Lack of Correlation between Activation of Jun–NH2-terminal Kinase and Induction of Apoptosis after Detachment of Epithelial Cells

Detachment of epithelial cells from the extracellular matrix leads to induction of programmed cell death, a process that has been termed “anoikis.” It has been reported recently that detachment of MDCK cells from matrix results in activation of Jun–NH₂-terminal kinases (JNKs) and speculated that these stress activated protein kinases play a causal role in the induction of anoikis (Frisch, S.M., K. Vuori, D. Kelaita, and S. Sicks. 1996. J. Cell Biol. 135:1377–1382). We report here that although JNK is activated by detachment of normal MDCK cells, study of cell lines expressing activated signaling proteins usually controlled by Ras shows that stimulation of JNK fails to correlate with induction of anoikis. Activated phosphoinositide 3-OH kinase and activated PKB/Akt protect MDCK cells from detachment-induced apoptosis without suppressing JNK activation. Conversely, activated Raf and dominant negative SEK1, a JNK kinase, attenuate detachment-induced JNK activation without protecting from apoptosis. zVAD-fmk, a peptide inhibitor of caspases, prevents MDCK cell anoikis without affecting JNK activation. p38, a related stress-activated kinase, is also stimulated by detachment from matrix, but inhibition of this kinase with SB 203580 does not protect from anoikis. It is therefore unlikely that either JNK or p38 play a direct role in detachment-induced programmed cell death in epithelial cells.     

Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop

A sequential kinase cascade culminating in activation of c-Jun N-terminal kinases (JNKs) plays a fundamental role in promoting apoptotic death in many cellular contexts. The mechanisms by which this pathway is engaged in response to apoptotic stimuli and suppressed in viable cells are largely unknown. Here, we show that apoptotic stimuli increase endogenous cellular levels of pathway components, including POSH, mixed lineage kinases (MLKs), and JNK interacting protein 1, and that this effect occurs through protein stabilization and requires the presence of POSH as well as activation of MLKs and JNKs. Our findings suggest a self-amplifying, feed-forward loop mechanism by which apoptotic stimuli promote the stabilization of JNK pathway components, thereby contributing to cell death.     

Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs

A sequential pathway (the JNK pathway) that includes activation of Rac1/Cdc42, mixed lineage kinases, MAP kinase kinases 4 and 7, and JNKs plays a required role in many paradigms of apoptotic cell death. However, the means by which this pathway is assembled and directed toward apoptotic death has been unclear. Here, we report that propagation of the apoptotic JNK pathway requires the cooperative interaction of two molecular scaffolds, POSH and JIPs. POSH (plenty of SH3s) is a multidomain GTP-Rac1-interacting protein that binds and promotes activation of mixed lineage kinases. JIPs are reported to bind MAP kinase kinases 4/7 and JNKs. We find that POSH and JIPs directly associate with one another to form a multiprotein complex, PJAC (POSH-JIP apoptotic complex), that includes all of the known kinase components of the pathway. Our observations indicate that this complex is required for JNK activation and cell death in response to apoptotic stimuli.     

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.     

c-Jun N-terminal kinase regulates apoptosis in endometrial cancer cells

c-Jun N-terminal kinases (JNKs) are important regulators of cell proliferation and apoptosis that have been implicated in tumorigenesis. We investigated the role of JNKs in apoptotic responses in Ishikawa and HEC-50 cells, models of type I and type II endometrial cancer, respectively. Etoposide treatment or UV irradiation resulted in sustained activation of JNK, correlating with the induction of apoptosis. Inhibition of JNK, or MAP kinase kinase 4 (MKK4), selectively suppressed apoptotic responses in both Ishikawa and HEC-50 cells. Knockdown of protein kinase C δ (PKCδ) also attenuated apoptosis in endometrial cancer cells and inhibited the sustained, UV-mediated JNK activation in HEC-50, but not Ishikawa cells. Etoposide-induced JNK phosphorylation was unaffected by PKCδ knockdown, implying that JNK can regulate apoptosis by PKCδ-dependent and independent pathways, according to stimulus and cell type. Thus, expression and activity of JNK and PKCδ in endometrial cancer cells modulate apoptosis and sensitivity to chemotherapeutic agents and may function as tumor suppressors in the endometrium. Elaine M. Reno and James M. Haughian are first authors.     

Ultraviolet light-induced apoptotic death is impaired by the HMG-CoA reductase inhibitor lovastatin

HMG-CoA reductase inhibitors (i.e., statins) attenuate C-terminal isoprenylation of Rho GTPases, thereby inhibiting UV-C-induced activation of c-Jun-N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs). Inhibition of UV-C-triggered JNK/SAPK activation by lovastatin is due to inhibition of Rac-SEK1/MKK4-mediated phosphorylation of JNKs/SAPKs at Thr183/Tyr185. UV-C-stimulated phosphorylation of p38 kinase (Thr180/Tyr182) is also impaired by lovastatin. Cell killing provoked by UV-C irradiation was significantly inhibited by lovastatin. This was paralleled by a reduced frequency of chromosomal aberrations, accelerated recovery from UV-C-induced transient replication blockage, inhibition of Chk1 kinase activation and impaired cyclinB1 expression. Furthermore, UV-C-induced activation of caspases and apoptotic death was largely reduced by lovastatin. Inhibition of JNK/SAPK by transient overexpression of dominant-negative JNK1/SAPK1 also conferred resistance to UV-C light and attenuated activation of caspase 3. Based on the data, we suggest that lovastatin-provoked resistance to UV-C light is due to the inhibition of UV-C-inducible Rac-SEK1/MKK4-JNK/SAPK-dependent signal mechanisms regulating cell cycle progression and activation of caspases and apoptotic death.     

Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.     

Amplification loop cascade for increasing caspase activity induced by docetaxel

The hierarchy of events accompanying induction of apoptosis by the microtubule inhibitor docetaxel was investigated in HL-60 human leukemia cells. Treatment of HL-60 cells with docetaxel resulted in the production of reactive oxygen species (ROS), activation of caspase-3 (-like) protease, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation, bcl-2 phosphorylation and apoptosis. Docetaxel elicited ROS production from NADPH oxidase as demonstrated by specific oxidase inhibitor diphenylene iodonium (DPI). ROS mediated the caspase-3 activation and apoptosis in HL-60 cells. The caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) effectively inhibited JNK/SAPK activation, bcl-2 phosphorylation and partially attenuated the ROS production induced by docetaxel. Docetaxel-induced bcl-2 phosphorylation was completely blocked by expression of dominant negative JNK or the JNK/SAPK inhibitor SP600125. Overexpression of bcl-2 partially prevented docetaxel-mediated ROS production and subsequent caspase-3 activation, thereby inhibiting apoptotic cell death. It is thus conferred that such sequent events as ROS production, caspase activation, JNK/SAPK activation, bcl-2 phosphorylation and the further generation of ROS should be parts of an amplification loop to increase caspase activity, thereby facilitating the apoptotic cell death process.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

Hyperglycemia induces apoptosis of pancreatic islet endothelial cells via reactive nitrogen species-mediated Jun N-terminal kinase activation

Hyperglycemia significantly stimulates pancreatic islet endothelial cell apoptosis; however, the precise mechanisms are not fully understood. In the present study, treating pancreatic islet endothelial (MS-1) cells with high glucose (30 mmol/l) but not mannitol significantly increased the number of apoptotic cells as compared with a physiological glucose concentration (5.5 mmol/l). Hyperglycemia significantly stimulated the expression of inducible nitric oxide synthase (iNOS) and production of NO and peroxynitrite (ONOO⁻), relevant to MS-1 cell apoptosis. Moreover, induced reactive nitrogen species (RNS) significantly increased the expression of bax, cleaved caspase-3 and poly adenosine diphosphate (ADP)-ribose polymerase (PARP) via JNK activation, but the expression of bcl-2 was not altered. Furthermore, SP600125 (a specific inhibitor of JNK) and 1400W (a specific inhibitor of iNOS) significantly attenuated cell apoptosis induced by high glucose. Therefore, hyperglycemia triggers MS-1 cell apoptosis by activating an intrinsic-dependent apoptotic pathway via RNS-mediated JNK activation.     

Effect of Reactive Oxygen Species Generation in Rabbit Corneal Epithelial Cells on Inflammatory and Apoptotic Signaling Pathways in the Presence of High Osmotic Pressure

It is generally accepted that high osmotic pressure (HOP) of lacrimal fluid is the core mechanism causing ocular inflammation and injury. However, the association between HOP and the regulation of cell inflammatory response and apoptotic pathways remains unclear. In the present study, we used HOP to interfere with in vitro cultured rabbit corneal epithelial cells, and found that HOP increased the generation of reactive oxygen species (ROS) in rabbit corneal epithelial cells, and increased ROS in turn induced the activation of JNK inflammatory signaling pathway, which further promoted the expression of pro-inflammatory factor NF-κβ and induced the generation of inflammatory factor IL-1β and TNF-α. In addition, HOP-induced ROS in rabbit corneal epithelial cells regulated the CD95/CD95L-mediated cell apoptotic signaling pathway by activating JNK inflammatory signaling pathway. These findings may serve as new theoretical basis and a new way of thinking about the treatment of ocular diseases, especially dry eye.     

The impact of trisomy 21 on early human hematopoiesis

Mitogen-activated protein kinases (MAPKs) are components of signaling cascades regulated by environmental stimuli. In addition to participating in the stress response, the MAPKs c-Jun N-terminal Kinases JNK1 and JNK2 regulate the proliferation of normal and neoplastic cells. JNKs contribute to these processes largely by phosphorylating c-Jun and thus contributing to the activation of the AP-1 complex. We here report that JNKs control entry into mitosis. We have observed that JNK activity and phosphorylation of c-Jun become elevated during the G2/M transition of the cell cycle in immortalized fibroblasts and ovarian granulosa cells. Pharmacological inhibition of JNK causes a profound cell cycle arrest at the G2/M transition in both cell types. This effect is specific as it occurs with two distinct small molecule compounds. Inactivation of JNK prior to mitosis prevents expression of Aurora B and phosphorylation of Histone-H3 at Ser 10. Silencing of JNK1 and 2 causes a similar effect, whereas overexpression of JNK1 and 2 causes the opposite effect. Inhibition of JNK delays activation of cdc-2 and prevents downregulation of Cyclin B1. We conclude that JNK signaling promotes entry into mitosis by promoting expression of Aurora B and thereby phosphorylation of Histone-H3.     

