Mark Evers, MD: The Endocrine Gene Neurotensin: Molecular Mechanisms and a Model of Endocrine Differentiation

What I'd like to talk to you about is a particular... one of the focuses in my laboratory has been examining the neurotensin gene.

[ Slide 01 ]   Progression of Neurotensin Studies

And what I'd like to do is to present this more as a story. Given the time it will be a short story. But kind of the progression of our studies and how we've come from some of the simple studies made in Dr. Thompson's laboratory to where we are today. Basically we've started out, some of the studies performed in large animals with this gene looking at the effects of neurotensin in the human and canine studies. We've taken it to the rat model, we've gone down to the cell, we've gone down to the gene, and now we're trying to come back out again. We're coming back out to the cell, we're using now transgenic models as animal models and hopefully as we all hope one day to bring it back to human applications.

[ Slide 02 ]   Neurotensin (NT/N)

A bit about neurotensin, abbreviated here as NT/N. It's a tridecapeptide, which was initially characterized by Bob Carraway and Susan Leeman back in 1973 from the brain. It has its greatest abundance in the GI tract, predominantly in the small bowel. It's released by intraluminal fats. Many of the prior studies, many of which were performed in Dr. Thompson's lab in the early '80s, mid-80s, that characterized the physiologic functions of this peptide, which includes the ability to facilitate fatty acid translocation, it stimulates pancreatic and biliary secretions, it stimulates colonic motility and it decreases small bowel and gastric motility.

[ Slide 03 ]   Trophic Effects of NT/N

What we have been, I guess, most focused on, most interested in, are the trophic effects of neurotensin, which many investigators in our laboratory as well as others have characterized. And in the normal GI tract, other investigators have shown that neurotensin can stimulate the growth of pancreas in the gastric antrum, it stimulates the growth of the small intestinal mucosa directly, it can reverse small intestinal mucosal atrophy, it augments intestinal regeneration after resection, and can also stimulate the growth of colonic mucosa. In addition to the effects in normal gut, investigators both in our laboratory and others have shown that neurotensin is present in about 50 percent of human small cell lung cancers and it stimulates in vitro growth. It's known to stimulate colon carcinogenesis in rats and it stimulates the growth of colonic and pancreatic cancers with neurotensin receptors. Now again, many of these studies were performed in large animals but there's just so many times that you can inject this peptide into an animal and see an effect, so what we were interested in is now taking these studies to the next step, to really understand what turns this gene on and which we think is a very important peptide and gene in the intestinal tract.

[ Slide 04 ]   NT/N Gene Expression

So some of our early studies, we looked at neurotensin gene expression, we characterized the localization in the GI tract, we looked at developmental expression, we looked at the effect of environmental influences, and the expression in colon cancers. And basically what we found from these studies, I will show briefly, but we also found that this gene not only is important as an important peptide in the intestinal tract, but what we're currently doing is using this as a model to look at mechanisms of intestinal cell differentiation.

[ Slide 05 ]   Localization of Neurotensin in the GI Tract

So first of all this is a Northern blot at the top which demonstrates the localization of neurotensin in the GI tract. And very quickly, the take-home message from this, what we've done, we've taken the small bowel from the rat from the ligament of Treitz to the ilio-cecal valve, divided it into 8 equal sections, and then extracted RNA to look at neurotensin expression. We basically found that it's expressed at a low level in the proximal intestine but is abundantly expressed in the distal small bowel. And in the adult the small bowel is really the organ where we see neurotensin expression.

[ Slide 06 ]   Developmental State of Neurotensin

What we've done, we've characterized the developmental state of neurotensin. We looked first at the rat model where we find that there's a low level of expression in the fetus. It increases greatly after birth of the rat. These are our human studies. The first two lanes at the top in A show small intestine taken from an 18 week fetus. We see a low level of neurotensin expression. It increases dramatically with adulthood as you see in lanes three and four. What was interesting to us, is that the colon, as you all know, it goes through a fetal stage where it has villi. And the villi disappear toward midgestation. And what we found, we found expression of neurotensin in the early fetal human colon and the first two lanes in B at the bottom shows neurotensin expression in an 18 week old fetus. Lane 3 is from a newborn that died of Sudden Infant Death Syndrome and lanes 4 and 5 are from the adult colon. So it's expressed at the time that the colon phenotypically looks like small bowel.

