Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.     

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

Although prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. We previously established Drosophila oogenesis, in particular nurse cell dumping, as a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. Here we provide the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion.     

Paxillin: A cytoskeletal target for tyrosine kinases

Paxillin is a recently identified member of the complex of cytoskeletal proteins that is found concentrated in cultured cells and in vivo at the cytoplasmic face of regions of cell attachment to the extracellular matrix. These sites, in view of their close proximity to the extracellular matrix, are well positioned to act as signal-transducing centers to ‘report on’ changes in the cells, immediate environment. Recent findings indicate that such signals are in part mediated through the activation of tyrosine kinases concentrated at the sites of adhesion. Changes in the phosphotyrosine content of paxillin accompanying this elevation in kinase activity suggest that paxillin may be an important intermediary in these pathways.     

Identification of cellular target proteins for signaling cyclic phosphates

Cyclic glycerophosphates and their deoxy analogs were previously found to induce intracellular tyrosine and threonine phosphorylation in Chinese hamster ovary (CHO) cells. Further studies have indicated that these compounds induce neuronal outgrowth in PC-12 cells, as well as elevation of the state of cellular differentiation in human breast cancer cell lines. The mechanism by which these cyclic phosphates operate is not yet fully delineated. Using an affinity labeling approach we probed for possible cyclic phosphate target proteins in CHO cells. A 170 kDa protein that was labeled by an affinity cyclic phosphate reagent was identified by mass spectrometry as the largest subunit of the eukaryotic initiation factor 3 (eIF3). Using In-Gel kinase assays allowed the detection of a approximately 70 kDa target kinase directly activated by cyclic phosphates. Identification of these proteins may provide a basis for deciphering the mechanisms, by which cyclic phosphates exert their effects.     

Metallothionein is a crucial protective factor against Helicobacter pylori-induced gastric erosive lesions in a mouse model

Infection with the gastric pathogen Helicobacter pylori (H. pylori) causes chronic gastritis, peptic ulcer, and gastric adenocarcinoma. These diseases are associated with production of reactive oxygen species (ROS) from infiltrated macrophages and neutrophiles in inflammatory sites. Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that can act not only as a metal-binding protein, but also as a ROS scavenger. In the present study, we examined the role of MT in the protection against H. pylori-induced gastric injury using MT-null mice. Female MT-null and wild-type mice were challenged with H. pylori SS1 strain, and then histological changes were evaluated with the updated Sydney grading system at 17 and 21 wk after challenge. Although the colonization efficiency of H. pylori was essentially the same for MT-null and wild-type mice, the scores of activity of inflammatory cells were significantly higher in MT-null mice than in wild-type mice at 17 wk after challenge. Histopathological examination revealed erosive lesions accompanied by infiltration of inflammatory cells in the infected MT-null mice but not in wild-type mice. Furthermore, activation of NF-kappaB and expression of NF-kappaB-mediated chemokines such as macrophage inflammatory protein-1alpha and monocytes chemoattractant protein-1 in gastric cells were markedly higher in MT-null mice than in wild-type mice. These results suggest that MT in the gastric mucosa might play an important role in the protection against H. pylori-induced gastric ulceration.     

beta-Catenin-mediated signaling: a novel molecular target for chemoprevention with anti-inflammatory substances

Inflammation is thought to play a role in the pathophysiology of cancer. Accumulating evidence from clinical and laboratory-based studies suggests that substances with anti-inflammatory activities are potential candidates for chemoprevention. Recent advances in cellular and molecular biology of cancer shed light on components of intracellular signaling cascades that can be potential molecular targets of chemoprevention with various anti-inflammatory substances. Although cyclooxygenase-2, a primary enzyme that mediates inflammatory responses, has been well recognized as a molecular target for chemoprevention by both synthetic and natural anti-inflammatory agents, the cellular signaling mechanisms that associate inflammation and cancer are not still clearly illustrated. Recent studies suggest that beta-catenin-mediated signaling, which regulates developmental processes, may act as a potential link between inflammation and cancer. This review aims to focus on beta-catenin-mediated signaling pathways, particularly in relation to its contribution to carcinogenesis, and the modulation of inappropriately activated beta-catenin-mediated signaling by nonsteroidal anti-inflammatory drugs and chemopreventive phytochemicals possessing anti-inflammatory properties.     

