Dr. Timothy A. McCaffrey
Dr. McCaffrey, a very elaborate and very intriguing study of yours was published in ATVB this summer. It seems like you generated some findings which were unexpected at the beginning of the experimental journey, is this right? Would you mind giving us a short summary of your study and the key findings?
A: As is often the case, we didn’t actually start out to address this particular question of how interferon regulates apoptosis in vascular lesion cells. We were, and still are, principally interested in the question of how these cells are resistant to death induced by fas ligation, transforming growth factor-ß (TGF-ß), and glucocorticoids. The overarching theory is that cells accumulate in diseased arteries because they fail to respond to the signals that tell them to stop repairing the artery. This theme recurs in many fibrotic diseases where wound repair seems to initiate normally, but it is never turned off. In this case, we knew that lesion cells could be sensitized to respond to fas ligand by prior treatment with interferon-. We assumed, incorrectly, that the mechanism of interferon was well known and thought that this would provide an important clue to how these cells were resistant to different types of inhibitors. Ultimately, we decided to conduct a microarray analysis of interferon’s targets, to see if any of them overlapped with candidate genes involved in the resistance to TGF-ß and fas ligation. We then systematically knocked down, or overexpressed about 75 different genes to determine which ones were actually involved in making lesion cells sensitive to apoptosis. The answer surprised us because it pointed clearly to the relatively obscure ‘immunoproteasome’ as being necessary for interferon’s ability to sensitize cells to fas-induced apoptosis. The immunoproteasome has mainly been studied in the context of interferon-induced antigen presentation on MHC. However, it seems it has an additional function of rapidly degrading prosurvival factors such as Mcl-1, a member of the Bcl-2 family. Thus, the immunoproteasome is an unexpected, but important connection between inflammation and the control of vascular cell survival, possibly explaining why there has been a long-standing connection between viral infections and heart attacks. Q: You ran these studies on cells derived from carotid endarterectomy-derived carotid artery plaques. How are these cells harvested, how much of them survive, and is there a selection at the end of a particular cell type or will it be a mix of endothelial cells, smooth muscle cells, and inflammatory cells? This is also important at the end if we can apply the study findings to all cell types in atherosclerotic plaques or only a certain cell population? A: Good question. We obtained in the vicinity of 400 human endarterectomy specimens from our very generous and patient vascular surgeons. About 30% on the samples will show some outgrowth of cells from the explanted human lesions, but often the cultures are relatively short-lived, and possibly 5-10% of lesions produce truly useful cultures that will survive 5-8 passages. The cells that grow from lesions are usually termed ‘smooth muscle cells’ because they exhibit the smooth muscle actin isoform, but that marker is common to myofibroblasts and other repair cells, so I prefer the term “lesion-derived cells” or myofibroblasts. Some of these cultures produced long-term cultures, that appear to be spontaneous transformations, but retain many of the smooth muscle markers in the original culture. In short, the cells are highly selected, and may represent a small fraction, possibly a progenitor fraction, of the overall lesion cell population. Based on the mechanisms involved, and an extensive literature, we expect that many cell types would show induction of the immunoproteasome and sensitivity to fas-induced death, although we’ve conducted the siRNA studies only in the human lesion cells. We’ll rely on experts in other disease states to determine whether the immunoproteasome is involved in their particular model.
Q: Very interesting to notice from the beginning is the impact of mitochondrial pathway on the apoptotic effect of FasL on atherosclerotic lesion-derived cells. While traditionally there been the separation between the extrinsic (Fas) and intrinsic (mitochondria) pathway of apoptosis, your results suggest some synergy. Is this correct and how does IFN-gamma play into this? A: True. In our prior papers, we observed that a major mode of resistance to death in these cells is via upregulation of Bcl-Xl, another member of the Bcl-2 family. While the extrinsic and intrinsic systems are often drawn as parallel and distinct paths, in our experience, they are elaborately connected in a ‘feed forward’ cascade. In most cells that we’ve studied, the extrinsic path, triggered by fas ligation and caspase 8 activation, requires amplification in the mitochondria to produce a significant apoptotic response. Our initial hypothesis was that interferon probably suppressed Bcl-Xl, but extensive studies showed no effect on Bcl-Xl or Bcl-2 in these cells. In fact, interferon regulates the third major member of the family, Mcl-1, but not by the direct transcriptional path that we expected, but by activating the system that degrades it.
