miRNA let-7e targeting MMP9 is involved in adipose-derived stem cell differentiation toward epithelia.

miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.

The role of TRKA signaling in IL-10 production by apoptotic tumor cell-activated macrophages.

Tumor-associated macrophages (TAMs) are a major supportive component within neoplasms. Mechanisms of macrophage (MΦ) attraction and differentiation to a tumor-promoting phenotype, which is characterized by pronounced interleukin (IL)-10 production, are under investigation. We report that supernatants of dying cancer cells induced substantial IL-10 release from primary human MΦs, dependent on signaling through tyrosine kinase receptor A (TRKA or neurotrophic tyrosine kinase receptor type 1 (NTRK1)). Mechanistically, sphingosine-1-phosphate (S1P) release from apoptotic cancer cells triggered src-dependent shuttling of cytosolic TRKA to the plasma membrane via S1P receptor signaling. Plasma membrane-associated TRKA, which was activated by constitutively autocrine secreted nerve growth factor, used phosphatidylinositol 3-kinase (PI3K)/AKT and p38 mitogen-activated protein kinase (MAPK) signaling to induce IL-10. Interestingly, TRKA-dependent signaling was required for cytokine production by TAMs isolated from primary murine breast cancer tissue. Besides IL-10, this pathway initiated secretion of IL-6, tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein-1 (MCP-1), indicating relevance in cancer-associated inflammation. Our findings highlight a fine-tuned regulatory system including S1P-dependent TRKA trafficking for executing TAM-like cell function in vitro as well as in vivo.

The interaction of superoxide with nitric oxide destabilizes hypoxia-inducible factor-1alpha.

In renal carcinoma cells (RCC4) hypoxia inducible factor-1 (HIF-1) is constitutively expressed due to a von Hippel Lindau protein deficiency, but can be degraded by calpain, independently of the 26S proteasome, when exposed to hypoxia/nitric oxide (NO). In this study we examined molecular mechanisms to explain calpain activation. The inability of hypoxia/NO to degrade HIF-1alpha in respiratory-deficient RCC4-rho0 cells pointed to the requirement for mitochondria-derived reactive oxygen species. A prerequisite for O(2)(-) in combination with NO to destabilize HIF-1alpha was corroborated in RCC4-rho0 cells, when the redox cycler 2,3-dimethoxy-1,4-naphthoquinone was used as a source of superoxide. Degradation of HIF-1alpha required intracellular calcium transients and calpain activation. Using uric acid to interfere with signal transmission elicited by NO/O(2)(-) blocked HIF-1alpha degradation and attenuated a calcium increase. We conclude that an oxidative signal as a result of NO/O(2)(-) coformation triggers a calcium increase that activates calpain to degrade HIF-1alpha, independently of the proteasome.

Recognition of apoptotic cells by macrophages activates the peroxisome proliferator-activated receptor-gamma and attenuates the oxidative burst.

It is appreciated that phagocytosis of apoptotic cells (AC) is an immunological relevant process that shapes the pro- versus anti-inflammatory macrophage phenotype. It was our intention to study the respiratory burst, a prototype marker of macrophage activation, under the impact of AC. Following incubation of RAW264.7 macrophages with AC, we noticed attenuated production of reactive oxygen species (ROS) in response to PMA treatment, and observed a correlation between attenuated ROS formation and suppression of protein kinase Calpha (PKCalpha) activation. EMSA analysis demonstrated an immediate activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) following supplementation of AC to macrophages. In macrophages carrying a dominant-negative PPARgamma mutant, recognition of AC no longer suppressed PKCalpha activation, and the initial phase of ROS formation was largely restored. Interference with actin polymerization and transwell experiments suggest that recognition of AC by macrophages suffices to attenuate the early phase of ROS formation that is attributed to PPARgamma activation.

Activation of PPARgamma is not involved in butyrate-induced epithelial cell differentiation.

Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor gamma (PPARgamma) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPARgamma in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPARgamma in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPARgamma ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPARgamma ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPARgamma mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPARgamma is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation.

