Involvement of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and its inhibitors in HuR-mediated cytokine mRNA stabilization in lung epithelial cells

Increasing evidence demonstrates that Na⁺, K⁺-ATPase plays an important role in pulmonary inflammation, but the mechanism remains largely unknown. In this study, we used cardiotonic steroids as Na⁺, K⁺-ATPase inhibitors to explore the possible involvement of Na⁺, K⁺-ATPase in pulmonary epithelial inflammation. The results demonstrated that mice after ouabain inhalation developed cyclooxygenase-2-dependent acute lung inflammation. The in vitro experiments further confirmed that Na⁺, K⁺-ATPase inhibitors significantly stimulated cyclooxygenase-2 expression in lung epithelial cells of human or murine origin, the process of which was participated by multiple cis-elements and trans-acting factors. Most importantly, we first described here that Na⁺, K⁺-ATPase inhibitors could evoke a significant Hu antigen R nuclear export in lung epithelial cells, which stabilized cyclooxygenase-2 mRNA by binding with a proximal AU-rich element within its 3′-untranslated region. In conclusion, HuR-mediated mRNA stabilization opens new avenues in understanding the importance of Na⁺, K⁺-ATPase, as well as its inhibitors in inflammation.     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Structural variations in wheat HKT1;5 underpin differences in Na<Superscript>+</Superscript> transport capacity

An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na⁺) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na⁺-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (K _m) for the Na⁺ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D⁴⁷¹/a gap and D⁴⁷⁴/G⁴⁷³ that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na⁺ exclusion in wheat that leads to an improved salinity tolerance in the field.     

C-peptide stimulates Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na⁺, K⁺-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na⁺, K⁺-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37°C for 10 min stimulated ⁸⁶Rb⁺ uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive ⁸⁶Rb⁺ uptake, C-peptide increased subunit phosphorylation and basolateral membrane (BLM) abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits. The increase in BLM abundance of the Na⁺, K⁺-ATPase ₁ and ₁ subunits was accompanied by depletion of ₁ and ₁ subunits from the endosomal compartments. C-peptide action on Na⁺, K⁺-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na⁺, K⁺-ATPase activation, phosphorylation of ₁-subunit and translocation of ₁ and ₁ subunits to the BLM were abolished by a MEK1/2 inhibitor (20 M PD98059). C-peptide stimulation of ⁸⁶Rb⁺ uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 M GF109203X). C-peptide stimulated phosphorylation of human Na⁺, K⁺-ATPase subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the subunit of Na⁺, K⁺-ATPase.Received 15 June 2004; received after revision 14 September 2004; accepted 14 September 2004     

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na⁺, K⁺-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.     

Memory CD4<Superscript>+</Superscript> T Cells: fate determination,positive feedback and plasticity

Naïve CD4⁺ T cells undergo massive cell proliferation upon encountering their cognate ligand. This proliferation depends upon appropriate cues from the antigen-presenting cells that have processed the antigen and present the peptide to the T cells, and requires the establishment of a cytokine environment that can support such proliferation. Expansion of antigen-specific CD4⁺ T cells needs to be coupled with differentiation into one of several effector/regulatory phenotypes if the priming event is to result in cells that can initially act to control the particular pathogen that elicited the response, and later to serve as memory cells to insure an appropriate response upon reintroduction of the pathogen. Here, we discuss the initiation of T helper lineage commitment, the positive feedback regulation by the cytokine environment to enhance and stabilize the differentiation into distinct T helper subsets, and the biological significance of CD4⁺ T cell plasticity and long-term CD4⁺ T cell memory.     

Nicotinamide is an inhibitor of SIRT1 in vitro,but can be a stimulator in cells

Nicotinamide (NAM), a form of vitamin B₃, plays essential roles in cell physiology through facilitating NAD⁺ redox homeostasis and providing NAD⁺ as a substrate to a class of enzymes that catalyze non-redox reactions. These non-redox enzymes include the sirtuin family proteins which deacetylate target proteins while cleaving NAD⁺ to yield NAM. Since the finding that NAM exerts feedback inhibition to the sirtuin reactions, NAM has been widely used as an inhibitor in the studies where SIRT1, a key member of sirtuins, may have a role in certain cell physiology. However, once administered to cells, NAM is rapidly converted to NAD⁺ and, therefore, the cellular concentration of NAM decreases rapidly while that of NAD⁺ increases. The result would be an inhibition of SIRT1 for a limited duration, followed by an increase in the activity. This possibility raises a concern on the validity of the interpretation of the results in the studies that use NAM as a SIRT1 inhibitor. To understand better the effects of cellular administration of NAM, we reviewed published literature in which treatment with NAM was used to inhibit SIRT1 and found that the expected inhibitory effect of NAM was either unreliable or muted in many cases. In addition, studies demonstrated NAM administration stimulates SIRT1 activity and improves the functions of cells and organs. To determine if NAM administration can generate conditions in cells and tissues that are stimulatory to SIRT1, the changes in the cellular levels of NAM and NAD⁺ reported in the literature were examined and the factors that are involved in the availability of NAD⁺ to SIRT1 were evaluated. We conclude that NAM treatment can hypothetically be stimulatory to SIRT1.     

<Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> supports Th17 differentiation and limits de novo regulatory T cell induction by directly interfering with T cell receptor signaling

Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4⁺ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4⁺ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3⁺ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4⁺ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4⁺ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3⁺ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4⁺ T cell subsets by altering their TCR downstream signaling.     

RIPK4 activity in keratinocytes is controlled by the SCF<Superscript>β-TrCP</Superscript> ubiquitin ligase to maintain cortical actin organization

RIPK4 is a key player in epidermal differentiation and barrier formation. RIPK4 signaling pathways controlling keratinocyte proliferation and differentiation depend on its kinase activity leading to Dvl2, Pkp1 and IRF6 phosphorylation and NF-κB activation. However, the mechanism regulating RIPK4 activity levels remains elusive. We show that cultured keratinocytes display constitutive active phosphorylated RIPK4 while PKC signaling can trigger RIPK4 activation in various non-keratinocyte cell lines, in which RIPK4 is present in a non-phosphorylated state. Interestingly, we identified the SCF^β-TrCP ubiquitin E3 ligase complex responsible for regulating the active RIPK4 protein level. The SCF^β-TrCP complex binds to a conserved phosphodegron motif in the intermediate domain of RIPK4, subsequently leading to K48-linked ubiquitinylation and degradation. The recruitment of β-TrCP is dependent on RIPK4 activation and trans-autophosphorylation. β-TrCP knock-down resulted in RIPK4-dependent formation of actin stress fibers, cell scattering and increased cell motility, suggesting that tight control of RIPK4 activity levels is crucial to maintain cell shape and behavior in keratinocytes.     

CD8<Superscript>+</Superscript> Tregs in autoimmunity: learning “self”-control from experience

Autoreactive CD8⁺ regulatory T cells (Tregs) play important roles as modulators of immune responses against self, and numerical and functional defects in CD8⁺ Tregs have been linked to autoimmunity. Several subsets of CD8⁺ Tregs have been described. However, the origin of these T cells and how they participate in the natural progression of autoimmunity remain poorly defined. We discuss several lines of evidence suggesting that the autoimmune process itself promotes the development of autoregulatory CD8⁺ T cells. We posit that chronic autoantigenic exposure fosters the differentiation of non-pathogenic autoreactive CD8⁺ T cells into antigen-experienced, memory-like autoregulatory T cells, to generate a “negative feedback” regulatory loop capable of countering pathogenic autoreactive effectors. This hypothesis predicts that approaches capable of boosting autoregulatory T cell memory will be able to blunt autoimmunity without compromising systemic immunity.     

pHi regulatory ion transporters: an update on structure, regulation and cell function

Intracellular pH (pH_i) is a major regulator of various and critical cellular functions. A close regulation of pH_i is thus mandatory to maintain normal cellular activity. To this end, all cells express ion transporters that carry across their plasma membrane H⁺ or equivalent H⁺ into and out of the cell. Besides pH_i, these ion transporters are under the regulation of neurohormonal stimuli. This review summarises the molecular identity, regulation and function of the main membrane pH-regulatory ion transporters. Received 30 December 1998; received after revision 4 February 1999; accepted 9 February 1999     

<Emphasis Type="Italic">C</Emphasis>-<Emphasis Type="Italic">Raf</Emphasis> deficiency leads to hearing loss and increased noise susceptibility

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K⁺ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf ^+/? mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.     

Intracellular organelles in the saga of Ca<Superscript>2+</Superscript> homeostasis: different molecules for different purposes?

An increase in the concentration of cytosolic free Ca²⁺ is a key component regulating different cellular processes ranging from egg fertilization, active secretion and movement, to cell differentiation and death. The multitude of phenomena modulated by Ca²⁺, however, do not simply rely on increases/decreases in its concentration, but also on specific timing, shape and sub-cellular localization of its signals that, combined together, provide a huge versatility in Ca²⁺ signaling. Intracellular organelles and their Ca²⁺ handling machineries exert key roles in this complex and precise mechanism, and this review will try to depict a map of Ca²⁺ routes inside cells, highlighting the uniqueness of the different Ca²⁺ toolkit components and the complexity of the interactions between them.     

