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Novel Mechanisms of Reproductive Hormone Action
Novel Mechanisms of Reproductive Hormone Action
Novel Mechanisms of Reproductive Hormone Action
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All right, welcome everyone to this session on Novel Mechanisms of Reproductive Hormone Action. My name is Eric Nelson. I'm from the University of Illinois, and I'm filling in. Both the moderators that were supposed to be here couldn't make it, so please bear with me. The good news is we've got three fantastic speakers lined up for you. And just a reminder, after this session, you can head on down to the Society's reception. All right, without delaying anything further, to start we've got Dr. Frank Clossens from KU Leuven. And he's going to be talking about rodent models that show differential roles of the three dimerization models of the androgen receptor in male development. Start. Thank you for the introduction. So I'm going to talk about the dimerization of the androgen receptor. At this point of the meeting, we don't need to introduce the action mechanism of steroid receptors. I'm just showing you the androgen receptor, and the most well-known dimerization of steroid receptor happens via the DNA binding domain, right? Binds to the androgen response elements. For the AR, there are other mechanisms of dimerization. One is the so-called NC interaction. That's the interaction of an FQNLF motif in the amino terminal end that binds to the AF2 of the ligand binding domain. It's specific for the androgen receptor. And then the third mechanism of dimerization happens via the LBD. That was discovered by the group of Eva Estebanes. But LBD dimerization, of course, is well-known for estrogen receptor and other nuclear receptors. So I'm going to talk about these three dimerization modes and what we found about them, the novelties. And then, if I have time, the fourth part will be on a dimerization inhibiting molecule that we are developing. So first, the DBD dimerization. This is a biochemistry textbook that steroid receptors, nuclear receptors, they have a DNA binding domain that consists of two zinc fingers. First zinc finger has this B box, which enters into the major groove and binds to the sequence. It recognizes the sequence. The second zinc finger has this D box, which is important for the dimerization. And indeed, this is reflected on the androgen response elements that we see always organized as inverted repeats. Even when in the AR chip, this consensus of the AREs sometimes is very hard to recognize the inverted repeat nature. And many people have been hypothesizing that the AR could be binding or acting via a monomeric site. So to study that, we made a mutation in the D box of the androgen receptor binding domain. And this mutation we made or we were inspired by the work from Martin Van Royen, who already eight years ago, I think, showed that the double mutation in the D box kills the androgen receptor dimerization. So we introduced this mutation. And when we do the reporter assays, as you do, the wild type receptor is nicely inducing this reporter gene, but the D box mutant doesn't. But that's not true for all AREs, as I already showed you here. For reporter genes that have the C31 ARE, the D box is not needed, apparently, or the mutation of the D box has no effect. We were very surprised by this. So we tried different cell lines, different constructs, even mouse receptor and human receptor made the same mutations. What I show you here is a reporter where there's only one C3 ARE upstream of the TATA luciferase. And still there, the demon is still active, at least as good as wild type. So puzzled by this, we were wondering what is happening in vivo. So we made, by CRISPR-Cas, we made the double mutant in the AR, and we call this mice demon for monomeric D box mutation. And this is what we saw as a phenotype. So I can't see it, but the males have a very large intergenital distance, relatively large. The females have a very small intergenital distance, and our demon model has an intergenital distance that's in between. So externally, we see there's something happening. When we look at the urogenital tract of these mice, we see that male mice have these seminal vesicles. Seminal vesicles, you have the kidneys, of course. You have the bladder with the prostate underneath. You have the testis in the pericardial fat, and you have the epididymis. If we then look at the demon male, we don't see any seminal vesicle being developed. Also, the prostate, we need a microscope to see some differentiation in the prostate. We also see some hypospadia-like penis phenotype. When we look at the androgen levels, that's the first thing we look at. The testosterone levels in the circulation, we see there's no big difference. There is, sorry, in the demon, there is a reduced testosterone level. So when comparing the phenotypes of these mice, we wanted to make sure that we are not looking at different levels of testosterone. So we did an intervention study where we castrate the mice and at the same time provide them sticks, celestic sticks, releasing either cholesterol, fecal, or testosterone. We did that for wild type and for demon littermates. If we look at these testes that we take out at castration, and we look at androgen-regulated genes, specific for Sertoli cell is the ROX-5. You see in the demon, there's hardly any expression. Also for the Leydig cells, the INSUL3 is an androgen-regulated gene. It's also lost in the demon. Now if we look at the kidney, the kidneys are conserved. They are well-developed in the demon, and they are androgen-responsive organs. So if we look at the kidney, we see that FKBP5... Now I'm struggling. We see first that the AR level, expression levels, are similar between the two models, the wild type and the demon. If we look at the CAP gene, the kidney androgen-regulated protein, you see that it's still androgen-responsive. But if we look at FKBP5, which is a well-known androgen-regulated gene, it's no longer androgen-regulated, or far less. And also the OGC1 is no longer androgen-regulated in these kidneys. So, conclusions on this debox. The demon mice seem to have a partial androgen insensitivity phenotype. Debox mutations have gene-specific effects in the reporter genes, but also in the mice. And of course, we need to do RNA-seq now, and chip-seq, and what have you. We're going to have crowdfunding for these analyses. And on the other hand, we also want to do crystallization studies for the DBDs to see whether the demon is still able to bind, and how does it bind to these elements. This is the first crystal we have, or the new crystals that we have of the ARDBD, but we don't have the demon crystal yet. For the demon prostates, important for those interested in prostates and prostate cancer. And for the demon prostates, we really need a microscope to be able to find them. So, the question is, is the debox dimerization a therapeutic target for prostate cancer? We can discuss it later. I want to go now to the second dimerization, this so-called NC interaction. Even the alpha fold, I looked it up, the alpha folds of Google, they will show you that for the AR, there is an FQNLF motif binding to the AF2 site in the LBD, once it's bound to ligand. If we introduce a mutation of the FQNLF motif, you see in the FRET assay in the intracellular, there is a strong reduction of the NC interaction. If we do the reported gene assay, you see the reduction in the potency of the receptor, the sensitivity is very similar. But again, this is in vitro, so we wanted to know what is happening in vivo, so we introduced this mutation in vivo by CRISPR-Cas in mice. We call this the NOC model. As you can see here, the intergenital distance of wild type and female are very different. In the complete knockout of the AR, the intergenital distance is very similar to female, but in our NOC, the distance is very similar to wild type. So the NOC doesn't seem to have an effect on the intergenital distance. When we look at the testosterone levels in these mice, we see no significant differences. Also, DLH has no significant difference, but there might be a tendency. To be sure that there's no possible effect of testosterone or DLH, we again did an intervention with the castration and the releasing celastic sticks. In this condition, we look at the testis at the moment of castration. The testis don't change in weight or are not different in weight. In the uretero-intergenital distance here, the testis are gone. But what you see is seminal vesicles, well-developed, and also prostate, well-developed, and