CME Slides Forum - A.S. Dusso

A joint Congress by ERA-EDTA and ISN

MECHANISMS UNDERLYING PARATHYROID HYPERPLASIA

Adriana S. Dusso, St. Louis, USA

Chair: Tilman Drueke, Paris, France

Markus Ketteler, Coburg, Germany

dusso

Dr. Adriana S. Dusso
Renal Division
Washington University School of Medicine
St. Louis, MO, USA

Good afternoon everyone. I would like to thank the organisers for the honour of being part of this symposium and what I’m going to talk about is the pathogenesis of parathyroid hyperplasia.

You know probably a lot better than me that in the course of kidney disease calcitriol deficiency, hyperphosphatemia due to phosphorous retention and hypocalcaemia secondary to calcitriol deficiency are the causes of the increase in parathyroid growth that precedes the decrease in VDR that induces the onset of resistance to vitamin D suppression of PTH and vitamin D suppression of parathyroid growth.

You know very well that the worst form of secondary hyperparathyroidism, nodular hyperplasia is characterised by very low levels of VDR expression that makes these patients refractory to therapy.

What I’m going to talk about today is what have we learned about the mechanisms by which calcium, phosphorous and 1, 25D regulate parathyroid cell growth. We have a limitation here and it is that we need to use an in vivo model because parathyroid cells in culture do not behave as they do in the parathyroid gland.

So we used our model, our rat model of secondary hyperparathyroidism that is induced kidney disease by 5, 6 nephrectomy and studied what happens in the parathyroid gland during the first week after the induction of kidney disease and also between week 2 and week 4 after 5/6 nephrectomy that we call established secondary hyperparathyroidism. Of course, we feed these animals with high phosphorous because as Jorge was mentioning it accelerates the progression of parathyroid hyperplasia.

We centre our attention on the established secondary hyperparathyroidism model because this reproduces nicely this lower rate of growth that we see at the beginning of the development of human secondary hyperparathyroidism.

The focus of our intention was transforming growth factor alpha, TNF-α because of a report by Doctor Gogusev in Doctor Drueke’s laboratory showing a couple of years ago that enhanced TGF-α expression in hyperplastic adenomatous human parathyroid glands. For those who are not familiar with TGF-α, TGF-α is released from its transmembrane precursors, activates the EGF receptor to undergo phosphorylation and that activates cell membrane and nuclear signals that induce growth. So we ask first are changes in TGF-α responsible for high phosphorous induction of parathyroid gland growth in experimental secondary hyperparathyroidism? To do these studies we checked what happened with TGF-α levels when we fed these animals with high phosphorous for one week. As you can see, there was a doubling of parathyroid gland size that was associated not only with enhanced TGF-α but also with its receptor, the EGFR. When this happens in cancers you have the worst form of growth and you have doubling of parathyroid gland size.

The same thing happens when you feed the animals with low calcium. So indeed we could see that high phosphorous and low calcium induced parathyroid hyperplasia associates with enhanced TGF-α- EGF receptor expression. Moreover what we saw also to our happiness was that the growth arrest induced by phosphorous restriction feeding this animal a high phosphorous diet or starting prophylactic administration of paracalcitol or 1, 25 or paracalcitol was due to or was associated with prevention of the increases in parathyroid TGF-α and EGFR.

This is part of the immunohistochemical data, you see doubling of the parathyroid gland size with high phosphorous that is surely due to high proliferative activity as measured by proliferating cell nuclear antigen and that is associated with marked increases in parathyroid TGF-α compared to the low phosphorous fed animals. Also with enhanced EGFR.

So what happens in established secondary hyperparathyroidism? I told you that there was a milder growth, indeed we saw that here also there was a direct correlation between parathyroid gland size and TGF-α content in experimental established secondary hyperparathyroidism between week 2 and week 4 after 5/6 nephrectomy and high phosphorous.