Lack of Na+,K+-ATPase expression in intercalated cells may be compensated by Na+-ATPase: A study on MDCK – C11 cells

The lack of Na⁺,K⁺-ATPase expression in intercalated cells (IC) is an intriguing condition due to its fundamental role in cellular homeostasis. In order to better understand this question we compared the activities of Na⁺,K⁺-ATPase and Na⁺-ATPase in two MDCK cell clones: the C11, with IC characteristics, and the C7, with principal cells (PC) characteristics. The Na⁺,K⁺-ATPase activity found in C11 cells is far lower than in C7 cells and the expression of its β-subunit is similar in both cells. On the other hand, a subset of C11 without α-subunit expression has been found. In C11 cells the Na⁺-ATPase activity is higher than that of the Na⁺,K⁺-ATPase, and it is increased by medium alkalinization, suggesting that it could account for the cellular Na⁺-homeostasis. Although further studies are necessary for a better understanding of these findings, the presence of Na⁺-ATPase may explain the adequate survival of cells that lack Na⁺,K⁺-ATPase. Received 09 July 2008; received after revision 03 August 2008; accepted 12 August 2008     

Identification of Treg-like cells in <Emphasis Type="Italic">Tetraodon</Emphasis>: insight into the origin of regulatory T subsets during early vertebrate evolution

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg cells) are critical for the maintenance of peripheral tolerance, and the suppression of autoimmune diseases and even tumors. Although Treg cells are well characterized in humans, little is known regarding their existence or occurrence in ancient vertebrates. In the present study, we report on the molecular and functional characterization of a Treg-like subset with the phenotype CD4-2⁺CD25-like⁺Foxp3-like⁺ from a pufferfish (Tetraodon nigroviridis) model. Functional studies showed that depletion of this subset produced an enhanced mixed lymphocyte reaction (MLR) and nonspecific cytotoxic cell (NCC) activity in vitro, as well as inflammation of the intestine in vivo. The data presented here will not only enrich the knowledge of fish immunology but will also be beneficial for a better cross-species understanding of the evolutionary history of the Treg family and Treg-mediated regulatory networks in cellular immunity.     

Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca<Superscript>2+</Superscript> fluxes in oxidative cell death

Oxidative DNA damage to cells activates poly(ADP-ribose)polymerase-1 (PARP-1) and the poly(ADP-ribose) formed is rapidly degraded to ADP-ribose by poly(ADP-ribose)glycohydrolase (PARG). Here we show that PARP-1 and PARG control extracellular Ca²⁺ fluxes through melastatin-like transient receptor potential 2 channels (TRPM2) in a cell death signaling pathway. TRPM2 activation accounts for essentially the entire Ca²⁺ influx into the cytosol, activating caspases and causing the translocation of apoptosis inducing factor (AIF) from the inner mitochondrial membrane to the nucleus followed by cell death. Abrogation of PARP-1 or PARG function disrupts these signals and reduces cell death. ADP-ribose-loading of cells induces Ca²⁺ fluxes in the absence of oxidative damage, suggesting that ADP-ribose is the key metabolite of the PARP-1/PARG system regulating TRPM2. We conclude that PARP-1/PARG control a cell death signal pathway that operates between five different cell compartments and communicates via three types of chemical messengers: a nucleotide, a cation, and proteins.     

Beta-carotene affects gene expression in lungs of male and female <Emphasis Type="Italic">Bcmo1</Emphasis><Superscript>−/−</Superscript> mice in opposite directions

Molecular mechanisms triggered by high dietary beta-carotene (BC) intake in lung are largely unknown. We performed microarray gene expression analysis on lung tissue of BC supplemented beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1 ^−/−) mice, which are—like humans—able to accumulate BC. Our main observation was that the genes were regulated in an opposite direction in male and female Bcmo1 ^−/− mice by BC. The steroid biosynthetic pathway was overrepresented in BC-supplemented male Bcmo1 ^−/− mice. Testosterone levels were higher after BC supplementation only in Bcmo1 ^−/− mice, which had, unlike wild-type (Bcmo1 ^+/+) mice, large variations. We hypothesize that BC possibly affects hormone synthesis or metabolism. Since sex hormones influence lung cancer risk, these data might contribute to an explanation for the previously found increased lung cancer risk after BC supplementation (ATBC and CARET studies). Moreover, effects of BC may depend on the presence of frequent human BCMO1 polymorphisms, since these effects were not found in wild-type mice.     

Na+,K+-ATPase as a docking station: protein–protein complexes of the Na+,K+-ATPase

The Na⁺,K⁺-ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na⁺ ions out of the cell and of K⁺ ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na⁺,K⁺-ATPase, recent work has suggested additional roles for Na⁺,K⁺-ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na⁺,K⁺-ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na⁺,K⁺-ATPase as a signal transducer, but also briefly discuss other Na⁺,K⁺-ATPase protein–protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme.