Here we show some of the specific SubAB receptors in HeLa cells which directly contribute to cell death. Analysis of SubAB-binding proteins in HeLa cells using immunoprecipitation, purification using lectin, and identification by LC-MS/MS led us to conclude that the membrane proteins responsible for binding to SubAB were α2β1 ITG, NG2, Met, and L1CAM. We previously reported that SubAB bound to α2β1 ITG, leading to vacuole formation in Vero cells (76
). However, we could not demonstrate that α2β1 ITG-dependent signaling induces cell death. It is possible that Neu5Gc-modified proteins differ between Vero cells and HeLa cells. Recently, it was reported that SubAB had a strong preference for binding to glycans terminating in Neu5Gc (7
). It was postulated that a monosaccharide, Neu5Gc, was the SubAB receptor, even though Neu5Gc is not synthesized in humans and protein-bound Neu5Gc is generated by metabolic incorporation of the Neu5Gc contained in food products. In fact, Neu5Gc-modified proteins are abundant in Vero cells compared to their prevalence in HeLa cells (data not shown). Thus, we hypothesize that following β1 ITG knockdown, Vero cells still have low-affinity SubAB-binding proteins containing Neu5Gc which are responsible for cell death. Therefore, we are unable to conclude that α2β1 ITG-dependent signaling induces cell death since β1 ITG-knockdown Vero cells may still have an alternative SubAB-dependent signaling pathway(s). Regarding structural features of receptor proteins required for SubAB-binding activity, neuraminidase treatment of HeLa cell lysate resulted in inhibition of SubAB binding (data not shown). Lectin blot analysis indicated that L1CAM contained O
-glycan and terminal SAα(2-3)-Gal, while NG2, α2β1 ITG, and Met were modified by N
-glycan-linked Gal-β(1-4)-GlcNAc and terminal sialic acids, and the terminal sialic acids of these binding proteins are essential for interaction with SubAB.
At the purification step using M. amurensis
lectin-agarose, the effluents contained not only L1CAM but also NG2, α2β1 ITG, and Met, suggesting the possibility that NG2, α2β1 ITG, or Met bound to M. amurensis
lectin under these undenatured conditions or that these receptors might form a complex in HeLa cells. In fact, these membrane proteins are known to interact or cross talk with each other and regulate various cellular functions. NG2 interacts with the galectin/α3β1 ITG complex on the cell surface, resulting in enhanced β1 ITG signaling, with effects on cell motility, endothelial tube formation in vitro
, and angiogenesis (21
). L1CAM functionally interacts with β1 ITG to potentiate neuronal migration toward extracellular matrix proteins through endocytosis and mitogen-activated protein kinase signaling (69
), and the RGD site in L1CAM involved in the interaction with ITG in tumors is known to play an important role in cell-cell binding, cell motility, invasiveness, and tumor growth (22
). Further, phosphorylated Met formed a complex with β1 ITG and was colocalized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src (33
). Since β1 ITG is a key protein in these cellular pathways, binding of SubAB to β1 ITG may induce interaction or cross talk with other proteins and affect cell movement, focal adhesion, or signal transduction.
To define which binding protein is associated with SubAB-induced apoptosis, we examined in HeLa cells the effect of gene silencing of SubAB-binding proteins on Bax/Bak activation, cytochrome c
release, and caspase-7 activation. We found that SubAB-induced Bax/Bak activation, cytochrome c
release, and apoptosis were significantly and most effectively decreased in β1 ITG-knockdown cells; SubAB-induced BiP cleavage, however, was observed at a level similar to that in control siRNA-transfected cells at the early and late time points, suggesting that SubAB-induced BiP cleavage might be mediated by other SubAB-binding proteins in Triton X-100-insoluble fractions or by terminal sialic acid-modified minor proteins. Similarly, NG2 and L1CAM but not Met siRNA treatment also slightly but significantly inhibited SubAB-induced cell death, cytochrome c
release, and Bax activation but not BiP cleavage. In addition, even siRNA knockdown of four proteins (NG2, β1 ITG, L1CAM, and Met) also significantly inhibited SubAB-induced caspase activation but not BiP cleavage (Fig. ). There are several reports that these SubAB-binding proteins regulate cell death. Stimulation of β1 ITG signal- ing markedly upregulated anchorage-dependent apoptosis (anoikis) of adenocarcinoma cells (52
). NG2, a novel proapoptotic receptor (35
), regulated anoikis of fibroblasts via changes in FAK phosphorylation through a protein kinase Cα-dependent pathway. Further, treatment with L1CAM monoclonal antibody inhibited tumor growth (5
). Thus, these findings suggest that these proteins regulate cell proliferation and death. Our results indicate that SubAB recognizes these proteins as functional receptors and thereby regulates cell death.