Regulation of the protein stability of POSH and MLK family

Sequential activation of the JNK pathway components, including Rac1/Cdc42, MLKs (mixed-lineage kinases), MKK4/7 and JNKs, plays a required role in many cell death paradigms. Those components are organized by a scaffold protein, POSH (Plenty of SH3’s), to ensure the effective activation of the JNK pathway and cell death upon apoptotic stimuli. We have shown recently that the expression of POSH and MLK family proteins are regulated through protein stability. By generating a variety of mutants, we provide evidence here that the N-terminal half of POSH is accountable for its stability regulation and its over-expression-induced cell death. In addition, POSH’s ability to induce apoptosis is correlated with its stability as well as its MLK binding ability. MLK family’s stability, like that of POSH, requires activation of JNKs. However, we were surprised to find out that the widely used dominant negative (d/n) form of c-Jun could down-regulate MLK’s stability, indicating that peptide from d/n c-Jun can be potentially developed into a therapeutical drug.     

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.     

Induction of Apoptosis after Expression of PYK2, a Tyrosine Kinase Structurally Related to Focal Adhesion Kinase

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell–ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase β (CAKβ) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFα and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH₂-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.     

RIP5 is a RIP-homologous inducer of cell death

Members of the RIP serine/threonine kinase family are involved in activation of NF-kappaB, JNK, and p38, and induction of apoptosis. Here we report the identification of a novel RIP-homologous protein designated as RIP5. The C-terminus of RIP5 contains a kinase domain, which is mostly homologous with the kinase domain of RIP. RIP5 also contains a large unconserved N-terminal domain. Overexpression of RIP5 induces cell death with characteristic apoptotic morphology. Overexpression of RIP5 also induces DNA fragmentation and this is blocked by the caspase inhibitor crmA. However, RIP5-induced apoptotic morphology is not blocked by crmA. These findings suggest that RIP5 may induce both caspase-dependent apoptosis and caspase-independent cell death.     

Two independent signaling pathways mediate the antiapoptotic action of macrophage-stimulating protein on epithelial cells

In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a growth and motility factor for epithelial cells. The growth and survival of epithelial cells generally require two signals, one generated by interaction with extracellular matrix via integrins, the other initiated by a growth factor. Therefore we investigated the effect of MSP on epithelial cell survival. Survival of epithelial cells cultured overnight in serum-free medium was promoted by adhesion, which activated both the phosphatidylinositol 3'-kinase (PI3-K)/AKT and mitogen-activated protein kinase (MAPK) pathways, operating independently of one another. The number of apoptotic cells resulting from inhibition of either pathway alone was approximately doubled by simultaneous inhibition of both pathways. This shows that each pathway made a partial contribution to the prevention of apoptosis. In the presence of an inhibitor of either pathway, MSP increased the activity of the other pathway so that the single uninhibited pathway alone was sufficient to prevent apoptosis. In contrast to the results with adherent cells, although MSP also prevented apoptosis of cells in suspension (anoikis), its effect was mediated only by the PI3-K/AKT pathway. Despite activation of MAPK by MSP, anoikis was not prevented in suspended cells with a blocked PI3-K/AKT pathway. Thus, activation of MAPK alone is not sufficient to mediate MSP antiapoptotic effects. Cell adhesion generates an additional signal, which is essential for MSP to use MAPK in an antiapoptotic pathway. This may involve translocation of MSP-activated MAPK from the cytoplasm into the nucleus, which occurs only in adherent cells. Our results suggest that there is cross talk between cell matrix adhesion and growth factors in the regulation of cell survival via the MAPK pathway. Growth factors induce MAPK activation, and adhesion mediates MAPK translocation from the cytoplasm into the nucleus.     

Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway

Normal epithelial cells undergo apoptosis when they are denied contact with the extracellular matrix, in a process termed "anoikis." Conversely, malignant epithelial cells typically acquire anchorage independence, i.e., the capacity to survive and grow in the absence of matrix interaction. Here we asked the question whether anoikis is affected by signaling through the EGF receptor (EGFR). We focused on the EGFR because EGFR signaling is frequently deregulated in malignant epithelial cells. We demonstrate that EGFR activation markedly alleviated the requirement of matrix engagement for survival of primary and immortalized human keratinocytes in suspension culture. Protection of epithelial cells through EGFR activation against anoikis was associated with and required sustained MAPK phosphorylation during the early phase of suspension culture. Interestingly, high levels of MAPK phosphorylation were not only required for EGFR-mediated protection against anoikis but also occurred as a consequence of caspase activation at later stages of suspension culture. These results demonstrate that EGFR activation contributes to anchorage-independent epithelial cell survival and identify MAPK activation as an important mechanism in this process.