We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide-and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50 -60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.

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 ␣ -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 .

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 (catagen). 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.

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.—Roloff, B., Fechner, K., Slominski, A., Furkert, J., Botchkarev, V. A., Bulfone‐Paus, S., Zipper, J., Krause, E., Paus, R. Hair cycle‐dependent expression of corticotrophin‐releasing factor (CRF) and CRF receptors in murine skin. FASEB J. 12, 287–297 (1998)