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  • Sherin Thomas

Influence from the cellular “grave” through metabolites

Written for Ours, Scientifica for the NUS Science Journalism Club

The terrifying spectre of Banquo, the haunting presence of King Hamlet, and the ghoulish bodach of Marley from Charles Dickens’ novel, A Christmas Carol; what do these iconic characters have in common? Well, firstly, they are all dead. As summarised in the opening lines of the novel A Christmas Carol, Marley, like the other characters, is “dead, to begin with. There is no doubt whatever about that”. This sentence conveys the attitude that we have towards the demise of cells. Dying cells are believed to be, in Dickens’ words, “as dead as a doornail”. However, just as these characters were able to influence the lives of the living beyond the grave, the surrounding living cells are also subtly influenced by the metabolites secreted by dying apoptotic cells. The underlying metabolic processes and significance of the event were uncovered recently in a paper published by Medina et al.

It has long been recognised by the classical embryologists of the nineteenth century that tissue cell loss and replacement is implicit in the cellular nature of life and occurs during the substantial structural changes that occur in a multicellular organism. During the 1970s, one such cell death mechanism called apoptosis was identified by Wylie and colleagues. Apoptosis is a form of cellular suicide orchestrated by cysteine proteases called caspases, which are enzymes that cleave a large number of intracellular proteins. Such regulated cleavage of intracellular proteins effectively “packages” and dismantles the cell in an orderly manner. For instance, the DNA in the nucleus is cut into large 50- to 300- kilobase pieces, cytoskeletal proteins are broken down and undergoes cross-linking, cytoskeletal filamentous actin proteins are remodelled to break the cell down into smaller pieces, and certain lipids such as phosphatidylserine are exposed on the outer layers of the plasma membrane of these cells which signals to immune cells, such as macrophages, to engulf and digest the apoptotic cells.

From the first description of apoptosis made by Wylie and colleagues, it has been established that this cell death mechanism does not elicit an inflammatory response, which is common in other cell death mechanisms such as necrosis, in which inflammation is caused by damage-associated molecular patterns (DAMPs) released by necrotic cells. While many studies have since confirmed that cell death via apoptosis is indeed anti-inflammatory, the mechanisms underlying the anti-inflammatory properties of apoptotic cells is not sufficiently well characterised. Nevertheless, the immunologically ‘silent’ and anti-inflammatory properties of apoptosis have led to proposals that injection of apoptotic cells could perhaps be used to control inflammatory diseases as the engulfment of apoptotic cells by phagocytes such as macrophages promotes tissue reconstruction and repair. Little is known about the apoptosis-associated molecules responsible for this effect.

In the recent paper published by Medina and colleagues, it was discovered that macrophages were stimulated to express particular genes involved in the inhibition of inflammation and tissue repair during the apoptosis of mammalian cells due to the release of specific small molecules such as metabolites. The authors began by profiling the metabolite secretome of different cell types undergoing apoptosis due various triggers, after which six metabolites consistently released by dying cells were identified. The specificity in the release of the metabolites can be attributed to the selectivity of pannexin 1 (PANX1), a protein channel found on the surface of cells. Under normal conditions, PANX1 is closed and only opens when it is cleaved by caspases during apoptosis. Interestingly, the paper reported that in cells designed to lack PANX1 undergoing apoptosis, the metabolites associated with apoptosis were not released. These metabolites were also identified by the authors and, notably, found that they were not able to alter the gene-expression profile of macrophages individually. However, when all six metabolites, or a mixture of three specific metabolites such as spermidine, guanosine monophosphate, and inosine monophosphate, were administered together, significant increases were observed in the expression of anti-inflammation and tissue-repair genes in macrophages. Additionally, when tested in vivo in a murine model of inflammatory arthritis and lung transplantation, the aforementioned three metabolites were found to have inhibited disease progression, preventing graft rejection, and were thus observed to have significant anti-inflammatory effects.

