Functional role of death-associated protein 3 (DAP3) in anoikis

Detachment of adherent epithelial cells from the extracellular matrix induces apoptosis, known as anoikis. Integrin stimulation protects cells from anoikis, but the responsible mechanisms are not well known. Here, we demonstrated that a pro-apoptotic GTP-binding protein, DAP3 (death-associated protein 3), is critical for induction of anoikis. Down-regulation of DAP3 expression by antisense oligonucleotides inhibited anoikis. Conversely, overexpression of DAP3 augmented cell death and caspase activation induced by cell detachment. Furthermore, the association of DAP3 with FADD and the activation of caspase-8 were induced by cell detachment. We also showed that DAP3 is phosphorylated by kinase Akt (PKB), and active Akt can nullify apoptosis induction by DAP3. Mutation of a consensus Akt phosphorylation site in DAP3 renders it resistant to suppression by active Akt in cells. Integrin ligation stimulates Akt activation and phosphorylation of DAP3 in intact cells, as well as suppresses the ability of DAP3 overexpression to augment anoikis. Involvement of DAP3 in anoikis signaling demonstrates a novel role for this GTP-binding protein in apoptosis induction caused by cell detachment.     

Identification of DELE,a novel DAP3-binding protein which is crucial for death receptor-mediated apoptosis induction

Death associated protein 3 (DAP3) is known to be a highly conserved protein, and is responsible for regulating apoptosis induced by various stimuli. To understand the molecular mechanism of how DAP3 induces apoptosis, we performed yeast two-hybrid screening, and identified a novel DAP3-binding protein termed death ligand signal enhancer (DELE). In this report, we show that DELE actually binds to DAP3 in mammalian cells. We found that the cells stably expressing DELE are susceptible to apoptosis induction by the stimulation of TNF-α and TRAIL. In addition, knockdown of DELE expression rescued the HeLa cells from apoptosis induction by these stimuli. Moreover, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF-α, anti-Fas or TRAIL was significantly inhibited by the knockdown of DELE expression. These results demonstrated the biological significance of DELE for apoptosis signal mediated by death receptors.     

Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha.

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon-gamma- and Fas/APO-1-induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF-alpha-induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady-state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well-known endoprotease plays an active role in cytokine-induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.     

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.     

Autophagy gets a brake: DAP1, a novel mTOR substrate,is activated to suppress the autophagic process

