Imaging Ca<sup>2+</sup> Responses During <em>Shigella</em> Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Université Paris Sud

Liver regeneration after partial hepatectomy is a plastic process during which the mechanisms that coordinate liver mass restoration compensate one another through a complex regulatory network of cytokines, growth factors, and hormones. Vasopressin, an agonist that triggers highly organized Ca2+ signals in the liver, may be one of these factors, although little in vivo evidence is available in support of this hypothesis. We provide evidence that hypothalamic vasopressin secretion is stimulated early after partial hepatectomy. Although hepatocytes were fully responsive to vasopressin during the first hours of regeneration, they became desensitized and exhibited slow oscillating Ca2+ responses to vasopressin on the following days. On the first day, hepatocyte V1a receptor density decreased and its lobular gradient increased in hepatectomized rats. By antagonizing the V1a receptor in vivo, we demonstrated that vasopressin contributes to NF-kappaB and cyclin (D1 and A) activation, to hepatocyte progression in the cell cycle, and to liver mass restoration. Finally, vasopressin exerted a choleretic effect shortly after hepatectomy, both in the isolated perfused liver and in the intact rat. In conclusion, we provide compelling in vivo evidence that vasopressin contributes significantly to growth initiation and bile flow stimulation in the early stages of liver regeneration.

Hypothalamic vasopressin release and hepatocyte Ca2+ signaling during liver regeneration: an interplay stimulating liver growth and bile flow.

The aim of this work was to study the effects of different bile acids on the permeability of gap junction channels (PGJC). We also looked at the effects of some bile acids on the coordination of intercellular calcium oscillations.

Cholestatic bile acids inhibit gap junction permeability in rat hepatocyte couplets and normal rat cholangiocytes.

During liver regeneration, a network of cytokines and growth factors interact with hepatocytes, helping to restore the liver mass and functions after partial tissue loss. Agonists that trigger Ca2+ signals in the liver contribute to this process, although little is known about calcium signalling during liver regeneration.

Remodelling of calcium signalling during liver regeneration in the rat.

Extracellular ATP regulates many hepatic functions by stimulating purinergic receptors. Only the G protein-coupled P2Y receptors have been studied in hepatocytes. We investigated the functional expression of P2X receptors, the ATP-gated channels in rat hepatocytes. P2X4 and P2X7 transcripts and proteins were detected by RT-PCR and by both Western blotting and immunocytochemistry. High concentrations of ATP, and 2'-and 3'-O-(4-benzoylbenzoyl)-ATP the preferring agonist of P2X7, induced membrane blebbing and significant uptake of 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide, both of which were inhibited by oxidised ATP, a blocker of P2X receptors. These results provide evidence that P2X4 and P2X7 receptors are expressed and functional on rat hepatocytes, possibly playing an important role in the purinergic signaling complex in these cells.

Rat hepatocytes express functional P2X receptors.

Signal-induced Ca(2+) oscillations have been observed in many cell types and play a primary role in cell physiology. Although it is the regular character of these oscillations that first catches the attention, a closer look at time series of Ca(2+) increases reveals that the fluctuations on the period during individual spike trains are far from negligible. Here, we perform a statistical analysis of the regularity of Ca(2+) oscillations in norepinephrine-stimulated hepatocytes and find that the coefficient of variation lies between 10% and 15%. Stochastic simulations based on Gillespie's algorithm and considering realistic numbers of Ca(2+) ions and inositol trisphosphate (InsP(3)) receptors account for this variability if the receptors are assumed to be grouped in clusters of a few tens of channels. Given the relatively small number of clusters ( approximately 200), the model predicts the existence of repetitive spikes induced by fluctuations (stochastic resonance). Oscillations of this type are found in hepatocytes at subthreshold concentrations of norepinephrine. We next predict with the model that the isoforms of the InsP(3) receptor can affect the variability of the oscillations. In contrast, possible accompanying InsP(3) oscillations have no impact on the robustness of signal-induced repetitive Ca(2+) spikes.

