Jeffrey B. Matthews, MD: Epithelial Secretion and Na-K-2Cl Cotransport: Molecular Insights

So my research... actually I'm flattered to be called a molecular biologist because I don't consider myself one. I really started... focus in mostly on physiology and epithelial transport, which has begun to creep into the worlds of molecular biology and how it relates to the cell biology of this important function.

[ Slide 01 ]   Diseases of Mucosal Secretion

Basically, all epithelial cells, all mucosal surfaces throughout the body, are moist. They have to hydrate themselves and it's a primitive defense mechanism by which epithelial cells are able to dilute and flush away noxious injury. It's widely conserved across species and in numerous organ systems throughout the body. My interest is in the gut, but the remarks that I'm going to make could be made virtually about every organ system. There are a number of important diseases that result from alterations in mucosal transport. Diseases that are on the too much side, too much epithelial secretion, ranging from infectious diarrhea to a variety of other syndromes that we as surgeons certainly see, and on the too little side, to the classic molecular disease of cystic fibrosis, which is due to a mutation in a gene encoding a cyclic AMP regulated chloride channel. Implications for this for surgeons go beyond the disease cystic fibrosis as we're recognizing that alterations in the mechanism of chloride transport underlie idiopathic recurrent acute pancreatitis and probably most cases of male infertility.

[ Slide 02 ]   Epithelial Salt and Water Secretion

This is a conventional model of epithelial secretion as it occurs in many epithelia throughout the body. There are four main transport pathways that are involved to produce this secretory flush. There's an apical chloride channel, actually several populations of chloride channels, and these are driven by the concerted actions of a variety of basilateral membrane transport pathways that load salt and then dump salt across the surface. It's a very important process to understand because basically it's a dramatic example of what all cells have to do to control their internal environment. The flush through that happens here basically exchanges the cell volume, cell electrolyte composition, within an extremely short period of time. All cells need to be able to regulate cell volume and cell ion composition. Intestinal epithelial cells and other epithelial cells do it in an extreme way. And virtually every process that we've heard about, talked about this morning, won't occur if your cells don't do this properly. Your cells won't grow properly, salt influx is a key process that goes on with cells that need to grow and divide. If your cell volume is altered, your cells can't migrate and metastasize. And basically every process that we've talked about today will be influenced by salt transport.

[ Slide 03 ]   Classical Model: Primary Regulation by Aprical Cl- Channels

The particular interest that I have is on actually one element of this transport pathway, which is at the basolateral membrane, a salt transporter that moves sodium, potassium and chloride together and is the main salt loading step. And we're interested in a very fundamental and deceptively simple question. Classically, it's always been thought that the primary regulation of transport events occurs at the apical membrane where the regulated opening of ion channels causes salt to dump out of the apical membrane into the lumen of a hollow viscous.

[ Slide 04 ]   Apical-basolateral Membrane "Crosstalk" During Secretion

Well the obvious question, although one that has really not been addressed very fully is the fact that, the observation that, chloride lost from the cell has to be balanced by chloride uptake across the basilateral membrane. But how that salt replenishment occurs was really not well understood until we began to, in the last few years between our group and a variety of other groups, to understand how this crosstalk occurs. So the secondary events that cause salt replenishment turn out to be quite complex, can be mediated by a variety of possible mechanisms including passive changes and driving force, which turn out not to be the case, but in turn phosphorylation events, changes in cell ion composition themselves, as well as changes in cell structure.

[ Slide 05 ]   Na-K-2Cl Cotransporters

Now the transporter that we focus on is called NKCC1, sodium potassium chloride co-transporter isoform 1. This is a widely distributed protein that's in virtually every cell of the body. Every mammalian cell possesses this. It's possessed in a high degree in salt transporting epithelial cells, where it's the so-called secretory isoform that's responsible for salt uptake. There are a number of members of this family, including NKCC2 which is kidney restricted and is the so-called absorptive isoform.

[ Slide 06 ]   Na-K-2Cl Cotransporters (NKCC1): Levels if Regulation

Our research is focused on how this transporter is regulated at multiple levels in the function of a cell, how the protein itself is able to transport salt and what factors regulate it, what controls how much a cell is going to express at the cell surface and what happens in the long-term to gene expression programs that affect its regulation and what consequences that has for salt transport in an epithelium. And I'm going to very quickly show you some research that focuses on all three levels.

