Désirée Griesemer

Differential Redox Regulation of ORAI Ion Channels: A Mechanism to Tune Cellular Calcium Signaling

Redox sensitivity of T cells decreases through ORAI Ca2+ channel subunit switching during T cell differentiation. Adapting to Oxidizing Environments Reactive oxygen species (ROS) were thought for many years to be only detrimental, causing damage to DNA and proteins. However, it has become clear that ROS, particularly H2O2, can act as intracellular signaling molecules that link cellular redox state to such processes as proliferation and differentiation. Bogeski et al. have uncovered a role for ROS in regulating calcium channel activity—and intracellular Ca2+ signals crucial to the immune response—in T lymphocytes. They found that activity of ORAI1 calcium channels was blocked by H2O2, whereas that of the related ORAI3 channels was not. Redox sensitivity decreased as naïve human T helper lymphocytes differentiated into effector T helper lymphocytes, which was associated with an increase in the abundance of mRNA encoding the insensitive ORAI3 protein. The authors suggest that changes in the specific complement of ORAI channels, and thereby sensitivity to ROS, could enable T lymphocytes to fine tune cellular responses in oxidizing environments such as those found during inflammation. Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca2+) influx through Ca2+ release–activated Ca2+ (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca2+ imaging, patch-clamp recordings, and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H2O2) on ORAI channel activity and human T helper lymphocyte (TH cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H2O2. The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. TH cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector TH cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector TH cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector TH cells may tune cellular responses under oxidative stress.

Calcium-dependent activation of T-lymphocytes

Activation of T-lymphocytes requires stimulation of T-cell receptors (TCR) and co-stimulatory signals. Among different signalling cascades, TCR engagement induces Ca2+ entry through plasma membrane Ca2+ channels, which is an indispensable step for T-cells to expand clonally and to acquire effector functions. The Ca2+ channels are activated by depletion of Ca2+ stores and are called Ca2+ release-activated Ca2+ (CRAC) channels. Ca2+ influx through CRAC channels is also controlled, directly or indirectly, by K+ channels, Ca2+-ATPases, mitochondria, endoplasmic reticulum and Ca2+ buffers. We review the functional implications of these transporters, organelles and buffers and develop a model of Ca2+ signal generation that depends mainly on their relative mutual localization. This model offers the possibility of controlling amplitude and kinetics of Ca2+ signals in T-cells. Decoding of various Ca2+ signals allows differential activation of the transcription factor families nuclear factor of activated T-cells (NFAT), nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Variation of amplitude and kinetics of Ca2+ signals thus is an important mechanism for modulating the specificity of T-cell responses.

Calcium dependence of T cell proliferation following focal stimulation

Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca2+ dependence of proliferation and apoptosis in primary human CD4+ T cells following stimulation with anti‐CD3/anti‐CD28‐coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL‐2 production and proliferation than non‐focal TCR stimuli. Surprising little Ca2+ entry through Ca2+ channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca2+]i) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4+ T cells. We also show that proliferation was very Ca2+ sensitive in the range 90–120 nM, whereas apoptosis was basically constant for [Ca2+]i levels of 90–120 nM. We conclude that very small changes in [Ca2+]i can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.

Thapsigargin Induces Expression of Activating Transcription Factor 3 in Human Keratinocytes Involving Ca2+ Ions and c-Jun N-Terminal Protein Kinase

Thapsigargin is a specific inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase of the endoplasmic reticulum. Here, we show that stimulation of human HaCaT keratinocytes with nanomolar concentrations of thapsigargin triggers expression of activating transcription factor (ATF) 3, a basic-region leucin zipper transcription factor. ATF3 expression was also up-regulated in thapsigargin-stimulated glioma cells, hepatoma cells, retinal pigment epithelial cells, and airway epithelial cells. Thapsigargin-induced up-regulation of ATF3 expression in keratinocytes was attenuated by BAPTA-acetoxymethyl ester or by expression of the Ca2+-binding protein parvalbumin in the cytosol of HaCaT cells but not by a panel of pharmacological agents that chelate extracellular Ca2+ (EGTA) or inhibit either ryanodine receptors (dantrolene) or voltage-gated Ca2+ channels (nifedipine). Hence, elevated levels of intracellular Ca2+, released from intracellular stores, are essential for the effect of thapsigargin on the biosynthesis of ATF3. The thapsigargin-induced signaling pathway was blocked by expression of either mitogen-activated protein kinase phosphatase-1 or -5. Experiments involving pharmacological and genetic tools revealed the importance of c-Jun N-terminal protein kinase (JNK) within the signaling cascade, whereas inhibition of extracellular signal-regulated protein kinase or p38 protein kinase did not attenuate thapsigargin-induced expression of ATF3. Functional studies showed that treatment of HaCaT keratinocytes with thapsigargin led to a 2-fold induction of caspase-3/7 activity. The up-regulation of caspase-3/7 activity in thapsigargin-stimulated HaCaT cells was attenuated by inhibition of JNK. Together, these data show that stimulation of HaCaT cells with thapsigargin induces a specific signaling pathway in keratinocytes involving activation of JNK, biosynthesis of ATF3, and up-regulation of caspase-3/7 activity.