Q: As we see next in your paper, IFN-gamma effects the expression of a number of factors and you did a remarkable work confirming this with qRT-PCR. At the end, would you say that there are three major groups serving as “the effectors” of IFN-gamma: the transcription factors involved in mediating IFN-gamma-induced gene expression, members of the apoptotic cascade, and subunits of the immunoproteasome? A: We were struck by the tremendous transcriptional signature of interferon on these cells, and made identifying the apoptotic mediators very difficult there were easily 75 credible targets. Dr. Yang had the clever idea to overexpress IRF1, which is thought to mediate many of interferon’s effects, and this would narrow the field. To our surprise, the IRF1 overexpressing cells did not become sensitive to apoptosis, and so we could actually use this to this discount, or falsify, the involvement of particular genes. About half of the candidates could be excluded because qRT-PCR showed that they were strongly induced in the IRF1 overexpressors, and yet the cells were not sensitized by IRF1. This allowed us to systematically walk through the remaining candidates. If I had to simply interferon signaling, we might view it as having 1) a very large transcriptional impact, mediated by STATs and IRFs, 2) a significant translational effect mediated by PKR/eIF2 effects on initiation, and RNA editing via ADAR/APOBECs, and 3) a substantial rearrangement of the proteasomal degradation pathway to more effectively present antigens and make the cell more susceptible to death in case the invading pathogen cannot be controlled.
Q: There are different types of the proteasome, which mediates the degradation of 80 to 90% of all proteins in eukaryotic cells. Usually, when the name “proteasome” is mentioned, the constitutive proteasome comes to mind and several studies have been published on this topic in atherosclerosis. Now, it seems like the first time the immunoproteasome comes more clearly into view – was this a surprising finding? A: It was very surprising to us, and we almost didn’t test the immunoproteasome components because even though they were very strongly induced by interferon, there was so little known about what they did, and no plausible reason to think that they were involved in apoptotic control. However, when we did knock down PSMB8, which encodes the LMP7 protein, there was no doubt that it was involved. We repeated it a half-dozen times to convince ourselves it wasn’t a fluke, and it worked every time.
Q: Seemingly, IRF-1-overexpression did not have a much as an inducing impact on the expression of proteasome subunits as IFN-gamma did and also IRF-1 overexpression did not sensitize lesion-derived cells to apoptosis. Did this constellation indicate a possible causal connection? Also, if not primarily through IRF-1, via which pathway does IFN-gamma induce the expression of proteasome subunits? A: This gave us some hesitation because there was published data that PSMB8/LMP7 is induced by IRF1 in other cell types. In our hands, strong overexpression of IRF1 produced only a very small increase in PSMB8 mRNA, and when we looked at the promoter sequence, there was only a single consensus IRF1 site, but there were 5 STAT sites, which would be directly activated from interferon receptor phosphorylation of STAT1. Thus, it looks like PSMB8 is a high priority, and direct target of STATs, probably STAT1, although we have not directly tested that.
Q: After an impressive series of experiments you identified interference with PSMB8/LMP7 expression as the intervention that would block the apoptosis-sensitization effect IFN- on lesion-derived cells. Please tell us more about this fascinating discovery, PSMB8/LMP7 and also the fact that PSMB8/LMP7 interference blocked sensitivity to apoptosis of atherosclerotic plaque cells despite high Fas levels. A: After we examined 10-20 of the most plausible candidates, and yet got no effect on apoptotic sensitivity, Dr. Yang and I would frequently discuss how badly we really wanted to know the answer. While we used several methods to narrow and prioritize the list of candidates, we concluded that sometimes brute force was the best method to gain insight into a process. The important aspect of our assay system was that it was relatively high-throughput and very reliable, which allowed us to test multiple candidates simultaneously, and include controls to be certain that we could see reversal if it occurred. We have since further streamlined this process by producing our own siRNAs which increased the speed and reduced the cost by about a factor of 10. The discovery of PSMB8 as a mechanism of interferon action is really exciting to us, and it also established a very agile system in which we can perform microarray profiling in conjunction with functional testing, and then systematically screen up to a 100 potential candidate genes for their functional role.