the epididymis is also well-developed. When we look at these ventral prostates for androgen-regulated genes, you see that the androgen regulation is still conserved for all the genes that we looked at. So the conclusion here, this FQNLF motif, although there are so many papers giving it functions or giving coactivators functions in this NC interaction, for instance, when we do this mutation in the mice, we see in vivo that this NC interaction is not relevant for normal male development. Of course, here too, we need to do RNA-seq and ARCHIP and what have you. And again, the question, is this a therapeutic target in prostate cancer, can be discussed afterwards. The third dimerization mode is happening via the ligand-binding domain. Here, we zoomed in at the center of this ligand-binding domain dimerization, and we selected a tryptophan-to-arginine mutation at position 752 in humans. We selected this mutation because it was found in androgen insensitivity patients. In the FRET, again, in live cells, we see a reduction of the dimerization. And in the reporter assay, we saw that the sensitivity is lower, but the maximal induction is the same as the wild type. Again, in vitro data, what is happening in vivo, long-lived CRISPR-Cas. We made the lemon mutation, the LBD monomerics, and these lemon mice, as you can see here, they have an anogenital distance that is very similar to the wild type females. When we look at the urogenital tract, we see no seminal vesicles, we see no epididymis, we see no prostates. We do see the testes, but they are much smaller, and the perigonal fat is also still present. When we look, this time, at testosterone levels in circulation, the testosterone levels are sky high. Our mouse andrologist has never seen such high levels of androgens, which is in contrast to the global androgen receptor knockout, which has very low levels of testosterone. When we look at the LH, we see the LH is very high in both the ARCO as well as the lemon. So the HPG axis is corrupted. Again, these high differences in testosterone level, they might be confounding our measurements and our observations, so we did the intervention study again with testosterone-releasing celestic sticks. Again, we look at the kidney. The other organs are not there. Seminal vesicles are prostates. In the kidney, we see that the androgen receptor is going into the nucleus, and the lemon androgen receptor is also going into the nucleus in a hormone-dependent way. When we look at whole extracts of these kidneys, we see similar expression levels. When we look at the chromatin level, however, also we see that lemon is still binding to chromatin. When we do the RNA-seq analysis of these kidneys, and we look at the androgen-regulated genes that are androgen-regulated in the wild type, we see no androgen regulation in the lemon. So there is no evidence of androgen receptor activity in the kidneys, despite the AR being in the chromatin fraction. Now, this RNA-seq we confirmed in qPCR, FKPP5, and CAP. FKPP5 and CAP are nicely androgen-regulated in wild type, but no longer regulated in the lemon kidneys. The observation is that when we do the chip assay on the androgen-receptor binding sites near the CAP and the FKPP5 gene, we see that the lemon AR is not binding anymore to these androgen-receptor binding sites. It's far less than the wild type AR. This is correlated with the active histone mark on the same androgen-receptor binding sites. For FKPP5 and for CAP, we don't see any changes on the TOX3 gene, which is a prostate-specific gene, so we didn't expect it there. So, very surprising, we see chromatin binding, but we don't see ARBS binding. So the conclusions here that is, first, that the lemon mice are feminized. There's no remaining lemon activity in the kidneys. The binding to the ARBS is impaired, so the LBD dimerization is necessary for the AR in vivo activity. Again, ChIP-seq and RIME and what have you are on our to-do list, and we want to know where the lemon is going, and we want to know who it is or not recruiting. Okay, so let's recapitulate. In the mouse model on the D-box, the DEMON shows partial androgen insensitivity, very similar to what we saw in the patients, and the double mutation were two mutations that are also found in, I forgot to say that, in patients with partial androgen insensitivity. For the NC interaction, we see a normal male phenotype, and for the LBD dimerization, the lemon model, we see a complete androgen insensitivity. So we decided, well, this last one is probably the most interesting one to develop dimer-inhibiting molecules. We gave this project, of course, to a structural biologist. He found a possible cavity. My finger is in front of the laser. There is this cavity where he was looking for pharmacophores, millions of compounds that he screened, molecular docking, visual inspection. Then we did a normal luciferase reporter assay. We came up with five hits. One of them we expanded. These three are very similar, so we expanded them, but the expansion didn't deliver better compounds. That's probably saying that our initial docking and pharmacophore search was optimal. But anyway, we have compounds that are in the micromolar range inhibiting AR function on these luciferase genes. What do they do? Well, in this luciferase gene, where you see here the DHT response, if you add DIM, the DIM20, you see a reduction of the potency, a reduction of the maximum level of induction. Similar to what is seen for TRIAC, which is a compound that binds to BF3, another surface of the LBD. If we then look at the effect of the DIMs on the hormone binding to the ligand binding domain, you see here a nice binding of DHT to the ligand binding domain in black. If you add enzalutamide, that's a competitive inhibitor, so it's normal that the curve shifts to the right. But if we add the DIMs, there's no effect on DHT binding. If it binds, it binds to another site. Does it bind? Well, in our BLI, in biolayer interferometry, we could show that the DIM20 is indeed binding to purified ligand binding domains of the androgen receptor that were coupled to the sensor. So the DIM20 is binding to the LBD, but not to the DHT binding site. In the nanobit system, we are analyzing the dimerization of the androgen receptor via ligand binding domain, and you see a nice hormone-responsive dimerization, which is inhibited by the DIMs. And then finally, we looked at the effect of the DIMs on the proliferation of AR-positive cancer cells, and admittedly, we have to use high concentrations at this moment, but we are able to inhibit the proliferation of VCAP cells, of LINCAP cells, but not of PC3 cells. I see I have still lots of time. So the conclusion of these DIMs is that they do inhibit AR transactivation, they bind to the LBD, but not competitively with DHT or TRIAC, they inhibit the AR dimerization in cells, and their action is different from enzalutamide, and they have an anti-proliferative effect on the LBD. And they have an anti-proliferative effect on AR-positive prostate cancer cells. Of course, this is to be continued. We want to make compounds that are in the nanomolar range active, and hopefully, next time, maybe I have something to declare. So that's what I wanted to tell you. These are the people that did the work, and not on that day. We had a Christmas party. The cat person here, that's Sarah, who did all the mouse work. This is Christine. She did most of the in vitro work. And Xiaoyin is doing the crystallization of the DNA binding domain and ligand binding domain of the androgen receptor. And Arnaud is our molecular structural guy. So thank you for your attention. All right, thank you very much. Mad respect for whoever names your mice. That was fantastic. And to the AV people, the iPad fell asleep, and it's no longer bringing up any questions. Go ahead. Hi, this is Mone Zayedi from Mount Sinai School of Medicine. Although it's not my field, but the data that you show is very relevant to prostate cancer and secondary resistance, right? So the patients who are on enzalutamide or arbitrone get secondary resistance due to upregulation of the AR or due to lineage plasticity. Do you think that your compound might be useful in overcoming the enzalutamide arbitrone resistance? And if so, could the drug be used in combination with, for example, enzalutamide? I'm hoping that it eventually will come to clinical use, yes. Um, well, if you look at the different modes or mechanisms of resistance, many of them still involve the dimerization of the AR. Exactly. So you need the LBD. Even if you have spice variants, there's still a full-size AR present. So my bet would be