This is the immunohistochemistry showing that data. You can see a marked increase in TGF-α, without changes in EGFR activation the increase in TGF-α results in a marked induction of parathyroid growth and as I showed to you before, this increase in parathyroid growth resulted in a marked reduction in parathyroid VDR.

So the important question is what percent of parathyroid growth is driven by TGF-α and EGFR?
So to answer this question we used the method that oncologists use. We shut down EGFR activation using small molecules, EGFR-TKI. What these molecules do is to block the activation site of the EGFR preventing growth signals.

So we asked are EGFR-TKI effective in blocking high phosphorous induced parathyroid hyperplasia in renal failure? Yes, indeed they do. If we blocked EGFR activation with these inhibitors during the first week, there was no increase in parathyroid growth in animals that were fed high phosphorus or low calcium diets.

Also when we did the same inhibition of EGFR activation from week 2 to week 4 with erlotinib there was no increase in parathyroid gland size.

This is the immunohistochemical data, so administration of erlotinib from week 2 to week 4 resulted in a prevention of EGFR activation that could indeed suppress growth. But that was not it erlotinib inhibition of EGFR activation not only prevents parathyroid cell growth but also prevents increases in TGF-α which indicates that TGF-α activation of EGFR is involved in TGF-α self-induction more TGF-α in the parathyroid gland and also prevents the reduction in VDR that indicated for the first time that there was a direct cause-effect relationship between EGFR activation and VDR reduction. Of course we wanted to know what were the mechanisms underlying these processes.

So the first question that we tried to answer was how essential is EGFR activation in normal parathyroid growth? In order to answer this question, we generated a transgenic mouse model with a parathyroid specific EGFR inactivation. So here is the cartoon of the EGFR. So in order to inactivate the EGFR, what we did was to truncate the receptor from the tyrosine phosphorylation site that is necessary for activation.

So we get a mutant EGFR that cannot be activated. In order to recognise it easily in the mouse parathyroid gland, we tagged with a small protein called V5. To direct that construct to the parathyroid gland we drew this construct by the parathyroid gene promoter and in order to be able to detect the parathyroid gland, we used a green fluorescent protein to be directed to the parathyroid gland.

So we were successful in targeting the dominant negative EGFR to the parathyroid. Here you see the parathyroid gland in the wild type, here the parathyroid gland in the transgenic. There is no green protein in this parathyroid, there is green protein in here and this coincides with V5 expression the target for the mutant EGFR and the localisation is fine, it’s in the cell surface. Ok but the most important question was, is this mutant EGFR capable of shutting down the endogenous EGFR in the parathyroid gland? So what we did is to give EGFR and EGFR ligand to the animals, wait for 20 minutes, sacrifice the animals, obtain parathyroid tissue in frozen sections and see what happened. In the normal EGFR, EGF binding will activate the receptor and induce ERK phosphorylation. In the mutant receptor that cannot happen but what makes this compound act as a dominant negative is that binding to the normal EGFR will shut down its activation. Did we achieve that? Of course, otherwise I would not be here showing it to you. So if you see the parathyroid gland in the wild type, you see phosphor-ERK in the parathyroid and you don’t see much phospho-ERK in the EGF and EGF mutant animal. So more importantly we could see when we measured parathyroid volume that there was a striking 7-fold reduction in parathyroid gland size in mice with a parathyroid specific EGFR inactivation.

Which led us to conclude that indeed EGFR activation is essential for normal parathyroid growth. So the second question we tried to address is how does EGFR activation reduce VDR? So we did it in vivo and in vitro and I’m going to summarise this in a cartoon. We know that TGF-α activation of the EGFR in the parathyroid gland leads to the synthesis of a molecule called LIP. LIP is a potent mitogen and inducer of transformation that leads to aggressive growth.

What is LIP? LIP is the truncated form of another transcription factor C/EBPβ- LAP, this is good. LIP is bad LAP is good. Why? Because LIP induces aggressive growth LAP is a potent growth suppressor of cycline 1 oncogen signature.

