Transcribed by Amy Maya 1.27.2014

Basic Tissues – Matrix Metalloproteinase Family by Ronald Craig

47 – Basic Tissues: The Matrix MetalloproteinaseWelcome back everybody. We covered a lot of stuff last Thursday wow. We started a discussion on extracellular matrix proteins, and we really focused in on the collagen gene family and one of the themes is structure/functions relationships. The collagen gene family is important because it confers tensile strength on a tissue so it is a prime determinant of tissue form. What confers that tensile strength is the primary amino acid sequence of glycine X and Y, and the X is frequently but not always proline and the Y is frequently but not always hydroxyproline. Glycine allows for a tight alpha helix, which allows interchange hydrogen bonding with hydroxyproline and I believe that’s in a right handed, who knows, its not important. Each alpha chain assembles with 2 other alpha chains coiled in the opposite direction, so it’s left-handed. This is hope you make rope. If you rotate in really important residues like a glycine, so if you mutate a glycine to another amino acid like a big one like tryptophan, that mutated protein won't be able to assemble into a triple helix and you’ll have a defective extracellular matrix. And we talked about Osteogenesis imperfecta being an example of that. Another property of having a glycine at every third position is the ability to make a very tight alpha helix, so tight that the R groups in the X and Y positions are thrown radially away from the peptide bond shielding it from protease attach. If you choose a molecule to determine tissue form, that molecule will be bathed in proteolytic activity, that molecule is going to need to be protease resistant. However, you look a lot different than the day you were born so we constantly develop. We heal and change as we age so we need to be able to turn over that collagen matrix. So today’s discussion is about the matrix metalloproteinase gene family and this is a family of proteases that have evolved to turn over proteins associated with the extracellular matrix. This is important to us as surgeons because most of our tissues are made of fibular collagen, but also the diseases we deal with involve the lysis, the destruction of collagen. And if you're in a surgical specialty, the holy grail is to get tissues to regenerate, so I want to be in control of regenerating lost collagen and that is the basis of our talk. So this is type 1 collage in an EM, electron micrograph, its predominant in the periodontal ligament.

48 – Rate of Collagen Turnover Vary Throughout the BodyAs it turns out collagens actually have different rates of turnover throughout the body. Sometimes I’ll give you an experiment and ill name people’s names, its not important for you to remember their names but we’re in a small profession and if you want you can actually meet these people and interact with them, which is pretty cool. So anyway, a person named Lorne Golub (sp?) is a periodontist although he doesn't want to admit it, at the other end of the LIE at Stony Brook. He is interested in how collagen gets turned over so early on this was a basic experiment done at Stony Brook. He fed some rats Purina rat chow that had radioactive proline labeled with tritium and 13% or so of the amino acid residues of type 1 collagen is proline so all the collagens made during that feeding period are highly radiolabeled with tritiated proline. And at time 0 he took away the radioactive rat chow and gave them

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Transcribed by Amy Maya 1.27.2014

Purina rat chow that was enriched in unlabeled proline. Then he sacrificed these animals at several times after t=0 then he processes them for histology like this slide then he does an extra step and takes the microscope slide and dips it into an emulsion and as the tritium breaks down it will release a beta particle and the beta particle will precipitate the silver halide particle present in the emulsion so while looking at the slide you see not only the tissue, but also see little silver grains over the cells or structures that have radioactive proline.

49 – Rates of Collagen Turnover Vary Among Anatomic LocationsSo you count those silver grains over the tissues of interest. So here’s t=0 and you count all the grains in a field so that’s the total amount of radioactivity that got incorporated and if you killed the animal 2 days layer there’s going to be less silver grains and another day it will be less and less because the radioactive collagen is destroyed and replaced with nonradioactive collagen. This is a classic pulse chase experiment. You keep on doing this until there is little silver grains left in these slides. Then you go back and you know where t=o was which was 100% then see what was half the number of silver grains so you go to the curve and drop down to this axis and read how long it took to do that. So that’s half life telling you how long it took radioactive collagen to turn into nonradioactive collagen or the half life of that tissue. And what he found was something interesting.

