Apoptotic and necrotic mechanisms of stress-induced human lens epithelial cell death

Exposure to ultraviolet radiation (UVR) and reactive oxygen species (ROS) can damage the human lens and contribute to cataract formation. Recent evidence suggests that apoptosis in lens epithelial cells (LEC) is an initiating event in noncongenital cataract formation in humans and animals. The present study examines the cellular and molecular mechanisms by which environmental (ultraviolet B [UVB]) and chemical (hydrogen peroxide [H(2)O(2)], t-butyl hydroperoxide [TBHP]) stress induces cell death in an SV-40 immortalized human lens epithelial (HLE) cell line. Treatment of HLE cells with UVB, H(2)O(2), and TBHP significantly decreased cell density with LD50 values of 350 J/m(2), 500 muM, and 200 muM, respectively. Cellular morphology, DNA fragmentation, and annexin/propidium iodide staining consistent with apoptosis was observed only in UVB-treated cells, whereas lactate dehydrogenase (LDH) release was significantly higher in H(2)0(2)- and TBHP-treated cells. In addition, activation of apoptotic stress-signaling proteins, including c-Jun NH2-terminal kinase (JNK), caspase-3, and DNA fragmentation factor 45 (DFF45) was observed only in UVB-treated cells. Inhibition of JNK activity increased UVB-induced cell death, suggesting that this pathway may serve a prosurvival role in HLE cells. These findings suggest UVB predominantly induces apoptosis in HLE cells, whereas H(2)O(2) and TBHP induce necrosis.     

As(III) inhibits ultraviolet radiation-induced cyclobutane pyrimidine dimer repair via generation of nitric oxide in human keratinocytes

Inorganic arsenic enhances skin tumor formation when combined with other carcinogens including ultraviolet radiation (UVR). The inhibition of DNA damage repair by arsenic has been hypothesized to contribute to the cocarcinogenic activities of arsenic observed in vivo. Cyclobutane pyrimidine dimers (CPDs) are an important mutagenic UVR photoproduct and implicated in the genesis of nonmelanoma skin cancer. The current study demonstrates that low concentrations of arsenite (As(III)) inhibit UVR-induced CPD repair in a human keratinocyte cell line via nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Following As(III) treatment, NO production and iNOS expression are elevated. Little is known about regulation of iNOS by As(III) and further investigations indicated that p38 mitogen-activated protein kinase (p38 MAPK) and NF-kappaB are required for As(III) induction of iNOS expression. This As(III)-stimulated signaling cascade was involved in inhibition of UVR-induced CPD repair as disruption of p38 MAPK activity and NF-kappaB nuclear translocation counteracted the effects of As(III) on CPD repair. Selective inhibition of iNOS ameliorated As(III) inhibition of CPD repair, thereby suggesting that iNOS is a downstream mediator of As(III) activity. These findings provide evidence that an As(III)-stimulated signal transduction cascade culminating in elevated iNOS expression and NO generation is an underlying mechanism for inhibition of UVR-induced DNA damage repair by arsenic.     

Molecular mechanisms of ultraviolet radiation-induced immunosuppression

Solar ultraviolet radiation (UVR) is well known for its immunosuppressive properties. UVR can suppress immune reactions both in a local and a systemic fashion. One of the major molecular mediators of photoimmunosuppression is UVR-induced DNA damage. In contrast to immunosuppressive drugs, UVR does not act in a general but antigen-specific fashion. This is due to the induction of regulatory T cells. Epidermal Langerhans cells harboring UVR-induced DNA damage appear to be essentially involved in the induction of these cells. Cytokines including interleukin (IL)-12, -18 and -23 exert the capacity to reduce UVR-induced DNA damage via induction of DNA repair. Accordingly, these cytokines prevent UVR-mediated immunosuppression. In contrast to IL-18, IL-12 and IL-23 can also inhibit the suppressive activity of regulatory T cells by a mechanism which still needs to be determined. Clarification of the molecular mechanisms underlying UVR-induced immunosuppression will help to develop new immunosuppressive therapeutic strategies by utilizing UVR-induced regulatory T cells for the treatment of immune-mediated diseases. In addition, these insights will contribute to a better understanding of photocarcinogenesis since suppression of the immune system by UVR essentially contributes to the induction of skin cancer.     