that they still inhibit the AR. Even in the presence of the spice variants, actually, the dim did it. So yeah, it's an alternative mechanism of inhibiting the AR, alternative from the enzalutamide. So any mutations in the LBD that make enzalutamide an agonist, for instance, or make other steroids agonists or other compounds agonist, they all depend on the dimerization of the AR in order to be active. And the combination, yeah, it's possible. So one of the models you could test this in is the RBP10 TP53 deleted mouse, where there's androgen resistance. And you could actually use this drug. And that would be my suggestion. Yes. Well, first, we want to get lower than the micromolar range to be able to transfer it to mice. So DeMeo from NIEHS. So I may have missed the explanation, but your lemon mouse had a higher than, significantly higher than the knockout, right? So what was the explanation for that? And are you worried that by developing a targeted drug for that, it may be activating an off-target effect, and that's why you're getting that? Because you can't get less than zero, right? I mean, right? You can't get less than zero. Well, we were very surprised by these high levels of testosterone. And another thing is that when they miss testosterone, they can also show androstenedione. And androstenedione was also very high in the circulation of these mice, which is abnormal for mice. That's why we looked at the enzyme that is important for the conversion of androstenedione into testosterone, HSD17B3. And that's an androgen-regulated gene. So the last gene that is involved, or the gene, the enzyme that is involved in the last step of making testosterone is itself regulated by the receptor. Why is testosterone then high? It must be conversion outside of the testis, because the enzyme is gone. And the 17-beta-HSD3 is gone in the testis. So it's similar, actually, to the HSD17B3 knockout mice. They also have high androstenedione and also high testosterone. Because of the high LH in circulation, all the enzymes that are before this last step, they are all up-regulated because of the LH. So that's why the androstenedione is so high. And by comparison, it's a heart attack. Yeah, yeah, yeah. I mean, that's an issue, right? It's an issue because you want knockdown androgen activity. Well, you're getting some activity with this, but you can't get less than zero. No, but yeah. I'm thinking that we're going to treat the patients with ADT and then this compound. So maybe we're killing the synthesis of the precursor as well. Hi, Vincent Giger from McGill. Maybe I missed it. Any of those mutants have a phenotype on the prostate itself? The lemon don't have any prostate. The demons, they have residual prostate. So if we make microscopy slides, then we see some seminal vesicle-like starts, invaginations, prostates too. The knock, they have normal prostates as well, as far as we can see. So if the lemon one doesn't have prostate, then how can you get the model of prostate cancer if you cross it with the P10 or P53 or knockout? No, no. Then we have to use the inhibitors, right? Or an inducible lemon mutilation, I don't think that's very realistic. Theoretically, it's possible, I think. I have a quick question. So you measure testosterone and whatnot. But in all the mice, are they equally fertile? What's their sperm count, sperm motility, that sort of thing? And for that matter, have you looked at the females? Sorry. If you don't want to answer it, you don't have to. Spermatogenesis in the lemon, we did the RNA-seq. And we saw the spermatogenesis stopped at the second meiotic division. For the knock, we see normal fertility. So the NC interaction, no effect. For the demon, when we look in the epididymis, we see mature sperm. Whether these sperms are capable of fertilizing, we have to do in vitro fertilization because of the hypospadia. So that's on the spermatogenesis. What happens with female mice is that the breeding is so difficult. If the males are infertile, you cannot get homozygous females. Only for the knock, that would be possible. But because the male phenotype is wild type, we were less interested in going into females. That's a lot of money. Last question. Hi, Katie Basham from the University of Utah. I'm interested if you can comment on any phenotypes in the adrenal gland. In the? Adrenal gland, especially based on the hormone changes that you're seeing. On the? Sorry. The adrenal. On top of the kidney. The gland sitting on top of your kidney that makes stress hormones. I'm trying to remember. I don't think we looked at them. There's pretty high AR expression throughout, particularly the adrenal cortex. So it'd be very interesting to look in some of your models. I'm trying to remember. In the ARCO, there is this X zone, which is not degraded or involuted. In the Lemon, the adrenals were larger, similar to the ARCO, the global knockout. And for the others, I don't think we looked at it. OK, great. Thank you. All right. Thank you very much. All right, next up, we have Maria Schuler-Almeida from UAMS. And she'll be chatting about the role of ER-alpha in osteoblast mitochondria. Hi. Good afternoon. Good afternoon. So I had to have these slides, but I guess it doesn't matter now, because there's no question and answer. Yeah, that's right. Can we pull up the slides, please? All right, so as you all know, loss of estrogens at the menopause causes changes in multiple tissues. And perhaps one of the most significant changes is loss of bone mass, or osteoporosis. Because it places women at higher risk for fractures. So how does estrogen protect our bones? Throughout life, our skeleton is remodeled by the work of these cells called the osteoclasts, which are cells that are specialized in removing the bone matrix. And so they create these little cavities that are then refilled by these cells called the osteoblasts. They produce new bone and fill up the cavities. So when the work of these two types of cells is balanced, our bones are maintained. However, in certain situations, like with estrogen deficiency, the number of osteoclasts increases, creating an imbalance, and loss of bone mass occurs. We've been interested for quite some time in understanding how estrogens control bone cells. And so osteoclasts, these cells that reserve the bone matrix that are impacted by estrogens, they are cells of the hematopoietic lineage. They differentiate from macrophages upon binding of this cytokine called Rank-Ligand, which is essential for the development of osteoclasts. And in doing so, it recruits that protein called TRAF6 and initiates several signaling cascades. So during osteoclast differentiation, cells, these monocyte or osteoclast precursors, they fuse to form these giant cells that, at some point, sit in the bone surface and secrete acid to degrade the bone matrix. We know for quite some time that estrogens, one of the mechanisms by which estrogens decrease osteoclasts is by acting directly on these cells. And this is via the alpha present in this cell lineage. So the work I'm going to talk about concerns direct effects of estrogens on these cells. What could be the effects that really decrease these cells directly? And so to understand a little bit better about these effects, we performed an experiment in which we added estrogen at different time points during the culture of bone marrow monocytes, which is the, I would say, primary culture. This is the most used model to study osteoclasts. And what we found was that the presence of estrogen, just for the first 24 hours of the cultures, is sufficient to cause this inhibitory effect on the formation of the cells, as you can see. And so one of the features of osteoclasts or osteoclast differentiation is an increase in the number of mitochondria and also their size. As you can appreciate here, we have bone marrow macrophages on this side. And you can appreciate the large mitochondria in the osteoclasts. And this most likely is to provide the energy necessary for the function of resolving the bone matrix, of course. And so most of the cellular energy is made at the mitochondria by the passage of electrons through the electron transport chain. And this electron transport chain is formed by five big protein complexes. Complex one is the largest one of all of them. It's the entry point of the electrons, which are donated by NADH, which gets oxidized to NAD. And so, of course, the major function of these complexes is to form ATP. However, there are other subproducts of this production of energy, which is the formation of reactive oxygen species, and also the oxidized form of NAD, which also acts as a co-factor