TRP Channels in Lymphocytes

TRP proteins form ion channels that are activated following receptor stimulation. Several members of the TRP family are likely to be expressed in lymphocytes. However, in many studies, messenger RNA (mRNA) but not protein expression was analyzed and cell lines but not primary human or murine lymphocytes were used. Among the expressed TRP mRNAs are TRPC1, TRPC3, TRPM2, TRPM4, TRPM7, TRPV1, and TRPV2. Regulation of Ca2+ entry is a key process for lymphocyte activation, and TRP channels may both increase Ca2+ influx (such as TRPC3) or decrease Ca2+ influx through membrane depolarization (such as TRPM4). In the future, linking endogenous Ca2+/cation channels in lymphocytes with TRP proteins should lead to a better molecular understanding of lymphocyte activation.

P2X7 receptor stimulation upregulates Egr‐1 biosynthesis involving a cytosolic Ca2+ rise, transactivation of the EGF receptor and phosphorylation of ERK and Elk‐1

The P2X7 receptor is an ATP‐gated ionotropic receptor that is permeable for small cations including Ca2+ ions. Using 293 cells expressing P2X7 receptors, we show that the P2X7 receptor‐specific ligand 2′,3′‐O‐(4‐benzoyl‐benzoyl)‐ATP (BzATP) induces a signaling cascade leading to the biosynthesis of biologically active Egr‐1, a zinc finger transcription factor. BzATP‐triggered Egr‐1 biosynthesis was attenuated by the mitogen‐activated protein kinase kinase inhibitor PD98059, by BAPTA‐AM, the acetoxymethylester of the cytosolic Ca2+ chelator BAPTA, and by an epidermal growth factor (EGF) receptor‐specific tyrosine kinase inhibitor (AG1478). These results indicate that phosphorylation and activation of extracellular signal‐regulated protein kinase ERK, elevated levels of intracellular Ca2+ and the transactivation of the EGF receptor are essential for BzATP‐induced upregulation of Egr‐1. The requirement of Ca2+ within the signaling cascade was upstream of Raf kinase activation. Lentiviral‐mediated expression of MAP kinase phosphatase‐1 (MKP‐1), a dual‐specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, inhibited Egr‐1 biosynthesis following BzATP stimulation, indicating that MKP‐1 functions as a nuclear shut‐off device. Furthermore, the ternary complex factor Elk‐1 was phosphorylated and the transcriptional activation potential of Elk‐1 was enhanced following P2X7 receptor stimulation. Expression of a dominant‐negative mutant of Elk‐1 impaired BzATP‐induced upregulation of Egr‐1 biosynthesis. Thus, Elk‐1 connects the intracellular signaling cascade elicited by activation of P2X7 receptors with the transcription of the Egr‐1 gene. J. Cell. Physiol. 213: 36–44, 2007.

Disruption of the cortical actin cytoskeleton does not affect store operated Ca2+ channels in human T-cells

Lymphocyte signaling and activation leads to the influx of extracellular Ca2+ via the activation of Ca2+ release activated Ca2+ (CRAC) channels in the plasma membrane. Activation of CRAC channels occurs following emptying of the endoplasmic reticulum intracellular Ca2+ stores. One model to explain the coupling of store‐emptying to CRAC activation is the secretion‐like conformational coupling model. This model proposes that store depletion increases junctions between the endoplasmic reticulum and the plasma membrane in a manner that could be regulated by the cortical actin cytoskeleton. Here, we show that stabilization or depolymerization of the actin cytoskeleton failed to affect CRAC activation. We therefore conclude that rearrangement of the actin cytoskeleton is dispensable for store‐operated Ca2+ entry in T‐cells.

Two-photon analysis of calcium signals in T lymphocytes of intact lamina propria from human intestine.

Lamina propria (LP) T cells of the human intestinal mucosa usually do not develop systemic immune responses despite permanent exposure to foreign antigens. The mechanisms maintaining this hyporeactivity in the normal gut are poorly understood. It is, at present, not clear what role the microenvironment of the mucosa plays for low T cell reactivity and in the pathogenesis of mucosal inflammation. Despite the importance of cytosolic Ca2+ signals for T lymphocyte activation, intracellular Ca2+ concentration measurements have so far only been performed in dissociated T cells, following disruption of the microenvironment. We used two‐photon technology to measure Ca2+ signals in identified T lymphocytes within the intact mucosa to minimize impact on tissue integrity while preserving the cellular microenvironment. We show that Ca2+ signals in LP T cells correlate with the hyporeactivity of T cells in the intestinal immune system and furthermore link Ca2+ signals with inflammatory bowel disease. Our data implicate that Ca2+ signals in LP T cells do not depend on the microenvironment of the intact mucosa, since they are very similar to Ca2+ signals in dissociated LP T cells.

Differential Redox Regulation of ORAI Channels: A Mechanism to Tune T-Cell Responses

Phagocytes play an essential role in host defence against pathogens by generating reactive oxygen species (ROS). Effector T helper (Th) cells migrating to sites of infection will be exposed to this highly oxidative environment. Here we show how Th-cells respond and adapt to ROS. Oxidation affects different Ca2+-signalling pathways essential for T-cell function. ORAI1 channels are inhibited with an IC50 of ∼40 μM H2O2, but ORAI3 channels are insensitive. We identify cysteine (C195) of ORAI1, absent in ORAI3, as the major redox sensor. A reduced sensitivity of effector Th-cells towards oxidation is due to upregulation of Orai3 and of cytosolic antioxidants. The differential redox regulation of ORAI channels is a novel mechanism to tune Th-cell based immune responses during clonal expansion and inflammation.