Neuropeptides and neuroendocrine hormones in ultraviolet radiation-induced immunosuppression

Exposure of the skin to ultraviolet radiation (UVR) can lead to deleterious effects such as sunburn, photoaging, and the development of skin cancer. UVR has also been shown to reduce local and systemic immune responses in humans and animals. In the recent past it has become clear that neuropeptides mediate some of the effects of UVR-induced immunosuppression. Among the neuropeptides released from cutaneous nerves after exposure to UVR, calcitonin gene-related peptide (CGRP) has been examined most extensively. It appears to lead to a reduction of contact hypersensitivity by inducing mast cells to degranulate and thus release tumor necrosis factor alpha (TNF-alpha) and, most likely, interleukin (IL)-10. Nitric oxide, which is coreleased with CGRP, seems to also play a role in immunosuppression through a yet undiscovered mechanism of action, while substance P may have counterregulatory effects. New evidence suggests that the release of neuropeptides from cutaneous sensory c-fibers after UVR is induced by keratinocyte-derived nerve growth factor. UVR can also induce epidermal and some dermal cells, such as melanocytes, keratinocytes, and dermal microvascular epithelial cells, to produce proopiomelanocortin (POMC) and its derivatives. The POMC product alpha-melanocyte-stimulating hormone (alpha-MSH) has been implicated in suppression of contact hypersensitivity and induction of hapten-specific tolerance, most likely by inducing keratinocytes and monocytes to produce the anti-inflammatory cytokine IL-10. Other POMC derivatives have not yet been investigated with regard to a possible role in UVR-induced effects on immunity.     

Poly(ADP-ribose) Contributes to an Association between Poly(ADP-ribose) Polymerase-1 and Xeroderma Pigmentosum Complementation Group A in Nucleotide Excision Repair

Exposure to ultraviolet radiation (UVR) promotes the formation of UVR-induced, DNA helix distorting photolesions such as (6-4) pyrimidine-pyrimidone photoproducts and cyclobutane pyrimidine dimers. Effective repair of such lesions by the nucleotide excision repair (NER) pathway is required to prevent DNA mutations and chromosome aberrations. Poly(ADP-ribose) polymerase-1 (PARP-1) is a zinc finger protein with well documented involvement in base excision repair. PARP-1 is activated in response to DNA damage and catalyzes the formation of poly(ADP-ribose) subunits that assist in the assembly of DNA repair proteins at sites of damage. In this study, we present evidence for PARP-1 contributions to NER, extending the knowledge of PARP-1 function in DNA repair beyond the established role in base excision repair. Silencing the PARP-1 protein or inhibiting PARP activity leads to retention of UVR-induced photolesions. PARP activation following UVR exposure promotes association between PARP-1 and XPA, a central protein in NER. Administration of PARP inhibitors confirms that poly(ADP-ribose) facilitates PARP-1 association with XPA in whole cell extracts, in isolated chromatin complexes, and in vitro. Furthermore, inhibition of PARP activity decreases UVR-stimulated XPA chromatin association, illustrating that these relationships occur in a meaningful context for NER. These results provide a mechanistic link for PARP activity in the repair of UVR-induced photoproducts.     

Inhibition of Rho-kinase induces alphaB-crystallin expression in lens epithelial cells