for several proteins that are so-called NAD-dependent proteins, like, for example, SIRT3, which is present in the mitochondria. So SIRT3 is a deacetylase that is very important for mitophagy. So we had some clues before about the connection between the actions of estrogens and the mitochondria, because we were interested in understanding the role of ROS in osteoclasts. And we did some work using a mouse model in which we overexpress a transgene that puts catalase in the mitochondria of osteoclast lineage cells. And this suppresses the amount of H2O2 that is made by osteoclasts. And what we found was that by doing that, the loss of bone mass that occurs that you see here in control mice with ovariectomy is greatly attenuated, suggesting that mitochondrial ROS is an important factor for the increase in bone resorption with estrogen deficiency. More recently, a colleague in the lab has become very interested in understanding the role of SIRT3, this mitochondrial deacetylase. And he used mice that are knockout for SIRT3. And when he performed a similar experiment, ovariectomy, he sees again that in the SIRT3 knockout mice, the loss of bone mass is attenuated. So these findings support the idea that mitochondria might be involved in this loss of bone caused by estrogen deficiency. However, these factors could be simply permissive and might not be part of the mechanisms via which estrogen regulate these cells. So we wanted to explore more this idea. Another very critical role of the mitochondria is in mediated programmed cell death. So the mitochondrial death pathway, when in the presence of stress signals that cause cell death, there is the dimerization of these two proteins of the BCL2 family called BACs and BAC, which promote the release of cytochrome c, activation of caspase-9 and caspase-3. And we know for quite some time that estrogens promote osteoclast apoptosis. And so it has been proposed a while ago that this effect of estrogens on apoptosis is mediated by an upregulation of fast ligand. And fast ligand, as you see in the cartoon here, is a stimulator of cell death via the death receptor pathway. And this happens by stimulating a caspase-8 and caspase-3. However, this increase in fast ligand could not be replicated by us and others. And we actually performed an experiment in which we use now mice that do not have fast ligand. So they are knocked out for fast ligand. And we find that in this case, the fast ligand knockouts lose as much bone as the control mice, suggesting that fast ligand doesn't play, indeed, a major role in the effects of estrogen. But we wanted to pursue what are the mechanisms via which estrogens promote apoptosis. And so we did some studies to examine whether this could be mediated by the mitochondrial death pathway. And we showed the different steps of this pathway. For example, the release into the cytosol of cytochrome c in the presence of estrogen and an increase in caspase-9 activity. So to test the functionality of this pathway, we also used some cells. This was all in vitro from mice that do not express backs and back, the two essential proteins in this pathway. And when we use these cells, we see that the effects of estrogen on apoptosis, as you see here, an increase in caspase-3, are prevented in cells lacking backs back. And the decrease in osteoclast number that estrogen causes is prevented. We actually saw an increase in the number of these cells, most likely because we're promoting their lifespan. And these are all very early effects, again, of estrogens. So we wanted to know what could be the signals that are initiating this pro-apoptotic effect. And so we performed an unbiased approach where we used cells from control, mice with ER alpha and mice lacking ER alpha. And we found that a lot of the terms that were altered were related to the mitochondria. And when we looked closer to the genes that were being impacted, we see that most of these genes belong to complex I. So these are pre-regulated in the absence of the estrogen receptor. So we performed several set of experiments to understand whether this change in gene expression could have any functional relevance. And what we found was that the stimulation of complex I activity by roentgen is attenuated by estrogen. And the same thing for the basal respiration measured by the oxygen consumption rate using a seahorse flux analyzer, so activation by roentgen, inhibition by estrogen, and same thing on ATP levels. And please note that the effects of estrogen are seen only in the presence of roentgen. These effects are dependent on roentgen being in the cultures. So suggesting that estrogen is inhibiting an early rapid effect of roentgen on activation of mitochondria. So what would be this signal that estrogen is attenuating? So we looked into this protein in macrophages. Activation or inflammatory signals, as shown here exemplified by LPS and binding to TLR4, they recruit TRAF6, similar to roentgen. And this causes the recruitment of this protein called evolutionary conserved signaling intermediate in toll pathways, or EXIT for short, which is an important protein for complex I assembly and stability and to promote mitochondrial respiration and an increase in mitochondrial ROS production that the macrophages do in response to these inflammatory signals. So because, as I told you, TRAF6 is also recruited by roentgen, we thought this mechanism could be responsible for this early effect that we see in osteoclasts. And so what we found when we did some immunoprecipitation studies was that in response to roentgen, there was an increase of association between TRAF6 and EXIT, and that this effect was greatly attenuated by the presence of estrogen. And also, accumulation of EXIT in this mitochondrial fraction now is also greatly attenuated by estrogen, suggesting that this, indeed, could be a mechanism mediating this effect. So we next silenced exit and treat the cells just with MCSF or MCSF plus Rank-Ligand. And we found that silencing of exit, indeed, compromised complex I activity, as it has been described. And it also decreased the NAD plus NADH ratio, which is, again, a measurement of the mitochondria energy production. And we found that the stimulatory effect of Rank-Ligand on any of these measurements was prevented in the apps or with silencing of exit. So in line with these findings, measuring, again, basal respiration, we saw a similar effect. So there is a decrease in the absence of exit, and the stimulatory actions of Rank-Ligand are not prevented. And the same thing for ATP production. When we culture these cells that are silenced for exit for five days to make mature osteoclasts, we see a decrease in cell number, as you would anticipate. But more surprisingly, what we found now is that in the absence of or with silencing of exit, now Rank-Ligand gives the cells a pro-apoptotic effect. So suggesting that when we attenuate exit, the pro-survival effect of Rank-Ligand switches into a pro-apoptotic effect. So finally, we wanted to understand also whether the changes in NAD that you see when you attenuate complex I and mitochondria activity are relevant for the effects of estrogen on cells. So what we saw was that to test the relevance of NAD, we used this nicotinamide riboside, which is a precursor for NAD. So it causes an increase in NAD levels and the NAD-NADH ratio, which were suppressed by estrogen. And you can see that SIRT3 activity, which, again, is suppressed by estrogen, is now pre-regulated, or it's recovered by addition of NAD, suggesting that this is, let's say, a downstream mechanism of these effects on complex I and the mitochondria. But when we culture the cells now to five days, we saw that while there is some rescue of the decline in cell number by estrogen, this is mostly related to the multinucleation of the cells, because it's the large cells that get rescued. And in contrast, the pro-apoptotic effect of estrogen is modestly impacted by the presence of NAD, suggesting that these changes in NAD might affect resorption activity more than the cell number. So in conclusion, we are testing these ideas that estrogen attenuates Rank-Ligand-Induced Complex I activity and mitochondrial respiration by preventing exit translocation to the mitochondria, that inhibition of exit switches Rank-Ligand pro-survival signals into pro-apoptotic, and might mediate the stimulation of the mitochondrial apoptotic pathway by estrogens. And finally, that other biology of the mitochondria related to reactive oxygen species and NAD also contribute to the anti-osteoclastogenic actions of estrogens. And so these are the people that did the