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.

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.

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.

A novel model of surgical injury in adult rat kidney: a "pouch model".

Regenerative mechanisms after surgical injury have been studied in many organs but not in the kidney. Studying surgical injury may provide new insights into mechanisms of kidney regeneration. In rodent models, extrarenal tissues adhere to surgical kidney wound and interfere with healing. We hypothesized that this can be prevented by wrapping injured kidney in a plastic pouch. Adult rats tolerated 5/6 nephrectomy with pouch application well. Histological analysis demonstrates that application of the pouch effectively prevented formation of adhesions and induced characteristic wound healing manifested by formation of granulation tissue. Additionally, selected tubules of the wounded kidney extended into the granulation tissue forming branching tubular epithelial outgrowths (TEOs) without terminal differentiation. Tubular regeneration outside of renal parenchyma was not previously observed, and suggests previously unrecognized capacity for regeneration. Our model provides a novel approach to study kidney wound healing.

IgM to S-nitrosylated protein is found intrathecally in relapsing-remitting multiple sclerosis.

This study has established the presence of IgM against S-nitrosylated proteins in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients using S-nitrosocysteine epitope (anti-SNOcys) as previously shown in serum. Anti-SNOcys IgM increased significantly in CSF during relapsing-remitting MS compared to milder neurological conditions. Evidence from albumin, IgG and IgM suggest that the production of anti-SNOcys IgM is intrathecal rather than the result of ingress from serum. Two correlations during relapse: between CSF level of anti-SNOcys IgM and time elapsed since relapse onset; and between CSF and serum anti-SNOcys IgM levels, suggest that this antibody may have potential as a biomarker.

Olig1 is expressed in human oligodendrocytes during maturation and regeneration.

Myelin repair is inhibited in multiple sclerosis (MS), ultimately leading to axonal damage and disability. We aimed to understand the transcriptional mechanisms of regeneration in primary human oligodendrocyte cultures isolated from white matter of medically intractable epilepsy patients. Cultures at isolation contained 84% mature oligodendrocytes and 16% oligodendrocyte progenitor cells (OPC). The two populations showed a protracted regeneration of membranes expressing myelin proteins after 2-3 weeks in culture, and were kept long-term to study membranes maintenance. We profiled by quantitative PCR (qPCR) the sequential mRNA expression of transcription factors Olig1, Olig2, Nkx2.2, Sox10, PPARδ, PPARγ, cyclic nucleotide phosphodiesterase (CNP), myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG). In summary, Olig1 was not expressed in freshly isolated oligodendrocytes, but was expressed from the beginning of process extension until membranes maintenance. In contrast, Olig2 expression was restricted to isolation and during membranes production. We show for the first time PPARδ expression and absence of PPARγ in human oligodendrocytes. Nkx2.2, Sox10, PPARδ, CNP, MBP and MOG messengers were expressed at any time, while MAG messenger was expressed at mature stage only. Myelin proteins CNP, MBP, MAG, and MOG were confirmed by immunocytochemistry. Our findings point to different roles of Olig1 and Olig2 in regeneration of cultured adult human oligodendrocytes. Noticeably, the transcriptional profiles found in cultured neonatal rodent OPC are different. More studies are necessary to elucidate myelin repair in the adult human brain.