The extracellular pH (pHe) of tumor tissues is often acidic, which can induce the expression of several proteins. We previously showed that production of matrix metalloproteinase-9 (MMP-9) was induced by culturing cells at acidic pHe (5.4 -6.5). Here we have investigated the signal transduction pathway by which acidic pHe induces MMP-9 expression. We found that acidic pHe (5.9) activated phospholipase D (PLD), and inhibition of PLD activity by 1-butanol and Myr-ARF6 suppressed the acidic pHe-induced MMP-9 expression. Exogenous PLD, but not phosphatidylinositol-specific PLC or PLA 2 , mimicked MMP-9 induction by acidic pHe. Western blot analysis revealed that acidic pHe increased the steady-state levels of phosphorylated extracellular signal-regulated kinases 1/2 and p38 and that the PLD inhibitors suppressed these increases. Using 5-deletion mutant constructs of the MMP-9 promoter, we found that the acidic pHe-responsive region was located at nucleotide ؊670 to ؊531, a region containing the NFB binding site. A mutation into the NFB binding site reduced, but not completely, the acidic pHe-induced MMP-9 promoter activity, and NFB activity was induced by acidic pHe. Pharmacological inhibitors specific for mitogen-activated protein kinase kinase 1/2 (PD098059) and p38 (SB203580) attenuated the acidic pHe-induced NFB activity and MMP-9 expression. These data suggest that PLD, mitogen-activated protein kinases (extracellular signal-regulated kinases 1/2 and p38), and NFB mediate the acidic pHe signaling to induce MMP-9 expression. A transcription factor(s) other than NFB may also be involved in the MMP-9 expression.Proteolytic degradation of extracellular matrix is important for tumor metastasis and angiogenesis. The matrix metalloproteinases (MMPs) 1 constitute a family of extracellular matrixdegrading enzymes. Among these enzymes, MMP-9/gelatinase B plays an important role in tumor invasion and metastasis because of its specificity for type IV collagen. Because the promoter region of the MMP-9 gene contains a TATA box and nuclear factor-B (NFB) and activator protein-1 (AP-1) binding sites, expression of MMP-9 mRNA can be up-regulated by stimuli such as interleukin (IL)-1 and tumor necrosis factor-␣ (1). In addition, MMP-9 expression can be up-regulated by phorbol 12-O-tetradecanoate 13-acetate through phospholipase D (PLD) (2, 3).Mitogen-activated protein kinases (MAPKs) are crucial enzymes in the receptor-mediated signaling cascade. There are three major groups of MAPKs, extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK) 1/2. PD98059 and SB203580, which are specific inhibitors of MAPK kinase (MEK) 1/2 and p38, respectively, repress MMP-9 expression and in vitro tumor cell invasion (4, 5). Moreover, the JNK inhibitor SP600125 has been shown to attenuate phorbol 12-O-tetradecanoate 13-acetate-induced MMP-9 expression (6).The extracellular pH (pHe) of solid tumors is acidic due to the presence of anaerobic glucose metabolites such as lactate. For example, the basal pHe of xenograft...

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.

Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34 ؉ differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cellto-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34 ؉ cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 ␤ or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. STEM CELLS

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Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblastlike cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [؊50% in silenced, scrambled (SiScr) controls vs. ؊15% for SiRac1], the number of F-actin fibers (؊60% in SiScr controls vs. ؊10% for SiRac1), and the depletion of matrix-bound VEGF (؊40% in SiScr controls vs. ؊8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects.-Guignandon, A., Faure, C., Neutelings, T., Rattner, A., Mineur, P., Linossier, M.-T., Laroche, N., Lambert, C., Deroanne, C., Nusgens, B., Demets, R., Colige, A., Vico, L. Rac1 GTPase silencing counteracts microgravityinduced effects on osteoblastic cells. FASEB J. 28, 4077-4087 (2014). www.fasebj.org