The small heat shock protein, alphaB-crystallin, has been shown to interact with actin and intermediate filament proteins. However, little is known regarding the cellular mechanisms regulating such interactions. In this study, we explored the role of the Rho/Rho-kinase pathway in alphaB-crystallin distribution and expression in porcine lens epithelial cells. alphaB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit any unique redistribution in response to actin depolymerization induced by Rho/Rho-kinase inhibitors (C3-exoenzyme or Y-27632) or by overexpression of the dominant negative mutant of Rho-kinase (DNRK) in porcine lens epithelial cells. Interestingly, alphaB-crystallin levels markedly increased in lens epithelial cells treated with the inhibitors of Rho/Rho-kinase proteins (lovastatin, Y-27632 or DNRK) while a protein kinase C inhibitor (GF109203x) was found to have no effect. Further, Y-27632 showed a dose (2-50 microM) response effect on alphaB-crystallin induction. Nocodazole, a microtubule-depolymerizing agent, elicited an increase in alphaB-crystallin levels but latrunculin, an actin depolymerizing agent, did not show any significant effect. Pretreatment with cycloheximide or genistein blocked the Rho-kinase inhibitor-induced increase in alphaB-crystallin protein levels. Rho-kinase inhibitor-induced increases in alphaB-crystallin levels were found to be associated with activation of P38 mitogen-activated protein kinase (MAPK). These results suggest that Rho/Rho-kinase negatively regulates alphaB-crystallin expression, and this response appears to be dependent on tyrosine-protein kinase and P38 MAPK function. Finally, alphaB-crystallin induction appears to be better correlated with the direct inhibition of Rho/Rho-kinase than with actin depolymerization per se.     

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.     

Ultraviolet stimulated melanogenesis by human melanocytes is augmented by di-acyl glycerol but not TPA

Epidermal melanocytes (MC) synthesise melanin in response to ultraviolet radiation (UVR). The mechanisms mediating the UV-induced activation of melano-genesis are unknown but since UVR induces turnover of membrane phospholipids generating prostaglandins (PGs) and other products, it is possible that one of these might provide the activating signal. We have examined the effects of prostaglandins (PGs) E₁, E₂, D₂, F_2α, and di-acyl glycerol upon the UV-induced responses of cultured human MC and the Cloudman S91 melanoma cell line. The PGs had little effect on unirradliated cells and did not alter the response to UVR in either human MC or S91 melanoma cells. However, a synthetic analogue of di-acyl glycerol, 1-oleyl 2-acetyl glycerol (OAG), caused a significant (P<0.0001), dose-related augmentation of melanin content both in human MC (seven-fold) and S91 cells (three-fold). UVR caused a significant augmentation of the OAG-induced melanognesis of both human MC and S91 cells. Since OAG is known to activate protein kinase C, it was possible that the observed modulation of the UVR signal could be via that pathway. Di-octanoyl glycerol, another di-acyl glycerol, which activates kinase C, caused a small (70%) increase in melanogenesis in MC which was not altered by UVR. However, 12-0 tetradecanoyl phorbol 13-acetate (TPA), a potent activator of protein kinase C, had no significant effect on either basal or UV-induced melanin synthesis in either cell type. These data suggest that the UV-induced signal activating melanogenesis could be mediated by di-acyl glycerol. Furthermore, they imply that the signal is transduced via an alternative, pathway that might be independent of protein kinase C.     

UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells

Epidermal growth factor receptor (EGFR) is overexpressed in human pancreatic cancer and is one of the clinical targets in its treatment. In the present study we investigated the mechanism underlying ultraviolet C (UVC)-radiation-induced cell growth inhibition and downregulation of EGFR in human pancreatic cancer cells (Panc1 and KP3). The cell proliferation assay indicated that Panc1 and KP3 cells were more sensitive to UVC radiation, and their growth was significantly inhibited compared to cells of the normal human pancreatic epithelial cell line, PE. Although EGFR levels was extremely low in PE cells, EGFR were highly overexpressed in Panc1 and KP3 cells, and UVC radiation downregulated the expression of EGFR in a time-dependent manner and induced phosphorylation of EGFR at Ser1046/1047 (S1046/7) in Panc1 and KP3 cells. UVC radiation induced activation of p38 mitogen-activated protein kinase (MAPK), and EGFR phosphorylation at S1046/7 induced by UVC radiation was markedly attenuated by the inhibition of p38 MAPK. Moreover, fluorescence microscopy revealed that p38 MAPK activated by UVC radiation triggered EGFR internalization and that this was not correlated with c-Cbl, an ubiquitin ligase, which plays an important role in EGF-induced EGFR downregulation. Taken together, our results suggest that in pancreatic cancer cells UVC radiation induced desensitization of the cells to EGFR stimuli via phosphorylation of EGFR at S1046/7 by activation of p38 MAPK, independent of c-Cbl.     

