CSB327 Lecture 15 Notes

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Department
Cell and Systems Biology
Course
CSB327H1
Professor
Maurice Ringuette
Semester
Fall

Description
CSB327 Lecture 15 Notes (November 21, 2012) – Sibling family and tissue mineralization 2 – The SIBLING family of calcium binding glycoproteins • I want to talk about key events in osteogenesis and tie it back to what you already learned about some of these proteins and expand their role in osteogenesis and bone remodelling. • BSP is correlated with the onset of tissue mineralization. OPN appears to have the opposite biological effect. While BSP is found primarily in mineralized tissues, OPN has a much broader distribution. It may contribute to preventing our soft tissues from undergoing abnormal mineralization. Do we have a modified nucleator, or are tissues prone to mineralize and it is more important to prevent their mineralization? o We know ectopic mineralization occurs. One example is smooth muscle cell in the cardiovascular system can promote mineralization, which is not good because you eventually form a plaque. Video • http://www.youtube.com/watch?v=0dV1Bwe2v6c • Estrogen promotes an increase in bone mass. When you go through menopause, it is very controversial if you should be taking estrogen therapy because it can also promote metastatic behaviour of cells. 3 – Two types of bone structure: compact and spongy (cancellous) bone • This is to show you that there are two major types of bone. • The cortical bone builds like plywood sheets added onto each other as it builds. • The spongy bone does not grow in that way and is less dense. They consist of plates. 4 – Small integrin-binding ligand, N-linked glycoproteins (SIBLING) and tissue mineralization • I will only focus on BSP and OPN. • They are called the sibling family because they are small integrin-binding ligands. They have small calcium glycoproteins that bind to integrins. They are structurally diverse in their biological activity. Many of them are expressed in tissues that undergo mineralization. However, they are also found in other tissues. This is not the case with BSP. 5 – BSP and OPN • BSP and OPN are small calcium binding glycoproteins. • BSP can be extensively phosphorylated and sulfated. There are a lot of post-translational modifications. OPN shares some of the post-translational modifications. This will dictate their biological activity to some degree. • BSP and OPN bind to HA, which is calcium phosphate. • Even though they bind to integrins, OPN tends to block mineralization and BSP promotes mineralization. BSP is bone specific. OPN has broad tissue distribution. Those are the key facts that I want you to remember. • They are both integrin binding proteins. • If you look at OPN in solution, it does not seem to have any regular structure. It looks like it is flopping in solution. How many interactions can it form specifically with the components of the ECM or cell surface receptors? One of the emerging things is that by having regions of protein with no regular 3D rigid structure greatly enhances their repertoire of molecular interactions that it can form. It seems counter-intuitive. OPN binds to a huge number of different ligands and molecules. If you don’t have a 3D, how can you have a lock and key analogy? This may be the wrong way to think about these molecules. They bind then adapt a 3D organization depending on the specific molecule or receptor that it interacts with. 6 – Cellular and molecular composition of bone • You should be aware of these values. • If you look at bone, this is the average value. It varies in our body from one bone to the next. Almost 70%, on average, of the inorganic component of bone is mineral (HA). • About 25%, on average, of the organic component of bone is made up of these molecular components. The star is type I collagen and its partner, type V collagen. o Less than 5% of the bone is cells. o What tissue has such a low percentage of cell and the rest is ECM and water? Cartilage. You can see that this formation is highly dependent on the cross-talk between these molecules and their ability to influence cell signalling and so on. • Bone is a highly dynamic tissue. It is vascularized, innervated and undergoes continuous remodelling. We remodel throughout life. 7 – Structure of calcium hydroxyapatite crystals • This is for your information only. • This is what the structure looks like in three dimension. • This is what the crystals look like in bone. • Where does HA form? What is its relationship to collagen? • Why is BSP perceived as one of the key nucleators? • As you will see when we go into how you initiate crystal formation, there is reasonable doubt that BSP may not be a nucleator, but promote the event not by nucleation. 8 – Bone type I collagen fibrils and crystals • The crystals form between and within the collagen fibrils. • These images are a little deceiving. I will explain why during matrix vesicles. 9 – Temporal expression of bone-associated genes during osteogenic differentiation in vitro • OPN is expressed very early in progenitor cells and pre-osteoblasts. • As the cells are expanding and becoming more cuboidal, BSP expression correlates with the onset of mineralization. • Osteocytes remain dynamic. They are sensors of bone stress and remodelling. • OPN is critical for the promotion of bone resorption. ALP is the source of phosphate that can breakdown glycerophosphate to create more phosphate to bind to calcium. 10 – Osteoclastogenesis and osteogenesis • Osteoclasts are derived from blood cells. They originate as monocytes. They eventually become multi-nucleated phagocytic cells. This is different from macrophages, which are mono-nucleated phagocytic cells. • The lining cells are quiescent cells osteoblasts that can be re-recruited depending on the event to transform back into osteoblasts. • This diagram emphasizes the osteoclast cohort for remodeling and the osteoblast for promotion or secretion of matrix elements that promote mineralization and crystallization. 