David A. Joyce

The Canonical NF-κB Pathway Governs Mammary Tumorigenesis in Transgenic Mice and Tumor Stem Cell Expansion

The role of mammary epithelial cell (MEC) NF-κB in tumor progression in vivo is unknown, as murine NF-κB components and kinases either are required for murine survival or interfere with normal mammary gland development. As NF-κB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-κB, the importance of MEC NF-κB to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-κB (IκBαSR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-κB in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demonstrated MEC NF-κB enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed that IκBαSR inhibited tumor stem cell pathways. IκBαSR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. MEC NF-κB contributes to mammary tumorigenesis. As we show that NF-κB contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-κB-dependent mammary tumorigenesis.

Glucocorticoid modulation of human monocyte/macrophage function: Control of TNF-alpha secretion

Abstract. Glucocorticoids suppress many functions in activated monocyte/macrophages, including the release of TNF-α. This is likely to contribute to the efficacy of glucocorticoids in some inflammatory diseases, such as rheumatoid arthritis, where TNF-α contributes to pathogenesis. Glucocorticoids suppress the activity of reporters which include TNF-α promoter regions and modify the activity of NF-κB family transcription factors in activated human monocytic cell lines, suggesting effects of glucocorticoids on TNF-α gene transcription. In addition, glucocorticoids have been reported to antagonise the enhanced translational efficiency of TNF-α mRNA which occurs at least after stimulation of murine monocytic cells. It is likely, therefore, that glucocorticoids act at several points in stimulated monocyte/macrophages to reduce TNF-α secretion. Understanding glucocorticoid control of TNF-α secretion may explain some of the variability in response to GC in inflammatory diseases and may reveal means of inducing glucocorticoid-like anti-inflammatory effects in monocyte/macrophages without exposing other tissues to the adverse effects of glucocorticoids.

12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits osteoclastogenesis by suppressing RANKL-induced NF-κB activation

The mechanism by which TPA‐induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW264.7 cell culture system and assays for NF‐κB nuclear translocation, NF‐κB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL‐induced NF‐κβ activation.

Sesquiterpene Lactone Parthenolide Blocks Lipopolysaccharide‐Induced Osteolysis Through the Suppression of NF‐κB Activity

Effective treatment for bacteria‐induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF‐κB inhibitor found in medicinal herbs, can block LPS‐induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF‐κB activity.

Calcium/calmodulin-dependent kinase activity is required for efficient induction of osteoclast differentiation and bone resorption by receptor activator of nuclear factor kappa B ligand (RANKL).

Calcium/calmodulin‐dependent protein kinase (CaMK) is a major down stream mediator of Ca2+ signaling in a wide range of cellular functions, including ion channel and cell cycle regulation and neurotransmitter synthesis and release. Here we have investigated the role of the CaMK signaling pathway in osteoclast differentiation and bone resorption. We observed that the CaMKI, CaMKII γ isoforms were present in both bone‐marrow derived macrophages and RAW264.7 murine macrophage cell line, and that expression persisted during osteoclast differentiation in the presence of receptor activator of nuclear factor kappa B (NF‐κB) ligand (RANKL). RANKL‐induced differentiation was accompanied by increased cyclic AMP response element transcriptional activity, and ERK phosphorylation, which are both downstream targets of CaMK. Two selective inhibitors of CaMKs, KN‐93 and KN‐62, inhibited osteoclastogenesis in a time and concentration‐dependent manner. This was accompanied by suppression of cathepsin K expression and osteoclastic bone resorption, which are markers for differentiated osteoclast function. KN‐93 and KN‐62 both inhibited RANKL‐induced ERK phosphorylation and CREB transcriptional activity. These findings imply a role for CaMK in osteoclast differentiation and bone resorption. J. Cell. Physiol. 212:787–795, 2007.

