Strolling through the Porticoes: A Conversation with Prof. Dr. Lucio Cocco

Interviewer: Dr. Stephen B. Shears, Inositol Signaling Group, Signal Transduction Laboratory, National Institute of Environmental, Health Sciences, Research Triangle Park, NC, 27709 USA

I am delighted to have been given the opportunity to interview Prof. Dr. Lucio Cocco for the Special Issue of Biomolecules, “Versatility of a Cellular Signaling Scaffold: The Inositol Ring Rules! – Honorary Special Issue Commemorating the Work of Prof. Lucio I. M. Cocco”, that honors his seminal work, which began in the late 1970s with his identification of nuclear phospholipids. During the following decade, Lucio pursued a new concept that, within the nuclear matrix, there are pools of inositol lipids with distinct cell-signaling activities. As noted elsewhere [1], this hypothesis was initially greeted with some skepticism; at that time, the inositol lipid field was preoccupied with the canonical functions of these signaling molecules at the plasma membrane. But Lucio persisted. He and his colleagues subsequently published a considerable and highly influential body of work that has furthered the understanding of the role of this nuclear signaling pathway during myogenic differentiation, giving new insight into its role in myotonic dystrophies, as well as during haemopoietic differentiation, and envisaging the pathophysiological significance of nuclear phosphoinositide signaling in myelodysplastic syndromes.

Dr. Stephen B. Shears (SBS): Hi, Lucio, thanks for agreeing to this interview. Can I begin by asking you to describe the career path that introduced you to phospholipid research?

Prof. Dr. Lucio Cocco (LC): In the mid-1970s I was an undergraduate at the University of Chieti Medical School. As part of my studies in medicine and surgery, Antonio Manzoli became my mentor in the Histology Laboratory. Others [2] had published studies stating that the phospholipid content of active chromatin was 5-fold higher compared to repressed chromatin. We were interested in pursuing the biological significance of this observation with regard to cell cycle regulation, but the stumbling block was the persisting controversy over the extent to which these phospholipids might arise by contamination from non-nuclear membranes. Antonio and I investigated this problem, and we concluded that non-contaminating phospholipids were genuinely associated with non-histone chromosomal proteins (NCHP), perhaps regulating DNA stability and replication. We also reported that NHCP fractions prepared from leukemic B-lymphocytes contained 40% less sphingomyelin and 4-fold higher levels of phosphatidylcholine compared to normal B-cells [3]. This work is also the origin of my interest in cancer research.

SBS: And after you graduated?

LC: I remained at Chieti and studied for a Ph.D. in sports medicine. It seemed such a qualification might provide good employment prospects. I must confess I was also tempted by the possibility of sharing time with famous football players: one of my friends was Leonardo Vecchiet, a university physician who worked with Fino Fini. They were the doctors for the Italian National Football Team, and Fini was also the Director of the Specialty in Sports Medicine.

But my passion for studying the biochemistry and functionality of nuclear lipids prevailed, and in 1978 I accepted a tenured position at the University of Bologna.

SBS: This was a significant change in your academic lifestyle; you swapped an institution that had not yet reached its 20^th birthday for a position at the oldest university in the Western world.

LC: Bologna was a very attractive opportunity; to live in a medieval city that is so rich in history, science and culture, and yet is compact and walkable, in no small part due to its 38 kilometers of porticoes, which were recently added to the UNESCO World Heritage List. It is a wonderful walk to and from the laboratory.

SBS: But almost immediately you took off to Glasgow, Scotland.

LC: In 1979 I began a series of sabbaticals at the Beatson Institute for Cancer Research. The late John Paul was Director at the time. Under his leadership, a small research team, originally housed in the one-time servant’s quarters of a local cancer hospital, had been transformed into a newly constructed, purpose-built institute within the picturesque grounds of Glasgow University; it had attracted several internationally reputed scientific teams and was considered the top place to study gene regulation.

SBS: Our paths almost crossed in the late 1970s; I worked just 74 km from you, at the Edinburgh Medical School. I found it challenging to follow the Edinburgh version of the Scottish accent, but Glaswegian can be incomprehensible.

LC: I had assumed I would be better prepared. English is my second language, courtesy of my father having placed me in an English primary school. However, my wife Erminia and I both struggled at first. Nevertheless, eventually we successfully adapted to this new way of communicating.

SBS: Stewart Gilmour was your colleague at the Beatson; how did he come to share your enthusiasm for nuclear phospholipids?