Nitric oxide-induced F-actin disassembly is mediated via cGMP, cAMP, and protein kinase A activation in rat mesangial cells.

Glomerular mesangial cells contain actin and myosin, and in analogy to vascular smooth muscle cells, they can contract and relax to regulate the glomerular filtration rate. A key molecule that determines hemodynamic properties is nitric oxide, which is produced by nitric oxide synthase isoenzymes located in individual cells of the kidney. The contractility of mesangial cells is based on the interaction of actin microfilament bundles (F-actin) with myosin. We had the notion that nitric oxide influences the shape change of mesangial cells, so we analyzed the signal transduction involved. Chemically unrelated nitric oxide donors induced F-actin dissolution, which was mediated by cGMP but was unrelated to protein kinase G activation. Actin disassembly was achieved with inhibitors of phosphodiesterase-3 and -4 or forskolin-evoked cAMP generation. We assumed that signal transmission involves activation of protein kinase A, and we went on to attenuate F-actin disassembly by protein kinase A inhibition. In conclusion, we found evidence that nitric oxide triggered F-actin dissolution via cGMP generation, inhibition of cAMP-hydrolyzing phosphodiesterase-3, and subsequent protein kinase A activation.

Regulation of the hypoxia-inducible factor 1alpha by the inflammatory mediators nitric oxide and tumor necrosis factor-alpha in contrast to desferroxamine and phenylarsine oxide.

Hypoxic/ischemic conditions provoke activation of the hypoxia-inducible factor-1 (HIF-1), which functions as a transcription factor. HIF-1 is composed of the HIF-1alpha and -beta subunits, and stability regulation occurs via accumulation/degradation of HIF-1alpha with the notion that a prolyl hydroxylase accounts for changes in protein level. In addition, there is evidence that HIF-1 is up-regulated by diverse agonists during normoxia. We investigated the impact of inflammatory mediators nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) on HIF-1alpha regulation. For comparison, LLC-PK(1) cells were exposed to hypoxia, stimulated with desferroxamine (DFX, known to mimic hypoxia), and the thiol-cross-linking agent phenylarsine oxide (PAO). Although all stimuli elicited HIF-1alpha stabilization with differences in the time-dependent accumulation pattern, significant variations appeared with regard to signaling. With the use of a superoxide anion (O(2-)) generator, we established an O(2-)-sensitive pathway that blocked HIF-1alpha stabilization in response to NO and TNF-alpha while DFX- and PAO-evoked HIF-1alpha stabilization appeared O(2-)-insensitive. NO and TNF-alpha signaling required phosphorylation events, especially activation of the phosphatidylinositol 3-kinase/Akt, which is in contrast to DFX and PAO. Based on HIF-1-dependent luciferase reporter gene analysis, it was found that, in contrast to NO and TNF-alpha, PAO resembled a stimulus that induced a dysfunctional HIF-1 complex. These data indicate that diverse agonists activate HIF-1alpha under normoxic conditions by employing different signaling pathways.

Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice.

Liver resident NK1.1+ T cells are supposed to play a pivotal role in the onset of inflammatory liver injury in experimental mouse models such as concanavalin A (Con A)-induced hepatitis. These cells, expressing the adhesion receptor, CD44, are largely depleted from the liver by a single intravenous injection of low-molecular-weight fragments of hyaluronic acid (LMW-HA). Here, we report that LMW-HA pretreatment protected mice from liver injury in several models of T-cell- and macrophage-dependent, tumor necrosis factor alpha (TNF-alpha)-mediated inflammatory liver injury, i.e., from liver injury induced by either Con A or Pseudomonas exotoxin A (PEA) or PEA/lipopolysaccharide (LPS). Interestingly, apart from inhibition of cellular adhesion, pretreatment of mice with LMW-HA was also capable of preventing hepatocellular apoptosis and activation of caspase-3 induced by direct administration of recombinant murine (rmu) TNF-alpha to D-galactosamine (GalN)-sensitized mice. LMW-HA-induced hepatoprotection could be neutralized by pretreatment with the nuclear factor-kappaB (NF-kappaB) inhibitor, pyrrolidine dithiocarbamate (PDTC), demonstrating the involvement of NF-kappaB in the observed protective mechanism. Indeed, injection of LMW-HA rapidly induced the production of TNF-alpha by Kupffer cells and the translocation of NF-kappaB into hepatocellular nuclei. Both LMW-HA-induced TNF-alpha production and NF-kappaB translocation were blocked by pretreatment with PDTC. Our findings provide evidence for an unknown mechanism of LMW-HA-dependent protection from inflammatory liver disease, i.e., induction of TNF-alpha- and NF-kappaB-dependent cytoprotective proteins within the target parenchymal liver cells.

