Egr-1 inhibits apoptosis during the UV response: correlation of cell survival with Egr-1 phosphorylation.
(65/12422)
UV irradiation of normal or immortalized cells induces a rapid increase in the expression of several transcription factors and is thought to serve a protective function. The human fibrosarcoma cell line, HT1080 clone H4, expresses almost undetectable levels of Egr-1 and does not respond to UV-C irradiation by the induction of Egr-1. The H4 cells are hypersensitive to UV which induces apoptosis and reduces clonogenicity. The introduction of exogenous Egr-1 into H4 (H4E9 and H4E4 cell-lines) confers protection from UV damage as measured by a number of assays. In both NIH3T3 (with inducible Egr-1) and H4E9 (constitutive Egr-1) cells, UV irradiation gave enhanced transactivation of Egr-1 reporters that correlated with phosphorylated Egr-1. Studies using inhibitors indicated that protein kinase-C and tyrosine kinases are involved in the anti-apoptotic effects of Egr-1 after UV damage. This is the first description of a biological effect of phosphorylated Egr-1. (+info)
UVA exposure of human skin reconstructed in vitro induces apoptosis of dermal fibroblasts: subsequent connective tissue repair and implications in photoaging.
(66/12422)
The skin reconstructed in vitro has been previously shown to be a useful model to investigate the effects of UVB exposure (Bernerd and Asselineau, 1997). The present study describes the response to UVA irradiation. Major alterations were observed within the dermal compartment. Apoptosis of fibroblasts located in the superficial area of the dermal equivalent was observed as soon as 6 h after irradiation, leading to their disappearance after 48 h. This effect was obtained without major alterations of epidermal keratinocytes suggesting a differential cell type sensitivity to UVA radiations. In addition, collagenase I was secreted by dermal fibroblasts. The UVA dermal effects could be observed even after removal of the epidermis during the post irradiation period, demonstrating that they were independent of the keratinocyte response. The analysis of the tissue regeneration during the following 2 weeks revealed a connective tissue repair via fibroblasts proliferation, migration and active synthesis of extracellular matrix proteins such as fibronectin and procollagen I. This cellular recolonization of the superficial part of the dermal equivalent was due to activation of surviving fibroblasts located deeply in the dermal equivalent. The direct damage in the dermis and the subsequent connective tissue repair may contribute to the formation of UVA-induced dermal alterations. (+info)
Alterations in cell death and cell cycle progression in the UV-irradiated epidermis of bcl-2-deficient mice.
(67/12422)
The effect of bcl-2 gene ablation on epidermal cell death induced by UV-B irradiation was investigated in mice. Exposure of depilated back skin of bcl-2-/- mice to 0.5 J/cm2 UV-B caused a prolonged increase in the number of epidermal cells showing nuclear DNA fragmentation compared to wild-type littermates. Consistently, skin explants from bcl-2-deficient mice exhibited a higher number of sunburn cells per cm epidermis (16.6+/-2.1 vs 7.0+/-1.5) following exposure to 0.1 J/cm2 UV-B in vitro. Furthermore, UV irradiation failed to increase pre-melanosomes in skin explants from mutant animals, and primary menalocyte cultures derived from bcl-2 null mutants were highly susceptible to UV-induced cell death compared to cultures from wild-type littermates. An accelerated reappearance of proliferating cells, showing nuclear immunoreactivity for Ki-67 and c-Fos, was observed in the UV-irradiated epidermis of bcl-2-deficient mice. Taken together, these findings suggest that effects of UV radiation on epidermal cell death and cell cycle progression are influenced by survival-promoting Bcl-2. (+info)
Activation of a low pH-dependent nuclease by apoptotic agents.
(68/12422)
Intracellular acidification caused by agents such as UV(C), etoposide or ceramide accompanies the progression of apoptosis. It is suggested that cellular acidosis may set favorable conditions for a dormant, low pH-dependent (acidic) nuclease, which could be involved in intranucleosomal genome degradation, a hallmark of programmed cell death. Here we show that exposure of HL-60 cells to acidotic/apoptotic agents results in the several-fold activation of a novel low pH-dependent (acidic) nuclease activity, as revealed by zymography. Its activity, which resides in nuclei, is associated with four polypeptides with apparent Mr of 56, 48, 45 and 40 kDa. Treatment of HeLa cells with UV(C) or ceramide causes also the up-regulation of an acidic nuclease activity which is represented by 70 and 62 kDa polypeptides. These observations suggest that acidic nuclease activation can be induced by the same apoptotic agents in different cell types. In HL-60 cells, acidic nuclease up-regulation triggered by acidotic agents follows the induction of AP-1 transcription factor active complexes and accompanies the progression of apoptosis. Inhibition of AP-1 factor activity caused by either anti-caspase/anti-acidotic agent Zn2+ or curcumin, an inhibitor of AP-1 binding to DNA and c-jun synthesis, protects cells from genome destruction. Acidic nuclease activation, however, is only partially inhibited by these factors. We propose that (i) the up-regulation of an acidic nuclease activity is governed by a regulatory pathway different from that responsible for AP-1 factor induction, caspases activation and intracellular acidification, and (ii) activation of an acidic nuclease does not cause any deleterious effects when AP-1 transcription factor induction, caspases activation and intracellular acidification are down-regulated. Thus, the acidic nuclease up-regulation alone is not a sufficient prerequisite for apoptosis. (+info)
The iron regulatory protein can determine the effectiveness of 5-aminolevulinic acid in inducing protoporphyrin IX in human primary skin fibroblasts.