50 – Rates of Collagen TurnoverSo this is for rats who have shorter life spans so they do things different but the relative rates are the same for us. If he took skin from the back of the rat it took 50 days for half of that collagen to turn over. If he takes hard tissue from the hard palate and looks at that half-life it’s more than a half faster, huh that’s kind of interesting. Maybe because of the huge amount of bacterial challenge in the oral cavity, the tissue is turned over much faster because of the constant presence of bacteria. Then the gingiva is even faster still. And then this is the best part because I'm a periodontist. If you look at the PDL it’s even faster still which is kind of amazing. It kind of fits if you think about it. You know that disease Scurvy we talked about. Newly synthesized collagen in the absence of vitamin C and ascorbic acid, Lysyl hydroxylase can't form that hydroxyl so the newly formed collagen turns into gelatin rather than to collagen and those sailors that had scurvy has loose teeth and spontaneous hemorrhage from their gingiva so that kind of makes sense.

51 – Periodontal Ligament is Richly SuppliedNow if we look at the periodontal ligament, if you look you see these big structures here, here’s one, here’s a second one…these are arterial and I can see some muscle fibers around it, some veins, another vein. So this tissue is highly vascularized which implies that it has a high metabolic rate and a high rate of collagen turnover.

52 – Collagens are Degraded by CollagenasesSince fibular collagen are pretty resistant to proteases then how do they get turned over? There are 2 mechanisms of turnover of fibular collagens. So bacterial collagenases cleave every triplet at the beginning of each glycine from one end of the

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Transcribed by Amy Maya 1.27.2014

triplet. After bacteria start chewing on that fibular collagen, what’s left is a bunch of free amino acids and triplets. You don’t do that. Eukaryotes make it more complex. We have vertebrate collagenases and ¼ of the way down the molecule, the triple helix has some give to it, it’s called the hinge region where vertebrate collagenases bind to and make one cut in each alpha chain, becoming gelatin, denatured collagen. There are then other proteases, gelatinases, that come in and finish it off.

53 – Vertebrate Collagenases Make a Single Cut…I have a little cartoon in here that shows this. let me walk you through it. Here is a collagen molecule with telopeptides and this sequence is blown up here. Here is an isoleucine that connects into the glycine. This is the little hinge region where the one cut happens through each alpha chain allowing the collagen to unravel. Now it is not native but denatured and the proteases can come in and clean this up. And one of the principle ones is gelatinase. When I was a dental student this is all I was taught and things have become much more complex

54 – Periodontal Ligament Fibroblasts Both Synthesize and Degrade Collagen FibrilsIn the periodontal ligament, there’s a population of cells. Morphologically called PDL fibroblasts. Here’s an EM of a human happy PDL fibroblast and here’s its nucleus and I know it’s very biosynthetically active because I can see some chromatin that is dense, electron-dense, and some that is kind of open, electron-lucent, and these are genes that are being transcribed then the mRNA comes out into the ER then the Procollagen gets packaged into secretory vesicles then pumped out into these growing collagen fibrils. And look how these incorporate and go around growing collagen fibrils, and they are really actively making these collagen fibrils. Here’s some ER, and here’s some vacuoles. And all of a sudden we see something weird. This lysozyme, a phagocytitic vacuole, and inside it looks like there's bits of collagen that still has a banded array but its getting chewed up so the initial excisions were made extracellularly and then these were formed into phagocytitic vesicles and fused with lysozymes and they are ingested intracellularly.

55 – Periodontal Ligament Fibroblasts Both Synthesize and Degrade Collagen FibrilsHere’s a higher power, here’s the nucleus, here’s a phagocytitic vesicle chewing up the collagen.

56 - Periodontal Ligament Fibroblasts Both Synthesize and Degrade Collagen FibrilsSo PDL fibroblasts are not only synthesized but also responsible for degradation of the collagen matrix that they synthesized

57 – Vertebrate Collagenases are members of the MMP familySo as it turns out, you guessed it (giggle), vertebrate collagenases and gelatinases belong to the gene family and this slide is WRONG! There are 25 MMPs in the human genome, they degrade proteins and they are present in the ECM and all require a divalent zinc at the active site to effect the proteolysis. Most share a homologous active site that has been spliced in with various other coding domains. All require divalent zinc. They also require divalent calcium for enzyme

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Transcribed by Amy Maya 1.27.2014

conformation and stability so if you wanna get rid of MMP activity like in a biopsy, one way to do that is get rid of divalent calcium.