[ Slide 07 ]   Effect of [Cl]i on NKCC Function

Some of our real interesting recent studies have looked at what actually controls the function of a salt transporter. And we have identified that actually the key element here is cell chloride. Cell chloride turns out to regulate in a non-thermodynamic way but really in a regulatory sense, the function of the NKCC co-transporter. This is a slide that shows basically the function of a co-transporter. The steeper the slope here, the more chloride is entering the cell. We measure it actually as potassium entry but we can see that there is a very strong role, this is intracellular chloride concentration, which is controlled in a cell permeablization model. And if we look at what happens to salt uptake through the co-transporter, we see that it is strongly dependent upon cell chloride within a very narrow window of chloride concentration. So that if cell chloride is in the physiologic range, we find it sort of in this area here, as cell chloride falls just a tiny degree, there's a marked increase in the function of this co-transporter. If salt increases to a small degree in the other direction, there's a profound inhibition. The thermodynamics under this situation actually do not... are not explained simply by alterations in driving force. This turns out to be due to changes in protein phosphorylation.

[ Slide 08 ]   Effect of Forskolin and Genistein on NKCC at [Cl]i = 40 mM

And what's more interesting is we find that the ability of the co-transporter to be regulated by kinases is entirely dependent upon the level of chloride that's existing in the cell, so that in a physiologic range of chloride, about 40 millimolar, you can stimulate chloride secretion by typical cyclic AMP agonists and you can inhibit it by various other pathways which I won't go into.

[ Slide 09 ]   Non-regulation of NKCC by Forskolin and Genistein at [Cl]i = 20 mM and 70 mM

But if you look for the same kind of regulatory patterns, at different levels of internal chloride, you no longer see this. So if chloride is reduced to 20 millimolar inside the cell you can no longer see evidence of regulation of chloride transport by these stimuli. Conversely if you increase cell chloride to a high enough level, you can completely fail to stimulate chloride transport by this transporter.

[ Slide 10 ]   NKCCl and [Cl]i

And basically this has led to a model that whereby basically chloride is controlling its own entry pathway through a mechanism that involves chloride dependent kinases and phosphatases within the cell which have now just begun to be identified. These are novel kinases that are distinct from protein kinase A that are regulated in a physiologic range, within a very narrow range of chloride concentrations.

[ Slide 11 ]   PMA Activates PKC

Now the second phase of studies that we've been looking at is in looking at what controls the surface expression of the NKCC co-transporter because this turns out to be important in terms of the capacity of the cell to bring chloride into its cell and we focused on some studies that have looked at the role of protein kinase C in controlling cell surface expression. And a variety of studies from our labs and other have identified, using the protein kinase C agonist PMA - it's a phorbal ester, to stimulate protein kinase C, have identified a number of negative regulatory effects on the machinery for salt and water transport by epithelial cells. But one particularly interesting thing that we've observed is the effect of prolonged stimulation with phorbal esters or physiologic agonists of PKC on co-transporter expression at the cell surface. And what we noticed was that not only the function of this transporter but several other transport pathways that are localized on the basilateral membrane of salt transporting epithelial cells were potently down regulated by stimulation of protein kinase C.

[ Slide 12 ]   Membrane Recycling

And one of the models that we came up with to try to unify these disparate observations was perhaps that protein kinase C was causing a general internalization of proteins on the cell surface by altering the balance between endocytosis and exocytosis, the constitutive recycling of membrane and the proteins that are contained within that membrane at the cell surface.

[ Slide 13 ]   Effect of PMA on Endocytosis

So we studied this looking at markers for membrane trafficking and in fact have been able to show that activation of protein kinase C in epithelial cells markedly enhances internalization of plasma membrane and the proteins that it contains, specifically at the basilateral surface of polarized epithelial monolayers. This would explain the loss or the inhibition of co-transporter function over time.

