What discovery impressed you most during your career?
Live Conversation between Ron Rosenfeld and Cheri Deal
With contributions by Ze’ev Hochberg, Alan Rogol, Jan-Maarten Wit, Alicia Belgorosky, Paul Czernichow. David Dunger, Ralph Rappaport, Leo Dunkel, Martin Ritzen
Cheri Deal: I have the pleasure of being able to probe Ron Rosenfeld’s brain. The question posed to Ron is “What scientific discovery did you feel, in your career, has made the most impact”. So, go ahead and give us your answer.
Ron Rosenfeld: Thank you Cheri. All of us in our investigative careers have had the experience of coming across a paper or a lecture where you said, “Wow, this has just opened up a whole new area of thinking.” And for me there’s no question that the paper (series of papers) that most impacted me were Argiris Efstratiadis knock-out models, which I think opened the entire growth hormone IGF system (Nature. 1990;345:78-80, Cell. 1991;64:849-59)
Deal: And in what way did you feel they impacted you? Is it mostly because of IGF-1 genetics and biology? IGF-2 receptors and biology? The receptors? You say the knock-out papers, but carry that a little further for me.
Rosenfeld: Well, let me give a little bit of background. So, the first successful knock-out mouse was performed in 1989 by Evans and Smithies and Capecchi. They ended up getting the Nobel Prize for that in 2007. Efstratiadis is a Greek trained molecular biologist working at Columbia University at the time, had no particular interest in endocrinology or growth hormone or IGF, but thought that that would be an interesting question to use a knockout system. So, within 6-12 months of the initial in 1989, he had successfully knocked out a series of genes. The first one was IGF2, and it was a tour-de-force, because what he discovered, for those of you who don’t remember, was that in knocking out IGF2, you could result in a 40% loss of fetal growth, but no impact on postnatal growth. In addition, quite remarkably, he recognized that IGF2 was an imprinted gene, and this is in fact, the first endogenous human gene to be demonstrated, to be imprinted. [Deal: Mouse gene] Mouse gene, to be correct, thank you Cheri. So, it was an extraordinary tour-de-force. Shortly thereafter, he knocked out the gene for IGF-1, and in the case of IGF-1, again, there was a 40% reduction of fetal growth, but this time an impact on postnatal growth. He then knocked out IGF1 and IGF2 in combination, the IGF-1 Receptor, IGF-1 and the IGF-1 Receptor, IGF-2 and the IGF-1 receptor, demonstrating the specific role of IGF-1, IGF-2, IGF-1 receptor in both intrauterine and postnatal growth. Finally, he knocked out both IGF-1 and the growth hormone receptor, resulting in a mouse that was only about 17% of normal mouse size. I remember very well that that year he was invited to give an honorary lecture at the endocrine society. Not sure, you must have been at that meeting, Cheri. And he shows a picture of this tiny mouse, and he said: “This mouse has done more for my career than Mickey did for Walt Disney”. And he was right. Of course, all of this, as you say Cheri, was done in mice but it totally opened up the molecular genetics of human growth. And shortly thereafter we identified (or we collectively, the endocrine community identified) patients with IGF-1 receptor defects, IGF-1 defects, IGF-2 defects, growth hormone receptor defects, Stat 5A defects and all of this was laid out by Efstratiadis’ work. So, and then he left the field of endocrinology. Said “I’ve answered all the questions I’ve wanted to ask, I’m going to move on to cancer biology”. But really, in the series of a half a dozen papers written over the period of three years, he paved the way of much of the work for many of us.
Deal: I was going to take this a bit further with regards to the Nature paper, which was the 1990 DeChiara Efstratiadis’ paper (Nature. 1990;345:78-80) that extended further in The Cell, 1991 paper. With regards to IGF2, the IGF2 knockouts, I didn’t do a PubMed search to try to get a handhold on the amount of literature published subsequent to the IGF1 knockout, but I did do PubMed to try to see what, because Ron shared with me what he was going to talk about, what the notion of IGF2 imprinting spawned. And it is absolutely remarkable because it spilled over to not only the growth field, but to the cancer field. It spilled over to the whole notion of epigenetics and plasticity and fetal determinants of adult health. It spilled over into a huge number of ageing papers, and so. For me, it was actually the IGF2 papers, which I think have contributed the most to science. I think that somewhere down the road, people are going to have to get a prize for that work.
Now the question I have for you, carrying this forward: You’re an expert in Darwin and Wallace, who came quite a bit after Lamarck. How do you think Darwin, who recognized, I believe, this notion of Lamarck’s, that you could inherit acquired characteristics, even though Lamarck never thought that was an important idea in his own career; how do you think Darwin would relate to those articles of Efstratiadis’ on IGF2 imprinting? How would he have incorporated that into his Theory of Evolution?
