To correlate this with a physiological, developmentally controlled tissue remodeling process, we have analyzed CRF and CRF-R expression during defined stages of the murine hair cycle with its rhythmic changes between growth (anagen), regression (catagen), and resting (telogen). Using reversed-phase HPLC combined with two independent anti-CRF radioimmunoassays, we have identified CRF in murine skin. Maximal CRF levels were found in anagen III-IV skin, and minimal values were detected in catagen and telogen skin. By immunofluorescence, maximal CRF immunoreactivity (CRF-IR) was seen in the basal epidermis, nerve bundles of skin, the outer root sheath and matrix region of anagen IV-VI follicles, and in defined sections of their perifollicular neural network, whereas catagen and telogen skin displayed minimal CRF-IR. Using quantitative autoradiography and 125I-CRF as a tracer, high-affinity binding sites for CRF were detected in murine skin. The highest density of specific binding sites was detected in the panniculus carnosus, the epidermis, and the hair follicle. CRF-R type 1 (CRF-R1) IR was detected by immunohistology mainly in the outer root sheath, hair matrix, and dermal papilla of anagen VI follicles, as well as in the inner and outer root sheaths of early catagen follicles. CRF-R1 expression was also hair cycle dependent. Therefore, in normal murine skin, the CRF-CRF-R signaling system may operate as an additional neuroendocrine pathway regulating skin functions, possibly in the context of cutaneous stress responses.

As β-turn-inducing motif, 4-(N-Fmoc-2-aminoethyl)-6-dibenzofuranpropionic acid was synthesized and incorporated into the corticotropin-releasing factor. The effect on structural and biological properties was investigated.

Polychlorinated biphenyls are a group of industrial chemicals that are widely distributed in the environment. Since these compounds occur as mixtures, studies of their possible interactive effects are important. In order to determine whether an interaction of 2,5,2',5'-tetrachlorobiphenyl (TCB) with 3,4,3',4'-TCB occurs during multistage hepatocarcinogenesis in vivo, like that previously observed in lymphocytes in vitro (L. M. Sargent et al., Mutat. Res., 224: 79-88, 1989), we exposed rats to a single initiating dose of diethylnitrosamine (DEN), 10 mg/kg after a 70% partial hepatectomy, and subsequently to 0.1 ppm 3,4,3',4'-TCB and/or 10 ppm 2,5,2',5'-TCB in the diet for 1 year. Administration of each of the TCBs alone after DEN initiation resulted in a low incidence of chromosomal damage in hepatocytes; but when the two were given together after DEN initiation, there was a more than additive effect on this parameter at both 7 and 12 months which was highly significant. Administration of the TCBs alone or in combination in the absence of DEN initiation also resulted in chromosomal damage, approaching that seen in livers of animals initiated with DEN when sacrificed at 12 months. In animals receiving 0.05% phenobarbital for a 12-month period after initiation with DEN, a significant degree of chromosomal breakage and fragment formation occurred both in hepatocytes expressing the ectoenzyme gamma-glutamyltranspeptidase (GGT) and in those that were GGT negative. However, the GGT-negative cells showed a significantly lower incidence of chromosomal damage than the GGT-positive hepatocytes. Exposure to phenobarbital for 7 months after DEN initiation resulted in no significant chromosomal damage in hepatocytes, whether GGT positive or GGT negative. Some degree of specificity in chromosomal alterations was seen in hepatocytes of animals initiated with DEN and promoted either with a combination of TCBs or with phenobarbital. The most frequent alterations seen were a trisomy of chromosome 1 or of its long arm and a monosomy of chromosome 3 or its short arm. Some chromosome 7 aberrations were also seen. The highest frequency of specific aberrations occurred in hepatocytes from rats that also bore hepatocellular carcinomas, suggestive of the hypothesis that genes involved in the development of hepatic carcinoma may reside in chromosome 1 and/or 3 of the rat.

Rats who were fed low doses of single PCBs, either 2,5,2',5' or 3,4,3',4', did not demonstrate any chromosome breakage or mitotic changes in their bone marrow cells. However, there was a significant increase in chromosome damage observed in bone marrow cells of rats ingesting 10 ppm 2,5,2',5' plus 0.1 ppm 3,3,3',4' in combination. It is suggested that this PCB combination, previously found to cause superadditive chromosome damage in vitro, is also capable of causing chromosome damage in vivo, but these effects do not compromise cell proliferation because the mitotic index is not depressed.

Chromosome analysis of human lymphocyte cultures exposed in vitro to the herbicides cyanazine or metolachlor demonstrated chromosome damage at concentrations which did not inhibit cell growth, 1 μg/ml for cyanazine and 1 μg/ml or 0.1 μg/ml for metolachlor.

In order to study the possible mutagenic properties of polychlorinated biphenyls (PCBs), human lymphocyte cultures were examined for chromosome breakage, rearrangements, sister-chromatid exchange, and mitotic delay. The present study, which used cyclophosphamide as a positive control, shows that one planar PCB congener, 3,4,3′,4′-tetrachlorobiphenyl, caused dose-related chromosome breakage in human lymphocytes exposed in vitro to 0.1–10−4 μg/ml. In contrast, the non-planar PCB, 2,5,2′,5′, did not cause chromosome damage in comparable tests even at concentrations as high as 1 μg/ml. However, when 3,4,3′,4′ at a concentration lower than that which causes chromosome breakage (10−5 μg/ml) was combined with a non-clastogenic concentration of 2,5,2′,5′, the chromosomal damage observed was far in excess of what one would expect from higher doses of 3,4,3′,4′ alone. These results suggest that some PCB congeners may interact to cause synergistic genotoxic effects.