Syndecan-4 is a signaling molecule for stromal cell-derived factor-1 (SDF-1)/ CXCL12

Stromal cell-derived factor-1 (SDF-1)/CXCL12, the ligand for CXCR4, induces signal transduction. We previously showed that CXCL12 binds to high- and low-affinity sites expressed by primary cells and cell lines, and forms complexes with CXCR4 as expected and also with a proteoglycan, syndecan-4, but does not form complexes with syndecan-1, syndecan-2, CD44 or beta-glycan. We also demonstrated the occurrence of a CXCL12-independent heteromeric complex between CXCR4 and syndecan-4. However, our data ruled out the glycosaminoglycan-dependent binding of CXCL12 to HeLa cells facilitating the binding of this chemokine to CXCR4. Here, we demonstrate that CXCL12 directly binds to syndecan-4 in a glycosaminoglycan-dependent manner. We show that upon stimulation of HeLa cells by CXCL12, CXCR4 becomes tyrosine phosphorylated as expected, while syndecan-4 (but not syndecan-1, syndecan-2 or beta-glycan) also undergoes such tyrosine phosphorylation. Moreover, tyrosine-phosphorylated syndecan-4 from CXCL12-stimulated HeLa cells physically coassociates with tyrosine phosphorylated CXCR4. Pretreatment of the cells with heparitinases I and III prevented the tyrosine phosphorylation of syndecan-4, which suggests that the heparan sulfate-dependent binding of SDF-1 to this proteoglycan is involved. Finally, by reducing syndecan-4 expression using RNA interference or by pretreating the cells with heparitinase I and III mixture, we suggest the involvement of syndecan-4 and heparan sulfate in p44/p42 mitogen-activated protein kinase and Jun N-terminal/stress-activated protein kinase activation by action of CXCL12 on HeLa cells. However, these treatments did not modify the calcium mobilization induced by CXCL12 in these cells. Therefore, syndecan-4 behaves as a CXCL12 receptor, selectively involved in some transduction pathways induced by SDF-1, and heparan sulfate plays a role in these events.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.     

Heparan sulfate chains of syndecan-1 regulate ectodomain shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.     

Cooperation of syndecan-2 and syndecan-4 among cell surface heparan sulfate proteoglycans in the actin cytoskeletal organization of Lewis lung carcinoma cells

Syndecan-2 cooperates with integrin alpha 5 beta 1 in cell adhesion to a fibronectin substratum and regulates actin cytoskeletal organization in an expression level-dependent manner; Lewis lung carcinoma-derived P29 cells with high expression form stress fibers, whereas the same tumor-derived low expressers, LM66-H11 cells, form cortex actin [Munesue, S., Kusano, Y., Oguri, K., Itano, N., Yoshitomi, Y., Nakanishi, H., Yamashina, I., and Okayama, M. (2002) BIOCHEM: J. 363, 201-209]. In this study we examined the participation of other cell surface heparan sulfate proteoglycans in this signaling. The two clones expressed syndecan-1, -2 and -4, and glypican-1 at similar levels except for syndecan-2. Treatment of cells with phosphatidylinositol-specific phospholipase C or immobilized anti-syndecan-1 antibodies demonstrated that neither glypican-1 nor syndecan-1 was involved in this signaling, indicating that individual cell surface heparan sulfate proteoglycans have functional specificity. Stimulation with immobilized anti-syndecan-2 or -4 antibodies induced stress fiber formation in P29 cells but not in LM66-H11 cells, despite the similar levels of syndecan-4 expression, suggesting that stress fiber formation required a threshold expression level of syndecan-2 acting downstream of syndecan-4. This was confirmed by cells in which syndecan-2 expression was artificially suppressed by antisense mRNA oligonucleotide treatment or elevated by cDNA transfection. This is the first report demonstrating that syndecan-2 and -4 cooperate in situ in actin cytoskeletal organization.     