58 – Vertebrate Collagenases are members of the MMP familySo most are synthesized as proenzymes and are secreted into the extracellular environment and other proteases in the ECF that are active and cleave off the propeptide and activate the MMP. You really want to control this carefully because you don’t want to unleash an MMP that will start degrading type 1 collagen because you end up with an individual that will be Jell-O so this needs a lot of regulation. So most extracellular matrices will have incorporated into them something called TIMPS, Tissue Inhibitors of Matrix Metalloproteinases. So as the MMP gets out there and chews up the EC Matrix, it releases its inhibitory factor, which turns off the enzyme you must make more of it to continue proteolysis. We will see this over and over again. What happens if you have a REAL inflammatory lesion, in the serum there is alpha 2 macroglobin, a general protease inhibitor, in the blood it will get rid of any MMPs that leaked into the environment. There are 4 TIMPS present in various EC matrices. So locally MMP are inhibited by TIMPs and systemically by alpha 2 macroglobin.

59 – Matrix Metalloproteinase FamilyI just want you to get an idea of the array of MMPs that are present in our patients and ill underline what you have to know. So what used to be called collagenases, there are 3 of them now called MMP 1, 8 and 13. MMP-1 is constitutively expressed by all CT cells such as fibroblasts. Its substrate is collagen 1 and collagen 3. MMP 13 is a little different and that’s very active against cartilage, collagen type 2, and this is going to come back clinically. Inflammatory cells like neutrophils, monocytes, osteoclasts, those guys, they also express collagenase but its genetically distinct from MMP-1 and its called MMP 8. So this is an inducible MMP, it is only synthesized in an inflammatory cell under appropriate signaling. So now we’ve got the collagen fibril cleaved and in comes gelatinases so CT cells constitutively express MMP 2 and it has a very large range of substrate specificity and it cleans up the network. Stromelysins I'm going to pass. (He reads word for word off the slides for a moment).

60 – Matrix Metalloproteinase FamilyThis is an interesting one called MMP 20, used to be called enamelysin, I think it is only expressed in ameloblast and odontoblast and it seems to be present in enamel matrix formation and dentin formation. In fact if you knock out this gene you get Amelogenesis imperfecta so this is the only TOOTH SPECIFIC. Then there is a group of MMPs (membrane-type MMPs) and they're expressed on the cell surface and tethered by a hydrophobic tail, they are active during development and chew their way through the matrix to allow cells to migrate. They are also present during inflammation, perhaps a neutrophil has to chew its way through the matrix to make it to a bacterial infection. If a carcinoma cell wants to metastasize it will express these MMPs to chew itself through the matrix for metastasis.

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Transcribed by Amy Maya 1.27.2014

Ok what do I have to know Dr. Craig? So what you have to know is MMP 1, 8 (deal with in dentistry, and 8 is expressed by inflammatory cells and 1 by CT cells), MMP 2, 9, don’t need to know stromelysins or matrilysins, need to know 20 because we are dentist, and MMP 14 because that is expressed by PMNs during wound healing and inflammation

61 – Domains of the MMP FamilyThe MMP family seemed to arise through exchanging exons. I made a mistake here, the mt-MMP have a peptide then once its cleaved it sits in the cell membrane. They all have an active site, and it’s kind of in the same general location of the gene. The N and C terminal is what confers substrate specificity so they all have a similar body plan that reflects the way they evolve

62 – Regulation of MMP ActivityWe kind of went over this already. They are synthesized as a proenzyme then have to cleave that propeptide piece off to get the active MMP. There are a whole bunch that are present in different environments. They are inhibited locally by TIMPS and systemically by that alpha 2 macroglobin (if it leaks into circulation) then other proteases that are active will activate other MMPs. Some MMPs will actually activate other MMPs but I won't go into that at this point in time. What I do want you to remember is that MMP-8, expressed by inflammatory cells, they are up-regulated by cytokines associated with inflammation, so TNF-alpha, IL1 and IL6, these 3 together are important in inflammation. We’ll talk about those more in general pathology in the fall when we talk about inflammation.