LC: Stewart and John Paul were studying the regulation of beta-globin genes. I recall a meeting in which I convinced them it could be instructive to investigate if there could be a role for nuclear phospholipids. In 1980, Stewart and I published our first paper together, which described the analysis of Triton-treated nuclear material to remove nuclear membranes [4]. We felt this short paper presented an important finding that put to bed the criticism that phospholipids in our nuclear preparations must have arisen from contaminating membranes: now we had shown nuclear phospholipids need not even be associated with membranes.

SBS: After your return to Bologna in 1983, what specifically drew your attention to the inositol lipids?

LC: The emergence of inositol lipids as cellular signals [5] initially escaped my attention. After all, they are present at very low levels and at that time were canonical plasma membrane signals. However, my enthusiasm for these particular phospholipids changed following the description by Smith and Wells in 1983 [6] of nuclear inositol lipid synthesis.

SBS: And you returned to the UK to pursue this…

LC: In 1985, I visited Stewart, who had relocated to the Institute of Animal Physiology in Babraham, Cambridge, and through him I met Robin Irvine. I asked Robin if we could work together to study nuclear inositol lipids. That was despite us both having our doubts; Smith and Wells had concluded inositol lipid synthesis occurred in the nuclear envelope, which we both thought was probably contaminated by the endoplasmic reticulum. Our hesitancy evaporated when we used 0.4% Triton to prepare membrane-free nuclei that were able to synthesize PtdIns(4,5)P₂ in a cytoplasm-independent manner; this work was published in 1987 [7].

SBS: From looking at your resume, it seems this period was a very busy time: you were Chair of the Anatomy Department at the University of Chieti, and then you became the Director of their post-graduate School in Hygiene.

LC: It was a very interesting time. I took on a number of academic commitments while still remaining active in research, part of which was still going on in Bologna. During this time Stewart and I co-authored several follow-up papers that helped our ideas become more generally accepted, and then others also began to publish studies into nuclear phosphoinositide signaling [8,9].

I would also like to acknowledge Stewart’s continuing guidance as I performed more experiments with Alberto M. Martelli in our Bologna laboratory; we had identified a nuclear-specific species of Phospholipase PLCβ that was activated within 2 minutes’ treatment of 3T3 mouse fibroblasts with insulin-like growth factor-1. These data yielded a manuscript that we submitted to Nature in 1992. The reviewers were positive but demanded additional experiments. Alberto didn't think these were necessary, but I did. However, the journal office put us under pressure to return the manuscript promptly. It was the Easter weekend, which in Italy is the second most important holiday after Christmas. I remember spending Good Friday and Easter Eve in the lab making dozens of Western blots; we were all relieved and excited when the work was accepted for publication in early April of 1992 [10].

SBS: Were there any immediate benefits from the Nature publication?

LC: Naturally, all of the members of our lab were excited by the novelty of the observations, and by the recognition that we were performing research that was deemed to be important to the scientific world. The publication of this work in a journal as prestigious as Nature gave me visibility and subsequently the opportunity to successfully request that our Cellular Signaling Laboratory be set up in Bologna. The location of the laboratory has historical significance in that the space was once occupied by Professor Oliviero Mario Olivo, a distinguished embryologist and labmate of Nobel Laureate Rita Levi Montalcini at the renowned School of Giuseppe Levi in Turin.

Our new setup encouraged generous financial support from the Italian Ministry for Research. Sadly, fundraising in my country is now very difficult; support from private institutions has become very important.

SBS: Another of your important scientific contributions in Bologna has been to organize annual symposia, Advances in Biological Regulation.

LC: This symposium series—originally Advances in Enzyme Regulation—was founded in the United States in the 1960s by George Weber and Sir Hans Krebs. I gave my first presentation in 1978 on the possible role of phospholipids in the nucleus in normal and neoplastic cells [11]. Beginning in 1995, I collaborated with George in the organization of these symposia in Indianapolis. Then, in 2005, George asked me if I felt like continuing his work, and so the symposium was renamed and moved to Bologna.

My feeling is that the success of this so-called closed conference is to bring together a group of scientists that are obliged for two days to communicate with each other, exchange ideas and hopefully initiate new collaborations. I have also been honored that a number of Nobel Laureates accepted my invitations to present plenary lectures. They seemed to have enjoyed the experience, always warmly thanking me for having had the opportunity to participate in this "historic" symposium.

SBS: I would like to express my gratitude for the privilege to attend several of these meetings. I must also thank your lab staff that perform much organizational work behind the scenes, but my special thanks go to Erminia, who is the most generous and gracious hostess each time we have been invited to your apartment in downtown Bologna. I can also testify that an evening stroll through the porticoes is fun too!

