An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.
Both clinical and experimental evidence indicates that AIDS-related Kaposi's sarcoma (AIDS-KS) has a multifactorial pathogenesis with factors such as HIV viral load, latent virus induction, and opportunistic infections contributing to disease progression. However, a consistent feature that unites these apparently diverse putative etiologic agents is sustained serum elevations of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). While virtually every cell responds to TNF-alpha with gene activation, the extent of TNF-alpha-mediated cellular signaling is regulated by a delicate balance between signal activation and signal arresting events. Reactive oxygen intermediates (ROI), which are generated as a consequence of TNF-alpha membrane interaction, are part of this TNF-alpha-initiated cellular activation cascade. Previous studies in our laboratory have shown that AIDS-KS cells possess impaired oxygen intermediate scavenging capacities, thereby establishing conditions permissive for the intracellular retention of ROI. In this study, we used cellular capacity to upregulate the cytoprotective enzyme superoxide dismutase (SOD) to address the extent of cellular response to TNF-alpha. Concurrent with the SOD analyses, nucleotide profiles were obtained to assess cellular bioenergetic responses during TNF-alpha challenge. Proliferative growth levels of mitochondrial (Mn)SOD activities showed an activity spectrum ranging from lowest activity in AIDS-KS cells, to intermediate levels in matched, nonlesional cells from the AIDS-KS donors, to highest activities in HIV normal fibroblasts. In contrast, following TNF-alpha challenge, the AIDS-KS and KS donor nonlesional cells showed a 11.89- and 5.86-fold respective increase in MnSOD activity, while the normal fibroblasts demonstrated a 1.35-fold decrease. Subsequent thiol redox modulation studies showed that only the normal fibroblast cultures showed a potentiation of TNF-alpha-mediated MnSOD upregulation following GSH depletion. In addition, provision of the GSH precursor, N-acetylcysteine during TNF-alpha challenge only diminished MnSOD activity and mitochondrial compartmentalization in the AIDS-KS cells, a finding that likely reflects the lower levels of reduced thiols in this cellular population. Our data, which show that a perturbation in their cellular thiol redox status accentuates AIDS-KS cellular responsiveness to TNF-alpha, suggest a biochemical rationale for the recognized TNF-alpha AIDS-KS clinical correlation.
The chemotherapeutic, doxorubicin, is currently used empirically in the treatment of AIDS- related Kaposi's sarcoma (AIDS-KS). Although often employed in a chemotherapeutic cocktail (doxorubicin, bleomycin, vincristine) single-agent therapy has recently been attempted with liposome encapsulated doxorubicin. Although doxorubicin's mechanism of action against AIDS-KS is unknown, we hypothesized that doxorubicin's ability to undergo redox cycling is associated with its clinical efficacy. The current study was conducted to investigate the effects of doxorubicin on selected xenobiotic-associated biochemical responses of three cellular populations: KS lesional cells, nonlesional cells from the KS donors, and fibroblasts obtained from HIV- aged matched men. Our results show that during doxorubicin challenge, there are strong positive correlations between cellular glutathione (GSH) levels and viability (r = 0.94), NADPH levels and viability (r = 0.93), and GSH and NADPH levels (r = 0.93), and demonstrate that as a consequence of their abilities to maintain cellular thiol redox pools HIV- donor cells are significantly less susceptible to doxorubicin's cytotoxic effects relative to AIDS-KS cells. Additional studies further supported the contribution of reduced thiols in mediating doxorubicin tolerance. While pretreatment with the GSH precursor, N-acetylcysteine was cytoprotective for all cell groups during doxorubicin challenge, GSH depletion markedly enhanced doxorubicin's cytotoxic effects. Studies to investigate the effects of a hydroxyl scavenger and iron chelator during doxorubicin challenge showed moderate cytoprotection in the AIDS-KS cells but deleterious effects in the HIV control cells. Inactivation of the longer lived membrane generated ROI in the cytoprotective deficient AIDS-KS cells, as well as an impairment of endogenous defenses in the HIV- donor control cells, may account for these scavenger and chelator associated findings. In summary, our findings show that doxorubicin mediates, at least in part, its AIDS-KS cellular cytotoxic effects by a redox related mechanism, and provides a biochemical rationale for doxorubicin's clinical efficacy in AIDS-KS treatment.
Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.