(69/12422)
The level of endogenous photosensitiser, protoporphyrin IX (PPIX), can be enhanced in the cells by 5-aminolevulinic acid (ALA). We investigated the effect of critical parameters such as growth state of the cells and availability of intracellular iron in modulating the level of PPIX, in human primary cultured skin fibroblasts (FEK4) maintained either in exponentially growing or growth-arrested phase, following treatment with ALA. The addition of ALA to exponentially growing cells increased the level of PPIX 6-fold relative to control cells; however, in growth-arrested cells the same treatment increased the level of PPIX up to 34-fold. The simultaneous addition of the hydrophilic iron-chelator Desferal with ALA, boosted the level of PPIX up to 47-fold in growing cells and up to 42-fold in growth-arrested cells, suggesting that iron is limiting under the latter conditions. The strict dependence of PPIX enhancement on free available iron levels was examined by the level of activation of iron regulatory protein in band shift assays. This analysis revealed that the basal level of iron regulatory protein in growth-arrested cells was 6-fold higher than in growing cells, reflecting the influence of the free available iron pool in exponentially growing cells. Interestingly, the same ratio was found between the basal level concentration of PPIX in growing and growth-arrested cells. We propose that iron regulatory protein activation could serve as a marker for developing photodynamic therapy protocols because it identifies cells and tissues with a propensity to accumulate PPIX and it is therefore likely to predict the effectiveness of such therapies. (+info)
A substance p agonist acts as an adjuvant to promote hapten-specific skin immunity.
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Because substance p (SP) has been reported to be released from cutaneous sensory nerve endings after hapten application, we determined whether SP participates in contact hypersensitivity (CH) induction by using a SP agonist, GR73632 or delta-Aminovaleryl [Pro9, N-Me-Leu10]-substance P(7-11) and a SP antagonist, spantide I. When injected intradermally, SP agonist enhanced CH induced by conventional, but not optimal, sensitizing doses of hapten. By contrast, SP antagonist inhibited the induction of CH by optimal sensitizing doses of hapten. Moreover, SP agonist promoted CH induction and prevented tolerance when hapten was painted on skin exposed to acute, low-dose ultraviolet-B radiation. Intradermally injected SP agonist altered neither the density nor the morphology of epidermal Langerhans cells, implying that SP agonist enhanced the generation of hapten-specific immunogenic signals from the dermis. It is proposed that SP is a natural "adjuvant" that promotes the induction of CH within normal skin. Although exogenous SP agonist can prevent impaired CH and tolerance after ultraviolet-B radiation, the susceptibility of native SP to local neuropeptidases renders the neuropeptide unable to prevent the deleterious effects of ultraviolet-B radiation on cutaneous immunity. (+info)
UVB irradiation stimulates deposition of new elastic fibers by modified epithelial cells surrounding the hair follicles and sebaceous glands in mice.
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UVB irradiation stimulates the synthesis of elastin in the skin of humans and experimental animals. In this study we localized the site and the cells that are responsible for the synthesis of murine dermal elastic fibers. SKH-1 hairless mice were irradiated with UVB and the skin removed for light microscopy, electron microscopy, in situ hybridization, immunohistochemistry, and biochemical studies. In response to chronic low doses of UVB there was an initial moderate increase in tropoelastin mRNA in the papillary dermis. By contrast, there was a continuous marked elevation of collagen alpha1(I) message localizing to sites of inflammatory cell influx throughout the upper and lower dermis. After 25 wk of UV irradiation there was a 2-fold increase in skin elastin, yet total collagen remained unchanged. Serial desmosine analysis from en face sections indicated the increase in elastin content was due to dermal elastic fibers, an increase in the size and number of the dermal cysts, and an increase in subpanniculus elastic fibers. Elastin stains of en face sections suggested that the elastic fibers in the upper dermis were exclusively derived from cells lining the epithelial root sheath and sebaceous glands. In response to UV irradiation, the elastic fibers increased in number and size, wrapping around these structures and aligning in both directions as long fibers parallel to the body axis. Electron micrographs indicated that modified epithelial cells in close proximity to the flattened epithelial cells that encircled the root sheath and sebaceous glands were the source of the elastic fibers. (+info)
Failure of measles virus to activate nuclear factor-kappa B in neuronal cells: implications on the immune response to viral infections in the central nervous system.
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Neurons are postmitotic cells that foster virus persistence. These cells lack the HLA class I molecules required for clearance of infected cells. Previously, we showed that HLA class I is induced by measles virus (MV) on glial cells, which is primarily mediated by IFN-beta. In contrast, MV was unable to induce HLA class I or IFN-beta in neuronal cells. This failure was associated with lack of NF-kappa B binding to the positive regulatory domain II element of the IFN-beta promoter, which is essential for virus-induced IFN-beta gene activity. In this study, we demonstrate that the failure to activate NF-kappa B in neuronal cells is due to the inability of MV to induce phosphorylation and degradation of I kappa B, the inhibitor of NF-kappa B. In contrast, TNF-alpha induced degradation of I kappa B alpha in the neuronal cells, suggesting that failure to induce I kappa B alpha degradation is likely due to a defect in virus-mediated signaling rather than to a defect involving neuronal I kappa B alpha. Like MV, mumps virus and dsRNA failed to induce I kappa B alpha degradation in the neuronal cells, suggesting that this defect may be specific to viruses. Autophosphorylation of the dsRNA-dependent protein kinase, a kinase possibly involved in virus-mediated I kappa B alpha phosphorylation, was intact in both cell types. The failure of virus to induce I kappa B alpha phosphorylation and consequently to activate NF-kappa B in neuronal cells could explain the repression of IFN-beta and class I gene expression in virus-infected cells. These findings provide a potential mechanism for the ability of virus to persist in neurons and to escape immune surveillance. (+info)