PKCδ-dependent signaling mediates ethambutol-induced toxic effects on human retinal pigment cells.

PURPOSE: Our previous report demonstrated that ethambutol (EMB) might induce cytoplasmic vacuolization and reduce the uptake of photoreceptor rod outer segments (ROS) in retinal pigment epithelium (RPE) cells, which are mediated via a protein kinase C (PKC)-dependent pathway. In the present study, we sought to identify the PKC isozyme(s) involved. METHODS: EMB-induced cytoplasmic vacuolization and uptake of ROS were observed under a phase contrast microscope. Western blots were performed to observe the membrane translocation of PKC isozymes and cytoplasmic release of cathepsin D. Quantitative PCR were performed to analyze gene expression of PKCδ. Human RPE cell line RPE50 and ARPE19 cells were pretreated with specific inhibitors or transfected with shRNAs of various PKC isozymes, including PKCα, ß, ε, γ, and δ, to examine whether EMB-induced toxic effects were prevented. RESULTS: In RPE50 cells, gene expression of PKCδ on both mRNA and protein levels was induced by EMB within 30 min to 3 h. EMB-induced cytoplasmic vacuolization in both RPE50 and ARPE19 cells was prevented by pretreating the cells with a specific inhibitor of PKCδ, Rottlerin, or depletion of PKCδ by shRNA. EMB-triggered reduction of ROS uptake was also significantly suppressed by pretreatment with Rottlerin, or depletion of PKCδ by shRNA technology. In contrast, pretreatment of the cells with specific inhibitors of PKCα, ß, ε, or γ, or depletion of PKCα or ß didn't influence the aforementioned EMB-triggered toxic effects. In addition, in RPE50, EMB induced the release of lysosomal enzyme cathepsin D into cytosol within 30 min to 6 h, which was also prevented by Rottlerin. CONCLUSIONS: EMB-induced vacuole formation, cytoplasmic release of cathepsin D, and reduction of phagocytosis in RPE are intimately correlated and regulated by the PKCδ signal pathway.

Factors associated with in vitro interferon-gamma production in tuberculosis.

BACKGROUND/PURPOSE: Macrophage activation assisted by interferon-gamma (IFN-γ) is a primary mechanism by which Mycobacterium tuberculosis is killed, but IFN-γ (production is inhibited in tuberculosis (TB) patients. The production of IFN-γ is influenced by many factors, such as interleukin (IL)-10, IL-12, IL-18, and clinical diseases; but the relative importance of each factor is unclear. METHODS: We evaluated the effects of these factors in 46 healthy individuals, 81 patients with TB, and 88 patients with non-TB pneumonia. The responses of IFN-γ, IL-10, IL-12 and IL-18 were determined from phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs). RESULTS: General linear model analysis showed that disease status and IL-12 response were the independent factors associated with the IFN-γ response. The production of IFN-γ was not affected by IL-10 and IL-18. There was a significant relationship between the IFN-γ response and the IL-12 response among patients with non-TB pneumonia, patients with TB, and healthy participants (Pearson's correlation coefficients of 0.466, 0.483, and 0.464, respectively). CONCLUSION: Production of IFN-γ in PBMCs was associated with active pulmonary TB and IL-12 response.

Association of visual motor processing and social cognition in schizophrenia.

Patients with schizophrenia have difficulties in social cognitive domains including emotion recognition and mentalization, and in sensorimotor processing and learning. The relationship between social cognitive deficits and sensorimotor function in patients with schizophrenia remains largely unexplored. With the hypothesis that impaired visual motor processing may decelerate information processing and subsequently affects various domains of social cognition, we examined the association of nonverbal emotion recognition, mentalization, and visual motor processing in schizophrenia. The study examined mentalization using the verbal subset of the Chinese version of Theory of Mind (CToM) Task, an equivalent task of the Faux Pas Test; emotion recognition using the Diagnostic Analysis of Nonverbal Accuracy 2-Taiwan version (DANVA-2-TW), and visual motor processing using a joystick tracking task controlled for basic motor function in 34 individuals with chronic schizophrenia in the community and 42 healthy controls. Patients with schizophrenia had significantly worse performance than healthy controls in social cognition, including facial, prosodic emotion recognition, and mentalization. Visual motor processing was also significantly worse in patients with schizophrenia. Only in patients with schizophrenia, both emotion recognition (mainly in prosodic modality, happy, and sad emotions) and mentalization were positively associated with their learning capacity of visual motor processing. These findings suggest a prospective role of sensorimotor function in their social cognitive deficits. Despite that the underlying neural mechanism needs further research, our findings may provide a new direction for restoration of social cognitive function in schizophrenia by enhancing visual motor processing ability.

Ethambutol induces PKC-dependent cytotoxic and antiproliferative effects on human retinal pigment cells.

Ethambutol (EMB)-induced ocular side effects may involve the influence on functions of retinal pigment epithelium (RPE), in addition to EMB-induced optic neuropathy. To address this issue, the molecular and cellular effects of EMB on RPE including growth regulation, morphological responses, phagocytic activity, and the relevant signaling pathways were investigated. EMB (at optimal concentration 8.0mM) can trigger cell cycle arrest in both RPE50 and ARPE19 cells, accompanied by reduced DNA synthesis. EMB also induced cytoplasmic vacuole formation in both RPE cell lines. Under transmission electric microscope, the phagosomes were replaced by vacuoles and the number of microvilli was reduced in EMB-treated cells. Animal experiments also demonstrated the vacuole formation within RPE of the EMB-treated rats. On the other hand, by in vitro phagocytosis assay using rod outer segment (ROS) as the target, we found EMB suppressed phagocytosis in the cultured RPE, which is consistent with the decreased rhodopsin uptake in the RPE of the EMB-treated rats. Furthermore, inhibitor of protein kinase C but not MAPK, prevented the EMB-induced phenotypical changes. Using a non-radioactive PKC assay, we also demonstrated the PKC activity in both RPE cell lines can be induced by EMB. In conclusion, EMB may exert toxic effects in RPE including suppression of cell growth, formation of cytoplasmic vacuoles and reduction of phagocytic functions via PKC signal pathway.

Enhanced PKCδ and ERK signaling mediate cell migration of retinal pigment epithelial cells synergistically induced by HGF and EGF.

Proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) are characterized by the development of epi-retinal membranes which may exert a tractional force on retina. A lot of inflammatory growth factors may disturb the local ocular cells such as retinal pigment epithelial (RPE) cells, causing them to migrate and proliferate in the vitreous cavity and ultimately forming the PVR membrane. In this study, the signal pathways mediating cell migration of RPE induced by growth factors were investigated. Hepatocyte growth factor (HGF), epidermal growth factor (EGF) or heparin-binding epidermal growth factor (HB-EGF) induced a greater extent of migration of RPE50 and ARPE19 cells, compared with other growth factors. According to inhibitor studies, migration of RPE cells induced by each growth factor was mediated by protein kinase C (PKC) and ERK (MAPK). Moreover, HGF coupled with EGF or HB-EGF had synergistic effects on cell migration and enhanced activation of PKC and ERK, which were attributed to cross activation of growth factor receptors by heterogeneous ligands. Furthermore, using the shRNA technique, PKCδ was found to be the most important PKC isozyme involved. Finally, vitreous fluids from PVR and PDR patients with high concentration of HGF may induce RPE cell migration in PKCδ- and ERK- dependent manner. In conclusion, migration of RPE cells can be synergistically induced by HGF coupled with HB-EGF or EGF, which were mediated by enhanced PKCδ activation and ERK phosphorylation.