Serge Arnaudeau

Functional specialization of calreticulin domains

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

STIM1- and Orai1-dependent Store-operated Calcium Entry Regulates Human Myoblast Differentiation

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca2+]in involving store-operated Ca2+ entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca2+ sensor activating Orai Ca2+ channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca2+]in and intracellular Ca2+ store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K+ channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release

A newly identified splice variant of STIM1 called STIM1L forms constitutive clusters that interact with actin and Orai1 and allows fast repetitive Ca2+ release.

Subplasmalemmal mitochondria modulate the activity of plasma membrane Ca2+-ATPases.

Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling.

Membrane Hyperpolarization Triggers Myogenin and Myocyte Enhancer Factor-2 Expression during Human Myoblast Differentiation

It is widely thought that myogenin is one of the earliest detectable markers of skeletal muscle differentiation. Here we show that, during human myoblast differentiation, an inward rectifier K+ channel (Kir2.1) and its associated hyperpolarization trigger expression and activity of the myogenic transcription factors, myogenin and myocyte enhancer factor-2 (MEF2). Furthermore, Kir2.1 current precedes and is required for the developmental increase in expression/activity of myogenin and MEF2. Drugs or antisense reducing Kir2.1 current diminished or suppressed fusion as well as expression/activity of myogenin and MEF2. In contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase (a pathway controlling initiation of the myogenic program) that inhibited both myogenin/MEF2 expression and fusion, did not affect Kir2.1 current. This non-blockade by LY294002 indicates that Kir2.1 acts upstream of myogenin and MEF2. We propose that Kir2.1 channel activation is a required key early event that initiates myogenesis by turning on myogenin and MEF2 transcription factors via a hyperpolarization-activated Ca2+-dependent pathway.

Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners, STIM1 and STIM2

Our recent work identified store-operated Ca2+ entry (SOCE) as the critical Ca2+ source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370–5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca2+ mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca2+ content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca2+-content during repeated KCl-induced Ca2+ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.

In vitro activated human T lymphocytes very efficiently attach to allogenic multipotent mesenchymal stromal cells and transmigrate under them

The regulatory effect of human multipotent mesenchymal stromal cells (MSC) on allogenic T lymphocytes is extremely powerful and of important clinical relevance, but the mechanisms underlying this process are not fully elucidated. We report here that T lymphocytes activated with a sub‐mitogenic stimulus such as phytohemaglutinin alone (PHA), or with mitogenic stimuli such as PHA + interleukin‐2 (P‐IL2), or immobilized anti‐CD3 + anti‐CD28 mAb (a3‐28), tightly bound allogenic MSC and transmigrated within 4 h under them, where they remained for approximately 60 h. Allogenic MSC induced T cell proliferation in cultures containing sub‐mitogenic PHA concentrations, and inhibited the mitogenic effect of P‐IL2 or a3‐28. Anti‐γ‐IFN mAb or L‐tryptophan complementation partially restored proliferation in P‐IL2 and a3‐28 cultures, whereby γ‐IFN‐synthesizing CD3+ cells were detectable. MSC‐lymphocyte contact hindrance using transwells abrogated proliferation in PHA cultures, restored it integrally in P‐IL2 cultures, and partially in a3‐28 cultures. These data suggest that MSC‐induced T lymphocyte regulation results from the combination of various processes. Allogenic cell–cell contact, as demonstrated by the PHA co‐cultures is per se stimulatory, whereas γ‐IFN synthesized by activated T lymphocytes, which activates indolamine 2,3‐dioxygenase in MSC, and L‐tryptophan depletion, which is induced by this enzyme, are inhibitory. Transmigration is nevertheless pivotal for the establishment of the inhibition by these mediators because it targets lymphocytes under the stroma in small extracellular spaces surrounded by MSC, where L‐tryptophan is efficiently destroyed, leading to T lymphocyte proliferation arrest. In conclusion lymphocyte transmigration under allogenic MSC potentiates the inhibitory effect of soluble mediators generated by these cells. J. Cell. Physiol. 214: 588–594, 2008.

Calcium sources used by post-natal human myoblasts during initial differentiation.