Transcription factors p53 and HIF-1alpha as targets of nitric oxide.

It is widely recognized that the production of nitric oxide (NO) from L-arginine metabolism is an essential determinate of diverse signalling cascades throughout the body, with a major impact during nonspecific host defence. Biological actions of NO and derived species comprise physiological as well as pathological entities, with an impressive and steadily growing number of signalling pathways and/or protein targets being involved. It is now appreciated that NO not only acts as an effector molecule but also as an autocrine as well as paracrine modulator of rapid and delayed cellular responses. Among multiple targets the tumour suppressor p53 and the hypoxia inducible factor-1alpha (HIF-1alpha) emerged. Accumulation of p53 in response to NO delivery may account for an interference in cell cycle progression and/or initiation of apoptosis that is found in close correlation with inducible NO synthase (NOS) expression. Quite similarly, accumulation of HIF-1alpha not only occurs during hypoxia, but also under conditions of NO delivery, thus mimicking a situation of reduced oxygen availability. Interestingly, p53 and HIF-1alpha share regulatory elements that cause protein stabilization in part as a result of impaired ubiquitin-evoked protein degradation. Here, we summarize current knowledge on the impact of NO on p53- and HIF-1alpha-stabilization and we will discuss pathophysiological consequences. These examples may help to shape and refine current concepts of NO action with an emphasis on transcription factor regulation.

Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation.

The toxic reactive aldehyde lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) is thought to be a major contributor to oxidant stress-mediated cell injury. HNE induced apoptosis in RAW 264.7 murine macrophage cells in a dose-dependent manner within 6-8 h after exposure. Expression of the antiapoptotic protein Bcl-2 in stably transfected RAW 264.7 cells prevented HNE-induced internucleosomal DNA fragmentation and apoptosis, and these cells resume growth after a temporary (24-48 h) growth delay. While parental RAW 264.7 cells released mitochondrial cytochrome c within 3 h after HNE exposure, expression of Bcl-2 prevented cytochrome c release. In control cells, p53 protein levels peaked at 6-9 h after HNE exposure and then declined, while in Bcl-2 expressing cells, p53 levels were maximal at 6-9 h and remained elevated up to 96 h. Expression of SV40 large T-antigen, which forms a stable complex with p53 protein, via stable transfection-blocked transactivation of the p53-regulated gene p21(WAF1/CIP1), but did not affect induction of apoptosis by HNE, suggesting that p53 function is not important in HNE-induced apoptosis. These results suggest that cytochrome c release, but not p53 accumulation, plays an essential role in HNE-induced apoptosis in RAW 264.7 cells.

Accumulation of HIF-1alpha under the influence of nitric oxide.