Docosahexaenoic acid reverts resistance to UV-induced apoptosis in human keratinocytes: involvement of COX-2 and HuR

The dramatic increase in the incidence of nonmelanoma skin cancer over the last decades has been related to the augmented exposure to ultraviolet (UV) radiation (UVR). It is known that apoptosis is induced as a protective mechanism after the acute irradiation of keratinocytes, whereas apoptotic resistance and carcinogenesis may follow the chronic exposure to UVR. We found that not all the human keratinocytes lines studied underwent apoptosis following acute exposure to UVR (10-60 mJ/cm²). Whereas UVR induced apoptosis in the HaCaT cells, NCTC 2544 and nr-HaCaT cells showed apoptosis resistance. The cytokeratin pattern of the apoptosis-resistant cells indicated that they possessed a degree of differentiation lower than that of HaCaT cells. They also showed an enhanced expression of cyclooxygenase-2 (COX-2), an early marker of carcinogenesis in various tissues, including skin. n-3 polyunsaturated fatty acids have drawn increasing interest as nutritional factors with the potential to reduce UVR carcinogenesis, and since they are apoptosis inducers and COX-2 inhibitors in cancer cells, we investigated the ability of n-3 polyunsaturated fatty acids to influence the resistance to UVR-induced apoptosis in keratinocytes. We observed that docosahexaenoic acid (DHA) reverted the resistance of nr-HaCaT cells to UVR-induced apoptosis, increasing the Bax/Bcl-2 ratio and caspase-3 activity, and reduced COX-2 levels by inhibiting the expression of the human antigen R (HuR), a known COX-2 mRNA stabilizer in keratinocytes. The transfection of nr-HaCaT cells with HuR siRNA mimicked the proapoptotic effect of DHA. Overall, our findings further support the role of DHA as a suitable anticarcinogenic factor against nonmelanoma skin cancers.     

Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.     

Effects of solar UV radiation and temperature on morphology and photosynthetic performance of Chaetoceros curvisetus

This study investigated the effect of solar ultraviolet radiation (UVR) and temperature on a chain length and photosynthetic performance of diatom Chaetorceros curvisetus. The cells were cultured in large quartz tubes and exposed to PAR, PAR + UV-A (PA), or PAR + UV-A + UV-B (PAB) radiation at 20°C and 28°C for six days, respectively. After recovery for 1 h, the cells were exposed again to three different radiations for 1 h. Then, a change in the photochemical efficiency (F_PSII) was examined and UVR-induced photoinhibition was calculated. The percentage of long chains (more than five single cells per chain) in C. curvisetus significantly increased from 8.2% (PAR) to 38.9% (PAB) at 20°C; while it was not notably affected at 28°C. Mycosporine-like amino acids (MAAs) concentration obviously increased by irradiance increment from PAR to PAB at 20°C. Chlorophyll (Chl) a concentration significantly declined with increasing irradiance at 20°C. Both MAAs and Chl a concentrations were not obviously changed by irradiance at 28°C. Before and after reexposure, F_PSII was significantly reduced both at 20°C and 28°C. UVR-induced photoinhibition at 20°C (39%) was higher than that at 28°C (30.9%). Solar UV radiation, especially UV-B, could significantly influence the percentage of long chains of C. curvisetus, especially at low temperature. UVR-induced photoinhibition can be alleviated by higher temperatures.     

