The Src Family Tyrosine Kinases yes and src Have Divergent Effects on Cytokine‐Induced Apoptosis in Pulmonary Endothelial Cells Through Divergent Downstream Effects on PI3K and ERK Pathways

Abstract

In pulmonary endothelial cell culture studies we found that the Src family tyrosine kinase (STK) yes activated caspase 3, while the STK src inhibited caspase 3 activation. We hypothesized that the divergent effects of yes and src were due to their differential effects on the downstream activation of extracellular signal‐regulated kinase (ERK) and/or phosphatidylinositol‐3‐kinase (PI3K) pathway. To test this hypothesis, human pulmonary microvascular endothelial cells (hPMVEC) were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls for 24 hours. After a 24 hour recovery period the hPMVEC were treated with cytomix (LPS, IL‐1β, TNF‐α, and IFN‐γ). Cytomix treatment increased levels of phospho‐ERK (pERK). Treatment with siRNAyes enhanced cytomix‐induced pERK levels, while siRNAsrc blunted the cytomix‐induced pERK levels. Inhibiting the ERK pathway using U0126 prevented the cytomix‐induced increase in pERK and increased cytomix‐induced caspase 3 activity in hPMVEC. Treating hPMVEC with cytomix induced Akt activation which was inhibited by siRNAsrc, while siRNAyes had little effect on Akt activity. Inhibiting PI3K using LY294002 prevented cytomix‐induced ERK activation and augmented cytomix‐induced cleaved caspase 3 levels. Taken together our data demonstrate that yes activation blunts the ERK cascade in response to cytomix resulting in greater apoptosis, while cytomix‐induced src activation induces PI3K pathway which in turn activates Akt and ERK to inhibit apoptosis.