Induced expression of STIM1 sensitizes intestinal epithelial cells to apoptosis by modulating store-operated Ca2+ influx.
ABSTRACT Apoptosis plays a critical role in the maintenance of gut mucosal epithelial homeostasis and is tightly regulated by numerous factors including intracellular Ca(2+). Canonical transient receptor potential channel-1 (TRPC1) is expressed in intestinal epithelial cells (IECs) and functions as a store-operated Ca(2+) channel. We have recently demonstrated that increased TRPC1 activity sensitizes IECs to apoptosis, but the upstream signaling initiating TRPC1 activation remains elusive. The novel protein, stromal interaction molecule 1 (STIM1), is shown to act as a store Ca(2+) sensor, and it can rapidly translocate to the plasma membrane where it directly interacts with TRPC1. The current study determined whether STIM1 plays an important role in the regulation of IEC apoptosis by activating TRPC1 channel activity.
Studies were conducted in IEC-6 cells (derived from rat intestinal crypts) and stable TRPC1-transfected IECs (IEC-TRPC1). Apoptosis was induced by tumor necrosis factor-α (TNF-α)/cycloheximide (CHX), and intracellular free Ca(2+) concentration ([Ca(2+)](cyt)) was measured by fluorescence digital imaging analysis. Functions of STIM1 were investigated by specific siRNA (siSTIM1) and ectopic overexpression of the constitutively active STIM1 EF-hand mutants.
Stable STIM1-transfected IEC-6 cells (IEC-STIM1) showed increased STIM1 protein expression (~5 fold) and displayed a sustained increase in Ca(2+) influx after Ca(2+) store depletion (~2 fold). Susceptibility of IEC-STIM1 cells to TNF-α/CHX-induced apoptosis increased significantly as measured by changes in morphological features, DNA fragmentation, and caspase-3 activity. Apoptotic cells were increased from ~20% in parental IEC-6 cells to ~40% in stable IEC-STIM1 cells 4 h after exposure to TNF-α/CHX (p<0.05). In addition, stable IEC-TRPC1 cells also exhibited an increased sensitivity to TNF-α/CHX-induced apoptosis, which was prevented by STIM1 silencing through siSTIM1 transfection. STIM1 silencing by siSTIM1 also decreased Ca(2+) influx after store depletion in cells overexpressing TRPC1. Levels of Ca(2+) influx due to store depletion were decreased by ~70% in STIM1-silenced populations. Similarly, exposure of IEC-STIM1 cells to Ca(2+)-free medium also blocked increased sensitivity to apoptosis.
These results indicate that (1) STIM1 plays an important role in the regulation of IEC apoptosis by altering TRPC1 activity and (2) ectopic STIM1 expression sensitizes IECs to apoptosis through induction in TRPC1-mediated Ca(2+) influx.
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ABSTRACT: Store-operated channels (SOCs) mediate Ca(2+) entry signals in response to endoplasmic reticulum (ER) Ca(2+) depletion in most cells. STIM1 senses decreased ER luminal Ca(2+) through its EF-hand Ca(2+)-binding motif and aggregates in near-plasma membrane (PM) ER junctions to activate PM Orai1, the functional SOC. STIM1 is also present in the PM, although its role there is unknown. STIM1-mediated coupling was examined using the stable EF20 HEK293 cell line expressing the STIM1-D76A/E87A EF-hand mutant (STIM1(EF)) deficient in Ca(2+) binding. EF20 cells were viable despite constitutive Ca(2+) entry, allowing study of SOC activation without depleting ER Ca(2+). STIM1(EF) was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1(WT). STIM(EF)-expressing cells had normal ER Ca(2+) levels but substantially reduced ER Ca(2+) leak. Expression of antiapoptotic Bcl-2 proteins (BCl-2, MCL-1, BCL-XL) were increased 2-fold in EF20 cells, probably reflecting survival of EF20 cells but not accounting for decreased ER Ca(2+) leak. Surface biotinylation and streptavidin pull-down of cells expressing STIM1(WT) or STIM1(EF) revealed strong PM interactions of both proteins. Although surface expression of STIM1(WT) was clearly detectable, STIM1(EF) was undetectable at the cell surface. Thus, the Ca(2+) binding-defective STIM1(EF) mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1(WT), is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIM(EF)-mediated Ca(2+) entry, but only in cells expressing endogenous STIM1(WT) and not in DT40 STIM1 knock-out cells devoid of STIM(WT). This suggests that PM-STIM1 may play a regulatory role in SOC activation.Journal of Biological Chemistry 09/2008; 283(38):26252-62. · 4.65 Impact Factor
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ABSTRACT: Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs), and induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-kappaB activation. However, the exact mechanism by which increased TRPC1 results in NF-kappaB inactivation remains elusive. Protein phosphatase 2A (PP2A) is a widely conserved protein serine/threonine phosphatase that is implicated in the regulation of a wide array of cellular functions including apoptosis. The present study tests the hypothesis that induced TRPC1 expression inhibits NF-kappaB activation by increasing PP2A activity through Ca2+ influx in IECs. The expression of TRPC1 induced by stable transfection with the wild-type TRPC1 gene increased PP2A activity as indicated by increases in levels of PP2A proteins and their phosphatase activity. Increased levels of PP2A activity in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1) were associated with decreased nuclear levels of NF-kappaB proteins and a reduction in NF-kappaB-dependent transcriptional activity, although there were no changes in total NF-kappaB protein levels. Inhibition of PP2A activity by treatment with okadaic acid or PP2A silencing with small interfering RNA not only enhanced NF-kappaB transactivation but also prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis induced by treatment with tumor necrosis factor-alpha (TNF-alpha)/cycloheximide (CHX). Decreasing Ca2+ influx by exposure to the Ca2+-free medium reduced PP2A mRNA levels, destabilized PP2A proteins, and induced NF-kappaB activation, thus blocking the increased sensitivity of IEC-TRPC1 cells to TNF-alpha/CHX-induced apoptosis. These results indicate that induced TRPC1 expression increases PP2A activity through Ca2+ influx and that increased PP2A sensitizes IECs to apoptosis as a result of NF-kappaB inactivation.AJP Cell Physiology 06/2008; 294(5):C1277-87. · 3.71 Impact Factor
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ABSTRACT: We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.Biochemical and Biophysical Research Communications 11/2007; 361(4):934-40. · 2.41 Impact Factor