I won’t forget one year when attendees were also invited to the anatomical wax museum in Bologna; fortunately, its visitors are no longer treated to a public dissection of freshly executed criminals! I recently learned of this 16^th-century practice from reading your 2000 account of the history of this museum [12].

LC: I was involved in writing this article because as a doctor I had acquired a deep knowledge of anatomy, which is also one of the fundamental disciplines of our department. It was instructive to go back in history and discover that research activities in the eighteenth century took place not in the University but at the Academy of Sciences of the Bologna Institute.

SBS: Another collateral benefit of my attending your Bologna meetings has been to keep me informed of the significant body of work that you have published on the links between nuclear inositol lipid signaling and myelodysplastic syndromes.

LC: In this area of work, Matilde Follo’s paper in the Journal of Clinical Oncology [13] has been particularly important. She and her co-workers determined that mono-allelic deletion of PLCβ1 is associated with worse clinical outcomes in myelodysplastic syndromes. This study captured the attention of hematologists, while also paving the way for further epigenetic analysis that demonstrated how PLCβ1 mRNA levels are predictive of whether the response to azacitidine therapy will be either clinically favorable or lead to a worsening condition; this work was published in PNAS and the Journal of Leukocyte Biology [14,15].

SBS: Recent years have seen considerable documentation of the extent to which women continue to be under-represented in senior scientific positions. Any who might not appreciate your efforts to counter this bias will find it instructive to direct their attention to the list of authors on the publications that you just mentioned.

LC: Throughout my career, I have simply tried to recruit the best scientists. It just worked out that many of these were female. The reward for me has been to mentor a number of female scientists who have attained important and successful academic positions.

SBS: Your most recent publications confirm that you continue to explore new areas of phosphoinositide pathogenesis.

LC: In addition to hematological diseases, my current work is focused on the possible role of abnormalities of PLCs in the pathogenesis of glioblastoma: it is the most common and most malignant of adult gliomas, and hence it also carries the worst prognosis. The first data are very encouraging [16].

SBS: As I reflect upon our brief conversation, I am led to guess you must be deeply satisfied that your early championing of the importance of nuclear inositol lipids has been rewarded with a wealth of data describing their regulation of chromatin remodeling, DNA repair, RNA processing, and gene transcription. Nevertheless, I find it intriguing that recent reviews by you and your colleagues (e.g., [17]) pose some of the same fundamental questions about inositol lipids that you were asking 40 years ago; for example, concerning their origins (nuclear delivery vs. nuclear synthesis?), their biophysical presentation (protein bound and/or present in biomolecular condensates?), and, of course, their mechanisms of action.

LC: We know so much more than we knew in the 1980s, yet full understanding is yet to be achieved. I believe that the most important thing we should solve is the total comprehension of how inositol lipids and other signal transduction systems are responsible for cell growth and differentiation processes. It is the pursuit of this information and the hope of providing new weapons for medical therapy that keeps me inspired and enthusiastic.

SBS: Many of my scientific publications owe much to the collegiate nature of our field and the generosity of my many colleagues. Are there any that were particular sources of inspiration that you would like to acknowledge?

LC: Among the colleagues who have inspired me in the continuation of my research activity, I wish to make particular mention of Robin Irvine, Daniel Raben, John York, Vytas Bankaitis, and last but not least, Sue Goo Rhee and Pann-Ghill Suh, who shared with me their own antibodies against PLCs, which are far superior to any of the commercial ones. I am keen to thank the organizations that have supported me financially in my research: the Italian Ministry of Research, the Italian Association for Cancer Research, the CARISBO Foundation, the Intesa San Paolo Foundation, and the Italian National Research Council.

But the biggest thanks go to my wife Erminia, who has totally supported me since the early years of my career, who followed me to Glasgow and who continues to be a precious stone in the social part of the Advances in Biological Regulation Symposia.

SBS: Thank you very much, Lucio! 

Acknowledgment: This interview was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, USA. 

