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.
- SourceAvailable from: Bidhan C Bandyopadhyay[Show abstract] [Hide abstract]
ABSTRACT: Ca2+ entry is essential for regulating vital physiological functions in all neuronal cells. Although neurons are engaged in multiple modes of Ca2+ entry that regulates variety of neuronal functions, we will only discuss a subset of specialized Ca2+-permeable non-selective Transient Receptor Potential Canonical (TRPC) channels and summarize their physiological and pathological role in these excitable cells. Depletion of endoplasmic reticulum (ER) Ca2+ stores, due to G-protein coupled receptor activation, has been shown to activate TRPC channels in both excitable and non-excitable cells. While all seven members of TRPC channels are predominately expressed in neuronal cells, the ion channel properties, mode of activation, and their physiological responses are quite distinct. Moreover, many of these TRPC channels have also been suggested to be associated with neuronal development, proliferation and differentiation. In addition, TRPCs also regulate neurosecretion, long-term potentiation and synaptic plasticity. Similarly, perturbations in Ca2+ entry via the TRPC channels have been also suggested in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of TRPCs in neuronal function and in neurodegenerative conditions would presumably unveil avenues for plausible therapeutic interventions for these devastating neuronal diseases.Cells. 01/2014; 3(2):455-75.