Repeated exposure of epithelial cells to apoptotic cells induces the specific selection of an adaptive phenotype: Implications for tumorigenesis.

The consequences of apoptosis extend beyond the mere death of the cell. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits PTEC proliferation, growth, and survival. Here, we tested the hypothesis that continual exposure to apoptotic targets can induce a phenotypic change in responding PTECs, as in other instances of natural selection. In particular, we demonstrate that repeated exposure to apoptotic targets leads to emergence of a PTEC line (denoted BU.MPTSEL) resistant to apoptotic target-induced death. Resistance is exquisitely specific. Not only are BU.MPTSEL responders fully resistant to apoptotic target-induced death (∼85% survival versus <10% survival of nonselected cells) but do so while retaining sensitivity to all other target-induced responses, including inhibition of proliferation and growth. Moreover, the resistance of BU.MPTSEL responders is specific to target-induced apoptosis, as apoptosis in response to other suicidal stimuli occurs normally. Comparison of the signaling events induced by apoptotic target exposure in selected versus nonselected responders indicated that the acquired resistance of BU.MPTSEL cells lies in a regulatory step affecting the generation of the pro-apoptotic protein, truncated BH3 interacting-domain death agonist (tBID), most likely at the level of BID cleavage by caspase-8. This specific adaptation has especial relevance for cancer, in which the prominence and persistence of cell death entail magnification of the post-mortem effects of apoptotic cells. Just as cancer cells acquire specific resistance to chemotherapeutic agents, we propose that cancer cells may also adapt to their ongoing exposure to apoptotic targets.

Apoptotic cells activate AMP-activated protein kinase (AMPK) and inhibit epithelial cell growth without change in intracellular energy stores.

Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses.

Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis.

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular ß-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses.

Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses.

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (m). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence m survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like m, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and m. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and m. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in m, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from m. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.

Macrophages from lupus-prone MRL mice have a conditional signaling abnormality that leads to dysregulated expression of numerous genes.

Macrophages (mϕ) from pre-diseased mice of the major murine inbred models of spontaneous autoimmunity (AI), including multiple lupus-prone strains and the type I diabetes-prone NOD (non-obese diabetic) strain, have identical apoptotic target-dependent abnormalities. This characteristic feature of mϕ from AI-prone mice suggests that abnormal signaling events induced within mϕ following their interaction with apoptotic targets may predispose to AI. Such signaling abnormalities would affect predominantly the processing and presentation of self-antigen (i.e., derived from apoptotic targets), while sparing the processing and presentation of foreign antigen (i.e., derived from non-apoptotic sources). Here, we used DNA microarrays to test the hypothesis that mϕ from AI-prone mice (MRL/MpJ [MRL/+] or MRL/MpJ-Tnfrsf6 ( lpr ) [MRL/lpr]) differentially express multiple genes in comparison to non-AI mϕ (BALB/c), but do so in a largely apoptotic cell-dependent manner. Mϕ were stimulated with lipopolysaccharide, a potent innate stimulus, in the presence or absence of serum (an experimental surrogate for apoptotic targets). In accord with our hypothesis, the number of genes differentially expressed by MRL mϕ was significantly increased in the presence vs. the absence of serum, the apoptotic target surrogate (n = 401 vs. n = 201). Notably, for genes differentially expressed by MRL mϕ in the presence of serum, serum-free culture normalized their expression to a level statistically indistinguishable from that by non-AI mϕ. Comparisons of mϕ from AI-prone NOD and non-AI C57BL/6 mice corroborated these findings. Together, these data support the hypothesis that mϕ from MRL and other AI-prone mice are characterized by a conditional abnormality elicited by serum lipids or apoptotic targets.

The affirmative response of the innate immune system to apoptotic cells.

Growing evidence exists for a new role for apoptotic cell recognition and clearance in immune homeostasis. Apoptotic cells at all stages, irrespective of membrane integrity, elicit a signature set of signaling events in responding phagocytes, both professional and non-professional. These signaling events are initiated by receptor-mediated recognition of apoptotic determinants, independently of species, cell type, or apoptotic stimulus. We propose that the ability of phagocytes to respond to apoptotic targets with a characteristic set of signaling events comprises a second distinct dimension of innate immunity, as opposed to the traditional innate discrimination of self vs. non-self. We further propose that a loss or abnormality of the signaling events elicited by apoptotic cells, as distinct from the actual clearance of those cells, may predispose to autoimmunity.

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Altered cell-cell and cell-matrix interactions in the development of systemic autoimmunity.

MPhi of mice from the major inbred models of systemic lupus erythematosus (SLE) have an identical defect affecting the activity of the cytoskeletal regulator and G-protein Rho. This abnormality is triggered by apo cells. Strikingly, SLE-prone MPhi show normal Rho activity when cultured in the absence of apo cells. We used gene arrays to identify adhesion-related gene products that are abnormally expressed by MPhi from prediseased 4-6-week-old SLE-prone MRL mice in the presence of serum lipids mimicking apo cells (SL-Apo). MPhi of MRL mice differentially expressed 42 adhesion-related genes in the presence of SL-Apo. Of these, 32 were expressed normally in the absence of SL-Apo. As adhesive interactions play a major role in lymphocyte activation, the detected apo cell-dependent abnormality could predispose to the development of autoimmunity. Indeed, several recent genetic studies support a role for adhesion-related genes in the pathogenesis of chronic autoimmunity.

Apoptotic and necrotic cells as sentinels of local tissue stress and inflammation: response pathways initiated in nearby viable cells.

Virtually all cells in the body have the capacity to recognize and respond to dead cells. Viable cells discriminate apo from nec targets via distinct cell surface receptors. Engagement of these receptors induces "recognition-dependent" signaling events in viable responding cells that differ for apo vs. nec targets. Although "engulfment-dependent" signaling events also contribute to the response by viable cells, these events do not differ for apo vs. nec targets. While many signaling events are conserved across diverse cell lineages, other signaling events, especially those involving Akt, demonstrate lineage-specific variation. Whereas apo targets activate Akt in MPhi, they inhibit Akt in kidney epithelial cells. Differences in the responses to dead targets by viable migratory cells, such as MPhi, and viable fixed cells, such as kidney epithelial cells, permit cell-specific adaptations to local environmental change or stress. We propose that dead cells (apo and nec) act as sentinels to alert nearby viable cells to local environmental change or stress.