Lipopolysaccharide inhibits translation of iron chaperone PCBP1 to regulate inflammatory cytokine response in macrophage.

Macrophages play a key role in maintaining systemic iron homeostasis and immunity. During pro-inflammatory stage macrophages retain iron due to the decrease of the unique iron exporter ferroportin. Increased cellular iron is sequestered in to storage protein ferritin by iron chaperone poly(rC)-binding protein 1 (PCBP1). However, the fate of PCBP1 and its interaction with ferritin in pro-inflammatory macrophages has not been studied so far. Here we report that PCBP1 protein level is down-regulated in lipopolysaccharide (LPS) treated macrophages. LPS did not alter PCBP1 mRNA and protein stability suggesting inhibition of translation as a mechanism of PCBP1 down-regulation that was confirmed by 35S-methionine incorporation assay. PCBP1 interacts with ferritin-H (Ft-H) subunit to load iron into ferritin. We detected a decreased interaction between PCBP1 and Ft-H after LPS-stimulation. As a result iron loading in to ferritin was affected with simultaneous increase in labile iron pool (LIP). Pre-treatment of cells with iron chelator dampened LPS-induced expression of TNF-α, IL-1ß and IL-6 mRNA. Silencing of PCBP1 increased the magnitude of expression of these cytokines compared to control siRNA transfected LPS-treated macrophages. In contrast, overexpression of PCBP1 resulted a decrease in expression of these cytokines compared to vector transfected macrophages. Our results reveal a novel regulation of PCBP1 and its role in expression of cytokines in LPS-induced pro-inflammatory macrophages.

Intracellular pathogen Leishmania intervenes in iron loading into ferritin by cleaving chaperones in host macrophages as an iron acquisition strategy.

Iron (Fe) sequestration is one of the most important strategies of the host to control the growth and survival of invading pathogens. Ferritin (Ft) plays a pivotal role in the sequestration mechanism of mammalian hosts by storing Fe. Recent evidence suggests that poly(rC)-binding proteins (PCBPs) act as chaperones for loading Fe into Ft. Incidentally, modulation of host PCBPs in respect to storing Fe in Ft during any infection remains unexplored. Among PCBPs, PCBP1 and PCBP2 are present in every cell type and involved in interacting with Ft for Fe loading. Leishmania donovani (LD) resides within macrophages during the mammalian stage of infection, causing life-threatening visceral leishmaniasis. Here, we reveal the ability of LD to cleave PCBP1 and PCBP2 in host monocytes/macrophages. LD cleaves PCBP1-FLAG into two fragments and PCBP2-FLAG into multiple fragments, thus affecting their interactions with Ft and resulting in decreased Fe loading into Ft. LD-derived culture supernatant or exosome-enriched fractions are also able to cleave PCBPs, suggesting involvement of a secreted protease of the parasite. Using an immune-depletion experiment and transgenic mutants, we confirmed the involvement of zinc-metalloprotease GP63 in cleaving PCBPs. We further revealed that by cleaving host PCBPs, Leishmania could inhibit Fe loading into Ft to accumulate available Fe for higher intracellular growth. This is the first report of a novel strategy adopted by a mammalian pathogen to interfere with Fe sequestration into Ft by cleaving chaperones for its survival advantage within the host.

Clinical relevance of cyclooxygenase 2 and peroxisome proliferator-activated receptor γ in eyelid sebaceous gland carcinoma.

AIMS: Sebaceous gland carcinoma (SGC) is a malignancy associated with the pilosebaceous unit, and occurs at ocular or non-ocular sites. Cyclooxygenases (COXs) are enzymes that are crucial for lipid metabolism. COX-2 is overexpressed in various cancers, and its inhibition by non-steroidal anti-inflammatory drugs is known to reduce the risk of many cancers. Peroxisome proliferator-activated receptor (PPAR)-γ is a transcription factor involved in adipogenesis. PPAR-γ is a potential therapeutic target for the treatment of malignant tumours, including colon carcinoma. The aim of this study was to explore the status of COX-2 and PPAR-γ as prognostic markers in human eyelid SGC. METHODS AND RESULTS: The immunohistochemical expression of COX-2 and PPAR-γ was evaluated in 31 SGC cases. Cytoplasmic expression of COX-2 was detected in 80% of the SGC cases, and nuclear expression of PPAR-γ in 87%. There were significant correlations of PPAR-γ expression with well-differentiated SGC [19/21 (90%)] and of COX-2 overexpression with reduced disease-free survival (P = 0.0441, log rank analysis). COX-2 expression [odds ratio (OR) 3.82, 95% confidence interval (CI) 1.02-14.33, P = 0.046] and lymph node metastasis (OR 0.17, 95% CI 0.04-0.65, P = 0.009) emerged as significant risk factors in the univariate analysis. However, COX-2 expression did not emerge as a significant independent prognostic factor in multivariate analysis. CONCLUSIONS: COX-2 is a potential marker for identifying high-risk SGC patients. Expression of PPAR-γ in eyelid SGC cases reflects terminal sebaceous differentiation. Inhibitors of COX-2 signalling and PPAR-γ agonists are both prospective novel therapeutic targets in the management of eyelid SGC patients.