work. Adriana was a PhD student that did most of this work. My colleague, Henry Kim, the expert on osteoclasts, and our funding. Thank you. Thank you very much for a great talk, AV people. The iPad fell asleep again. So I don't know if anyone's actually asking questions online, but yeah, go ahead. I'm Mone Zadeh again. I think I know a little more about osteoclasts than I knew about prostate cancer. Let me ask you a couple of questions. So it's very elegant work that you've shown. The osteoclast makes hydrogen peroxide, and we know that from our work way back in 1990. I think the elegance of your work stems from the fact that it's the mitochondria that makes it. But the question here is, it is a highly reactive species. And the fact is, there's a bunch of nitric oxide there. So it's going to make an even more highly reactive peroxynitrate and hydroxyl ions. So how do you tease out the effect between these various reactive oxygen species? That's a great question. I think it's probably difficult to tease apart all these different effects. We are trying to do that, obviously. But in this work, we are more focused on understanding the upstream signal, really, that leads to these changes. My second question is, how convinced are you that estrogen has a direct effect on apoptosis of osteoclasts? Because the cell paper at that time was pretty controversial. And you haven't shown us any data on separating effects on osteoclast precursor transitioning to mature osteoclasts versus osteoclast survival. So if you tell me that in your hands there is an effect on osteoclast survival, I should believe that. So you're referring to the recent cell paper saying that the osteoclasts do not die, right? Or you're referring to the older one, the Fast Ligand? Years ago, yeah, the Fast Ligand one. You're right. I mean, we need to do more work to test these ideas. I mean, the Bax-Bac, we're trying to do it in vivo now, using the mouse model to address these ideas. Your point about early effects versus late is great, because there is no really animal model where we can distinguish those effects, early versus late. Now we're trying, we're doing, we're complementing all this work with some single cell analysis. And that hopefully will shed some light on that point. Thank you very much, Elie. Thank you. Just following up, in some of your models here, so you looked maybe at apoptosis, but have you looked at subsequent activity of those osteoclasts, like bone resorption activity? We have, I mean, we need to do those studies. Although it's in vitro and in vivo, it's hard to tease apart, because we know that estrogens decrease the cell number, right? So after that, it's really hard to tease apart whether it's resorption or then that's, but we'll try. We'll try the hard. Hi there. Really nice talk. I'm interested in the multinucleation and the effect of E2 on that. Is that ligand dependent, or what's known about the factors that promote that, those multinucleated cells? And how do you think estrogen is affecting that? Well, that's a great question. Multinucleation is another hard thing to study in osteoclasts, because there are a few factors that have been proposed to be important for the multinucleation, but it's, indeed, hard to tease apart. It's easier when there is no effect on cell number. Sometimes we don't see effects on cell number, and we blame these effects on resorption activity. But these are really hard, particularly in the mouse studies, because it's really hard to count nuclei, because those osteoclasts are not that big. But yeah, it's a great point. So a follow-up on that, is the estrogen effect, I thought I may have misunderstood, is it affecting only the multinucleation or also cell number? Well, all these effects we see are very early. These are actually, most of these effects are six hours after addition of rung ligand and estrogen. So this is actually before the number of mitochondria increased by rung ligand. So we think these are, I mean, it's really early events. We don't know, probably some of them will have implications in the resorption, like the NAD effect. We think it's mostly related to resorption and not as much as the number. But yeah, you're right, it's a work in progress. Hi, Matt Sikora, Colorado. Really nice talk. So this convergence of estrogen receptor and rung ligand on the mitochondria, is this unique to osteoclasts? In particular, it's got me thinking about the progesterone induction of rung ligand in the mammary gland, and then whether estrogen is doing something in a paracrine model there. I think I can reply to the first part of that question. The estrogen effects on osteoclasts are particular, I think. Because in most of the other cells, estrogens are stimulatory, right? They promote proliferation, they promote. This is an inhibitory effect, because we think it's this particular interference with this rung ligand signal, which is, I mean, to me, one of the questions is, does this happen in other tissues where rung ligand also has other functions? But yeah, we don't know. Thank you. Thank you. Nice work. My name is Asma Knaoui from Moffitt Cancer Center. So I would like to ask about the men, the osteoclasts in men. Since we don't have estrogen, does rung ligand have an opposed effect on osteoclasts, maybe leading to metastasis of prostate cancer in prostate cancer patient? Is that something you're aware of? Thank you. I don't know how to address the cancer question. I'm sorry. But I can address the idea that, at least in bone cells, estrogens have effect in men. I mean, I don't think there's a doubt about that. Now, the direct effect of estrogens on osteoclasts is not, we don't think it happens in males. At least work done in mice with the deletion of these receptors. So I have a couple of questions to follow up. And I'm going to take my power as chair to ask them. So I haven't followed the literature for probably 10 years. So you showed some work on sirtuin, sirtuin 3. Do we yet have a selective inhibitor of SIRT3? As far as I know, not. OK, and then a question you can ask, or you can answer maybe. So you showed that estradiol is disrupting the E-cyst something. Do you know, is ER directly binding to that? Or do you know how that's happening? We're performing now some proteomic studies and unbiased studies to try to understand that aspect. We don't know. We don't really know. Excellent work. Thank you. Thank you. All right. To close out the session, we have James Sigaras from Johns Hopkins University. And he'll be chatting about the progesterone receptor actions in uterine fibroids and characteristics and treatment. Take it away. So thank you very much. Thank you, everybody, for staying to the last talk here today. My presentation today will focus on progesterone action and progesterone signaling, specifically in uterine fibroids. And I'll also talk about selective progesterone receptor modulators and how fibroids respond to these drugs. My disclosures are shown here. I'll mention two compounds by Bayer and one by AbbVie. But note that none are approved for use in the United States. And I have no financial interest on those compounds. The outline of my presentation, I'm sorry, learning objectives are shown here. And the outline of my presentation is shown here. I'll talk about progesterone signaling, the clinical problem of uterine fibroids, the response of uterine fibroids to selective progesterone receptor modulators or sperms, as I may call them, and activation of PRB biomechanical signaling. So progesterone is a very powerful steroid hormone. And it is, of course, the progestational hormone. Under the influence of progesterone, the uterus increases 1,000-fold in volume and 20-fold in size. Its effects are not limited to reproduction. As you know, it has effects on brain mood and many other tissues in the body. But I'm going to focus a little bit to start on the uterus. And progesterone is a steroid compound that affects mechanical signaling. In fact, the uterus exhibits enormous tissue plasticity, compliance, and remodels during the course of pregnancy. And these involve mechanical signaling. Now, as endocrinologists, we often think about soluble signaling. But we don't think about mechanical signaling or solid-state signaling, as it's known here. And this results from the extracellular matrix impacting on the cell membrane, leading to phosphorylation of FAC, ultimately activation of rho, rho kinase, and actin nucleation. Now, the observation that progesterone altered mechanical properties of the uterus led to the question, then, is there a feedback loop involving mechanical signaling and possibly a direct effect of