Cleavage of syndecan-4 by ADAMTS1 provokes defects in adhesion

Syndecan-4 is a membrane-bound heparan sulfate proteoglycan that participates in cell-cell and cell-matrix interactions and modulates adhesion and migration of many cell types. Through its extracellular domain, syndecan-4 cooperates with adhesion molecules and binds matrix components relevant for cell migration. Importantly, syndecan-4 is a substrate of extracellular proteases, however the biological significance of this cleavage has not been elucidated. Here, we show that the secreted metalloprotease ADAMTS1, involved in angiogenesis and inflammatory processes, cleaves the ectodomain of syndecan-4. We further showed that this cleavage results in altered distribution of cytoskeleton components, functional loss of adhesion, and gain of migratory capacities. Using syndecan-4 null cells, we observed that ADAMTS1 proteolytic action mimics the outcome of genetic deletion of this proteoglycan with regards to focal adhesion. Our findings suggest that the shedding of syndecan-4 by ADAMTS1 disrupts cell adhesion and promotes cell migration.     

Diverse repertoire of HIV-1 p24-specific,IFN-gamma-producing CD4+ T cell clones following immune reconstitution on highly active antiretroviral therapy

HIV-1 Ag-specific CD4(+) T cell proliferative responses in human subjects with advanced, untreated HIV-1 disease are often weak or undetectable. Conversely, HIV-1-specific CD4(+) T cell proliferation is occasionally detected following suppression of HIV-1 replication with highly active antiretroviral therapy (HAART). These observations suggest that unchecked HIV-1 replication may lead to depletion or dysfunction of HIV-1-specific CD4(+) T cells, and that these defects may be partially corrected by viral suppression and subsequent immune reconstitution. However, the impact of this immune reconstitution on the repertoire of HIV-1-specific CD4(+) T cells has not been thoroughly evaluated. To examine the HIV-1-specific CD4(+) T cell repertoire in this clinical setting, we established HIV-1 p24-specific CD4(+) T cell clones from a successfully HAART-treated subject whose pretreatment peripheral CD4 count was 0 cells/ micro l. Eleven different p24-specific CD4(+) T cell clonotypes were distinguished among 13 clones obtained. Most clones produced both IFN-gamma and IL-4 upon Ag stimulation. Clones targeted eight distinct epitopes that varied in their conservancy among HIV-1 strains, and responses were restricted by one of three MHC II molecules. Clones showed a range of functional avidities for both protein and peptide Ags. Additional studies confirmed that multiple HIV-1 p24-derived epitopes were targeted by IFN-gamma-producing CD4(+) cells from subjects first treated with HAART during advanced HIV-1 disease (median, 4.5 peptides/subject; range, 3-6). These results suggest that in HAART-treated subjects whose peripheral CD4(+) T cell pools were once severely depleted, the HIV-1-specific CD4(+) T cell repertoire may include a diverse array of clonotypes targeting multiple HIV-1 epitopes.     

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.     

HIV-1 Nef enhances dendritic cell-mediated viral transmission to CD4+ T cells and promotes T-cell activation

HIV-1 Nef enhances dendritic cell (DC)-mediated viral transmission to CD4(+) T cells, but the underlying mechanism is not fully understood. It is also unknown whether HIV-1 infected DCs play a role in activating CD4(+) T cells and enhancing DC-mediated viral transmission. Here we investigated the role of HIV-1 Nef in DC-mediated viral transmission and HIV-1 infection of primary CD4(+) T cells using wild-type HIV-1 and Nef-mutated viruses. We show that HIV-1 Nef facilitated DC-mediated viral transmission to activated CD4(+) T cells. HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+) T cells. However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+) T cells. Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+) T cells and in the activation and proliferation of resting CD4(+) T cells, which likely contribute to viral pathogenesis.     

Human immunodeficiency virus type 1 induces apoptosis in CD4(+) but not in CD8(+) T cells in ex vivo-infected human lymphoid tissue