Stochastic aspects of oscillatory Ca2+ dynamics in hepatocytes.

Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study.

ATP release after partial hepatectomy regulates liver regeneration in the rat.

Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES-expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES-expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells.

Cytosolic calcium regulates liver regeneration in the rat.

In many cell types, specific and robust signalling relies on a high level of spatiotemporal organization of Ca(2+) dynamics. In response to external stimulation, Ca(2+) signals ranging from a small increase of a few tens of nanomolar concentrations at the mouth of an inositol 1, 4, 5-trisphosphate receptor to the periodic propagation of waves invading an organ or a tissue, can be observed. Here, we review our combined experimental and computational approach of Ca(2+) dynamics, which has been mainly carried out on liver hepatocytes. We focus in particular on the understanding of the relationship between elementary Ca(2+) increases, Ca(2+) oscillations and intra- or intercellular Ca(2+) waves. The physiological impact of such signalling on liver function is also discussed.

[Experimental and computational approach of calcium signaling].

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca(2+) handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca(2+) dynamics in this cell type. We found that in hepatocytes isolated from Hint2(-/-) mice, the frequency of Ca(2+) oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca(2+) pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2(-/-) mice; we found that Hint2 accelerates Ca(2+) pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca(2+) in suspensions of mitochondria. This prediction was then confirmed experimentally. 

Characterization of the effect of the mitochondrial protein Hint2 on intracellular Ca(2+) dynamics.

Hepatocytes, which perform the main functions of the liver, are particularly vulnerable to toxic agents such as cadmium, an environmental pollutant. To identify the molecular targets for cadmium in hepatocytes, we have studied the effects of CdCl2 on the hybrid cell line WIF-B9 that exhibits stable structural and functional hepatocytic polarity.

Cadmium disorganises the scaffolding of gap and tight junction proteins in the hepatic cell line WIF B9.

Cholangiocytes are biliary epithelial cells, which, like hepatocytes, originate from hepatoblasts during embryonic development. In this study we investigated the potential of human embryonic stem cells (hESCs) to differentiate into cholangiocytes and we report a new approach, which drives differentiation of hESCs toward the cholangiocytic lineage using feeder-free and defined culture conditions. After differentiation into hepatic progenitors, hESCs were differentiated further into cholangiocytes using growth hormone, epidermal growth factor, interleukin-6, and then sodium taurocholate. These conditions also allowed us to generate cholangiocytes from HepaRG-derived hepatoblasts. hESC- and HepaRG-derived cholangiocyte-like cells expressed markers of cholangiocytes including cytokeratin 7 and osteopontin, and the transcription factors SOX9 and hepatocyte nuclear factor 6. The cells also displayed specific proteins important for cholangiocyte functions including cystic fibrosis transmembrane conductance regulator, secretin receptor, and nuclear receptors. They formed primary cilia and also responded to hormonal stimulation by increase of intracellular Ca(2+) . We demonstrated by integrative genomics that the expression of genes, which signed hESC- or HepaRG-cholangiocytes, separates hepatocytic lineage from cholangiocyte lineage. When grown in a 3D matrix, cholangiocytes developed epithelial/apicobasal polarity and formed functional cysts and biliary ducts. In addition, we showed that cholangiocyte-like cells could also be generated from human induced pluripotent stem cells, demonstrating the efficacy of our approach with stem/progenitor cells of diverse origins.

Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells.