One of the three metabolites that were found to have significantly influenced the gene-expression of neighbouring cells such as macrophages is spermidine which is a type of polyamine. This molecule is a product of a metabolic pathway that converts arginine to ornithine and then to putrescine and finally to spermidine. Each metabolic conversion step is regulated by enzymes such as spermidine synthase (SRM) which converts putrescine to spermidine (Figure 1). In the paper, the authors noted that the synthesis of spermidine and its precursor, putrescine, is upregulated in apoptotic cells before dying by tracing the conversion of arginine to spermidine in apoptotic cells. Nevertheless, only spermidine is released by apoptotic cell and not putrescine, and the release of spermidine is dependent on PANX1. While this phenomenon was observed after using only ultraviolet radiation to induce apoptosis, this finding is indeed promising and raises the possibility that the induction of apoptosis upregulates this spermidine producing metabolic pathway. Interestingly, although previous studies have noted that spermidine can reduce inflammation when supplemented exogenously, the apoptoc release of spermidine is the first observed physiological or natural extracellular source of the molecule.

Figure 1. In healthy human cells, the amino acid arginine is converted to ornithine, which is then either converted to putrescine, a precursor of spermidine, or transported and used in subsequent metabolic processes into the mitochondrion, a cellular organelle.

Another interesting finding of the paper is that when the authors administered a drug called a BH3 mimetic which induces mitochondrial outer membrane permeabilisation (MOMP), thereby triggering a key step in apoptosis. The level of spermidine released by cells treated with the drug was comparable to that released by apoptotic cells. This might be because the transport of ornithine to the mitochondria, after which it would then be metabolised to citrulline (Figure 2), might be interrupted by MOMP, leading to the utilisation of ornithine in the cytoplasm, thereby inducing spermidine production. Future studies can test this hypothesis by exposing cells designed to lack the necessary cellular components needed for MOMP to BH3 mimetic.

Figure 2. In apoptotic cells, the apoptotic machinery in the cell activates caspases which then open PANX1 channels and inhibit the mitochondrial transport of ornithine. In addition, spermidine and putrescine production increases such that the level of these molecules in the cell is higher than normal. Subsequently, spermidine and other metabolites, which are not shown here, are released from the cell through the open PANX1 channels.

One limitation of the paper is that the authors did not determine if urea, an inflammatory DAMP released by cells undergoing necrosis and a by-product of the arginine to spermidine metabolic pathway, was released by apoptotic cells via PANX1. Nevertheless, the increase in arginine metabolism observed by the authors during apoptosis might account for the anti-inflammatory nature of apoptosis, provided that urea is not released through PANX1.

Indeed, if the three metabolites – inosine monophosphate, guanosine monophosphate, and spermidine – were able to alter the gene-expression profile of macrophages, what are the mechanisms underlying this process? Also, why do apoptotic cells need to release all three metabolites together in order to manifest the desired effect? Well, firstly, inosine monophosphate which can be converted to inosine which has anti-inflammatory effects and was found to significantly reduce inflammation in mice exposed to a bacterial toxin. Additionally, studied have shown that inosine monophosphate and guanosine monophosphate can signal through adenosine receptors and anti-inflammatory signalling pathways induced by the activation of adenosine receptors can be possibly enhanced in the presence of spermidine.

In all, findings by Medina and colleagues provide new opportunities to investigate how metabolic processes change during apoptosis in dying cells and how these metabolites are able to influence the neighbouring cells and tissues. Other forms of cell death such as necrosis are also able to influence the surrounding cells profoundly and the means through which metabolic changes induced in these dying cells influence other tissues is unknown. While Medina’s work offers new possibilities in treating human diseases, efforts to use metabolites to treat such conditions might prove difficult due to the limited extrapolation of results obtained from mice models owing to differences in adenosine receptor expression between species. Nevertheless, more research is warranted in this field which has only begun to uncover the complexities of apoptosis and the effects of this process on the surrounding cells. After all, the dead need not be quiescent. Just as the spectres of Banquo, King Hamlet, and Marley are able to permeate the lives of living, messages from the grave and beyond can influence the living still.


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