Progression of human immunodeficiency virus (HIV) disease is associated with massive death of CD4(+) T cells along with death and/or dysfunction of CD8(+) T cells. In vivo, both HIV infection per se and host factors may contribute to the death and/or dysfunction of CD4(+) and CD8(+) T cells. Progression of HIV disease is often characterized by a switch from R5 to X4 HIV type 1 (HIV-1) variants. In human lymphoid tissues ex vivo, it was shown that HIV infection is sufficient for CD4(+) T-cell depletion. Here we address the question of whether infection of human lymphoid tissue ex vivo with prototypic R5 or X4 HIV variants also depletes or impairs CD8(+) T cells. We report that whereas productive infection of lymphoid tissue ex vivo with R5 and X4 HIV-1 isolates induced apoptosis in CD4(+) T cells, neither viral isolate induced apoptosis in CD8(+) T cells. Moreover, in both infected and control tissues we found similar numbers of CD8(+) T cells and similar production of cytokines by these cells in response to phorbol myristate acetate or anti-CD3-anti-CD28 stimulation. Thus, whereas HIV-1 infection per se in human lymphoid tissue is sufficient to trigger apoptosis in CD4(+) T cells, the death of CD8(+) T cells apparently requires additional factors.     

Syndecan-4 mediates the coinhibitory function of DC-HIL on T cell activation

Receptor-ligand interactions between APCs and T cells determine whether stimulation of the latter leads to activation or inhibition. Previously, we showed that dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) on APC can inhibit T cell activation by binding an unknown ligand expressed on activated T cells. Because DC-HIL binds heparin/heparan sulfate and heparin blocks the inhibitory function of DC-HIL, we hypothesized that a heparin/heparan sulfate proteoglycan on activated T cells is the relevant ligand. Screening assays revealed that syndecan-4 (SD-4) is the sole heparan sulfate proteoglycan immunoprecipitated by DC-HIL from extracts of activated T cells and that blocking SD-4 abrogates binding of DC-HIL to activated T cells. Moreover, cell-bound SD-4 ligated by DC-HIL or cross-linked by anti-SD-4 Ab attenuated anti-CD3 responses, whereas knocked-down SD-4 expression led to enhanced T cell response to APC. Blockade of endogenous SD-4 using specific Ab or soluble SD-4 receptor led to augmented T cell reactions to syngeneic and allogeneic stimulation in vitro and exacerbated contact hypersensitivity responses in vivo. We conclude that SD-4 is the T cell ligand through which DC-HIL mediates its negative coregulatory function.     

Characterization of syndecan-4 expression in 3T3-F442A mouse adipocytes: link between syndecan-4 induction and cell proliferation.

Heparan sulfate proteoglycans are found on the surface of most cells. Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan. Using quantitative RNase protection assays and immunoblotting, syndecan-4 expression was characterized in 3T3-F442A mouse adipoblasts. These cells exhibit dramatic changes in their biological and morphological characteristics during differentiation to adipocytes. During this process, the levels of syndecan-4 protein and mRNA expression changed dramatically. They peaked at the time when quiescent cells reentered the cell cycle before differentiation. Serum depletion-repletion also replicated the syndecan-4 mRNA induction when the cells were released back into proliferation, and a cycloheximide treatment abolished the peak of induction. In addition, inhibiting syndecan-4 induction with antisense oligonucleotides inhibited the proliferation of 3T3-F442A cells. In the terminally differentiated adipocytes characterized by the loss of proliferation capability, the serum inducibility of syndecan-4 is repressed, emphasizing the link between syndecan-4 induction in 3T3-F442A cells and cell proliferation.     

CCL2 increases X4-tropic HIV-1 entry into resting CD4+ T cells

During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4(+) T cells, we infected purified resting CD4(+) T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4(+) T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection.     

Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes

The accessory protein Nef plays a crucial role in primate lentivirus pathogenesis. Nef enhances human immunodeficiency virus type 1 (HIV-1) infectivity in culture and stimulates viral replication in primary T cells. In this study, we investigated the relationship between HIV-1 replication efficiency in CD4(+) T cells purified from human blood and two various known activities of Nef, CD4 downregulation and single-cycle infectivity enhancement. Using a battery of reporter viruses containing point mutations in nef, we observed a strong genetic correlation between CD4 downregulation by Nef during acute HIV-1 infection of activated T cells and HIV-1 replication efficiency in T cells. In contrast, HIV-1 replication ability was not significantly correlated with the ability of Nef to enhance single-cycle virion infectivity, as determined by using viruses produced in cells lacking CD4. These results demonstrate that CD4 downregulation by Nef plays a crucial role in HIV-1 replication in activated T cells and underscore the potential for the development of therapies targeting this conserved activity of Nef.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.     

