title: Induced expression of STIM1 sensitizes intestinal epithelial cells to apoptosis by modulating store-operated Ca2+ influx.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cell Biology Group, Department of Surgery, School of Medicine, University of Maryland, Baltimore, MD 21201, USA. jtimmons@smail.umaryland.edu
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Timmons
authors.forename: Jaladanki N
authors.initials: JN
authors.lastname: Rao
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Turner
authors.forename: Tongtong
authors.initials: T
authors.lastname: Zou
authors.forename: Lan
authors.initials: L
authors.lastname: Liu
authors.forename: Lan
authors.initials: L
authors.lastname: Xiao
authors.forename: Peng-Yuan
authors.initials: PY
authors.lastname: Wang
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Wang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology; D006706:Homeostasis / Q000502:physiology; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051381:Rats; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha
chemical_list: D015415:Biomarkers; D008562:Membrane Glycoproteins; C518857:Stim1 protein, rat; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; D014409:Tumor Necrosis Factor-alpha; C096351:transient receptor potential cation channel, subfamily C, member 1; D003513:Cycloheximide; C505410:Casp3 protein, rat; D053148:Caspase 3; D002118:Calcium