Overexpression of cytosolic sequestered caspase 3 increased human lung microvascular endothelial cell migration and proliferation, an effect that was abolished by YAP knockdown.
Does cytosolic caspase 3 increase human lung microvascular endothelial cell migration and proliferation through YAP signaling?
Cytosolic caspase 3 promotes human lung microvascular endothelial cell proliferation and migration via YAP signaling, suggesting a potential reparative mechanism following endothelial injury in acute respiratory distress syndrome.
Abstract Rationale Endothelial apoptosis is a major contributor to the breakdown of the lung capillary membrane during the acute respiratory distress syndrome (ARDS). Previously, we reported nuclear translocation of caspase 3 is required for endothelial apoptosis, whereas programed cell death does not occur when caspase 3 is sequestered in the cytoplasm. After apoptosis, endothelial migration and proliferation are critical for repairing the endothelial barrier. Intriguingly, preliminary data from our lab suggests cytosolic caspase 3 is important for migration and proliferation of human lung microvascular endothelial cells (hLMVECs), although the mechanism of how this occurs is unknown. We hypothesize that cytosolic caspase 3 supports cell migration, and proliferation by activating YAP, a major regulator of pro-reparative signaling. Methods Caspase 3 was inhibited with DEVD in hLMVECs. For gain of function experiments, cells were transduced with an adenovirus expressing either an eGFP control, wild type caspase 3, or cytosolic sequestered caspase 3. A BrdU assay was used to examine proliferation, and a scratch wound assay was performed to assess cell migration. Scratch wound gap area was measured after a 5-hour period using ImageJ. Knockdown of YAP was achieved by treating cells with si-scramble or si-RNA targeting YAP. For immunofluorescence (IF) experiments, caspase 3 and YAP were co-stained and examined for overlap. Co-immunoprecipitation (Co-IP) of caspase 3 was done from lung tissue and protein binding partners were identified using mass spectrometry. Results Inhibition of caspase 3 reduced hLMVEC migration and proliferation. Consistent with this, overexpression of wild type caspase 3 led to an increase in migration and proliferation compared to eGFP control. The proliferative phenotype of caspase 3 was further enhanced with overexpression of cytosolic sequestered caspase 3 and was abolished when YAP was knocked down. Moreover, co-IP data from lung tissue found caspase 3 binds with several proteins in the YAP signaling cascade, and IF staining showed overlap between caspase 3 and YAP in hLMVECs, suggesting caspase 3 may localize with YAP signaling proteins. Conclusion Caspase 3 increases hLMVEC proliferation and migration, likely through the activation of YAP. Future studies are warranted to examine the mechanism of how caspase 3 localizes and activates YAP signaling. This abstract is funded by: T32HL007534; R01HL173981
Stevens et al. (Fri,) conducted a other in Acute respiratory distress syndrome (ARDS). Caspase 3 overexpression or inhibition vs. eGFP control or YAP knockdown was evaluated on Cell migration and proliferation. Overexpression of cytosolic sequestered caspase 3 increased human lung microvascular endothelial cell migration and proliferation, an effect that was abolished by YAP knockdown.