Endogenous attenuation of allergic lung inflammation by syndecan-1

The airway plays a vital role in allergic lung diseases by responding to inhaled allergens and initiating allergic inflammation. Various proinflammatory functions of the airway epithelium have been identified, but, equally important, anti-inflammatory mechanisms must also exist. We show in this study that syndecan-1, the major heparan sulfate proteoglycan of epithelial cells, attenuates allergic lung inflammation. Our results show that syndecan-1-null mice instilled with allergens exhibit exaggerated airway hyperresponsiveness, glycoprotein hypersecretion, eosinophilia, and lung IL-4 responses. However, administration of purified syndecan-1 ectodomains, but not ectodomain core proteins devoid of heparan sulfate, significantly inhibits these inflammatory responses. Furthermore, syndecan-1 ectodomains are shed into the airway when wild-type mice are intranasally instilled with several biochemically distinct inducers of allergic lung inflammation. Our results also show that syndecan-1 ectodomains bind to the CC chemokines (CCL7, CCL11, and CCL17) implicated in allergic diseases, inhibit CC chemokine-mediated T cell migration, and suppress allergen-induced accumulation of Th2 cells in the lung through their heparan sulfate chains. Together, these findings uncover an endogenous anti-inflammatory mechanism of the airway epithelium where syndecan-1 ectodomains attenuate allergic lung inflammation via suppression of CC chemokine-mediated Th2 cell recruitment to the lung.     

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

Beyond help: direct effector functions of human immunodeficiency virus type 1-specific CD4(+) T cells

The immune correlates of protection in human immunodeficiency virus type 1 (HIV-1) infection remain poorly defined, particularly the contribution of CD4(+) T cells. Here we explore the effector functions of HIV-1-specific CD4(+) T cells. We demonstrate HIV-1 p24-specific CD4(+)-T-cell cytolytic activity in peripheral blood mononuclear cells directly ex vivo and after enrichment by antigen-specific stimulation. We further show that in a rare long-term nonprogressor, both an HIV-1-specific CD4(+)-T-cell clone and CD4(+) T cells directly ex vivo exert potent suppression of HIV-1 replication. Suppression of viral replication was dependent on cell-cell contact between the effector CD4(+) T cells and the target cells. While the antiviral effector activity of CD8(+) T cells has been well documented, these results strongly suggest that HIV-1-specific CD4(+) T cells are capable of directly contributing to antiviral immunity.     

Commitment to apoptosis in CD4(+) T lymphocytes productively infected with human immunodeficiency virus type 1 is initiated by lysosomal membrane permeabilization, itself induced by the isolated expression of the viral protein Nef

Primary CD4(+) T lymphocytes, supporting in vitro human immunodeficiency virus type 1 (HIV-1) replication, are destined to die by apoptosis. We explored the initial molecular events that act upstream from mitochondrial dysfunction in CD4(+) T lymphocytes exposed to the HIV-1(LAI) strain. We tracked by immunofluorescence the cells expressing the p24 viral antigen and used Percoll density gradients to isolate a nonapoptotic CD4(+) T-cell subset with a high inner mitochondrial transmembrane potential (DeltaPsim) but no outer mitochondrial membrane (OMM) rupture. In most p24(+) (but not bystander p24(-)) cells of this subset, the lysosomes were undergoing limited membrane permeabilization, allowing the lysosomal efflux of cathepsins (Cat) to the cytosol. This was also induced by HIV-1 isolates from infected patients. Using pepstatin A to inhibit Cat-D enzymatic activity and Cat-D small interfering RNA to silence the Cat-D gene, we demonstrate that once released into the cytosol, Cat-D induces the conformational change of Bax and its insertion into the OMM. Inhibition of Cat-D activity/expression also conferred a transient survival advantage upon productively HIV-1-infected cells, indicating that Cat-D is an early death factor. The transfection of activated CD4(+) T lymphocytes with a Nef expression vector rapidly induced the permeabilization of lysosomes and the release of Cat-D, with these two events preceding OMM rupture. These results reveal a previously undocumented mechanism in which Nef acts as an internal cytopathic factor and strongly suggest that this viral protein may behave similarly in the context of productive HIV-1 infection in CD4(+) T lymphocytes.