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Extracellular acidification accompanies neoplastic transformation of tissues and increases with tumor aggressiveness [1, 2]. The intracellular signaling cascade triggered by this process remains poorly understood and may be linked to recently discovered proton-activated G protein-coupled receptors such as OGR1 and G2A [3, 4]. Here, we report that OGR1 and G2A are expressed in human medulloblastoma tissue and its corresponding neuronal cell line. We show that extracellular acidification activates phospholipase C, IP(3) formation, and subsequent Ca2+ release from thapsigargin-sensitive stores in neurons. The number of responsive cells and the amount of Ca2+ released from stores correlated positively with the extent of extracellular acidification. Ca2+ release recruited the MEK/ERK pathway, providing a mechanistic explanation for how acidification stimulates cell growth. In addition, acidification activated Ca2+-permeable ion channels through a mechanism dependent on phospholipase C but independent of store depletion or a cytoplasmic Ca2+ rise. Hence, extracellular acidification, to levels seen in tumor tissue, activates temporally and spatially distinct pathways that elevate Ca2+ and may be directly relevant for tumor cell biology.

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) is associated with enhanced invasion and metastasis in head and neck squamous cell carcinoma (HNSCC). Long Pentraxin PTX3 is involved in immune escape in cancer cells. Here, we identified PTX3 as a promoting factor that mediates EGF-induced HNSCC metastasis. EGF-induced PTX3 transcriptional activation is via the binding of c-Jun to the activator protein (AP)-1 binding site of the PTX3 promoter. PI3K/Akt and NF-κB were essential for the PTX3 activation. EGF-induced PTX3 expression was blocked in c-Jun- and NF-κB-knockdown cells. EGF-mediated PTX3 secretion resulted in the enhancement of cell migration and invasion, and interactions between cancer and endothelial cells. The tail-vein injection animal model revealed that depletion of PTX3 decreased EGF-primed tumor cell metastatic seeding of the lungs. In addition, fibronectin, matrix metalloproteinase-9 (MMP9) and E-cadherin were essential components in EGFR/PTX3-mediated cancer metastasis. In conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF. Autocrine production of EGF-induced PTX3 in turn induces metastatic molecules, activating inflammatory cascades and metastasis.

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The goal of this study was to elucidate whether triggering the sphingomyelin pathway modulates LPS-initiated responses. For this purpose we investigated the effects of N-acetylsphingosine (C2-ceramide) on LPS-induced production of NO and PGE2 in murine RAW 264.7 macrophages and explored the signaling pathways involved. We found that within a range of 10–50 μM, C2-ceramide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction accompanied by a reduction in NO and PGE2 formation. By contrast, a structural analog of C2-ceramide that does not elicit functional activity, C2-dihydroceramide, did not affect the LPS response. The nuclear translocation and DNA binding study revealed that ceramide can inhibit LPS-induced NF-κB and AP-1 activation. The immunocomplex kinase assay indicated that IκB kinase activity stimulated by LPS was inhibited by ceramide, which concomitantly reduced the IκBα degradation caused by LPS within 1–6 h. In concert with the decreased cytosolic p65 protein level, LPS treatment resulted in rapid nuclear accumulation of NF-κB subunit p65 and its association with the cAMP-responsive element binding protein. Ceramide coaddition inhibited all the LPS responses. In addition, LPS-induced PKC and p38 mitogen-activated protein kinase activation were overcome by ceramide. In conclusion, we suggest that ceramide inhibition of LPS-mediated induction of inducible NO synthase and cyclooxygenase-2 is due to reduction of the activation of NF-κB and AP-1, which might result from ceramide’s inhibition of LPS-stimulated IκB kinase, p38 mitogen-activated protein kinase, and protein kinase C.