Although SubAB-induced BiP cleavage was observed in NG2-, β1 ITG-, L1CAM-. or all four protein-knockdown cells, SubAB-induced apoptosis was significantly decreased to a different extent dependent on each receptor. These results raise the possibility that SubAB-induced cell death requires, in addition to BiP cleavage-induced ER stress, additional signaling pathways resulting from SubAB binding to surface receptors. We demonstrated that Tg-induced caspase activation was not inhibited in β1 ITG-knockdown cells (Fig. ). Thus, our results suggest that SubAB receptors may be regulated by a SubAB-induced apoptotic pathway, which is different from that regulated by Tg. The importance of β1 ITG signaling was demonstrated using MAb P5D2, which is known to inhibit β1 ITG signaling. SubAB-induced caspase-7 activation was significantly enhanced in cells pretreated with MAb P5D2 and clearly decreased in β1 ITG siRNA-treated cells, while BiP cleavage was at a level similar to that in control IgG-treated cells. Caspase-7 activation by mSubAB was not observed in cells pretreated with MAb P5D2, suggesting that β1 ITG-related signaling alone is not sufficient to induce caspase activation. β1 ITG-induced signaling needs to be coupled with BiP cleavage to enhance apoptosis. SubAB binding to NG2 and L1CAM also might alter signal transduction, leading to effects on apoptosis. Differences in SubAB-induced cytotoxicity in each receptor-knockdown cell might depend on the signaling pathway associated with the particular SubAB receptor.
There were several reports of apoptosis in response to ER stress (63
). Bax and Bak operate in both ER and mitochondria as part of an essential gateway for selected apoptotic signals. Interferon gene regulatory element 1 (IRE1) forms a heterotrimeric complex with tumor necrosis factor-associated factor 2 and apoptosis signal-regulating kinase 1 (ASK1), resulting in activation of c-Jun N-terminal protein kinase (JNK), leading to cell death (55
). Our previous work demonstrated that SubAB-induced cell death appears to be induced through Bax/Bak-dependent cytochrome c
release, which was not mediated by the IRE1 or JNK pathway (75
). Here we show that SubAB induced Bax conformational changes and that Bax/Bak complex formation was dramatically suppressed in β1 ITG-knockdown cells. However, it is unclear how, in SubAB-treated HeLa cells, β1 ITG regulates Bax/Bak conformational changes and their oligomerization, cytochrome c
release, and cell death. It was reported that (i) ITG regulates the apoptotic function of Bax through FAK activity, (ii) ITG-mediated positional control is required for maintaining homeostasis, and (iii) defective ITG signaling resulted in apoptosis (24
). We found that FAK was cleaved following BiP cleavage after 30 min of incubation with SubAB but not mutant SubAB (Fig. ). FAK regulates proliferation and migration of normal and tumor cells (12
). It has been proposed that FAK cleavage attenuates its autokinase activity, participates in disassembly of the focal adhesion complex, interrupts survival signals, and then finally promotes cell death (11
). ER-associated degradation (ERAD) has been known to mediate the ER stress-induced decrease in several membrane proteins (27
). Recently, it was reported that ER stress by cis
-hydroxyproline leads to activation of an intracellular proteolytic process, including caspase-independent FAK degradation, resulting in cell damage (53
). We found here that SubAB-induced FAK fragmentation was caused by a proteasome-dependent pathway. Interestingly, SubAB-induced apoptotic activation was inhibited by MG132 treatment at the early time points. Our data indicate that the inhibition of proteasomal degradation of SubAB receptor-related signal molecules including FAK may block SubAB-induced cell death. We proposed that ER stress by SubAB-induced BiP cleavage caused FAK proteolysis and thereby might alter FAK activity in an ITG-regulated signaling pathway, resulting in defective signaling, followed by Bax conformational changes and cell death.
Finally, a proposed model is shown in Fig. . First, SubAB interacts with terminal sialic acids of NG2, L1CAM, α2β1 ITG, or Met. After SubAB is delivered to the ER, SubAB directly cleaves BiP, leading to induction of ER stress signals, which induces activation of the proteasome pathway. At the same time, SubAB binding to NG2, L1CAM, α2β1 ITG, or Met induces signals from each receptor. It remains unclear how SubAB-induced ER stress signals and signals from the receptors affect mitochondria or modify receptor-mediated signaling pathways. However, β1 ITG in HeLa cells is the major receptor for SubAB-induced cell death, and NG2 and L1CAM are also involved, at least in part, in cell death.
Proposed model of SubAB-induced apoptosis signaling pathway in HeLa cells. See text for additional details.