Autophagy, a highly regulated catabolic process, is controlled by the action of positive and negative regulators. While many of the positive mediators of autophagy have been identified, very little is known about negative regulators that might counterbalance the process. We recently identified death-associated protein 1 (DAP1) as a suppressor of autophagy and as a novel direct substrate of mammalian target of rapamycin (mTOR). We found that DAP1 is functionally silent in cells growing under rich nutrient supplies through mTOR-dependent inhibitory phosphorylation on two sites, which were mapped to Ser3 and Ser51. During amino acid starvation, mTOR activity is turned off resulting in a rapid reduction in the phosphorylation of DAP1. This caused the conversion of the protein into a suppressor of autophagy, thus providing a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under conditions of nutrient deprivation. Based on these studies we propose the “gas and brake” concept in which mTOR, the main sensor that regulates autophagy in response to amino acid deprivation, also controls the activity of a specific balancing brake to prevent the overactivation of autophagy.Key words: DAP1, mTOR, autophagy, amino acid starvation, phosphorylationIn recent years, many of the genes controlling and executing the autophagic process have been identified. Most of these genes act as positive mediators of the various steps of the process, including the ULK1 complex, which regulates the induction step, the Vps34-Beclin 1 complex that participates in the vesicle nucleation step and two ubiquitin-like pathways, the Atg12-Atg5 and the LC3-phosphatidylethanolamine (PE) conjugation steps, which play a central role in the vesicle elongation process. To date, only a few negative regulators of autophagy have been identified, including mTOR and the anti-apoptotic Bcl-2 family members. mTOR Ser/Thr kinase is a central suppressor of autophagy acting at the initiating regulatory steps of the process. Many signaling pathways act to inhibit mTOR activity, thus relieving its inhibitory effects on autophagy. The anti-apoptotic Bcl-2 and Bcl-X_L proteins, on the other hand, act at the nucleation step, by directly binding to Beclin 1''s BH3 domain, thus reducing the activation of Vps34 and subsequent autophagy. This inhibition can be relieved through dissociation of the complex, following either JNK-1 mediated phosphorylation of Bcl-2 or DAP kinase-mediated phosphorylation of the BH3 domain of Beclin 1.DAP1 is a small (∼15 kDa), ubiquitously expressed protein, rich in prolines and lacking known functional motifs. DAP1 was isolated more than a decade ago in our laboratory using a functional approach to gene cloning aimed at identifying novel mediators of IFNγ-induced cell death in mammalian cell cultures. Until recently, very little was known about the cellular and molecular functions of DAP1, mainly due to the lack of homology to other known proteins and the lack of functional motifs that could indicate a possible cellular function and studies in mammalian systems were missing.Recently, we discovered that DAP1 is another negative regulator of autophagy; yet, interestingly, its suppressive activity is selectively turned on during the autophagic process. Moreover, we found that DAP1 suppressive activity is tightly linked to the status of mTOR kinase activity. Under nutrient-rich culture conditions, DAP1 is phosphorylated by mTOR on two sites, Ser3 and Ser51, resulting in its inactivation. In response to nutrient deprivation, mTOR is inhibited and DAP1 undergoes rapid dephosphorylation. By knocking down the endogenous DAP1 and introducing either the phosphomimetic or the nonphosphorylatible DAP1 mutants, we found that the dephosphorylation leads to activation of the autophagic suppressive function of DAP1, whereas the phophorylated form is inactive. These results led to a “gas and brake” model, in which at the same time that autophagy is induced, some brakes such as DAP1 are also activated to provide a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under nutrient-deprivation conditions (Fig. 1). Notably, balancing autophagy is extremely important, since deregulated or excessive autophagy has been implicated in the pathogenesis of diverse diseases, such as certain types of neuronal degeneration and cancer and also in cellular aging.Open in a separate windowFigure 1“Gas and brake” model. During nutrient-rich conditions, active mTORC1 phosphorylates and inactivates the components of the ULK1 complex, ULK1 and Atg13, thus preventing the induction of autophagy. DAP1 is also inactivated simultaneously by mTORC1-mediated phosphorylation on Ser3 and Ser51. In addition, mTORC1 phosphorylates and activates p70S6K and 4E-BP1, which mediate the protein translation and cell growth activities of mTOR. Upon nutrient starvation, mTORC1 activity is attenuated, leading to dephosphorylation and activation of ULK1. ULK1, in turn, undergoes autophosphorylation and phosphorylates Atg13 and FIP200 resulting in ULK1 complex activation and induction of autophagy. On the other hand, activation of DAP1 by dephosphorylation, results in suppression of autophagy, thus inserting a brake into the process of autophagy. Note that the inactive proteins/complexes are faded out.The current challenge is to identify the molecular basis of the suppressive functions of DAP1 on autophagy. We have recently shown that DAP1 knockdown enhances LC3 lipidation and autophagosome accumulation both during amino acid starvation and rapamycin treatment. In addition, preliminary data indicate that the knockdown of DAP1 has no effect on mTOR complex 1 (mTORC1) activity in cells, at least during the first hours of starvation. Accordingly, DAP1 may function between the mTORC1 and the LC3 conjugation systems. The potential targets may fall into one of the multiprotein complexes functioning downstream of mTOR such as the ULK1 complex, the Vps34-Beclin 1 complex and more. Future studies will be performed to identify the molecular mechanism by which DAP1 suppresses autophagy. The lack of known functional motifs in the DAP1 protein sequence suggests that this small proline-rich protein may function as an adaptor blocking autophagy by binding to critical protein partners that still await identification.Although autophagy is primarily a protective process for the cell, it can also play a role in cell death. In response to prolonged starvation, autophagy can act either as a cell survival mechanism or be recruited as a cell death executer. In the future it would be interesting to examine whether the autophagy enhancement resulting from DAP1 knockdown contributes to increased cell death in our system or even may convert the survival properties of autophagy into death induction. This will fit the “gas and brake” model, in which autophagy, which is initially recruited as a cell survival mechanism, is converted into cell death machinery when a certain threshold is crossed due to the loss of the “brake” by the knockdown of DAP1.To date, very little is known about the putative mechanisms that restrict the intensity of the autophagic flux to maintain the continuous benefits of this process under stress. Therefore, the ability of DAP1 to counterbalance and buffer the process in a manner that is tightly linked to the status of a central player in autophagy (i.e., mTOR) is an important discovery in this field and provides a target for future drug design.     