[ Slide 07 ]   Effects of Environmental Influences

We're also interested in looking at the effects of environmental influences. Could all of these effects be just simply due to the fact that luminal nutrients were causing the expression of neurotensin? Well, we performed really a nice model looking at athymic nude mice where we took the fetal intestine from the rat and we transplanted it into the backs of nude mice where these, it's amazing these xenografts actually grow - you can see peristalsis - it's a very nice model separate out intraluminal nutrients from other effects. Basically what we found is that it didn't have any effect. B shows in situ intestine, we see a low level of expression of neurotensin as shown in the fetus. We see a dramatic increase by day 14, and the same thing occurred in the transplanted intestine, given the same time constraints. So what these told us, what these results were telling us, is that the effect of environmental influences, at least luminal nutrients, did not have an effect on neurotensin expression.

[ Slide 08 ]   Expression of Neurotensin in Colon Cancers

The other thing that we've also been interested in, because to be a model of gut differentiation, a gene would have to be expressed when neoplastic cells become so-called de-differentiated. And so we looked then at various colon cancers whether they also expressed neurotensin. This is an RNase protection assay. The important points are that we found neurotensin expression in about 25 to 30 percent of all colorectal cancers but nothing in the normal mucosa.

[ Slide 09 ]   Summary of Studies

So the summary from these studies. We found neurotensin gene expression, it's expressed in the small bowel in a definite temporal and spatial specific pattern. And this strict expression pattern of neurotensin is not affected by the location or luminal contents, thus suggesting a predetermined program of expression. And finally it's expressed in the fetal colon, repressed after midgestation and then re-expressed in certain colon cancers.

[ Slide 10 ]   Cellular Factors

So from this we then decided to look more at the cellular level. What exactly are the mechanisms for this increase in expression, increase in transcription? So the question here is what are the cellular factors, i.e. transcription factors, that regulate neurotensin gene expression? This is basically a cartoon just showing transcription factors binding to the promoter region, which is the regulatory region of a gene. And so in order to make these successful, you need to do two things from the outset. You need to have an in vitro cell model which makes your gene of interest and you also... it's required that you clone the gene promoter.

[ Slide 11 ]   The BON Cell Line

Just briefly, we were fortunate enough to have Courtney Townsend at our place, in our group. Developed a cell line, which is called the BON cell line. It's a pancreatic carcinoid cell line, which has served as the basis for many of our grants. It's an interesting cell line in the fact that it produces neurotensin. These cells express the gene, produce the peptide, the peptide is secreted from the cells. Also produces a plethora of other intestinal hormones. It's been an excellent model for our group to look at. The bottom just shows a Northern blot where we've taken BON cell RNA and compared it to human ileum and we see an abundant message of neurotensin in the BON cell line.

[ Slide 12 ]   Cloning the Neurotensin Gene

So this cell line now has formed the basis for many of our molecular studies. The dog and rat genes have previously been cloned by a collaborator of ours, by the name of Paul Dobner, but we undertook cloning of the human neurotensin gene, because we would rather work with the human gene in many of our studies. And this is a busy slide but it basically shows from A at the top, that in cloning the neurotensin gene, neurotensin is a tridecapeptide, but it's also included in tandem array with another peptide, called neuromedin N, which is also shown in blue, which is a hexapeptide. And basically the sequence analysis when you compare bovine, dog, and rat, it's about 90... a little over 90 percent homologous to the bovine and dog sequences and about 75 percent homologous to the rat.

[ Slide 13 ]   Techniques

So in our studies, first of all we performed various binding assays. We performed DNase foot printing assays and then gel mobility shift and super shift assays because we were interested in what factors are binding to the neurotensin gene promoter to affect expression.