mechanical signaling on progesterone action? Progesterone signaling is complex. And I'll give just a brief review of that to remind you of that. The progesterone receptor gene has two major transcripts, PRB, the larger transcript, and PRA, the smaller transcript that lacks the N-terminal region, as shown. Multiple other transcripts are present. But in general, in the human, PRB activates, and PRA less so. And then PRA may antagonize PRB. And the net effect is due to both transcripts. The classical mechanism of progesterone action is shown here, with P4 leading to activation of progesterone through dimerization, as been mentioned before earlier for the androgen receptor, leading to transactivation and recruitment of cofactors, and ultimately, RNA transcription. However, other progesterone receptors have been described, for instance, the transmembrane receptor in PGRM1 and M2, as shown here in protein form. And signaling by these other progesterone receptors is often membrane or cytoplasmic and very rapid, leading to transactivation of factors or RNA transcription through other proteins that regulate the cell response. However, often, the two pathways are called classical and non-classical signaling. And some people think that they're distinct from one another. But that, I think, is not true. In fact, progesterone signaling is much more complex. As suggested here, there's interaction between the nuclear hormone receptors and the cytoplasmic factors. So with that brief introduction, returning to the question of whether mechanical factors, such as stiffness, could affect progesterone action, a number of investigators have examined this, particularly in the pregnant myometrium. For instance, the group led by Dr. Lai and Shinlova, as shown here, noted that mechanical stretch leads to ERK-mediated phosphorylation of progesterone receptor B, as shown in the top panel here. However, PRB levels did not change in this myometrium, as shown here, when exposed to stretch. So it's a little more complex than that. Furthermore, and interestingly from what I'll talk about with fibroids, that there's an effect of mechanical signaling that led to an increase in TGF-beta 3 and TGF-beta 1. And this is important because TGF-betas are important for fibrosis. And I'm not going to summarize the data with regard to progesterone signaling in myometrium. It would take way too much time. But I just want to point out that this fact in the pregnant myometrium has been confirmed in multiple laboratories by multiple investigators. So this begs the question, then, is this effect of mechanical signaling on progesterone action limited to the pregnant myometrium or not? So we were interested to examine this question in fibroids. And fibroids are highly prevalent tumors occurring in 70% to 80% of women. By age 25, 25% of African-American women will have fibroids, and 10% of white women will have fibroids. They result in immense financial burden to both the health care system and the patients that are affected. And on a personal level, fibroids can have a huge effect on health, as illustrated by this patient's story. This is Tanika Gray from the White Dress Project. Five blood transfusions, one surgery, nine weeks of recovery. Now, all fibroids have some shared characteristics. They are stiff tumors, if you look at them. They show an increase in smooth muscle actin. And the ultrastructure and extracellular matrix of fibroids is increased in amount and altered in composition. And if you measure them, you'll find that they're mechanically stiff tumors. But I should mention that despite these similarities, there are differences in mutations of genes within the fibroids. And this is shown in this proteogenomic analysis from Bateman, where they showed mutated genes and copy number variants in the heat map. Notably, about 60% of fibroids will have a mutation in the MED12 gene, specifically exon 2. And 10 to 15% will have abnormalities in HMG A2 gene. There are also syndromic fibroids, but I'm not gonna talk about them. I just wanted to point out the fact that they're different mutations. And as I mentioned, too, as well, there's a racial disparity in the instance of uterine fibroids. And I'd like to point to another report by Bateman, where they used a proteogenomic approach for fibroids. What they did was examined formalin-fixed paraffin-embedded tissues. They looked at 35 individuals that were European-American descent, 31 African-American, and confirmed this in a group of 26 validation cohort. And then they quantified the proteomic analysis and verified this by whole exome sequencing. Oh, I'm sorry, whole genome. So the important comparison is shown on this slide. And what you see is that there's a really clear distinction between the races and the fibroid composition using proteomics. In fact, there's little overlap between the two groups. And if you look on the right, you'll see the receiver operating characteristic curve shows there's almost a clear distinction between race based simply on the proteomic signatures of the fibroids. And I mention this because the treatments for fibroids may depend upon the racial admixture of the person you're treating. But returning then to fibroids themselves, this slide shows a common characteristic of fibroids, which is that the fibroids actually exert attention on the myomecium. So these stiff tumors actually sort of press out, if you will, on the myomecium. But it's peculiar because if you look at the cells within a fibroid, what you find is that they have a reduced response to mechanical signaling. And we've used a number of approaches to check for that. And one of the approaches I'm just gonna show here, one that's easy to grasp, and that is we used a device where we plated the fibroids on a silicon membrane, and then we could press the silicon membrane. Normal cells will orient perpendicular to the strain when you do this. And what we found, however, in the non-pregnant myometrial cells, as shown on the left, that the cells oriented perpendicular to the strain as you would expect. However, the fibroid cells did not orient perpendicular to the strain as in the bottom right. So with that brief introduction of fibroids, I'd like to turn then to how progestins have been used and mention a few pioneers of treatment of progestins with fibroids. Dr. Anna Alvarez Murphy was the first to treat progestins, sorry, use progestins to treat fibroids. And then Lynette Niemann did this later with CDB2914, which is now known as eulipristal acetate. And then Christoph Schwales was the first to show that azopresnil reduces fibroid size. The mechanism of action of sperms on fibroids is shown here. These bind to the agonist, or in the case of sperms, selective antagonism leading to an alteration and a different conformation, which leads to different co-receptor and co-activator binding at the level of the DNA, which of course explains the effects of these pharmacologic properties. This slide shows the development of sperms for uterine fibroids. And as you probably know, development of eulipristal acetate was suspended in the United States because of rare cases of fulminant hepatotoxicity. And it's been limited since January of 2021 overseas for that reason. A notable difference, however, of the structures for the sperms that I'm showing here is that for eulipristal acetate, what you see is an aniline ring. However, for veliprasan, there is no aniline ring there, so this still might be in play. None of the compounds are approved in the United States, but studies have been done in the United States using several compounds, as I mentioned. Uterine fibroids cause bleeding. That's one of the main things they do. And there is a significant reduction in uterine bleeding after treatment with these compounds. As you see, the time to amenorrhea on the Y axis, I'm sorry, on the X axis, and the amenorrheic patients show a considerable improvement that is a reduction in the time to amenorrhea with eulipristal acetate compared to luprolide. In the interest of time, I'm just gonna just briefly show the results for eulipristal acetate in uterine fibroids. And what you see, again, is this reduction in time to amenorrhea in the red bar in the middle from seven to 21 days. This slide shows similar results with viliprasan compared to eulipristal acetate. And what you see in this slide is, again, a clear reduction in bleeding and also a reduction in uterine size, which is greater for viliprasan than it was for eulipristal acetate, as I showed you. So it's clear that fibroids reduce, I'm sorry, that sperm reduce fibroid size, but the question then is how they do that. And