A novel form of DAP5 protein accumulates in apoptotic cells as a result of caspase cleavage and internal ribosome entry site-mediated translation

Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5' untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.     

Death-associated protein 3 localizes to the mitochondria and is involved in the process of mitochondrial fragmentation during cell death

Death-associated protein 3 (DAP3) was previously isolated in our laboratory as a positive mediator of cell death. It is a 46-kDa protein containing a GTP binding domain that was shown to be essential for the induction of cell death. DAP3 functions downstream of the receptor signaling complex, and its death-promoting effects depend on caspase activity. Recent reports have suggested that DAP3 is localized to the mitochondria, but no functional significance of this localization has been reported so far. Here, we study the sub-cellular localization and cellular function of human DAP3 (hDAP3). We found that hDAP3 is localized to the mitochondria and, in contrast to cytochrome c, is not released to the cytoplasm following several cell death signals. Overexpression of hDAP3 induced dramatic changes in the mitochondrial structure involving increased fragmentation of the mitochondria. Both the mitochondrial localization of hDAP3 and its GTP-binding activity were essential for the fragmentation. The punctiform mitochondrial morphology was similar to that observed upon treatment of HeLa cells with staurosporine. In fact, reduction of endogenous hDAP3 protein by RNA interference partially attenuated staurosporine-induced mitochondrial fission. Thus, hDAP3 is a necessary component in the molecular pathway that culminates in fragmented mitochondria, probably reflecting its involvement in the fission process. These results, for the first time, provide a specific functional role for hDAP3 in mitochondrial maintenance.     

TRAIL-induced apoptosis is independent of the mitochondrial apoptosis mediator DAP3

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and the adapter protein Fas-associated death domain protein (FADD). Recently, an adapter function for death-associated protein 3 (DAP3) between DR4/DR5 and FADD has been proposed. However, DAP3 has been reported to be a ribosomal protein localized to the mitochondrial matrix. To address these discrepancies, the intracellular localization of DAP3 after apoptosis induction in human T-lymphocytes with recombinant TRAIL was analyzed. DAP3, in contrast to cytochrome c, remained intra-mitochondrial during apoptosis. No interaction between FADD and DAP3 after cell fractionation could be detected as long as subcellular compartments remained intact. Only whole cell lysate co-immunoprecipitation revealed an ex vivo interaction between DAP3 and FADD. Therefore, DAP3 and FADD interact only in vitro after disruption of the cellular compartments. TRAIL-induced and DR4-mediated apoptosis in Jurkat cells is independent of DAP3.     

Death-associated protein 3 regulates cellular senescence through oxidative stress response

Death-associated protein 3 (DAP3) has been originally identified as a positive mediator of apoptosis. It has been revealed recently that the predominant localization of DAP3 to mitochondria implies its functional involvement in mitochondrial metabolism in addition to apoptosis. However, little is known about the molecular basis of these physiological functions of DAP3. Here, we demonstrate that DAP3 is reduced in both replicative and premature senescence induced by oxidative stress, and the DAP3 reduction induced by oxidative stress is observed mostly in a mitochondrial fraction. Using DAP3-specific short hairpin RNA (shRNA) in a clonogenic survival assay, we reveal that reduction of DAP3 induces resistance to oxidative stress and decreases intracellular reactive oxygen species (ROS) production. Furthermore, this strategy allows us to show that loss of DAP3 is involved in the avoidance of replicative senescence in mouse embryonic fibroblasts (MEFs). Thus, our study offers an insight into the potential regulatory function of mitochondrial DAP3 involved in cellular senescence.     