The key player for adaptation to reduced oxygen availability is the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of the redox-sensitive HIF-1alpha and the constitutively expressed HIF-1beta subunits. Under normoxic conditions, HIF-1alpha is rapidly degraded, whereas hypoxia, CoCl(2), or desferroxamine promote protein stabilization, thus evoking its transcriptional activity. Because HIF-1 is regulated by reactive oxygen species, investigation of the impact of reactive nitrogen species was intended. By using different nitric oxide (NO) donors, dose- and time-dependent HIF-1alpha accumulation in close correlation with the release of NO from chemically distinct NO donors was established. Intriguingly, small NO concentrations induced a faster but transient HIF-1alpha accumulation than higher doses of the same NO donor. In contrast, NO attenuated up-regulation of HIF-1alpha evoked by CoCl(2) in a concentration- and time-dependent manner, whereas the desferroxamine-elicited HIF-1alpha signal remained unaltered. To demonstrate an autocrine or paracrine signaling function of NO, we overexpressed the inducible NO synthase and used a coculture system of activated macrophages and tubular cells. Expression of the NO synthase induced HIF-1alpha accumulation, which underscored the role of NO as an intracellular activator for HIF-1. In addition, macrophage-derived NO triggered HIF-1alpha up-regulation in LLC-PK(1) target cells, which points to intercellular signaling properties of NO in achieving HIF-1 accumulation. Our results show that NO does not only modulate the HIF-1 response under hypoxic conditions, but it also functions as a HIF-1 inducer. We conclude that accumulation of HIF-1 occurs during hypoxia but also under inflammatory conditions that are characterized by sustained NO formation.

Delayed activation of PPARgamma by LPS and IFN-gamma attenuates the oxidative burst in macrophages.

Desensitization of macrophages is important during the development of sepsis. It was our intention to identify mechanisms that promote macrophage deactivation upon contact with endotoxin (LPS) and interferon-gamma (IFN-gamma) in vitro. Macrophage activation was achieved with 12-O-tetradecanoylphorbol 13-acetate (TPA), and the oxidative burst (i.e., oxygen radical formation) was followed by oxidation of the redox-sensitive dyes hydroethidine and dichlorodihydrofluorescein diacetate. Prestimulation of macrophages for 15 h with a combination of LPS/IFN-gamma attenuated oxygen radical formation in response to TPA. Taking the anti-inflammatory properties of the peroxisome proliferator-activating receptorgamma (PPARgamma) into consideration, we established activation of PPARgamma in response to LPS/IFN-gamma by an electrophoretic mobility shift, supershift, and a reporter gene assay. The reporter contains a triple PPAR-responsive element (PPRE) in front of a thymidine kinase minimal promoter driving the luciferase gene. We demonstrated that PPRE decoy oligonucleotides, supplied in front of LPS/IFN-gamma, allowed a full oxidative burst to recover upon TPA addition. Furthermore, we suppressed the oxidative burst by using the PPARgamma agonists 15-deoxy-Delta12,14-prostaglandin J2, BRL 49653, or ciglitazone. No effect was observed with WY 14643, a PPARalpha agonist. We conclude that activation of PPARs, most likely PPARgamma, promotes macrophage desensitization, thus attenuating the oxidative burst. This process appears important during development of sepsis.

Protein thiol modification of glyceraldehyde-3-phosphate dehydrogenase and caspase-3 by nitric oxide.

The regulation of enzyme activity function is a major factor in the cellular response to a changing environment. One mechanism of enzyme activity regulation includes post-translational protein thiol modification by nitric oxide (NO) or its redox species. Major routs used by NO to modify cysteine residues of proteins include S-nitrosation, oxidation, mixed disulfide formation with glutathione, and the covalent attachment of nucleotide cofactors, i.e NAD(+)/NADH. Critical thiol centers serve as recognition sites for NO, thus channeling the NO signal through post-translational modifications and oxidation into cellular functions. Here, we summarize current knowledge on active site thiol modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and caspase-3 by nitric oxide. Although very different in their cellular function, both enzymes contain highly reactive cysteines which represent sensitive targets for NO. Our studies are supportive of a potential role of S-nitrosation and mixed disulfide formation as a general signaling mechanism that allows sensing of nitrosative stress. At the same time, modification of GAPDH and caspase-3 by NO show the diversity of mechanisms (S-nitrosation versus oxidations) that we are confronted with as a result of NO delivery, especially comparing in vitro studies with cellular systems. In the future it will be challenging to dissect how nitrosative and oxidative signaling mechanisms overlap and how intracellular communication systems allow their activation in a selective way.