our group was interested in that question. And so what we did was we started by staining eulipristal acetate-treated fibroids for progesterone staining. What we found was a reduction in PRB staining and PRA staining, as shown here. And furthermore, what we found was that there was a reduction in phosphofac. The first upstream protein and mechanical signaling. And this led us to examine mechanical signaling with progesterone further in uterine fibroids. We wondered specifically whether the reduction in size might be due to a reduction in mechanical signaling and progesterone action in uterine fibroids. And specifically, we sought to determine whether there may be an interaction between mechanical signaling and progesterone signaling in the fibroids. This is represented by the non-classical signaling I showed you earlier, shaded in yellow. And while studies in pregnant myometrium had shown that there were effects that could be observed on mechanical tension and progesterone action, this has not been done in fibroids, and it's not been done in myometrial cells that were non-pregnant. So we tested this possibility with several approaches, and I'll show them now. We began with a patient-matched myometrium in fibroid cells that were plated on the substrates of different stiffness. And here we're using a soft versus stiff substrate. An analysis of PRA and PRB phosphorylation by Western showed that phosphorylation of PRB was greater in cells plated on the stiff compared to the soft substrate. Myometrial cells had somewhat of a different, a similar trend, but to a lesser degree. But what really was striking was that when we performed reporter assays looking at the stiff versus soft plates, there was a considerable difference in luciferase reporter activity in fibroids plated on polystyrene versus a soft substrate. And this effect was seen in multiple cell lines. This is for patient-matched primary cells. Here's for Hume cells. Again, a significant increase in progesterone reporter activity. And similarly, similar effects were observed here showing for the MMTV promoter. And looking upstream, we found that fibroid cells actually had a higher level of phosphorylated fat than did myometrial cells, and that the FACT inhibitor 573228 actually reduced this dependence on progesterone-responsive reporter activity. So inhibition of FACT reduced progesterone-responsive activity. And this activation was enhanced by PRB, but not PRA as shown by the experiments here where we added both, and then examined the luciferase change. Furthermore, if you knock down PRB, you eliminated the effect as shown here. This differential effect was seen not only in reporter assays, but also in RNA transcripts, and I'm just showing one example of an experiment shown here, looking at polystyrene and silicone subtrates again. And in this case, I wanted to point out that it was not always increased in the stiff, it occasionally was increased in the soft. So there is a differential effect of progesterone action shown here for LAT2 mRNA in primary cultures. To probe the pathway further, we performed pharmacologic inhibition using the regular suspects, if you will, of candidate proteins involved in progesterone signaling shown here for PD-98059 and SB-202-190 for ERK, and P38 MAP kinase, as well as the RTK inhibitor intended app. And what we found was that P38 MAP kinase did not have a significant effect, but the others actually did show reduction to a great degree in the mechanical signaling, or if you will, ligand dependent augmentation of progesterone action by mechanical signaling. Next, we tested for the effects of Rho inhibition, and we used two compounds, A13 that blocks the GEF binding ability to the activation of RhoA via GEF, and we used a second compound, Y27632, that blocks the Rho-associated kinase, or ROC. And what we found, again, was that both reduced the mechanical activation of progesterone signaling, which was ligand dependent. So our group had previously shown that the RhoGEF A13 enhanced activation of both estrogen receptor and GR, not mechanically, but just in cells, and we wondered whether the overexpression of this RhoGEF might further augment the effect. And the rationale was based on the fact that this is not only a RhoGEF that can nucleate actin, but it also has, in the carboxyl domain, an LXXLL motif, which is known to bind nuclear hormone receptors. And so we overexpressed this in the cells, and as you can see here, overexpression of ACAP13 augmented the effect. We examined this in several ways, but in the interest of time, just showing this assay, which was a binding assay, we wondered if this was true, maybe we could show that, or find, or illustrate whether ACAP13 bound directly to PRB. And so what we did was labeled this an old-fashioned GST binding assay, and precipitated the protein that had the LXXLL motif. Whoops, sorry about that. Yeah, I'm going back here. Yeah, you have to be really careful with that thing, don't you? So, here we go. So this area that had the LXXLL motif actually precipitated the GST protein. All right. Now, admittedly, these are very preliminary results, and our working model is shown here, and that is that there is an interaction, or an interdigitation, or crosstalk, between mechanical signaling and progesterone receptor activation, specifically with PRB. And this suggests, then, that perhaps that there is an ability of the progesterone receptor to respond to proteins that augment mechanical signaling. I've shown that the sperms reduce fibroid size and bleeding, and this is what led us to look at this. It suggests that perhaps the increased stiffness of uterine fibroids is antagonized by the sperms, and then that the stiffness of the fibroids actually promotes increased progesterone action in the fibroids, which then leads to production of extracellular matrix proteins. I mentioned TGF-beta and others, but this is what we see in the fibroids. I've shown that mechanical signaling was increased by ligand-dependent activation of PRB, and that PRB activation by mechanical signaling required Fosfac, Rho, and RTK. So, future research would be potentially to explore treatments based on progesterone and mechanical signaling, and we would like to have, obviously, something that would represent a cure for fibroids. If you treat fibroids with selective progesterone receptor modulators, the fibroids don't go away. They'll reduce to about 50% of their size, and so what's needed is some mechanism, if you will, to take that to completion, and what this gives us, then, is some sort of a direction, if you will, for exploring the role of progesterone in uterine fibroids. So, I'd like to thank you for, again, staying and point to the people that did the work, and I appreciate your attention. All right, thank you very much. For those of you who are online, now's your chance to ask a question. The iPad is working. Hello, Zeynep Madakar, University of Illinois, great talk. My question's about that striking disparity, actually, between white and also African-American women, in terms of the fibroids. Are there any differences in the stiffness of the fibroids, or did you have any chance to do any studies on at what level in that pathway there might be differences? So, let me, can you say it one more time again? I did. Yeah, so the, okay, so the difference between white and African-American women. Yeah, thank you very much, yeah. So, actually, it's not published yet, but there actually is a difference in terms of the, not so much the stiffness, but the composition of fibroids varies, as I showed you with the proteomic analysis, and the fibroids are actually a little bit different. So, the principal factors that make them are, differ in black versus white women, at least is what we understand, and that the fibroids that are in African-American women typically are more likely to have overexpression of genes involved in X-cellular matrix, and Caucasian women, more due to sort of cancer-driver genes like that you might think about that would be associated with tumors like PF3 kinase and that kind of thing. So, but the stiffness, per se, was the same in black versus white women for what we've done and measured. I didn't mention it, but most of the cell lines that I've talked about here were actually from African-American women, and so, but it's a great question, and I think that it may be that there will be differential responses to that. Does it know, is it known clinically if progestin treatment? So, what's known clinically is that they both respond, both races respond to selective progesterone receptor modulators, and it seems to be somewhat