Rosenfeld: Cheri, such a tough question. So first of all, you are correct that Darwin in fact, embraced many of Lamarck’s claims, including acquired characteristics. Something incidentally that Wallace took him to task about, because Wallace said there’s no evidence to support that. But certainly, neither of them would have had any thoughts about epigenetics at that point. That was far beyond them. But I think each of them would have looked at the question of “What do these genes and different regulatory processes in these genes contribute to survival of the fittest”. And you know, we should remember that, really, survival of the fittest, means survival of your genome. How does it help you transmit your genome? The way I always remember imprinting of the IGF2 system is the idea that the fathers want their children to be large, so they’re willing to endow the IGF2 gene, whereas the mothers want the fetus to be small, so she’s going to endow the IGF2 receptor to degrade the protein. So that’s something I think Darwin and Wallace would have embraced.
Deal: Okay, now I’m going to take it further. Are any of you aware of the other theories, if we’re going to extend evolution and the reasons for imprinting in terms of survival of the species?
So, epigenetics, what you’re saying is epigenetics. We should perhaps transpose that to the phenomenon of puberty, and I think there is evidence that it definitely occurs. But getting back to the evolutionary reasons for the development of imprinted genes or for the propagation of imprinted genes. We are very anthropocentric because the notion of imprinting has existed, or of epigenetics has existed for many years prior to the demonstration in mice of Efstratiadis, in angiosperms, in flowering plants. And there, it was clearly known that this phenomenon of selective gene inactivation occurred in the endosperm, which is an analogous organ to the placenta, where of course we have the most amount of imprinted genes. And so, I find it interesting that a paper that has so influenced, or papers that have so influenced the direction of a lot of our scientific thought in many fields actually had to be brought to mammalian biology and even human biology with a demonstration of imprinted genes in humans before it really took off. And other areas, for instance, look at breeding. When you look at the donkey-horse crosses, we know that getting a hinny or a mule, depends on whether the father is the horse, or the father is the donkey. And in phenomenon like that, which are analogous to puberty in actual fact, the phenomenon of going from a caterpillar to a butterfly, and I guess we can look at that as a sort of puberty, in caterpillars and butterflies. That’s very epigenetic, and there are imprinted genes in that. And the same with the epigenetics involved in the honeybee, depending on the amount of royal jelly that is eaten, so this brings in the concept of nutrition, you deviate either to get a queen bee or a worker bee. So, all I say is that I find it astounding that we as scientists need to bring a scientific discovery back to us as humans, or at least in our animal realm, before we really take it and run with it. And that says something about scientific ideas and how they’re born, but I would like to carry that on, maybe with comments from you.
Rosenfeld: Your points are absolutely well-taken Cheri, and it harks back to Ze’ev’s introductory comments about the fact that we speak to each other all the time in our papers and our meetings, but we don’t have the opportunities often to speak to people outside our own field and to be enriched by scientific or humanistic or sociological observations that are outside of the field of paediatric endocrinology, which can often greatly assist us in understanding issues that are important to us.
Deal: Yes.
Ze’ev Hochberg: Before the era of genomics, the center discussion in evolution used to be the calculus of energetic, and the saying was that a calorie used to growth cannot be stored, and it cannot be used for reproduction. In the center of all of this is growth Hormone. Clinical conditions where you have slow growth, such as growth hormone deficiency, are associated with deposition of fat, and clinical conditions were a child grows slower, such as for example, around juvenility, the age of adrenarche at 7-8, there is deceleration of growth and at the same time, we have the adipose rebound. So, at the center of this adiposity, there is low growth hormone. And with all of these genes that Ron mentioned, there is always a change in growth, but also a change in adiposity.
Deal: Maybe I can ask you another question now. If you were going to point out the next field within the growth hormone, IGF-1 axis, or within the IGF-1, IGF2 story as the groundwork being laid by Efstratiadis pointed out, what would you say would be the next big thing?
Rosenfeld: So, as I was preparing for tonight’s meeting, I was thinking about the fact that there are limitations to mouse knock-out studies, in terms of what they can tell us about human biology. They open up a lot of issues, but humans are not mice, and we shouldn’t expect that it’s necessarily the case. When Efstratiadis knocked out IGF1 and GH receptor, he got a cumulative, an additive effect on growth. So, his conclusion was that growth hormone has growth promoting actions that are independent of the IGF system. And that observation would seem to hold true when you look at IGF-1 therapy of Laron Dwarfs. We know that IGF-1 treatment of growth hormone receptor mutants cannot duplicate the effect of growth hormone treatment in growth hormone deficiency. But I personally think that the question of whether growth hormone has growth promoting actions independent of IGF-1, or whether IGF-1 is in fact a total mediator, or the IGF- system in total is the total mediator of growth hormones, growth promoting actions in humans remain unanswered. And I think that has important therapeutic implications, clinically. So, to me that remains a very interesting question.
