identification-intracellular-signaling-events-induced-viable-cells

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 ...

Kutztown University of Pennsylvania

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.

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

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.

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

Macrophages (mÏ•) 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 mÏ• from AI-prone mice suggests that abnormal signaling events induced within mÏ• 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 mÏ• from AI-prone mice (MRL/MpJ [MRL/+] or MRL/MpJ-Tnfrsf6 ( lpr ) [MRL/lpr]) differentially express multiple genes in comparison to non-AI mÏ• (BALB/c), but do so in a largely apoptotic cell-dependent manner. MÏ• 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 mÏ• was significantly increased in the presence vs. the absence of serum, the apoptotic target surrogate (nâ€‰=â€‰401 vs. nâ€‰=â€‰201). Notably, for genes differentially expressed by MRL mÏ• in the presence of serum, serum-free culture normalized their expression to a level statistically indistinguishable from that by non-AI mÏ•. Comparisons of mÏ• from AI-prone NOD and non-AI C57BL/6 mice corroborated these findings. Together, these data support the hypothesis that mÏ• from MRL and other AI-prone mice are characterized by a conditional abnormality elicited by serum lipids or apoptotic targets.

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

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

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.

Article	Year
Altered cell-cell and cell-matrix interactions in the development of systemic autoimmunity. Autoimmunity\| PubMed ID: 19811276	2009
Recognition of apoptotic cells by epithelial cells: conserved versus tissue-specific signaling responses. The Journal of biological chemistry\| PubMed ID: 19910463	2010
Macrophages from lupus-prone MRL mice have a conditional signaling abnormality that leads to dysregulated expression of numerous genes. Immunogenetics\| PubMed ID: 21229240	2011
Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis. The Journal of biological chemistry\| PubMed ID: 22396534	2012
Apoptotic cells activate AMP-activated protein kinase (AMPK) and inhibit epithelial cell growth without change in intracellular energy stores. The Journal of biological chemistry\| PubMed ID: 26183782	2015

Angelika Antoni

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