The R116C mutation in alpha A-crystallin diminishes its protective ability against stress-induced lens epithelial cell apoptosis.

alphaA-crystallin is a small heat-shock protein expressed preferentially in the lens and is detected during the early stages of lens development. Recent work indicates that the expression of alphaA-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R116C) in alphaA-crystallin has been associated with congenital cataracts in humans. However, the physiological consequences of this mutation have not been analyzed in lens epithelial cells. In the present study, we expressed wild type or R116C alphaA-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C alphaA-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the alphaA-crystallin aggregate in lens epithelial cells increased from 500 to 600 kDa for the wild type protein to >2 MDa in the R116C mutant. When cells were exposed to physiological levels of UVA radiation, wild type alphaA-crystallin protected cells from apoptotic death as shown by annexin labeling and flow cytometric analysis, whereas the R116C mutant had a 4- to 10-fold lower protective ability. UVA-irradiated cells expressing the wild type protein had very low TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining, whereas cells expressing R116C mutant had a high level of TUNEL staining. F-actin was protected in UVA-treated cells expressing the wild type alphaA-crystallin but was either clumped around the apoptotic cells or was absent in apoptotic cells in cultures expressing the R116C mutant. Structural changes caused by the R116C mutation could be responsible for the reduced ability of the mutant to protect cells from stress. Our study shows that comparing the stress-induced apoptotic cell death is an effective way to compare the protective abilities of wild type and mutant alphaA-crystallin. We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract.     

Rapid activation of ERK1/2 mitogen-activated protein kinase by corticosterone in PC12 cells

Although the nongenomic effects of glucocorticoids have been well acknowledged, its precise intracellular signal transduction pathway remains to be elucidated. The present study using Western immunoblot and protein kinase activity assay, for the first time, showed that corticosterone (B) can induce a rapid activation of Erk1/2 mitogen-activated protein kinase (MAPK) in PC12 cells. The dose-response curve was bell shaped, with the maximal activation at 10(-9) M in 15 min. The results from immunofluorescence staining also revealed that the activated Erk1/2 MAPK was translocated from cytoplasm to nucleus of PC12 cells in 15 min. Activation of Erk1/2 MAPK by B was apparently not mediated by the classical cytosolic steroid receptors, for B-BSA can induce the phosphorylation of Erk1/2 MAPK, but the antagonist (RU38486) cannot block the phosphorylation of Erk1/2 MAPK induced by B. Phosphorylation of Erk1/2 MAPK induced by B was not affected by a tyrosine kinase inhibitor (genistein), suggesting that the pathway did not involve the tyrosine kinase activity. On the other hand, protein kinase C activator (PMA) can activate and protein kinase C inhibitor (G?6976) can block the activation of Erk1/2 MAPK induced by B. Taken together, these data clearly demonstrated that B might act via putative membrane receptor and rapidly activate Erk1/2 MAPK through protein kinase C alpha in PC12 cells.     

Convergence of signaling pathways induced by systemin,oligosaccharide elicitors,and ultraviolet-B radiation at the level of mitogen-activated protein kinases in Lycopersicon peruvianum suspension-cultured cells

We tested whether signaling pathways induced by systemin, oligosaccharide elicitors (OEs), and ultraviolet (UV)-B radiation share common components in Lycopersicon peruvianum suspension-cultured cells. These stress signals all induce mitogen-activated protein kinase (MAPK) activity. In desensitization assays, we found that pretreatment with systemin and OEs transiently reduced the MAPK response to a subsequent treatment with the same or a different elicitor. In contrast, MAPK activity in response to UV-B increased after pretreatment with systemin and OEs. These experiments demonstrate the presence of signaling components that are shared by systemin, OEs, and UV-B. Based on desensitization assays, it is not clear if the same or different MAPKs are activated by different stress signals. To identify specific stress-responsive MAPKs, we cloned three MAPKs from a tomato (Lycopersicon esculentum) leaf cDNA library, generated member-specific antibodies, and performed immunocomplex kinase assays with extracts from elicited L. peruvianum cells. Two highly homologous MAPKs, LeMPK1 and LeMPK2, were activated in response to systemin, four different OEs, and UV-B radiation. An additional MAPK, LeMPK3, was only activated by UV-B radiation. The common activation of LeMPK1 and LeMPK2 by many stress signals is consistent with the desensitization assays and may account for substantial overlaps among stress responses. On the other hand, MAPK activation kinetics in response to elicitors and UV-B differed substantially, and UV-B activated a different set of LeMPKs than the elicitors. These differences may account for UV-B-specific responses.