Increases in cytoplasmic Ca2+ are crucial for inducing the initial steps of myoblast differentiation that ultimately lead to fusion; yet the mechanisms that produce this elevated Ca2+ have not been fully resolved. For example, it is still unclear whether the increase comes exclusively from membrane Ca2+ influx or also from Ca2+ release from internal stores. To address this, we investigated early differentiation of myoblast clones each derived from single post‐natal human satellite cells. Initial differentiation was assayed by immunostaining myonuclei for the transcription factor MEF2. When Ca2+ influx was eliminated by using low external Ca2+ media, we found that approximately half the clones could still differentiate. Of the clones that required influx of external Ca2+, most clones used T‐type Ca2+ channels, but others used store‐operated channels as influx‐generating mechanisms. On the other hand, clones that differentiated in low external Ca2+ relied on Ca2+ release from internal stores through IP3 receptors. Interestingly, by following clones over time, we observed that some switched their preferred Ca2+ source: clones that initially used calcium release from internal stores to differentiate later required Ca2+ influx and inversely. In conclusion, we show that human myoblasts can use three alternative mechanisms to increase cytoplasmic Ca2+ at the onset of the differentiation process: influx through T‐types Ca2+ channels, influx through store operated channels and release from internal stores through IP3 receptors. In addition, we suggest that, probably because Ca2+ elevation is essential during initial differentiation, myoblasts may be able to select between these alternate Ca2+ pathways. J. Cell. Physiol. 208: 435–445, 2006.

Local and global calcium signals associated with the opening of neuronal alpha7 nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChRs) are Ca(2+)-permeable ligand-gated channels widely expressed in the central and peripheral nervous system. One of the most Ca(2+) selective isoform is the homopentameric alpha7-nAChR implicated in schizophrenia. The activity of alpha7-nAChRs is usually recorded electrophysiologically, which limits the amount of information obtained. Here, we used fluorescence imaging to record Ca(2+) transients associated with activation of the alpha7-nAChR in neuroblastoma cells stably expressing human alpha7-nAChRs. Application of nicotine (50 microM) consistently evoked transient (30s), stereotyped Ca(2+) responses that were inhibited by the selective alpha7-nAChRs antagonists methyllycaconitine (MLA) and alpha-bungarotoxin, and greatly increased and prolonged by the allosteric modulator PNU-120596 (1 microM). Unexpectedly, brief (1-5s), repetitive Ca(2+) transients of sub-micrometric dimension were observed in filopodia of cells expressing alpha7-nAChR. PNU-120596 increased the frequency and slowed the decay kinetics of these miniature Ca(2+) elevations, which were insensitive to ryanodine, preserved during hyperpolarisation, and prevented by MLA, alpha-bungarotoxin, or Ca(2+) removal. Global Ca(2+) responses were also recorded in ganglion cells of embryo chicken retina during co-application of PNU-120596 and nicotine, together with whole-cell currents and brief current bursts. These data demonstrate that Ca(2+) signals generated by alpha7-nAChRs can be recorded optically both in cell lines and in intact tissues. The possibility to image miniature Ca(2+) signals enables to map the location of functional alpha7-nAChR channel clusters within cells and to analyze their single channel properties optically. Deciphering the rich pattern of intracellular Ca(2+) signals generated by the activity of the alpha7-nAChRs will reveal the physiological role of these receptor-channels.

Glucose‐regulated protein 78: A new partner of p53 in trophoblast

Although wild‐type p53 protein is overexpressed in first trimester trophoblast, it is inactive towards its target genes Metalloproteinase 2 and 9. This seems to be due to a complex mechanism of inactivation and stabilization of p53 relying on the formation of protein complexes involving the N‐terminus of p53. To detect the proteins associated with this sequence, we incubated biotinylated p53 N‐terminal peptide in cytotrophoblastic cell medium 24 h before lysis of cells. We purified the proteins retained on biotinylated peptide using a neutravidin affinity column. Proteins were then identified by peptide mass finger printing followed or not by peptide fragmentation sequencing. Among these proteins, we identified glucose‐regulated protein 78 (GRP78) and verified its interaction with p53 in trophoblastic cells by immunoprecipitation and Western blot analysis. Moreover, the decreased expression of GRP78 induced by GRP78siRNA or versipelostatin decreased the formation of high molecular weight p53 complexes and p53 monomer and increased trophoblastic invasion. These results suggest that GRP78 is involved in inactivation and stabilization of p53 and in the regulation of trophoblastic invasion.