[ Slide 14 ]   Footprinting Assay - 1

First of all, this is a footprinting assay, which what we've done, we've extracted nuclear protein from our BON cell line and then added it to a labeled probe or promoter of the neurotensin gene. And the important points here is that we see footprinting or binding of proteins to two specific regions, which are signified by the Roman numeral I and II. So what this indicated was that there are proteins binding to this region. At this point we didn't know which proteins or even if they had an effect.

[ Slide 15 ]   Footprinting Assay - 2

So what we then did was we synthesized oligonucleotides to these two footprinted regions and at the top is an oligonucleotide used in a gel shift super shift assay looking at that first footprinted site. And the message here is that within this footprinted site there's a near consensus CRE which is TGACATCA. So we were already clued in to look at AP-1 and CREB factors, and basically what we found is this is a crucial element. It binds both c-Jun, JunD. It also binds members of the CREB/ATF family, ATF-1, ATF-2, and CREB. So if you can imagine two different signaling pathways are coming together at this one promoter element. Also we looked at the second footprinted region and we found that it bound a member of the orphan steroid hormone family called ARP-1, which can act as a minor repressor element.

[ Slide 16 ]   Functional Assays

So with this information we then want to know, well sure you've got proteins binding, but are these proteins really a cause and effect on neurotensin expression and transcription? So we next looked at functional assays, where we took the promoter, linked it to luciferase, you can either link it to luciferase or catenin. In our instance we linked it to a luciferase vector and we either perform 5' deletions of the promoter or we perform mutations of specific promoter regions transfected into the BON cells and then we measure luciferase activity.

[ Slide 17 ]   Relative Luciferase Activity

The important point from this slide is to look at sequence -100 to -91 where you see this large increase in luciferase activity, which signifies that when you mutate this region, what you're seeing is your activity actually goes up. So this is the site of our footprinted site II, so the protein that is binding to the site is actually acting as a minor repressor element. The other important point is when you mutate the region that contains the AP-1 CRE factors, you see your expression go almost down to nothing. So this is a crucial site for the constitutive expression of neurotensin.

[ Slide 18 ]   Summary of Transcription Factors Binding to NT/N

And this just summarizes what we know to date about the regulation of neurotensin. This crucial proximal promoter element that binds both CREB, ATF and AP-1 binding proteins. This more distal promoter site that binds ARP-1, we have some preliminary studies now to suggest that in one of the introns there may also be a regulatory region.

[ Slide 19 ]   Transgenic Studies

So next we've gone now to... we're trying to come out now into animal models, and we're looking at transgenic studies.

[ Slide 20 ]   Control vs. Transgenic

Again these are very preliminary studies, but what we've done is we've linked 3000 base pairs of the human neurotensin promoter to the reporter gene, human growth hormone, and we've constructed various transgenic mice. And specifically this is a control or non-transgenic intestine and this is a transgenic. And what I'm trying to show by the arrows here, this brown staining is human growth hormone. So we have some transgenic animals now that we are interested in looking at. If these factors are really acting in vivo to give you the strict temporal and spatial specific expression of neurotensin. One other aspect that we hope to use with these transgenics is once we've fully characterized them, that they can actually direct expression to the gut, then we want to link the human neurotensin promoter to other genes to see if we can specifically get expression specific to the intestine.

[ Slide 21 ]   Conclusions/Future Perspecitives

So conclusions, future perspectives. Neurotensin has multiple functions in the GI tract, including effects on secretion, motility and growth. The neurotensin gene is a useful molecular model to define the complex differentiation pathways leading to gut development and maturation. Understanding the protein-DNA interactions that are required for restricting neurotensin expression will yield important information on the regulation and attainment of the intestinal phenotype.

[ Slide 22 ]   The Future

And I think as surgeons, performing molecular studies has been quite gratifying, you don't need some fancy statistical, some Kruskal-Wallace or whatever statistical analysis to tell you if your data is actually significant, it's usually black and white. And I think as surgeons in the future we may be like this surgeon here. We're going to have to be adept at gene splicing, understanding mechanisms. As adept at this as we are at cutting and sewing patients.

Thank you.