Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.

Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA–mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.

Mitochondria play a pivotal role during stress-induced apoptosis as several proapoptotic proteins are released to the cytosol to activate caspases. Smac/DIABLO is one of the proapoptotic proteins released from the mitochondria and has been shown to inactivate IAPs. However, gene knockout studies in mice revealed a redundant role for Smac during development and cell death. By applying RNA interference-mediated loss of function approach, we demonstrate that Smac/DIABLO is required for the activation of effector but not initiator caspases during stress and receptor-mediated cell death in HeLa cells. Cells with reduced Smac resist apoptosis and retained clonogenicity. Our results suggest an obligatory role for Smac/DIABLO in these tumor cells during several pathways of apoptosis induction.

Protein-tyrosine phosphatases (PTPases) regulate insulin signaling by catalyzing the tyrosine dephosphorylation of the insulin receptor and its substrate proteins. Previous studies have implicated a PTPase localized to a cell membrane fraction in the regulation of the insulin receptor in situ. LAR (leukoyte antigen related) is a transmembrane PTPase in insulin-sensitive tissues with in vitro catalytic specificity for the insulin receptor kinase domain. When transfected into Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-hlR), the LAR protein was processed as expected into an 85-kDa subunit containing the transmembrane and cytoplasmic domains. LAR was increased an average of 6-fold in clonal lines of stably transfected cells, and cell fractionation confirmed its localization in the cell membrane. After stimulation with 100 nM insulin, tyrosine phosphorylation of the insulin receptor was decreased by 31% at 1 min (P < 0.01) and by 42% at 10 min (P < 0.01), and that of IRS-1 was decreased by 34% (P < 0.01) at 1 min and by 56% (P < 0.01) at 10 min in the LAR-overexpressing cells compared with empty vector transfectants. LAR overexpression also blocked insulin-stimulated receptor kinase activation as well as thymidine incorporation into DNA. Quantitatively similar results were obtained in populations of CHO-hlR cells transfected transiently by electroporation. In contrast, overexpression of recombinant LAR cytoplasmic domain, detected as a 72-kDa protein in the cell cytosol, did not significantly affect the insulin-stimulated tyrosine phosphorylation of the insulin receptor or IRS-1 (99% and 93% of control at 10 min, respectively). These studies provide the first evidence that increased expression of LAR has negative regulatory effects at a proximal site in the insulin-signaling pathway. Since this effect occurs only when LAR is eutopically expressed at the cell membrane, these data further suggest that LAR requires a transmembrane localization to directly interact with the insulin receptor in situ.