The effects of the deep-sea environment on transmembrane signaling

Membrane-associated processes may be particularly susceptible to perturbation by the high hydrostatic pressures and low temperatures of the deep ocean. Transmembrane signaling by guanyl nucleotide binding protein (G protein) coupled receptors (GPCRs) is affected at a number of steps: (1) agonist activation of the GPCR; (2) the interaction of the receptor with the heterotrimeric G protein; (3) the G protein GTPase cycle; and (4) the activation and function of the effector element, adenylyl cyclase. The effects of low temperature and high hydrostatic pressures on the A₁ adenosine receptor–inhibitory G protein (G_i)–adenylyl cyclase signaling complex were examined in teleost fishes from three families, Scorpaenidae, Macrouridae and Moridae. In a comparison of teleost fishes, rat and chicken, species with body temperatures from 1 to 40 °C, at atmospheric pressure, A₁ adenosine receptor agonist binding is conserved at the body temperature of the species. In the marine teleost fishes examined, increased pressure decreases agonist efficacy. There are differences among species in the effects of increased hydrostatic pressure on G protein interactions with receptors, GTP binding to G protein α subunits and the intrinsic GTPase activity of α subunits. Adenylyl cyclase activity and modulation are affected by increased pressure in all the species examined, except Antimora rostrata which was unaffected by pressure changes. At pressures approximating those which the species experience in situ adenylyl cyclase activity retains its sensitivity to modulators. To understand the physiological consequences of impaired cell signaling several prototypical human diseases are discussed.     

Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes

The double-ring chaperonin GroEL and its lid-like cochaperonin GroES form asymmetric complexes that, in the ATP-bound state, mediate productive folding in a hydrophilic, GroES-encapsulated chamber, the so-called cis cavity. Upon ATP hydrolysis within the cis ring, the asymmetric complex becomes able to accept non-native polypeptides and ATP in the open, trans ring. Here we have examined the structural basis for this allosteric switch in activity by cryo-EM and single-particle image processing. ATP hydrolysis does not change the conformation of the cis ring, but its effects are transmitted through an inter-ring contact and cause domain rotations in the mobile trans ring. These rigid-body movements in the trans ring lead to disruption of its intra-ring contacts, expansion of the entire ring and opening of both the nucleotide pocket and the substrate-binding domains, admitting ATP and new substrate protein.     

Extracellular ATP promotes breast cancer chemoresistance via HIF-1α signaling

We have previously demonstrated that extracellular adenosine 5''-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.Subject terms: Breast cancer, Cell signalling     

The effects of membrane compartmentalization of csk on TCR signaling

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.     

ATP and cancer immunosurveillance

While intracellular adenosine triphosphate (ATP) occupies a key position in the bioenergetic metabolism of all the cellular compartments that form the tumor microenvironment (TME), extracellular ATP operates as a potent signal transducer. The net effects of purinergic signaling on the biology of the TME depend not only on the specific receptors and cell types involved, but also on the activation status of cis‐ and trans‐regulatory circuitries. As an additional layer of complexity, extracellular ATP is rapidly catabolized by ectonucleotidases, culminating in the accumulation of metabolites that mediate distinct biological effects. Here, we discuss the molecular and cellular mechanisms through which ATP and its degradation products influence cancer immunosurveillance, with a focus on therapeutically targetable circuitries.     