Noncanonical cell death programs in the nematode Caenorhabditis elegans

Genetic studies of the nematode Caenorhabditis elegans have uncovered four genes, egl-1 (BH3 only), ced-9 (Bcl-2 related), ced-4 (apoptosis protease activating factor-1), and ced-3 (caspase), which function in a linear pathway to promote developmental cell death in this organism. While this core pathway functions in many cells, recent studies suggest that additional regulators, acting on or in lieu of these core genes, can promote or inhibit the onset of cell death. Here, we discuss the evidence for these noncanonical mechanisms of C. elegans cell death control. We consider novel modes for regulating the core apoptosis genes, and describe a newly identified cell death pathway independent of all known C. elegans cell death genes. The existence of these noncanonical cell death programs suggests that organisms have evolved multiple ways to ensure appropriate cellular demise during development.     

Death-associated protein kinase-related protein 1, a novel serine/threonine kinase involved in apoptosis

In this study we describe the identification and structure-function analysis of a novel death-associated protein (DAP) kinase-related protein, DRP-1. DRP-1 is a 42-kDa Ca(2+)/calmodulin (CaM)-regulated serine threonine kinase which shows high degree of homology to DAP kinase. The region of homology spans the catalytic domain and the CaM-regulatory region, whereas the remaining C-terminal part of the protein differs completely from DAP kinase and displays no homology to any known protein. The catalytic domain is also homologous to the recently identified ZIP kinase and to a lesser extent to the catalytic domains of DRAK1 and -2. Thus, DAP kinase DRP-1, ZIP kinase, and DRAK1/2 together form a novel subfamily of serine/threonine kinases. DRP-1 is localized to the cytoplasm, as shown by immunostaining and cellular fractionation assays. It binds to CaM, undergoes autophosphorylation, and phosphorylates an exogenous substrate, the myosin light chain, in a Ca(2+)/CaM-dependent manner. The truncated protein, deleted of the CaM-regulatory domain, was converted into a constitutively active kinase. Ectopically expressed DRP-1 induced apoptosis in various types of cells. Cell killing by DRP-1 was dependent on two features: the status of the catalytic activity, and the presence of the C-terminal 40 amino acids shown to be required for self-dimerization of the kinase. Interestingly, further deletion of the CaM-regulatory region could override the indispensable role of the C-terminal tail in apoptosis and generated a "superkiller" mutant. A dominant negative fragment of DAP kinase encompassing the death domain was found to block apoptosis induced by DRP-1. Conversely, a catalytically inactive mutant of DRP-1, which functioned in a dominant negative manner, was significantly less effective in blocking cell death induced by DAP kinase. Possible functional connections between DAP kinase and DRP-1 are discussed.     

Death-associated Protein 3 Regulates Mitochondrial-encoded Protein Synthesis and Mitochondrial Dynamics

Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.     

DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain.

Death-associated protein (DAP)-kinase is a calcium/calmodulin regulated serine/threonine kinase that carries ankyrin repeats, a death domain, and is localized to the cytoskeleton. Here, we report that this kinase is involved in tumor necrosis factor (TNF)-alpha and Fas-induced apoptosis. Expression of DAP-kinase antisense RNA protected cells from killing by anti-Fas/APO-1 agonistic antibodies. Deletion of the death domain abrogated the apoptotic functions of the kinase, thus, documenting for the first time the importance of this protein domain. Overexpression of a fragment encompassing the death domain of DAP-kinase acted as a specific dominant negative mutant that protected cells from TNF-alpha, Fas, and FADD/MORT1-induced cell death. DAP-kinase apoptotic function was blocked by bcl-2 as well as by crmA and p35 inhibitors of caspases, but not by the dominant negative mutants of FADD/MORT1 or of caspase 8. Thus, it functions downstream to the receptor complex and upstream to other caspases. The multidomain structure of this serine/threonine kinase, combined with its involvement in cell death induced by several different triggers, place DAP-kinase at one of the central molecular pathways leading to apoptosis.     