Well fine but what does it have to do with growth control of VDR? What happens is that in the human VDR promoter there are C/EBP β binding sites and LAP the good protein to suppress growth induces VDR gene expression.
So what happens in the parathyroid glands of a uremic animal? TGF-α activation of the EGFR will induce LIP to compete with LAP for a C/EBPβ binding site leading to more growth and VDR reduction at the transcriptional level.

So the other question is how does TGF-α induce its own expression to aggravate growth and VDR reduction? What we have shown is that TGF-α activation of the EGFR will induce the synthesis of a protein called activator protein 2 that is a major transcription factor to induce the expression of the TGF-α gene and that will lead to the self-induction that I showed to you before.

Ok this is a cartoon, what is the evidence that this happens in humans? As you can see here, diffuse parathyroid glands have a lot less TGF-α than nodular glands that results in more activation of the EGFR as measured by phospho-ERK and also more AP2 levels. AP2 as I mentioned will further induce TGF-α expression to aggravate EGFR activation more AP2, more VDR reduction as measured by in situ hybridisation in diffuse and nodular and human parathyroid glands. When you measure mRNA levels for the VDR by real time PCR, there is a 20% reduction in mRNA. So to summarise what I have told you so far is that increases in parathyroid TGF-α and the release of TGF-α from its precursor activates the EGFR to induce two very harmful circles, one is TGF-α self-induction through an AP2 control of TGF-α gene transcription that induces more growth and more EGFR activation induction of LIP to exacerbate growth and reduce VDR gene transcription. So the critical question to target this effectively is what causes the initial release of TGF-α that initiates these feed forward loops for exacerbated growth and VDR reduction? We focus on an enzyme TACE, tumour necrosis alpha converting enzyme. Why? Because of the work of the oncologists that shows that we know that TACE is necessary to release mature TGF-α what we didn’t know and the oncologists didn’t know either was that increases in TACE are sufficient to cause the most aggressive signalling from TGF-α and the worst prognosis in human carcinomas. How much worse? So much worse that if you inhibit TACE, you can’t reverse malignancy in renal cell carcinomas. But we don’t know much and oncologists don’t either on how TACE is regulated but the most important thing for you to remember because Vicky will go through this in her presentation later, is that one way to control TACE activity is simply by controlling cytosolic activation and translocation to the surface to release TGF-α because once TGF-α is released EGFR activation feeds back on TACE activity and translocation generating another vicious cycle for more TACE, more TGF-α release, more EGFR activation.

Ok so the question that we tried to answer and that Vicky will be presenting in more detail is, what is the role of TACE in the regulation of parathyroid cell growth? What she’s going to be presenting and I’m going to summarise in this slide is that we have known for many years that dietary phosphorus has a very distinctive control of parathyroid cell growth and now I have shown to you that it’s through the control of TGF-α and what she’s going to show you is that whereas low phosphorus that shuts down growth, suppresses -- location, high phosphorous induces a TACE translocation and that is enough to initiate this vicious circle.
So this will be more TGF-α in the parathyroid, more growth, less VDR, more TACE activation.
So we are asking now is this a mechanism by which dietary phosphorus controls growth?
Are calcimimetics regulating TACE translocation? And what Vicky’s going to be presenting also is that there will be an advantage in the use of vitamin D as Jorge pointed out in the correction of vitamin D deficiency to increase the capacity of the parathyroid gland to synthesise 1, 25 (OH)2 D endogenously and activate the VDR because 1, 25 (OH)2 D has a direct effect on suppression of TACE at the transcriptional level.
So the take home message that I would like you to take home is that the degree of parathyroid TACE activation determines the celerity of the onset and the severity of the vicious TGF-α-EGFR driven cycle for severe parathyroid growth and VDR reduction. So TACE is a very novel target if you want to control the development of secondary hyperparathyroidism.