Wnt signaling in skin organogenesis

While serving as the interface between an organism and its environment, the skin also can elaborate a wide range of skin appendages to service specific purposes in a region-specific fashion. As in other organs, Wnt signaling plays a key role in regulating the proliferation, differentiation and motility of skin cells during their morphogenesis. Here I will review some of the recent work that has been done on skin organogenesis. I will cover dermis formation, the development of skin appendages, cycling of appendages in the adult, stem cell regulation, patterning, orientation, regional specificity and modulation by sex hormone nuclear receptors. I will also cover their roles in wound healing, hair regeneration and skin related diseases. It appears that Wnt signaling plays essential but distinct roles in different hierarchical levels of morphogenesis and organogenesis. Many of these areas have not yet been fully explored but are certainly promising areas of future research.Key words: morphogenesis, hair, feathers, tracts, epithelium-mesenchyme interactions, Wnt signaling pathwayThe integument forms the interface between an organism and its environment.^1,2 As such it protects against dehydration, infection, temperature extremes, etc while providing a means for display, camouflage and other functions.³ The skin can elaborate remarkable structural diversity producing specialized functions in a region-specific fashion to provide organisms with a selective advantage. For example, the development of feathers led to the acquisition of flight in birds and the formation of mammary glands enabled mammals to nurse their young.⁴ The advantage of these evolutionary developments can be seen by the number of birds and mammals present today.Skin appendages, such as skin, hairs, feathers, scales, glands and teeth grow from the epithelium as a result of epithelial-mesenchymal interactions,⁵ largely in response to common molecular signals with slight variations in their placement and timing during tissue morphogenesis.⁶ Theoretically, stem cells are totipotent and progressively can be guided toward their specific fates by exposure to specific regulatory signals. The juxtaposition of molecular signals or lack thereof may have a tremendous impact on cell fate decisions. Hence, the difference between skin appendages is due to the topological arrangement of the epithelia during developmental processes. These are presumably regulated by adhesion molecules whose expression is controlled by signaling molecules as well as by physical constraints.Hairs and feathers are attractive model systems for experimental research because of their ability for seasonal or periodic renewal. Obviously not all hairs or feathers are replaced at one time or birds would lose all of their feathers at once and fall from the sky in mid-flight; rather hairs and feathers are replaced over a period of time in a wave-like pattern.⁷ Yet this cycling behavior enables thousands of entirely new organs to be regenerated again and again throughout these animal''s lives. Hairs and feathers demonstrate an incredible diversity of forms arising in different locations over the body surface. For instance, hairs on the scalp, face and body differ in size, coarseness, color, etc. This regional specificity indicates that in each cycle skin stem cells are directed to form distinct structures through a series of molecular and cellular interactions.     

Hedgehog signaling in skin cancers

An increasing progress on the role of Hedgehog (Hh) signaling for carcinogenesis has been achieved since the link of Hh pathway to human cancer was firstly established. In particular, the critical role of Hh signaling in the development of Basal cell carcinoma (BCC) has been convincingly demonstrated by genetic mutation analyses, mouse models of BCCs, and successful clinical trials of BCCs using Hh signaling inhibitors. In addition, the Hh pathway activity is also reported to be involved in the pathogenesis of Squamous Cell Carcinoma (SCC), melanoma and Merkel Cell Carcinoma. These findings have significant new paradigm on Hh signaling transduction, its mechanisms in skin cancer and even therapeutic approaches for BCC. In this review, we will summarize the major advances in the understanding of Hh signaling transduction, the roles of Hh signaling in skin cancer development, and the current implications of “mechanism-based” therapeutic strategies.     

Wnt signaling in skin organogenesis

While serving as the interface between an organism and its environment, the skin also can elaborate a wide range of skin appendages to service specific purposes in a region-specific fashion. As in other organs, Wnt signaling plays a key role in regulating the proliferation, differentiation and motility of skin cells during their morphogenesis. Here I will review some of the recent work that has been done on skin organogenesis. I will cover dermis formation, the development of skin appendages, cycling of appendages in the adult, stem cell regulation, patterning, orientation, regional specificity, and modulation by sex hormone nuclear receptors. I will also cover their roles in wound healing, hair regeneration and skin related diseases. It appears that Wnt signaling plays essential but distinct roles in different hierarchical levels of morphogenesis and organogenesis. Many of these areas have not yet been fully explored but are certainly promising areas of future research.     

How to win ATP and influence Ca signaling

This brief review discusses recent advances in studies of mitochondrial Ca²⁺ signaling and considers how the relationships between mitochondria and Ca²⁺ responses are shaped in secretory epithelial cells. Perhaps the more precise title of this review could have been “How to win ATP and influence Ca²⁺ signaling in secretory epithelium with emphasis on exocrine secretory cells and specific focus on pancreatic acinar cells”. But “brevity is a virtue” and the authors hope that many of the mechanisms discussed are general and applicable to other tissues and cell types. Among these mechanisms are mitochondrial regulation of Ca²⁺ entry and the role of mitochondria in the formation of localized Ca²⁺ responses. The roles of Ca²⁺ signaling in the physiological adjustment of bioenergetics and in mitochondrial damage are also briefly discussed.     