63 – Periodontitis is Associated with Excessive Expression of MMP-8Gee, I’m on time. This is one disease you’ll deal with, severe periodontitis. These are terrible crown you won't make crowns like this. When these crowns were placed they were subgingivally so you can see how much tissue has been degraded, lots of bacterial plaque and we’ll talk about this next year. There is red inflamed tissue, destruction of the PDL, alveolar bone attaching the teeth into the head. The teeth are beginning to migrate, he has lost some teeth to this disease. The major ECM protein is type 1 collagen so in order to get this kind of destruction you need collagen to be destroyed. When I was doing my PhD, an individual on my thesis committee said Dr. Craig at any given time half of what you think are facts are wrong. The trouble is you don’t know which is wrong which is right so always keep an active mind. So Lauren Golub demonstrated that this tissue destruction was actually due to an inflammatory response from the host against these bacteria and in order to uncover bacteria from the ECM, it needs MMPS to expose the matrix and come in and clean out bacteria. They are kind of large and bacteria are real small so they can get into the brushwork of ECM proteins easily and in order to engulf and kill the bacteria, various inflammatory cells come in like monocytes and OMNS and elaborate MMP which gives tissue destruction. So as it turns out, that is MMP 8 and 1, genetically distinct proteins, and MMP 1 has a HIGHER affinity for divalent zinc at its active site than MMP 8 does. So Lorn Golub came up with this idea to get a molecule to bind to the active site of MMP 8 but leave the active site of MMP 1 in tact. And he found that

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Transcribed by Amy Maya 1.27.2014

tetracycline as a class of antibiotics that have 2 properties. They have an antibiotic effect but they also bind to divalent metal. So he was able to do a number of studies and showed that if you put a patent on very low levels of tetracycline like 20 mg twice a day, that tis really low, usually you give 250, if you do this you inhibit by competitive inhibition the binding of zinc2+ of MMP 8 and leave MMP 1 intact so you can inhibit this inflammatory tissue loss. So he came out with a drug he called it chemically modified tetracyclines. The product is Periostat, which is 20 mg of tetracycline in a gelatin capsule and its proven to be quite effective once you get rid of the bacteria. So with this profession (dentistry) we don’t like to pick up a prescription pad, we will for an antibiotic, maybe an analgesic, but not for something like Periostat. And Lorn (? I can't tell what this guys name is) tried to get the dental profession into this and dentists don’t think like a physician, we think like a surgeon. So he doesn't bother with dentistry anymore. And there are a number of diseases where inflammation plays a big role in the pathogenesis like atherosclerosis.

64 – Summary Are there questions on MMP-1 vs MMP-8? If in the clinic someone asks and says the patient benefitted from the Periostat, remember this is not inhibiting the bacteria because it is way too low for antibiotic effect. What your doing is you're inhibiting MMP 8 and keeping MMP 1 intact and there are folks who like Periostat and I’m one of them.So what did we talk about today? We mentioned fibril forming collagens especially 1 2 and 3 are resistant to most proteases but they are turned over. The bacterial collagenases cleave at each glycine residues and there are ones (vertebrate) that are successful ancestors evolved and they make one cleavage about ¼ the way down and that allows the molecule to unravel and another protease to come in, such as gelatinase. The vertebrate collagenases are part of the MMP family, and they all require divalent zinc at the active site and a drug such as low dose tetracycline because it turns out in heart attacks and strokes there is a higher expression of MMP-8 so we want to inhibit those. And I think that’s it. So questions on the collagen, non-collagen proteins, MMP lectures? You have the power points from NYU classes and the handouts. There is also a reference there. If you have questions you know where I live electronically and my office is on the 10th floor, and ill see you next month in craniofacial biology. Enjoy the Super bowl but be safe because this is the time that all types of weird people come into the city.

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