equally, so I'm not aware that it's a significant difference based on the data that I've seen, but this is one of the reasons why the FDA wanted the trials to be done not only overseas, but also in America and the U.S. so that they could see differences perhaps based on race. It's a great question, though, thank you. Thank you for the excellent talk. I'm Kiara, postdoc at NIEHS, and I'm new to the fibroids field, so I just wanted to get more information about whether or not the modulators and the mechanisms you're seeing are different or similar to contraceptives, and the mechanisms that act through that. I'm specifically thinking about depo shots and how fibroids can shrink, and the size of those can, over time, but then maybe reoccur after you stop usage, so I'm just wondering if you have any thoughts about that. So it's a really good question. So the first person that used these was Anna Murphy, who was using metformin for uterine fibroids and what you find and what we observed clinically, is what you said, is that occasionally, you'll treat women with long-acting progestins, LARCs, and they'll actually have a reduction in fibroid size and sometimes it'll come back. So the short answer is that I don't understand it totally, but I do think that some fibroids will respond to progestins in a way that's very salutary. However, oftentimes, like you said, they will come back. So I think we need to understand it better. So I think there's plenty for you to do. I'm glad you're at the NIHS. So that's great. It's a good place to be. Thank you. Diana Blythe, NICHD. You answered one of my questions, which is, does ulipristal acetate equally work in African-American as well as Caucasian women? And as you know, FDA won't let us do anything with ulipristal acetate in a chronic usage pattern because of what I know to be about nine women out of a million who had about, you know, had a severe liver injury that is very tenuously, if at all, linked to ulipristal acetate, but they couldn't rule it out. Do you know whether there was any racial difference? Were those all in Europe? Because Fibrostal is available in Canada and has, you know, we didn't see any of them in the clinical trial. Right, right, right, right. So it's a really good question. So I think here in the States, I think the people that were familiar with both the ulipristal acetates and viliprasone didn't observe anything like this. Of course, the numbers are still small. So, and with that dictum, I guess, by the European Medicines Association, or was it authority, in January of 2021, I think that sort of put the nail in the coffin for where things were right now for ulipristal in the United States. It may come back. I mean, so we'll see, we'll see. But I don't know that there was a racial difference with regard to that. And they said in that ruling that there was no way that they could have predicted those women that were gonna respond to it. And so far as I know from all the investigators that I know of that were in the States, I don't know of anyone in the States that had that problem. But there may have been some people that had some bumps in their liver function test with ulipristal acetate. We haven't seen it. And the FDA basically says, if you wanna do something chronically with ulipristal acetate, you have to show us the mechanism by which it causes this liver disease that we think doesn't actually cause. And I just, this is not a scientific question, but this is lots of women are suffering with fibroids and there isn't as good a treatment as ulipristal acetate would be. And is there any, I know in Europe, there's some pushback and they're beginning to prescribe it again a little bit more. In the US, there's been no pushback that I know of. And I just wonder if there couldn't be some more advocacy from folks who do treat fibroids because it's, you know. Right, I think there's, even overseas, what I understand is that the women overseas are actually advocating for this and getting it overseas. And right now, I think it's, you know, I don't wanna say it's relatively recent, but it's January 21. And so I think we'll have to kind of wait and see. The reason I, it sort of got us into this with the progesterone and mechanical signaling, which is an interesting thing. It's kind of a little bit awkward, if you will. But I do think it's important to keep the conversation going, which is one of the reasons I brought them up in the talk today. So thank you for asking. I'm not sure, I'll say this. I'm not sure they're dead yet. I'm not sure it's completely gone. And we'll see. All right. Julie Kim, Northwestern. Great talk, Jim. My question is, maybe we're talking about the same thing, I'm not sure. But is it really the substrate of silicon that is creating all of those responses? Or is it really mechanical? Are you talking about mechanical in terms of how it attaches to substrate? Or are there things, materials or receptors even on the silicon? Yeah, so thank you for asking that question. And so I went over it really pretty quickly and not too well, actually. And I appreciate your question. So the, part of the thing is that these membranes can be purchased with collagen or different substrates on them. So I said silicon, but actually they're not sitting on silicon. They're sitting on collagen or they're sitting on protonactin or something like that. So you can do that on either one. When you plate the cells on that softer substrate, you do see a phenotypic change in the way that the cells sit down. So like when you culture your cells, these fibroid spread out and you can see fibroid versus myometrial cells just on plastic, right? If it's coated with collagen or if it's coated, if it's uncoated, you can see differences. So the answer is that I think that it's not just, I don't think a reaction to silicon per se, but it may be the ability of these cells to reach out, if you will, and form these focal adhesions. And then there's a differential response in that. And I think your question brings up a lot of data that I didn't talk about in the interest of time. But this, we did some experiments to sort of drill down on this a little bit further using different substrate stiffnesses. You can engineer these and then test how it does and look at row signaling and all this kind of stuff. So we did some of that and I didn't show any of that. But that suggests that actually it is something to do with the stiffness of the cells and the cells interacting with that stiffness. At least that's what I conclude and I'd be happy to have your, talk about it later and see if you've got other suggestions. I know you have and I'd be glad to hear them. All right, thank you very much. Let's thank all of the presenters. Thank you.
Video Summary
In this video, the speaker discusses the role of estrogen receptor alpha (ER-alpha) in osteoblast mitochondria and its effect on bone mass. Estrogen deficiency at menopause leads to loss of bone mass, or osteoporosis, and the speaker aims to understand how estrogens protect bones. They focus on osteoclasts, which are cells that remove bone matrix, and osteoblasts, which produce new bone. The speaker presents findings showing that estrogen inhibits osteoclast formation within the first 24 hours of culture. They also discuss the role of mitochondria in osteoclasts and how loss of mitochondrial ROS and SIRT3 can attenuate the loss of bone mass caused by estrogen deficiency. The speaker further explains that estrogens promote osteoclast apoptosis through mitochondrial death pathway genes. Estrogens also inhibit complex I activity and basal respiration in osteoclasts, as well as attenuating the interaction of TRAF6 and EXD, a protein involved in complex I assembly. Silencing of EXD impairs the stimulatory effects of RankL on mitochondria function. The speaker highlights the relevance of NAD levels and SIRT3 activity in estrogen's effects on cells, showing that nicotinamide riboside can rescue the decline in cell number caused by estrogen. Overall, the study suggests that estrogen's effects on osteoclasts and bone mass are mediated through ER-alpha and mitochondria function. The video concludes by acknowledging the researchers and funding sources, and the speaker answers questions about the role of reactive oxygen species, the direct effect of estrogen on osteoclast survival, and the effect of estrogen on multinucleation in osteoclasts.
Keywords
estrogen receptor alpha
ER-alpha
osteoblast mitochondria
bone mass
osteoporosis
osteoclasts
osteoblasts
mitochondrial ROS
SIRT3
osteoclast apoptosis
complex I activity
TRAF6
EXD
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