Topoisomerase II is a cellular target for antiproliferative cobalt salicylaldoxime complex.

Topoisomerase II is a cellular target for a number of clinically relevant antitumor drugs. To elucidate the possible cellular target for the antiproliferation activity of cobalt salicylaldoxime (CoSAL), which inhibits 50% of leukemic cell proliferation at a concentration of 60 microM, DNA binding studies and studies of the action of this complex on topoisomerase II catalytic activities were carried out. The results from DNA binding studies show that CoSAL binds DNA strongly with a stoichiometric ratio of two drug molecules for five nucleotide bases and shows a mode of interaction similar to that of DNA groove binding agents. The results from topoisomerase II inhibition studies show that the complex inhibits the relaxation activity of topoisomerase II in a dose-dependent manner and poisons its activity through cleavage complex formation. To see if the hydroxyl group present on imine nitrogen is involved in topoisomerase II poisoning, we synthesized an analogue of CoSAL in which the hydroxyl group was replaced with semicarbazone. This complex too binds DNA with an affinity similar to that of CoSAL, but with a small difference in the mode of interaction; however, it marginally inhibits leukemic cell proliferation and does not inhibit topoisomerase II activity, which suggests the involvement of a hydroxyl group. An immunoprecipitation assay was conducted which showed that the cleavage complex formed in the presence of CoSAL contained 75% of the complex, while the other complex shows only 7. 65%. Cyclic voltametric spectra of the complexes in the presence of DNA show that they do not oxidize DNA. These results suggest that CoSAL shows a bidirectional mode of interaction with enzyme and DNA and inhibits topoisomerase II activity by forming a drug-mediated cleavage complex. Our data strongly suggest that topoisomerase II may be one of the cellular targets for antiproliferation activity of CoSAL.     

Autonomous rexinoid death signaling is suppressed by converging signaling pathways in immature leukemia cells.

On their own, retinoid X receptor (RXR)-selective ligands (rexinoids) are silent in retinoic acid receptor (RAR)-RXR heterodimers, and no selective rexinoid program has been described as yet in cellular systems. We report here on the rexinoid signaling capacity that triggers apoptosis of immature promyelocytic NB4 cells as a default pathway in the absence of survival factors. Rexinoid-induced apoptosis displays all features of bona fide programmed cell death and is inhibited by RXR, but not RAR antagonists. Several types of survival signals block rexinoid-induced apoptosis. RARalpha agonists switch the cellular response toward differentiation and induce the expression of antiapoptosis factors. Activation of the protein kinase A pathway in the presence of rexinoid agonists induces maturation and blocks immature cell apoptosis. Addition of nonretinoid serum factors also blocks cell death but does not induce cell differentiation. Rexinoid-induced apoptosis is linked to neither the presence nor stability of the promyelocytic leukemia-RARalpha fusion protein and operates also in non-acute promyelocytic leukemia cells. Together our results support a model according to which rexinoids activate in certain leukemia cells a default death pathway onto which several other signaling paradigms converge. This pathway is entirely distinct from that triggered by RAR agonists, which control cell maturation and postmaturation apoptosis.     

Paxillin is the target of c-Jun N-terminal kinase in Schwann cells and regulates migration

During development of the peripheral nervous system (PNS), Schwann cells migrate along axons, wrapping individual axons to form a myelin sheath. This process is all mediated by the intercellular signaling between neurons and Schwann cells. As yet, little is known about the intracellular signaling mechanisms controlling these morphological changes including Schwann cell migration. We previously showed that c-Jun N-terminal kinase (JNK) plays a key role in Schwann cell migration before the initiation of myelination. Here we show that JNK, acting through phosphorylation of the cytoskeletal protein paxillin, regulates Schwann cell migration and that it mediates dorsal root ganglion (DRG) neuronal conditioned medium (CM). Phosphorylation of paxillin at the Ser-178 position, the JNK phosphorylation site, is observed following stimulation with neuronal CM. Phosphorylation is also detected as a result of stimulation with each of growth factors contained in neuronal CM. Knockdown of paxillin with the specific small interfering RNA (siRNA) inhibits migration. The reintroduction of paxillin reverses siRNA-mediated inhibition of migration, whereas paxillin harboring the Ser-178-to-Ala mutation fails to reverse it. In addition, while JNK binds to the N-terminal region (called LD1), the deletion of LD1 blocks migration. Together, JNK binds and phosphorylates paxillin to regulate Schwann cell migration, illustrating that paxillin provides one of the convergent points of intracellular signaling pathways controlling Schwann cell migration.     

