Occludin is required for apoptosis when claudin-claudin interactions are disrupted

Abstract

Disruption of tight junctions is often seen during pathogen infection, inflammation, and tumor progression. Mislocalization of the tight junction proteins occludin and claudin in mammary epithelial monolayers leads to apoptosis through the extrinsic pathway. To further investigate the mechanism of this response, a normal mammary epithelial cell line (EpH4) as well as primary mammary epithelial cells were treated with a claudin-disrupting mimic peptide, DFYNP (aspartic acid-phenylalanine-tyrosine-asparagine-proline). Using fluorescent indicators, we found that caspase-3 activation, resulting from treatment with DFYNP, was restricted to EpH4 and primary mammary epithelial cells with mislocalized claudin-4. Mislocalized claudin-4 and occludin were colocalized in non-junctional puncta, and both molecules were found in the death-inducing signaling complex (DISC) where they colocalized with Fas, fas-associated protein with death domain (FADD), active caspase-8 and caspase-3 at distinct apical domains. Importantly, caspase-3 activation was totally repressed in primary mammary epithelial cells from occludin null mice. Thus, the apoptotic response appears to be initiated by the movement of occludin to the DISC suggesting that this molecule has signaling properties that initiate cell death when its tight junction location is disrupted.

Figure 1

Caspase-3 is activated in cells with mislocalized claudin. Confocal microscopy images show staining of EpH4 mammary epithelial monolayers for active caspase-3 (red), claudin-4 (green), and nuclei (blue). Caspase-3 is activated in cells treated with the DFYNP claudin mimic peptide where activation is restricted to cells with non-junctional claudin-4 localization

Figure 2

Colocalization of occludin and claudin. Confocal microscopy images show staining of EpH4 mammary epithelial monolayers for claudin-4 (green) and occludin (red) in the absence and presence of the claudin mimic peptide. Claudin-4 and occludin both localize to sites of tight junctions in untreated epithelium. After treatment with the claudin mimic peptide, bright puncta of non-junctional occludin colocalize with bright puncta of non-junctional claudin-4

Figure 3

Colocalization of occludin, claudin-4, and components of the death inducing signaling complex (DISC). Magnified ( × 100) confocal microscopy images show staining of EpH4 mammary epithelial monolayers for: (a) occludin (red), active caspase-8 (white), and FADD (green); (b) claudin-4 (red), active caspase-8 (white), and FADD (green) after treatment of cells with the claudin mimic peptide for 4 h. Claudin-4 and occludin both colocalize with active caspase-8 and FADD in response to treatment with the peptide. Z-stack images show: (c) EpH4 cells stained for nuclei (DAPI, blue), occludin (red), FADD (white), and active caspase-8 (green); (d) cells stained for nuclei (blue), claudin-4 (red), FADD (white), and active caspase-8 (green); or (e) cells stained for nuclei (blue), occludin (red), claudin-4 (white), and active caspase-8 (green). Occludin, claudin-4, FADD, and active caspase-8 colocalize to distinct apical domains in response to treatment with the claudin mimic peptide

Figure 4

Fas and caspase-3 colocalize with claudin and DISC components. Magnified ( × 100) confocal microscopy images show staining of EpH4 mammary epithelial monolayers for active caspase-8 (green), claudin-4 (white) and (a) Fas or (b) active caspase-3 (red) after 4 h of treatment with the claudin mimic peptide. Claudin-4 colocalizes with active caspase-8 as well as Fas and activated caspase-3 in response to peptide treatment

Figure 5

Claudin is not disrupted when occludin is disrupted. Confocal microscopy images show staining of EpH4 mammary epithelial monolayers for claudin-4 in the (a) absence and (b) presence of an occludin-disrupting LYHY mimic peptide. Claudin-4 remains at sites of tight junctions when occludin is disrupted

Figure 6

Claudin localization is disrupted in occludin null cells. (a) Western blot analysis of occludin protein expression was performed with tissue lysates from wild-type (+/+) and occludin null (−/−) mice to confirm the loss of occludin protein in occludin null cells. Confocal images show cultured monolayers of primary mammary epithelial cells isolated from occludin null mice stained for claudin-4 in the (b) absence and (c) presence of the claudin mimic peptide for 16 h before fixation. In the absence of occludin, claudin-4 is still able to change localization in response to the claudin mimic peptide

Figure 7

Occludin is required for peptide-induced caspase-3 activation. Cultured monolayers of primary mammary epithelial cells from wild-type and occludin null-transgenic mice were treated with the claudin mimic peptide. After 16 h, the number of caspase-3-positive cells was measured. Caspase-3 activation in response to the claudin mimic peptide was inhibited in cells that lacked occludin protein expression. Mean±S.E.M., n=3–6, ^*P<0.05 versus untreated