Myocyte Enhancer Factor 2 and Microphthalmia-associated Transcription Factor Cooperate with NFATc1 to Transactivate the V-ATPase d2 Promoter during RANKL-induced Osteoclastogenesis

The V-ATPase d2 protein constitutes an important subunit of the V-ATPase proton pump, which regulates bone homeostasis; however, currently little is known about its transcriptional regulation. Here, in an attempt to understand regulation of the V-ATPase d2 promoter, we identified the presence of NFATc1, microphthalmia-associated transcription factor (MITF)- and myocyte enhancer factor 2 (MEF2)-binding sites within the V-ATPase d2 promoter using complementary bioinformatic analyses, chromatin immunoprecipitation, and electromobility shift assay. Intriguingly, activation of the V-ATPase d2 promoter by NFATc1 was enhanced by either MEF2 or MITF overexpression. By comparison, coexpression of MITF and MEF2 did not further enhance V-ATPase d2 promoter activity above that of expression of MITF alone. Consistent with a role in transcriptional regulation, both NFATc1 and MITF proteins translocated from the cytosol to the nucleus during RANKL-induced osteoclastogenesis, whereas MEF2 persisted in the nucleus of both osteoclasts and their mononuclear precursors. Targeted mutation of the putative NFATc1-, MITF-, or MEF2-binding sites in the V-ATPase d2 promoter impaired its transcriptional activation. Additionally retroviral overexpression of MITF or MEF2 in RAW264.7 cells potentiated RANKL-induced osteoclastogenesis and V-ATPase d2 gene expression. Based on these data, we propose that MEF2 and MITF function cooperatively with NFATc1 to transactivate the V-ATPase d2 promoter during RANKL-induced osteoclastogenesis.

Targets of glucocorticoid action on TNF-α release by macrophages

Abstract. Glucocorticoid drugs affect virtually every cell type involved in inflammatory response, to some degree. Macrophage/monocytes (Mφ) are particularly sensitive, and glucocorticoids suppress release of most known Mφ inflammatory mediators, including TNF-α. In the case of TNF-α, several levels of regulation are already characterised and ongoing research hints at further glucocorticoid targets. The relative importance of transcriptional and post-transcriptional regulation is lineage-dependent and may also change during the course of Mφ differentiation. In human monocytic cell lines, glucocorticoids primarily suppress transcriptional activation through adjacent promoter binding sites for NF-κB transcription factor complexes and for complexes of c-Jun with activating transcription factor-2 (ATF-2). The goal of glucocorticoid research in inflammation is to develop drugs with the anti-inflammatory potential of glucocorticoids, but without the systemic toxicity. Each of the multiple targets for glucocorticoid action presents an opportunity for anti-inflammatory drug development. However, none of the known targets is unique to Mφ, and no single pathway is preeminent in all situations. Research is now directed at characterising targets and regulating them without systemic activation of the glucocorticoid receptor.

Caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL-induced NF-κB and NFAT activity.

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009.

Pharmacokinetics of ropivacaine following caudal analgesia in children

Ropivacaine has a favourable toxicity profile for epidural anaesthesia in adults, so it may also be an appropriate agent for epidural analgesia in children. We therefore designed this study to determine the pharmacokinetic variables of ropivacaine relevant to the risk of toxicity, after caudal administration in children. We studied nine healthy children, aged 1–6 years who received 1 ml.kg−1 of ropivacaine 0.25% for caudal analgesia. Venous blood samples were collected at intervals for 12 h after injection. Total plasma concentration of ropivacaine was assayed by high performance liquid chromatography, and pharmacokinetic descriptors were estimated from the plasma concentration‐time data. The median peak venous plasma concentration was 799 μg·l−1[interquartile range (IQR) 707–1044 μg·l−1], and was reached at a median time of 1.5 h (IQR 0.5–2 h). The mean elimination half‐life was 3.9 h (95% CI 2.7–5.0 h), and the mean apparent clearance and volume of distribution were 7.6±1.6 ml·min−1·kg−1 (95% CI 6.1–9.1 ml·min−1·kg−1) and 2.4±0.6 l·kg−1 (95% CI 1.9–3.0 l.kg−1), respectively. Analgesia was satisfactory in all cases and no systemic ropivacaine toxicity was observed. Caudal administration of weight‐adjusted doses of ropivacaine to children resulted in systemic exposure similar to that reported for adults. No systemic toxicity was observed. The findings strengthen predictions that the relative systemic safety of epidural ropivacaine in adults will apply to children. However, the pharmacokinetics and safety of epidural ropivacaine need to be studied further in children with circumstances that affect drug disposition and systemic tolerance.

Thapsigargin Modulates Osteoclastogenesis Through the Regulation of RANKL‐Induced Signaling Pathways and Reactive Oxygen Species Production

The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase‐3 activity, cytosolic Ca2+, and RANKL‐induced activation of NF‐κB and AP‐1 activities.