Enhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b(+) myelopoiesis

in Arthritis Research & therapy (2004), 6(3), 220-231

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b(+) myeloblasts. Both disease manifestations are more pronounced in interferon-gamma ... [more ▼]

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b(+) myeloblasts. Both disease manifestations are more pronounced in interferon-gamma receptor knock-out (IFN-gammaR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b(+) splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-gammaR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-kappaB ligand (RANKL) or with tumour necrosis factor-alpha (TNF-alpha). Significantly larger numbers of such cells could be generated from splenocytes of IFN-gammaR KO mice than from those of wildtype mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-alpha-induced osteoclast formation. Splenocyte cultures of IFN-gammaR KO mice also produced more TNF-alpha and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-gammaR KO mice contained comparatively low levels of pro-interleukin-1beta (pro-IL-1beta) and pro-caspase-1, indicating more extensive conversion of pro-IL-1beta into secreted active IL-1beta. These observations provide evidence that all conditions are fulfilled for the expanding CD11b(+) splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-gammaR KO mice to CIA. [less ▲]

Downregulation of ICAM-1 and VCAM-1 expression in endothelial cells treated by photodynamic therapy

in Oncogene (2004), 23(53), 8649-8658

Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell diseases that depends on the uptake of a photosensitizing compound followed by selective irradiation with ... [more ▼]

Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell diseases that depends on the uptake of a photosensitizing compound followed by selective irradiation with visible light. In the presence of oxygen, irradiation leads to the production of reactive oxygen species (ROS). A large production of ROS induces the death of cancer cells by apoptosis or necrosis. A small ROS production can activate various cellular pathways. Here, we show that PDT by pyropheophorbide-a methyl ester (PPME) induces the activation of nuclear factor kappa B (NF-kappaB) in HMEC-1 cells. NF-kappaB is active since it binds to the NF-kappaB sites of both ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) promoters and induces the transcription of several NF-kappaB target genes such as those of IL-6, ICAM-1, VCAM-1. In contrast, expression of ICAM-1 and VCAM-1 at the protein level was not observed, although we measured an IL-6 secretion. Using specific chemical inhibitors, we showed that the lack of ICAM-1 and VCAM-1 expression is the consequence of their degradation by lysosomal proteases. The proteasome and calpain pathways were not involved. All these observations were consistent with the fact that no adhesion of granulocytes was observed in these conditions. [less ▲]

Cell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers

in Biochemical Pharmacology (2003), 66(8), 1651-1659

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the ... [more ▼]

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the photosensitizers currently used in PDT localize in different cell compartments such as mitochondria, lysosomes, endoplasmic reticulum and generate cell death by triggering necrosis and/or apoptosis. Efficient cell death is observed when light, oxygen and the photosensitizer are not limiting ("high dose PDT"). When one of these components is limiting ("low dose PDT"), most of the cells do not immediately undergo apoptosis or necrosis but are growth arrested with several transduction pathways activated. This commentary will review the mechanism of apoptosis and growth arrest mediated by two important PDT agents. i.e. pyropheophorbide and hypericin. (C) 2003 Elsevier Inc. All rights reserved. [less ▲]

Potentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha

in Molecular and Cellular Biology (2003), 23(17), 6200-6209

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappaB. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium ... [more ▼]

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappaB. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-kappaB-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact kappaB sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-kappaB-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-kappaKB in the nucleus. We showed that the p65 subunit of NF-kappaB was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-kappaB after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the IkappaBalpha inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-kappaB. This delay was due neither to a defect in IkappaBalpha mRNA production nor to a nuclear retention of IkappaBalpha but was rather due to a persistent proteasome-mediated degradation of IkappaBalpha. A prolongation of IkappaB kinase activity could explain, at least partially, the delayed IkappaBalpha cytoplasmic reappearance observed in presence of TNF plus TSA. [less ▲]

Mechanisms involved in exogenous C2- and C6-ceramide-induced cancer cell toxicity

in Biochemical Pharmacology (2003), 65(10), 1633-1642

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. To determine whether ceramides can mediate the ... [more ▼]

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. To determine whether ceramides can mediate the apoptosis of HCT116 and OVCAR-3 cancer cells, exogenous C2-, C6-, and C16-ceramides were used to mimic the endogenous lipid increase that follows a large variety of stresses. C2- and C6-ceramides (cell-permeable ceramide analogs), but not C16-ceramide, induced nuclear factor-kappaB (NF-kappaB) DNA-binding, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and mitochondrial cytochrome c release, indicating that apoptosis occurs through the caspase cascade and the mitochondrial pathway. No difference in survival was observed between control cells and cells expressing mutated IkappaBalpha and treated with the permeable ceramides. This suggests that, at least in these cell lines, stable NF-kappaB inhibition did not modify the ceramide-induced cytotoxicity pathway. C6-ceramide also induced a double block in G1 and G2, thus emptying the S phase. [less ▲]

Mechanisms involved in exogenous C2- and C6-ceramide-induced cancer cell toxicity.

in Biochemical Pharmacology (2003), 65(10), 1633-42

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. To determine whether ceramides can mediate the ... [more ▼]

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. To determine whether ceramides can mediate the apoptosis of HCT116 and OVCAR-3 cancer cells, exogenous C2-, C6-, and C16-ceramides were used to mimic the endogenous lipid increase that follows a large variety of stresses. C2- and C6-ceramides (cell-permeable ceramide analogs), but not C16-ceramide, induced nuclear factor-kappaB (NF-kappaB) DNA-binding, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and mitochondrial cytochrome c release, indicating that apoptosis occurs through the caspase cascade and the mitochondrial pathway. No difference in survival was observed between control cells and cells expressing mutated IkappaBalpha and treated with the permeable ceramides. This suggests that, at least in these cell lines, stable NF-kappaB inhibition did not modify the ceramide-induced cytotoxicity pathway. C6-ceramide also induced a double block in G1 and G2, thus emptying the S phase. [less ▲]

Phosphorylation of varicella-zoster virus IE63 protein by casein kinases influences its cellular localization and gene regulation activity

in Journal of Biological Chemistry (2002), 277(23), 21050-21060

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined ... [more ▼]

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivo phosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or post-transcriptional level. [less ▲]

Activation of the NF-kappaB transcription factor by DNA damage

Poster (2002, January 07)

S Phase Dependence and Involvement of Nf-Kappab Activating Kinase to Nf-Kappab Activation by Camptothecin

in Biochemical Pharmacology (2001), 62(5), 603-16

Camptothecin (CPT) and derivatives are topoisomerase I poisons currently used as anticancer drugs. Their cytotoxicity is maximal for cells in S phase. Using asynchronous and S phase-synchronized HeLa ... [more ▼]

Camptothecin (CPT) and derivatives are topoisomerase I poisons currently used as anticancer drugs. Their cytotoxicity is maximal for cells in S phase. Using asynchronous and S phase-synchronized HeLa cells, we showed that both the nuclear factor-kappaB (NF-kappaB) activation and its transcriptional activity, induced by CPT treatment, are enhanced in S phase cells. After CPT treatment, NF-kappaB activation reached a maximum within 2-3 hr and was still detectable after 24 hr. The nature of the complex evolved with time, forming mostly p50/p65 after 2 hr to almost exclusively p52 after 24 hr. In HeLa cells, the different steps of the induction were readily observable in S phase synchronized cells, whereas they were barely noticeable in a randomly growing cell population. The signal progressed through the activation of the IKK complex, the phosphorylation of IkappaBalpha, and the degradation of phosphorylated-IkappaBalpha and -IkappaBbeta. The stable expression of wild-type HA-tagged-IkappaBalpha or mutated HA-tagged-IkappaBalpha (S32,36A) allowed us to confirm the essential role of Ser32 and Ser36. NF-kappaB-activating kinase (NIK) could play a role upstream of the IKK complex, as the transient expression of a kinase inactive mutant NIK(K429,430A) abolished the activation of NF-kappaB by CPT. A kinase inactive mutant of mitogen-activated protein/ERK kinase kinase 1 (MEKK1), another kinase susceptible of acting upstream of the signalsome, did not. Cytotoxicity studies with clonal populations expressing different amounts of wild-type or mutated IkappaBalpha revealed that the overexpression of wild-type IkappaBa in large amount increases the sensitivity of HeLa cells to CPT more efficiently than a lower level of expression of non-phosphorylable IkappaBalpha. [less ▲]

S phase dependance and involvement of the NF-kappaB activation kinase to NF-kappaB activation by Camptotheci

Poster (2001, July 04)