References

1. Irvine, R. Nuclear lipid signaling. Sci STKE 2000, 2000, re1, doi:10.1126/stke.2000.48.re1.
2. Rose, H. G.; Frenster, J. H. Composition and metabolism of lipids within repressed and active chromatin of interphase lymphocytes. Biochim Biophys Acta 1965, 106, 577-591, doi:10.1016/0005-2760(65)90073-1.
3. Manzoli, F. A.; Maraldi, N. M.; Cocco, L.; Capitani, S.; Facchini, A. Chromatin phospholipids in normal and chronic lymphocytic leukemia lymphocytes. Cancer Res 1977, 37, 843-849.
4. Cocco, L.; Maraldi, N. M.; Manzoli, F. A.; Gilmour, R. S.; Lang, A. Phospholipid interactions in rat liver nuclear matrix. Biochem Biophys Res Commun 1980, 96, 890-898, doi:10.1016/0006-291x(80)91439-4.
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6. Smith, C. D.; Wells, W. W. Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation. J Biol Chem 1983, 258, 9368-9373.
7. Cocco, L.; Martelli, A. M.; Gilmour, R. S.; Ognibene, A.; Manzoli, F. A.; Irvine, R. F. Rapid changes in phospholipid metabolism in the nuclei of Swiss 3T3 cells induced by treatment of the cells with insulin-like growth factor I. Biochem. Biophys. Res. Commun 1988, 154, 1266-1272.
8. Divecha, N.; Banfic, H.; Irvine, R. F. The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor (for IGF-I) in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C to the nucleus. EMBO J 1991, 10, 3207-3214, doi:10.1002/j.1460-2075.1991.tb04883.x.
9. Payrastre, B.; Nievers, M.; Boonstra, J.; Breton, M.; Verkleij, A. J.; Van Bergen en Henegouwen, P. M. P. A differential localization of phosphoinositide kinases, diacylglycerol kinase and phospholipase c in the nuclear matrix. J. Biol. Chem 1992, 267, 5078-5084.
10. Martelli, A. M.; Gilmour, R.S.; Bertagnolo, V.; Neri, L.M.; Manzoli, L.; Cocco, L. Nuclear localization and signalling activity of phosphoinositidase Cb in swiss 3t3 cells. Nature 1992, 358, 242-245.
11. Manzoli, F. A.; Capitani, S.; Maraldi, N. M.; Cocco, L.; Barnabei, O. Chromatin lipids and their possible role in gene expression. A study in normal and neoplastic cells. Adv Enzyme Regul 1978, 17, 175-194, doi:10.1016/0065-2571(79)90013-x.
12. Maraldi, N. M. M., G.; Cocco L.; Manzoli, F.A. Anatomical Waxwork Modeling: The History of the Bologna Anatomy Museum. The Anatomical Record 2000, 261, 5-10.
13. Follo, M. Y.; Finelli, C.; Clissa, C.; Mongiorgi, S.; Bosi, C.; Martinelli, G.; Baccarani, M.; Manzoli, L.; Martelli, A.M.; Cocco, L. Phosphoinositide-phospholipase C beta1 mono-allelic deletion is associated with myelodysplastic syndromes evolution into acute myeloid leukemia. J Clin Oncol 2009, 27, 782-790, doi:10.1200/JCO.2008.19.3748.
14. Follo, M.Y.; Finelli, C.; Mongiorgi, S.; Clissa, C.; Bosi, C.; Testoni, N.; Chiarini, F.; Ramazzotti, G.; Baccarani, M.; Martelli, A. M.; et al. Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to azacitidine in high-risk MDS. Proc Natl Acad Sci U S A 2009, 106, 16811-16816, doi:10.1073/pnas.0907109106.
15. Cocco, L.; Finelli, C.; Mongiorgi, S.; Clissa, C.; Russo, D.; Bosi, C.; Quaranta, M.; Malagola, M.; Parisi, S.; Stanzani, M.; et al. An increased expression of PI-PLCbeta1 is associated with myeloid differentiation and a longer response to azacitidine in myelodysplastic syndromes. J Leukoc Biol 2015, 98, 769-780, doi:10.1189/jlb.2MA1114-541R.
16. Ratti, S.; Marvi, M. V.; Mongiorgi, S.; Obeng, E. O.; Rusciano, I.; Ramazzotti, G.; Morandi, L.; Asioli, S.; Zoli, M.; Mazzatenta, D.; et al. Impact of phospholipase C beta1 in glioblastoma: a study on the main mechanisms of tumor aggressiveness. Cell Mol Life Sci 2022, 79, 195, doi:10.1007/s00018-022-04198-1.
17. Xian, J.; Owusu Obeng, E.; Ratti, S.; Rusciano, I.; Marvi, M. V.; Fazio, A.; De Stefano, A.; Mongiorgi, S.; Cappellini, A.; Ramazzotti, G.; et al. Nuclear Inositides and Inositide-Dependent Signaling Pathways in Myelodysplastic Syndromes. Cells 2020, 9, doi:10.3390/cells9030697.