Oscillations of cytosolic Ca(2+) concentration are a widespread mode of signalling. Oscillatory spikes rely on repetitive exchanges of Ca(2+) between the endoplasmic reticulum (ER) and the cytosol, due to the regulation of inositol 1,4,5-trisphosphate receptors. Mitochondria also sequester and release Ca(2+), thus affecting Ca(2+) signalling. Mitochondrial Ca(2+) activates key enzymes involved in ATP synthesis. We propose a new integrative model for Ca(2+) signalling and mitochondrial metabolism in electrically non-excitable cells. The model accounts for (1) the phase relationship of the Ca(2+) changes in the cytosol, the ER and mitochondria, (2) the dynamics of mitochondrial metabolites in response to cytosolic Ca(2+) changes, and (3) the impacts of cytosol/mitochondria Ca(2+) exchanges and of mitochondrial metabolism on Ca(2+) oscillations. Simulations predict that as expected, oscillations are slowed down by decreasing the rate of Ca(2+) efflux from mitochondria, but also by decreasing the rate of Ca(2+) influx through the mitochondrial Ca(2+) uniporter (MCU). These predictions were experimentally validated by inhibiting MCU expression. Despite the highly non-linear character of Ca(2+) dynamics and mitochondrial metabolism, bioenergetics were found to be robust with respect to changes in frequency and amplitude of Ca(2+) oscillations. 

Interplay Between Intracellular Ca(2+) Oscillations and Ca(2+)-stimulated Mitochondrial Metabolism.

Ca (2+) oscillations, a widespread mode of cell signaling, were reported in non-excitable cells for the first time more than 25 years ago. Their fundamental mechanism, based on the periodic Ca (2+) exchange between the endoplasmic reticulum and the cytoplasm, has been well characterized. However, how the kinetics of cytosolic Ca (2+) changes are related to the extent of a physiological response remains poorly understood. Here, we review data suggesting that the downstream targets of Ca (2+) are controlled not only by the frequency of Ca (2+) oscillations but also by the detailed characteristics of the oscillations, such as their duration, shape, or baseline level. Involvement of non-endoplasmic reticulum Ca (2+) stores, mainly mitochondria and the extracellular medium, participates in this fine tuning of Ca (2+) oscillations. The main characteristics of the Ca (2+) exchange fluxes with these compartments are also reviewed. 

Fine tuning of cytosolic Ca (2+) oscillations.

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca increases are transient and oscillatory, defining the so-called Ca code that links cell responses to specific Ca oscillatory patterns. During cell invasion,  induces atypical local and global Ca signals. Here, we show that by hydrolyzing phosphatidylinositol-(4,5)bisphosphate, the  type III effector IpgD dampens inositol-(1,4,5)trisphosphate (InsP) levels. By modifying InsP dynamics and diffusion, IpgD favors the elicitation of long-lasting local Ca signals at  invasion sites and converts -induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP-dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca-dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.

The  type III effector IpgD recodes Ca signals during invasion of epithelial cells.

Mitochondria play a significant role in shaping cytosolic Ca signals. Thus, transfer of Ca across the mitochondrial membrane is much studied, not only in intact cells but also in artificial systems such as mitochondrial suspensions or permeabilised cells. Observed rates of Ca changes vary by at least one order of magnitude. In this work, we investigate the relationship between the Ca dynamics observed in various experimental conditions using a computational model calibrated on experimental data. Results confirm that mitochondrial Ca exchange fluxes through the mitochondrial Ca uniporter (MCU) and the Na /Ca exchanger obey the same basic kinetics in cells and in suspensions, and emphasise the important role played by the high Ca levels reached in mitochondria-associated endoplasmic reticulum membranes in intact cells. Tissue specificity can be ascribed to the different modes of regulation of the MCU by Ca , probably related to the specific levels of expression of the Ca sensing regulator subunit of this channel. The model emphasises the importance of mitochondrial density and buffering in controlling the rate of Ca exchanges with mitochondria, as verified experimentally. Finally, we show that heterogeneity between individual mitochondria can explain the large range of amplitudes and rates of rise in mitochondrial Ca concentration that have been observed experimentally.

Laurent Combettes

Inserm,
UMRS1174,
Inserm, UMRS1174

Université Paris Sud

Imaging Ca²⁺ Responses During Shigella Infection of Epithelial Cells

Laurent Combettes

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Université Paris Sud

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Inserm,
UMRS1174,
Inserm, UMRS1174

Imaging Ca²⁺ Responses During Shigella Infection of Epithelial Cells