Alan Rogol: As many of you know, one of the things I deal with is doping in athletes. This is exactly what the issue is that at least among bodybuilders and other athletes, that they don’t consider those two compounds the same, and if they don’t do it, there’s something behind it, that’s our charge, is to find out what’s behind it.
Deal: And further to growth hormone actions and working through the growth hormone receptor, what is your feeling about the intersection of immunology with growth hormone biology? Because I think that too is something, if you’re talking about the importance of growth hormone effects.
Rosenfeld: You’re talking about immunology, Alicia might want to address this one also, well, the central issue is that the growth hormone receptor is a cytokine receptor. And that like many cytokine receptors it signals through the Jak system. There’s got to be an important message in that. It hasn’t been adequately explored of course, but one very real possibility is that the growth failure that one sees in so many chronic diseases, not just immune dysfunction diseases, but any chronic inflammatory disease can in fact be due to interference by immune modulators with growth hormone receptor signalling, and I think that’s clearly an area that’s ripe for research. Jan-Maarten, any thoughts about this?
Jan-Maarten Wit: I think it’s also interesting that Stat3 hyperactivity seems to give a downregulation of Stat5.
Deal: And there is actually a lovely symposium on Saturday that I have the honor of chairing, and one of the papers presented will be on Stat3 biology and genetics, so we’re looking forward to that.
Rosenfeld: Just to add to that, one of the things that is, I think, underappreciated by the endocrine community is that constitutive mutations of Jak2 and Stat5 have clear implications for cancer. And as we think about the potential ramifications of growth hormone or IGF-therapy in terms of malignancy, there’s got to be an important message there also.
Alicia Belgorosky: I wanted to say something about one question that we have to answer, Ron, this is how the IGFBP systems works, because it is something that we don’t know. There is a steady state function in some tissues, but not in other tissues. And I think that it’s a big question that we need to answer in the future. And in relation to Stat5 and the immune system, it’s something very interesting, because how, when Stat5b gene mutation, how the immune system is alternated is very interesting, and why we have the variability in phenotype in terms of immune system alteration is something very interesting to look for.
Deal: I actually wanted to turn this over to the rest of you. Because Ron had time to think about this, but when you received the agenda, all of you probably had a bit of a flashback as to what paper influenced you the most in your career, or you feel has maybe set the stage for a huge body of work and knowledge over your practicing years. So, I’d like to go around and make each one of you come forth with an idea. If you agree with Ron that’s fine too, but have you thought when you saw this agenda, did the lightbulb go on?
Belgorosky: The technique of PCR.
Deal: The technique of PCR. Yeah, I was so happy when I did my fellowship with Ron in California, my license plate started with PCR, and I kept that license plate, I’ll give you a photograph, I made it into a slide.
Rogol: Actually it’s quite recent and I’m suspected many of us who have had relatively long careers have been influenced by recent papers, and it’s the work of Karsenti (Cell. 2016;165:882-95). He has talked about how the hematologic system talks to the brain talks to the regulation of metabolism. It’s in terms of energy for the whole animal, and to me this was kind of, if you remember your neuroanatomy class, you were tested and if you could give 5 knife-cuts, you could get everything, but you really had to know your neuroanatomy where one knife-cut could do all of those things. I think he has really brought together in a very fundamental way, how many systems are organized, be it bone, be it energetics, be it the brain.
Hochberg: I got a present for my son’s birthday, Richard Dawkins’ the Selfish Gene. And this changed my thinking altogether. This was for me my career-changing book.
Paul Czernichov: You’re probably going to laugh at me, but when I was 24 and I discovered radioimmunoassay. We had a window open on the body and can measure the variation of hormones. And I thought that what a fantastic tool for clinical research, and it has been a fantastic tool, even though most people are using now ELISA. I was shocked when we were measuring blood glucose in kids with diabetes, and we discovered the disasters of our treatment, and it still is. And I thought that the next step would be measuring hormones and fluids inside of the cell, and that would be probably a great achievement when we complete our understanding of what we are doing. It is something that is left over on the caravan that I think that being able to merge our IGF-1 inside of the cell. That probably would be the further step in our ability to understand what we are doing.
Rosenfeld: I just want to add Paul, to your story, that Ray Hintz told me, when he was an assistant professor, Rosalyn Yalow, from Radioimmunoassay told him, “Why are you wasting your career on IGFs, what a total waste of time, can’t you go do a real hormone?”
Jan-Maarten Wit: A paper that I found very interesting was that in the cell, one hormone is changed to another hormone, for example by the enzyme aromatase, in chondrocytes. Hormones that we first only knew in the adrenal are actually present in a lot of cells.
