Discussion of Session 4

Ameloblast Gene Expression and CellBiology

Moderator: Tim Wright

Wright: I’d like to open it up for general discussionnow, so any questions you have germane to this topic.Clarkson: I wonder for any of the two cell lines that were

described, whether they’ve ever been grown on dentin?Li: We are trying to work on that. Stefan (Habelitz)

and one of our students prepared a dentin tissue andtried to grow these cells on dentin, but unfortunately wegot contamination, so are not successful yet.Brodarac: I have a question for Pierre (Moffatt). I

understand you did just the screening, but I was won-dering if you might have any idea, as you presentednicely, about signaling molecules. For example, whatmight be their role in enamel formation and the basis ofthe knowledge of what all of the known proteins areworking and —Moffatt: Many of the signaling molecules that we have

pulled from this screen have previously been identified intooth formation processes, for example. So, I chose tofocus on those novel genes to further try to elucidatetheir function, but it doesn’t mean that other knowngenes are not worth looking at also in this context.Hubbard: Can I go back to the beginning of the session

and the RhoGAP and amelogenin being produced at thesame time. Bill (Bonass), is there any idea of the relativeamounts of transcript for amelogenin and RhoGAP andmaybe their respective half lives? Do we have to thinkthat RhoGAP’s going to be seriously competing or mightit just get in there every now and again and be producedat a quite lower level?Bonass: That’s a very good question. I don’t have any

information on that, but perhaps Carolyn might com-ment on that.Gibson: Actually for nested genes, the big one is

usually expressed at a very low level, and the smallerone, the nested one, is usually the tissue specific oneand expressed at a high level. I think the competitioncould go in the other direction, that the RhoGAP maynot be expressed while amelogenin is highly expressed.That was my interest in this initially because many ofthese genes have been proposed to work together insome way when they are nested; there’s a functionalsignificance. Either they’re expressed similarly oroppositely, or they have a similar function, and my goalis to see if the RhoGAP is having some role in theregulation of cell shape, because it regulates the cyto-skeleton. There may be a relationship between them.There’s also the possibility of anti-sense RNA, which isanother level of regulation that is potential, if these twopolymerases get past each other.

Snead: I was just wondering if, in fact, the anti-senseorientation to the question has been evoked to considerwhether or not there is actually an anti-sense regulation,and whether or not the evolution then of the amelogeningene coincided with how one considers the linkage ofthese two regulatory states?Sire: We don’t know exactly. Yesterday, I said that

amelogenin has been probably translocated, a long timeago, on what is currently the chromosome X after theduplication from ameloblastin. The question is, did thistranslocation coincide with the translocation of a copy ofRhoGAP-6 or has amelogenin been translocated in thisregion separately? Now, we have to look at the evolu-tionary relationship of the various members of RhoGAPfamily genes.Robinson: Can I go back to Charlie Smith’s presenta-

tion and ask what his view is? We’ve talked about dis-posing of the protons, but at maturation the pH doesdrop, it oscillates in fact. And I wonder, in fact, whetherthis carbonic anhydrase may be part of the mechanism,which is able to drop the pH. That’s the first thing I’dlike your view on, and secondly, of course, you’re pro-ducing bicarbonate, which may well affect how thecrystals grow. If it is incorporated, it would increasesurface energy, and you would improve crystal growth.I’d like your views on that.Smith: One of the original theories that came from the

work that Lin et al. (1994) did in the 90s, and was rel-ative to their observation that a hydrogen ATPaseactivity and carbonic anhydrase-2 activity were presentin the maturation stage ameloblast, and they concludedfrom this observation that there may be a situation wherethe hydrogen ions are being pumped into the enamel,and that would satisfy; that interpretation is perhaps anexplanation for why the pH is dropping. We’ve counter-argued that in our recent paper in the Journal of Boneand Mineral Research (Smith et al., 2005), based oncalculations trying to account for internal componentsthat combine buffering, external components, permeab-ility and other factors. And we came to the conclusionfrom that work that there are likely so much hydrogenions being generated as the hydroxyapatite is forming,combining the calcium, the HPO4, and the watertogether that will lead to the generation of protons.There would be enough protons to potentially swamp thesystem unless we were getting an input of bicarbonateions, perhaps, from ameloblasts to counteract this. So,there are two opposing opinions at the moment. There’sno evidence as to which one is the correct at this moment

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in time. It’s my understanding that attempts that peoplehave made to correlate bicarbonate ion content andcarbonate content of the enamel; there’s not a directcorrelation that I’m aware of.Robinson: That would be a surface phenomenon, I

think.Smith: Correct.Robinson: Just to go back to the removal of protons,

you’re right in the sense that if you look at the bulk ofcrystals, there would be a lot of protons produced as aresult of that precipitation. But the maturation stage, atleast in the rat, occurs over quite a long period of time, 3or 4 days. Is there not plenty of time for that to diffuseout, at least in part? Do you really need that massivebuffering, do you think?Smith: Yes, we do, and for this reason. This is a

problem of trying to visualize how the modulation bandsare occurring. When you’re looking at an enamel surface,you will have some point over the surface of the enamelwhere there are smooth-ended ameloblast. Okay, andthere’s usually multiple of these around the crown. Thoseameloblasts at that moment in time are smooth-ended,and in the case of something like the rat system, that is a2-hour phenomenon, that they’re smooth-ended. And inthat 2 hours, they are leaky. Anything that’s in there candiffuse out, but what Kai Josephsen showed is that alongthe end, towards the incisal mature end, or towards theapical or proliferating end, this is totally sealed withlanthanum impenetrant junctions. Therefore hydrogenions that might be generating in the ruffle-ended areas,both in front and behind, are not able to diffuse into this

smooth-ended patch (Josephsen (1984), Tooth EnamelIV). When that smooth-ended area goes ruffled, it’s thegroup in front that are going to go smooth-ended.They’ve got a lot of hydrogen ions in there, those aregoing to jump right out, no argument. But the groupbehind it that was smooth is now ruffled, the protons aregenerating, that surface is sealed, and therefore we needbicarbonate to help –Robinson: Could the protons not diffuse laterally in the

enamel?Smith: Well, you’ve argued this for years –Robinson: That’s been said, thank you very much

Charlie.Smith: I have no answer.Wright:When they did those pH dye studies, they were

banded and pretty distinct. No, I think when they wentthrough in sections, they were distinct all the waythrough the thickness.Smith: They extend all the way through.Robinson: Maturing enamel is very porous all the way

through and is hydrated.Fukae: I want to correct your general conception of

kallikrein-4 expression. The secretory stage ameloblastsexpress the kallikrein-4 involved in the degradation of20 kDa amelogenin (which occurs in an inner layersecretory enamel), although it’s very high expression isfound in transition stage ameloblasts. It is confirmed inQiaomei’s (Yan) presentation; thank you.Wright: I appreciate your indulgence and your dis-

cussion, and a round of applause for all of our speakers.

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