[ Slide 14 ]   Short-term Effects of PKC on the NKCCl Internalization - 1

And we looked at that to see whether in fact surface expression - I knew I'd get to a blot - that surface expression of protein was in fact altered and what this represents here is actually in Lane A here is NKCC protein, co-transporter protein, that's at the cell surface and accessible for biotinilation. So the proteins that are on the cell surface are labeled with biotin and then immunoprecipitated and through a strep-avidin blot, you can light up proteins that are on the cell surface. And we see that under control circumstances, the bulk of co-transporter is at the cell surface but over time in response to phorbal esters, this decreases where the amount of unbiotinilated protein, the protein was never at the cell surface, increases. This is evidence that activation of protein kinase C causes an internalization.

[ Slide 15 ]   Short-term Effects of PKC on the NKCCl Internalization - 2

We've been looking recently at the mechanism whereby this occurs and we've been looking specifically at what isoform of protein kinase C is involved. And I won't go through the data. Suffice it to say that it is a novel calcium independent isoform of PKC, PKC epsilon which turns out to be an important downstream target of PI3 kinase and a variety of other important molecules in cell migration in cancer growth. That corresponds to the control of surface expression of these molecules.

[ Slide 16 ]   Effect of MARCKS "PSD" Peptide on PMA-Induced MARCKS Translocation

Moreover it turns out to involve the actin cytoskeleton and we have focused in particular on the ability of protein kinase C to alter the plasticity of the plasma membrane and we believe that this involves effects on the actin cytoskeleton and in particular, with an actin cross-linking protein that's known as MARCKS. And this is an experiment really that shows our ability to phosphorylate MARCKS in response to activation of protein kinase C in these epithelial cells. And this phosphorylation of the MARCKS protein causes and leads to an actin rearrangement event that allows endocytosis to occur. We've engineered some peptides that are able to block the phosphorylation of the MARCKS protein - and I won't go through the details of this slide because they're complex - but suffice it to say that we've been able to mechanistically link the effects of phorbal esters and protein kinase C epsilon on the MARCKS protein,

[ Slide 17 ]   Effect of MARCKS "PSD" Peptide on PMA-Induced Endocytosis

and [have] been able to use this to reverse the effects on endocytosis, where we can very clearly mechanistically link rearrangements in the actin cytoskeleton to membrane deformability to endocytosis to removal of co-transporters from the cell surface.

[ Slide 18 ]   Dietary Fiber and SCFA

The final level of regulation that we've looked at for the NKCC co-transporter is at the level of gene expression. And we've been struck by some interesting observations that compounds such as butyrate, which are the natural fermentation products of dietary fiber in the gut, are able to change the pattern of gene expression in a wide variety of tissues and they're also useful as agents to control colitis and in fact secretory diarrheas. We've done some studies in collaboration with Rich Hodin in Boston which show basically that sodium butyrate, the product of dietary fiber, is able to profoundly down regulate the NKCC protein... through a mechanism involving histone hyperacetylation. And the result of this is in fact a down regulation of the chloride secretory capacity of intestinal epithelial cells, independent of other transport sites and may be a mechanistic explanation for how butyrate may be used therapeutically in diarrheal states.

[ Slide 19 ]   Levels of Control

So just a very quick whirlwind tour through some of the stuff that's going on in the laboratory right now where we've looked at multiple levels of control of this ubiquitously expressed transport protein that is specially adapted for chloride secretory epithelial cells. We have looked at a variety of models in which we can specifically regulate either the function of these molecules in the plasma membrane, the number of molecules that are acutely present in the plasma membrane, and the gene expression of this protein and control chloride secretory capacity and thus diarrheal diseases.

[ Slide 20 ]   "Rheostat" Model of Secretion

The model that's emerging from our studies and from other studies is that we can envision the ability to transport salt and water as if it were a light, which is controlled by a dimmer switch. Basically most of the work in the field has focused on what turns the switch on and off and that turns out to be at level of the apical membrane. But what controls the brightness of the switch, what controls how much an epithelium can secrete, how much diarrhea you can make, or how much... how your mucosal surfaces can be moistened, is controlled at the level of the dimmer switch, and the dimmer switch turns out to be this very important sodium potassium chloride co-transporter molecule.

[ Slide 21 ]   Acknowledgements

And with that I'll just stop and thank my collaborators both in our own lab and particularly Chris Lytle out at UC-Riverside has provided a lot of the reagents for our studies. And I'll catch my breath and answer any questions.