David Dunger: Yes, I was going to mention someone named David Barker, who I disagreed with every time I met him, because I talked about genes, he refused to talk to me at all in the last few years of his work. I don’t think he had very good ideas about what he was talking about, but he did open up this whole area of making developmental biology and its factor in adult life respectable. And these spawned thousands of thousands of grants in lots of careers.
Deal: I was delighted when you said Efstratiadis, because I was going to say the IGF2 knockouts, and I will share personal information. I was a fellow in Ron’s lab when he actually presented that Cell paper by Efstratiadis. And that to me was a lightbulb moment, and I went back to Montreal, and wrote my first big grant, and the reviewer said: “Oh, but IGF2 imprinting has been shown in mice, why do you want to show it in humans?” And that really ****ed me off. And so I got together with Constantine and his graduate student came to my lab and actually we ended up doing it without any funding. It was all internal funds that I had, and it lead to the Nature Genetics paper showing the first imprinted gene in humans, and that actually is my most cited paper. But I think that says something very much about how science works. And not only is it really hard to overthrow scientific paradigms as Kuhn wrote in his Making of the Scientific Revolution, but it is also very hard to get innovative ideas through granting agencies. What I always teach students is, Don’t worry about money, actually that came from Ron, Don’t worry about money, if you have a good idea, go for it, and I appreciate you for having said that, because I’ve always done that.
Jean-Pierre Bourguignon: So, this is back in the early 80s, I had been in research for a few years, for it was about in the mid 70s I started. And this was the work of Knobil [unsure of name, 1:48:30], first using a primate as a model which is fantastic, and second showing that changing rithm of stimulation of a cell system would come to totally unexpected effects such as increasing the frequencies, reducing response. Well that really was a shock and I think it was quite an extraordinary finding, that has raised many points including treatment with GnRH agonists. So, I think it was quite a finding.
Ralph Rappaport: Just to go back to something which may sound less glamorous. It’s a type of psychosocial dwarfism which was described by Bob Blizzard and I was there at that time, and it was to me completely new. At that time Growth Hormone could barely be measured in blood. And soon after a paper came out which I haven’t reread, but I must go back to it, it was a paper in Cell showing that a plant would not grow if you don’t put your hand every day on it. And touching the plant every day would help it to grow. And the authors have shown that there is in the machinery which I have completely forgotten in that plant, something which makes a plant feel the hand every day and help her to grow. I thought that it was a vegetable psychosocial problem, [diffuse laughter] but who knows? Here we are coming close to social and environmental issues translated into hormone secretion and action.
Deal: Any other phenomenal scientific discoveries that have stimulated your thinking?
Leo Dunkel: So actually, I didn’t think about this in advance, but I have been really impressed with the latest developments in cellular biology and molecular genetics, especially cellular reprogramming combined with genome editing. I have been interested in GnRH neurons for years and now we are able to culture from stem cells of our patient fibroblasts, culture them in the GnRH neurons and even create monogenic mutations, specific mutations in the cell. And this is totally opening new fields, because I have never been able to test these types of cells like this, and especially like creating specific mutations, so I have really been sort of fascinated and blown away by these nice techniques.
Martin Ritzen: Well I think the real breakthrough since I have come through, is improved methodology. I have been old enough to see the development of modern molecular biology and PCR and whatever, and of course that has spread the whole field of science. So, this is fantastic, as humans with new methodology. But I also like to put a word for an observant clinician’s thoughts and conclusions. One paper that I was fascinated by was published in New England Journal of Medicine, October 16th 1994, Eric Smith’s paper on the ER-alpha knockout man, and where he could show completely unexpectedly that Estrogen (ER alpha at least) is extremely important for bone remodelling in males. Nobody had thought about that before. And he also showed that spermatogenesis is affected, which is again, estrogen directed? Estrogens needed for spermatogenesis? That again was new. So, an astute clinician that can go back and of course you remember that here, he involved good scientists from molecular biology, and that’s why he could show logical steps in the formation of this. So, if we as clinicians could find the key patients and then work them up properly, we’ll find new discoveries.
Deal: And that paper also hammers home the point, the subsequent papers that follows, that mice and men are really different, because when you look at all the knockouts, the ER Alpha, the ER Beta knockouts, the impact on growth and on puberty, gonadal development, are completely different from what you predict in the human. We have to close. So, it’s been delightful hearing from everybody, and I will turn this back to Ze’ev so he can give his comment.
Rosenfeld: Just to follow up on Michael’s earlier comments. So maybe this kind of question, “What scientific discovery impacted you”, is relevant to use because it puts what we do in a historical context, which is something that I think young researchers aren’t exposed to. Maybe this is something we should think about introducing into SP-programs, or PES programs or whatever. Have somebody talk about the historical background that impacted their research directions. I think that would be well received.
COMMENTS TO THIS CONVERSATION ARE TO BE MAILED TO JAN-MAARTEN [J.M.Wit@lumc.nl].