Oxidation resistance 1 is a novel senolytic target

The selective depletion of senescent cells (SCs) by small molecules, termed senolytic agents, is a promising therapeutic approach for treating age‐related diseases and chemotherapy‐ and radiotherapy‐induced side effects. Piperlongumine (PL) was recently identified as a novel senolytic agent. However, its mechanism of action and molecular targets in SCs was unknown and thus was investigated. Specifically, we used a PL‐based chemical probe to pull‐down PL‐binding proteins from live cells and then mass spectrometry‐based proteomic analysis to identify potential molecular targets of PL in SCs. One prominent target was oxidation resistance 1 (OXR1), an important antioxidant protein that regulates the expression of a variety of antioxidant enzymes. We found that OXR1 was upregulated in senescent human WI38 fibroblasts. PL bound to OXR1 directly and induced its degradation through the ubiquitin‐proteasome system in an SC‐specific manner. The knockdown of OXR1 expression by RNA interference significantly increased the production of reactive oxygen species in SCs in conjunction with the downregulation of antioxidant enzymes such as heme oxygenase 1, glutathione peroxidase 2, and catalase, but these effects were much less significant when OXR1 was knocked down in non‐SCs. More importantly, knocking down OXR1 selectively induced apoptosis in SCs and sensitized the cells to oxidative stress caused by hydrogen peroxide. These findings provide new insights into the mechanism by which SCs are highly resistant to oxidative stress and suggest that OXR1 is a novel senolytic target that can be further exploited for the development of new senolytic agents.     

PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex

Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1.     

Nodal/Activin signaling: a novel target for pancreatic cancer stem cell therapy

Targeting of cancer stem cells (CSCs) has the potential to address the recalcitrance of pancreatic cancer to chemotherapy. In this issue of Cell Stem Cell, Lonardo et al. (2011) demonstrate that Nodal/Activin signaling is crucial for the maintenance and tumor-initiating capacity of pancreatic CSCs.     

Leptin receptor signaling is required for vaccine-induced protection against Helicobacter pylori

Background:  A vaccine against Helicobacter pylori would be a desirable alternative to antibiotic therapy. Vaccination has been shown to be effective in animal models but the mechanism of protection is poorly understood. Previous studies investigating the gene expression in stomachs of vaccinated mice showed changes in adipokine expression correlated to a protective response. In this study, we investigate a well-characterized adipokine-leptin, and reveal an important role for leptin receptor signaling in vaccine-induced protection.
Materials and Methods:  Leptin receptor signaling-deficient (C57BL/Ks Lepr^db), wild-type C57BL/Ks m littermates and C57BL/6 mice were vaccinated, and then challenged with H. pylori . Levels of bacterial colonization, antibody levels, and gastric infiltrates were compared. The local gene expression pattern in the stomach of leptin receptor signaling-deficient and wild-type mice was also compared using microarrays.
Results:  Interestingly, while vaccinated wild-type lean C57BL/6 and C57BL/Ks m mice were able to significantly reduce colonization compared to controls, vaccinated obese C57BL/Ks Lepr^db were not. All mice responded to vaccination, i.e. developed infiltrates predominantly of T lymphocytes in the gastric mucosa, and made H. pylori -specific antibodies. A comparison of expression profiles in protected C57BL/6 and nonprotected C57BL/Ks Lepr^db mice revealed a subset of inflammation-related genes that were more strongly expressed in nonprotected mice.
Conclusions:  Our data suggest that functional leptin receptor signaling is required for mediating an effective protective response against H. pylori .