11 – Temporal expression of bone-associated genes during osteogenic differentiation in vitro • How did they study the process of mineralization in tissue culture? o If you want to study osteogenesis, you are going to go after intramembranous ossification. Cells go directly from a mesenchyme to formation of bone. • This is a Northern blot. You are running RNA samples from cells extracted from calvarial cultures at different days. This is a month-long experiment. You isolate RNA from different stages of progress in osteogenesis. o This is the temporal sequence of major events associated with this progress. • BSP is expressed at the onset of mineralization. • ALP comes on a bit earlier. • COL-1 may increase, but they are expressed all the way across. • OC is the other bone specific molecule. Its role in bone mineralization is correlated with actin as a chemoattractant for osteoclasts to promote the remodeling process. • OPN has a biphasic expression. The late stage is correlated with remodeling. • SPARC is not a nucleator. It is found in a lot of tissues, like OPN. • The loading control is to make sure that the amount of RNA that you load on the gel is comparable. At D11, you have more RNA loaded. So when you do your analysis, you standardize the amount of expression. 12 – The molecular arrangement of collagen and hydroxyapatite crystals in compact bone • Where do you find BSP? These are type I collagen fibrils. You tend to find bone crystals concentrated in the hole zone. This is also where BSP tends to be associated. Its association seems to be correlated with its ability to bind to the α2 of type I collagen. o It is anchoring by interaction with collagen. o You find crystals between the hole zone. You find crystals between collagen fibrils. You find crystals throughout the entire bone. • Does BSP promote crystal formation in the hole zones? 13 – Expression and deposition of BSP by osteoblasts in vivo and in vitro • BSP is observed in the Golgi apparatus because it is a secreted glycoprotein. o You see BSP in the nascent bone matrix and BSP expressed by osteoblasts. • In vitro, you see the same kind of event as in vivo when you look at early stages of osteogenesis in developing embryos. 14 – Crystal formation by mineralization-competent matrix vesicles (MVs) • How do you initiate bone formation? • What do I mean by matrix vesicles and HA formation? • The osteoblast is highlighted in red. If you take an osteoblast in vitro, you see these vesicles outside the osteoblast. There are vesicles that form inside the osteoblast that are released out into the matrix by exocytosis. • As the vesicles eventually fall apart into the matrix, you see needle-like HA associated with these vesicles. o There is a tremendous amount of calcium in our extracellular matrix compared to what we have intracellularly. The intracellar calcium concentration is 0.1 -10 µM. The ECM calcium concentration is 1 mM. • These matrix vesicles are made in osteoblasts, released and as they are coming out, they are forming these crystals. • This is a TEM showing needle-like projections of HA crystals forming on the vesicles. What is special about the vesicles that can promote the nucleation by HA? 15 – MV composition and the formation of intravesicular calcium-phosphate nanocrystals • This is the membrane of a vesicle. • In the cytoplasm, NPPs can break down the phosphodiesterase bond of NTPs and give you NMPs and PPi (bisphosphate). o PPi inhibits tissue mineralization. There are modifications on PPi in the treatment of excessive bone development. • Fortunately, TNAP on the vesicles can breakdown PPi into inorganic phosphates within the cytosol. • There is a type III Na+-dependent transporter that will bring the phosphate molecules inside the vesicle. • Annexin, a family of membrane-associated proteins, can act as Ca2+ transporter. o You have calcium inside. • Phosphatidylserine loves to interact with calcium. This brings the calcium and the inorganic phosphate in close proximity to each other. o You have the birth of small HA crystals within these intracellular vesicles. • In the process of mineralization, osteoblasts make these vesicles so that they can begin the process of HA formation within a confined space. • I will come back to the MMPs. 16 • This is a summary slide. • Nanocrystals are crystals in the nanometer range in size. • What is wrong with this slide? Is this calcified bone? If annexin can bind to collagens, where are you mineralizing? You are mineralizing in the hypertrophic zone of cartilage. You find type II collagen and type X collagen in the hypertrophic zone. Another member of annexin will promote the interaction with type I collagen. • The matrix vesicles initiate the formation of nanocrystals. They get secreted by osteoblast cells. If you imagine this surface being collagen and a cell is on top, the vesicles are coming in and they are seeding onto the collagen. It may well be that as the vesicles fall apart and release these nanocrystals, that you have already set the stage for a molecular interaction with BSP within the hole zone that helps further localize where mineralization can be enhanced. If I don’t have BSP, the matrix vesicles will form HA crystals but they are just slower at it. Our bodies, during development, you want to accelerate things. Hence, you build multi-component systems. It is important in the initial phase of extracellular mineralization of BSP, but the event is already set inside the cells themselves by the formation of these mineralization competent matrix vesicles. • When you start disregulating the smooth muscle cells in our vasculature, they also can turn to the production of mineralization competent vesicles, which will lead to mineralization of the arteries. The disregulating in smooth muscle cells is the underlying cause of many vascular pathologies. • There is a such a high level of calcium in our extracellular matrices that mineralization can occur independent of molecules (e.g., BSP) because collagen, by itself, can nucleate. The rate of nucleation and the type of nucleation is disregulated. o BSP seems to be along the scene at the right time at the onset of mineralization. Its biological activity is involved in the interplay of mineralization competent vesicles that form nanocrystals that get deposited onto the ECM (e.g., collagen). • How do you put the brakes on having osteoblasts keep making matrix unregulated? o You need to synchronize this by having remodeling by osteoclasts. How do you activate osteoclasts? This involves a ligand by RANK by osteoblasts that will signal to the progenitor osteoclast cells to undergo transformation into multi- nucleated osteoclasts. Video • http://www.youtube.com/watch?v=GpMV197xZXc • The parathyroid hormone and vitamin B promote the remodeling process. • You are not responsible for OPG, which is a decoy receptor for the ligand. 17 – Osteoblast differentiation • They are called master genes for osteoblast differentiation. Without the master genes, you don’t form mineralized bone because you don’t get proper osteoblast differentiation. 18 – Temporal expression of bone-associated genes during osteogenic differentiation in vitro
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