Critical function of death-associated protein 3 in T cell receptor-mediated apoptosis induction

Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.     

Down-regulation of statin,a nonproliferation-specific nuclear protein,and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G₁ phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G₁ phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley-Liss, Inc.     

Expression of Cell Cycle-Related Genes During Neuronal Apoptosis: Is there a Distinct Pattern?

An emerging hypothesis considers the process of neuronal apoptosis as a consequence of unscheduled and unsynchronized induction of cell cycle mediators. Induction of several cell cycle genes precedes neuronal apoptosis and may be involved in determination of cell fate. We have now characterized changes in expression of cell cycle genes during apoptosis induced by oxidative stress in chick post-mitotic sympathetic neurons. Induction of cyclin B occurred prior to the commitment of neurons to both dopamine- and peroxide-triggered apoptosis. Both the neuronal death and the rise in cyclin B were inhibited by antioxidant treatment, suggesting a functional role for cyclin B induction during neuronal apoptosis. Induction of the cyclin dependent kinase CDK5 protein coincided with the time point when neurons were irreversibly committed to die. Expression of other cell cycle mediators such as cyclin D1 and the cyclin dependent kinases CDC2 and CDK2 was undetected and not induced by exposure to oxidative stress. Comparative analysis of the profile of cell cycle mediators induced during neuronal apoptosis of different neuronal cell populations revealed no distinct pattern of events. There are no cell cycle stage-specific mediators that are ultimately stimulated during neuronal apoptosis, suggesting that multiple pathways of re-activating the dormant cell-cycle, converge to determine entry into apoptosis. Nevertheless, the existence of some cell cycle mediators, that were not reported so far to be induced in post mitotic neurons during oxidative stress, substantiate them as part of the strong differentiating forces.     

Tumor Necrosis Factor-α-Induced Apoptosis in a Human Keratinocyte Cell Line (HaCaT) Is Counteracted by Transforming Growth Factor-α

The integrity of the human epidermis is guaranteed by a regulated balance of proliferation, differentiation, and physiologic cell death of its main cellular constituent, the epidermal keratinocyte. Physiologic cell death is known as apoptosis and has been recognized as an active regulatory mechanism, complementary to, but functionally opposite of, proliferation. The regulators of the delicate balance between cell death and proliferation are only partially understood in human keratinocytes. Transforming growth factor-α (TGF-α) has been identified as a positive regulator of proliferation and growth, while tumor necrosis factor-α (TNF-α) induces apoptosis. Both mediators are thought to influence epidermal keratinocytes under various physiological and pathophysiological conditions. In the current study we have begun to investigate potential regulatory interactions between these two mediators in the human keratinocyte cell line HaCaT. We have found that, when the HaCaT cells were sensitized by the translation inhibitor cycloheximide, TNF-α induced apoptosis, as evidenced by nuclear disintegration, DNA fragmentation (“DNA laddering”), and the appearance of soluble DNA/histone complexes. Moreover, we found that the induction of apoptosis was reduced by preincubation of the cells with TGF-α. The protective effect of TGF-α was abrogated by translation inhibition, indicating that it depended onde novoprotein synthesis. Moreover, the protective effect was not accompanied by a reduced surface expression of TNF receptor molecules. We postulate that TNF-α-induced apoptosis in HaCaT cells is counteracted by constitutively produced suppressors of apoptosis, the synthesis of which can be downregulated by inhibition of translation and upregulated by the cytokine TGF-α.