So these are the people that helped me go through these processes through the years, it was like with a strong support from Edoardo at the beginning he was with us all the time, supporting us and NIH and Cedar. We have collaborators like Doctor Jorge Cannata who provided the gene arrays to examine VDR and TACE levels in human parathyroid glands exclusively diffuse. Doctor Basile and Doctor Lomonte from Italy who provided numerous parathyroid glands and we are very thankful to Doctor Arnold who provided us with the PTH promoter to get the transgenics. Thank you very much for your attention.

Chairman:Thank you doctor Dusso for sharing this very interesting data with us and for keeping very nicely in time. So we have space for questions.

Question: Thank you for this wonderful cell biology lecture. What are the molecular mechanisms of phosphorous induced change in trafficking of TACE? Do you have any clues about that?

Dr. Dusso: No, nobody knows that and we were the first to find that there was a translocation thing. The mechanism by which TACE is moved to the surface is along the secretory pathway, so that’s why I’m postulating that high calcium and low calcium are doing the same thing because I think that this TACE movement to the surface can put together under regulation of intracellular calcium and ialuronic acid that moves TACE and PTH along the secretory pathway will put together something that we have known for a long time that is that the regulation of PTH secretion accompanies what we see in the increase in cell expansion in the parathyroid gland. So it’s not for sure we’re going to test that.

Question: EGFR-TKI are current treatment in breast cancer cell metastasis for instance. Would you consider giving these kinds of drugs in severe hyperplasia in CKD patients?

Dr. Dusso: Yes my patients are rats but actually we are establishing a collaboration with the oncologist department because in triple negative breast cancer progesterone receptor negative, estrogen receptor negative and -- negative TACE inhibition is sufficient to reverse malignancy. There was a paper in JCI at the end of 2007 showing that increases in TACE that was my inspiration to look at TACE in the parathyroid gland. They postulated that TACE inhibitors will totally reverse malignancy and will be a great choice to synergise with EGFR activators in the control of cancer. What I said before very briefly is that in renal cell carcinoma also the combination of TACE inhibitors plus anti-EGFR therapy is really effective. Inhibition of TACE reverses malignancy in renal cell carcinoma in a mouse model of human renal cell carcinoma. What I want to point out to you is one problem is that TACE inhibitors that were developed to treat arthritis because TACE is TNF-α converting enzyme so it releases TNF-α and causes tremendous inflammation had very, very bad side effects. So that’s why I’m very happy to tell you please use vitamin D because we have demonstrated a very profound inhibition of TACE by vitamin D at the transcriptional level that in the case of parathyroid gland completely prevents TACE enhancement and the enlargement of the parathyroid gland just correcting vitamin D deficiency if we start it early. So it’s a very good tool as an anti-TACE therapy.

Question: Adriana every time that I hear your lecture I learn a piece more, so I was paying a lot of attention and one key issue here is the phosphorylation of ERK 1, 2. It seems to me that there’s an experiment missing have you ever tried to inhibit the activity by adding PD? It has to be in vitro of course and see that all this regulation of vitamin D receptors and TNF has been disrupted. I’m asking this for one simple question because you have ERK 1, 2 phosphorylation by EGFR activation and also by the calcium sensing receptor activation. I’m trying to put this together.

Dr. Dusso: You told me in our presentation of ASN when we show the mechanisms by which EGFR activation shuts down the VDR you said well how can we dissociate? Here we are, we made a transgenic mouse just to answer that question Mariano so I hope that I will be able to show that to you. But there are two important things that I want to say, there is another way that TACE can be stabilised in the cytosol and I think that everybody in the audience should be aware of that and it’s not just ERK through the EGFR but fibroblasts growth factor receptor. So there will be a mechanism by which FGF23 and klotho could modulate that. Not only that, TACE releases klotho in the distal tubular cells. So there is a lot more that we need to learn. Of course, we’re going to do the experiments in the transgenic mouse to see how is the VDR regulated and if they can be independent.