Metastasis is the primary cause of cancer death. The inflammatory tumor microenvironment contributes to metastasis, for instance, by recruiting blood and lymph vessels. Among tumor-infiltrating immune cells, tumor-associated macrophages (TAMs) take a center stage in promoting both tumor angiogenesis and metastatic spread. We found that genetic deletion of the S1P receptor 1 (S1pr1) alone in CD11bhi CD206+ TAMs infiltrating mouse breast tumors prevents pulmonary metastasis and tumor lymphangiogenesis. Reduced lymphangiogenesis was also observed in the nonrelated methylcholanthrene-induced fibrosarcoma model. Transcriptome analysis of isolated TAMs from both entities revealed reduced expression of the inflammasome component Nlrp3 in S1PR1-deficient TAMs. Macrophage-dependent lymphangiogenesis in vitro was triggered upon inflammasome activation and required both S1PR1 signaling and IL-1β production. Finally, NLRP3 expression in tumor-infiltrating macrophages correlated with survival, lymph node invasion, and metastasis of mammary carcinoma patients. Conceptually, our study indicates an unappreciated role of the NLRP3 inflammasome in promoting metastasis via the lymphatics downstream of S1PR1 signaling in macrophages.
The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μm, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 μm RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.
Ferritins are iron storage proteins, which maintain cellular iron homeostasis. Among these proteins, the ferritin heavy chain is well characterized, but the regulatory principles of mitochondrial ferritin (FTMT) remain elusive. FTMT appears to be cleaved from a 27 kDa to a 22 kDa form. In human macrophages, FTMT increased under hypoxia in a hypoxia-inducible factor 2-dependent manner. Occurrence of FTMT resulted from cleavage by thrombin, which was supplied by serum. Inhibition of thrombin as well as serum removal decreased FTMT, while supplementation of thrombin under serum-deprived conditions restored its expression. Besides hypoxia, thrombin facilitated FTMT expression after treatment with the ferroptosis inducer RSL3 and the pro-inflammatory stimulus lipopolysaccharide. This study provides insights into the regulation of FTMT under hypoxia and identifies thrombin as a FTMT maturation-associated peptidase.
Sodium nitroprusside is a vasodilator and an inhibitor of platelet activation. It is thought that these effects are mediated by the spontaneous release of nitric oxide and stimulation of cytosolic guanylate cyclase. We have found that sodium nitroprusside (5-200 μM) greatly increased a cytosolic ADP-ribosyltransferase that ADP-ribosylates a soluble 39-kDa protein. This activity causes the mono-ADP-ribosylation of the 39-kDa protein, since digestion with snake venom phosphodiesterase releases 5′-AMP. This enzyme is present in platelets, brain, heart, intestine, liver, and lung. The effect of sodium nitroprusside is not related to stimulation of soluble guanylate cyclase and the production of cyclic GMP because cyclic GMP, dibutyryl cyclic GMP, and 8-bromo-cyclic GMP are ineffective. 3-Morpholinosydnonimine (commonly known as SIN-1) (20-1000 μg/ml), another compound that acts through the spontaneous formation of nitric oxide as does sodium nitroprusside, also stimulates ADP-ribosylation of the 39-kDa protein. Hemoglobin, which binds nitric oxide, inhibits sodium nitroprusside's activation of the cytosolic ADP-ribosyltransferase. These studies demonstrate a novel action of nitric oxide related to the activation of an endogenous ADP-ribosyltransferase. The physiological role of this ADP-ribosylation needs further exploration.
Using conditions that produced chronic inflammation in rat liver, we were able to find a correlation between induction of nitric oxide production and inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). This enzyme is a tetramer composed of identical M(r) 37,000 subunits. The tetramer contains 16 thiol groups, four of which are essential for enzymatic activity. Our information indicates that four thiol groups are S-nitrosylated by exposure to authentic nitric oxide (NO) gas. Furthermore, NO decreased GAPDH activity while increasing its auto-ADP-ribosylation. Reduced nicotinamide adenine dinucleotide and dithiothreitol are required for the S-nitrosylation of GAPDH caused by the NO-generating compound sodium nitroprusside. Our results suggests that a new and important action of nitric oxide on cells is the S-nitrosylation and inactivation of GAPDH. S-Nitrosylation of GAPDH may be a key covalent modification of multiple regulatory consequences in chronic liver inflammation.