Extracellular ATP signaling and homeostasis in plant cells

Extracellular ATP (eATP) is now recognized as an important signaling agent in plant growth and defense response to environmental stimuli. eATP has dual functions in plant cell signaling, which is largely dependent on its concentration in the extracellular matrix (ECM). A lethal level of eATP (extremely low or high) causes cell death, whereas a moderate level of eATP benefits plant growth and development. Ecto-apyrases (Nucleoside Triphosphate-Diphosphohydrolase) help control the eATP concentrations in the ECM, and thus contributing to the mediation of plant growth and defense response upon environmental stress. In this review, we summarize eATP signaling in plants and highlight the correlation between eATP homeostasis control and programmed cell death.     

Kynurenic acid inhibits colon cancer proliferation in vitro: effects on signaling pathways

Kynurenic acid (KYNA), a tryptophan metabolite, inhibits proliferation of several cancer cell lines including colon cancer, renal cancer and glioblastoma cells. Previous studies reported that inhibitory properties of KYNA may be related to interactions of KYNA with cell cycle regulators and signaling proteins. However, the exact molecular interaction of KYNA with signaling pathways in colon cancer cells has not been studied to date. The molecular mechanism of KYNA activity towards colon cancer cells may be of great importance taking into consideration that KYNA is present in several tissues and physiological fluids, including gastrointestinal tract, and it is also present in various products of human diet. In this study, the inhibitory effect of KYNA on activation of phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways in colon adenocarcinoma HT-29 cells was revealed. KYNA decreased phosphorylation of Akt, ERK 1/2 and p38 kinases in HT-29 cells. Interestingly, the study revealed also unexpected effect of KYNA on Wnt pathway in HT-29 cells. KYNA in millimolar concentrations increased protein expression of β-catenin. However, the nuclear translocation of β-catenin in HT-29 cells exposed to KYNA was not observed. Moreover, KYNA 1 mM increased antiproliferative properties of inhibitors of signaling pathways: wortmannin, PD98059, SB202190 and IWR-1. Taking into consideration these results, KYNA may be seen as a potential chemopreventive agent in colon cancer or supportive agent in standard cancer chemotherapy. However, the interactions between KYNA, Wnt signaling pathway and β-catenin need further studies to exclude potential effect of KYNA on colon carcinogenesis.     

The effects of smoking on experimental skin flaps in hamsters

To study the effects of the inhalation of cigarette smoke on the survival of skin flaps, 30 Syrian Golden hamsters were divided into three groups of 10. Two of these groups were acclimatized to cigarette smoke in increasing increments for 9 weeks in standard Hamburg I smoking cages. The third group of 10 (group A) served as controls and were sham-smoked throughout the experiment. After acclimatization, one group of 10 (group B) was smoked for a further 6 weeks. A standard axial-pattern flap was then raised on the dorsum of the animals. Ten animals in group C were smoked for 6 weeks preoperatively and for 2 weeks postoperatively, at which time the animals in all groups were sacrificed. All animals survived the experiment. The flaps in control group A all survived without necrosis. Two of the 10 dorsal flaps sustained terminal necrosis in group B animals. Six of the 10 flaps resulted in significant terminal necrosis in group C animals. Statistical analysis of the results indicated a significant comparison between control group A and group C of those animals smoked throughout the experiment. We conclude from this experiment that the inhalation of cigarette smoke consistent with that of a heavy smoker (2 packs per day) has an adverse effect on wound healing of skin flaps in hamsters. Apparently, cessation of smoking even at the time of surgical preparation of the flap obviates much of the noxious effect and increase flap survival significantly.     

The direct physiological effects of mitoK(ATP) opening on heart mitochondria

The mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoK(ATP) opening that should be observable at the level of the mitochondrion. MitoK(ATP) opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoK(ATP) opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoK(ATP) openers and blockers with those of valinomycin, we show that four independent assays of mitoK(ATP) activity yield quantitatively identical results for mitoK(ATP)-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoK(ATP) opening for mitochondria.     

The effects of clozapine on the GSK-3-mediated signaling pathway

We investigated the effect of 10 μM clozapine on the activity of glycogen synthase kinase-3β (GSK-3β) and its upstream and downstream molecules in SH-SY5Y human neuroblastoma cells. Clozapine activates both Akt- and Dvl-mediated phosphorylation of GSK-3β through phosphorylation at Ser9, and increased total cellular and intranuclear levels of β-catenin. Pretreatment with the specific inhibitor of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, LY294002 (20 μM), prevented the phosphorylation of Akt but did not affect the phosphorylation of GSK-3β. These results suggest that clozapine regulates the phosphorylation of GSK-3β through Wnt signal pathways involving Dvl upstream but not through the PI3K-Akt pathway in SH-SY5Y cells.