Q: Looking at specific regulators that were modulated by PSMB8/LMP7, Mcl-1 appeared before as one of the proteins that should be up-regulated by IFN-gamma based on mRNA levels. Yet on protein level, its expression was low – was this constellation already a tip off for your group to focus on the proteasome? A: I think that when we didn’t see changes in Bcl-Xl or Bcl-2, we somewhat discounted the entire family, and that was a mistake, except that there are several members of that family, and different spice forms, and they can each be phosphorylated as a means of regulating there activity. So without knowing that the proteasome was involved, we might still be looking at splicing, translation, subcellular localization, or phosphorylation of the many Bcl members. Because of some related work on the CDK inhibitor flavopiridol, we were aware of Mcl-1’s susceptibility to proteasomal regulation, but we didn’t connect it to interferon until we saw the siRNA knockdown of PSMB8, and then it started to make sense.
Q: What are the functionally consequences of Mcl-1 on cells? What is the underlying cellular pathway of its activity? How is this activity regulated – primarily by the proteasome or the immunoproteasome in the cell depending on their exposure to IFNgamma? A: Mcl-1 is a very potent prosurvival factor in the Bcl-2 family. A number of molecular functions have been assigned to this family of anti-apoptotic factors, and so they may have multiple mechanisms of action. Reports suggest that they may directly inactivate the proapoptotic factor tBid, is cleaved and activated by caspase 8, or they may act more indirectly by neutralizing Bak/Bax in the mitochondrial membrane. Regardless of the specific molecular interaction, the common result is that mitochondrial membrane integrity is stabilized by Mcl-1, and other Bcl-2 members, resulting in resistance to depolarization and subsequent cytochrome C release and formation of ‘apoptosome’ which accelerates caspase activation. By increasing immunoproteasome activity, Mcl-1 is actively degraded and the mitochondria is more susceptible to depolarization. There may be additional effects of the immunoproteasome, for instance, it might be involved in caspase activation, or in degrading caspase inhibitors such as IAP family members.
Q: You provided a very nice illustrating figure at the end. Is the arrow also pointing into the direction of significance of the immunoproteasome versus the constitutive proteasome for atherosclerosis and more studies to be done in this area? What do you consider to be the next step? A: We’re very interested in whether this has translational utility in relation to atherosclerosis and restenosis in humans. The challenge is to test knockdown of PSMB8/LMP7 in a model that might reasonably mimic the complex inflammatory aspects of the human disease. Further, one may need to achieve stable knockdown over a sustained period to obtain a clear picture of the immunoproteasome’s role in disease, and that presents certain additional challenges. A recent Nature Medicine paper by Muchamuel and colleagues (Nat. Med. 15(7): p781, July 2009) identifies a new LMP7-specific small molecule inhibitor and shows that it is useful in ameliorating the inflammatory aspects of a mouse model of rheumatoid arthritis, and that may represent an interesting new strategy for testing in animal models of vascular disease. Finally, it will be important to determine whether there are any interesting genetic variations at the PSMB8 locus which might modulate the susceptibility to inflammatory activation of the immunoproteasome.
Q: Thank you so, so much Dr. McCaffrey for this exciting study and the privilege to dwell on it for a little bit longer in form of this interview. I deeply appreciate your time, insight, and interaction. If anyone would like to discuss it further, we will facilitate it via email@example.com.
Journal: Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1213-9.
European Society of Cardiology
European Heart HouseLes Templiers2035 Route des CollesCS 80179 Biot
06903Sophia Antipolis, FR
Our mission: To reduce the burden of cardiovascular disease
© 2017 European Society of Cardiology. All rights reserved