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130-401: 3SH4 , 3SH5 3339 15530 ENSG00000142798 ENSMUSG00000028763 P98160 Q05793 NM_001291860 NM_005529 NM_008305 NP_001278789 NP_005520 NP_032331 Perlecan (PLC) also known as basement membrane-specific heparan sulfate proteoglycan core protein (HSPG) or heparan sulfate proteoglycan 2 ( HSPG2 ), is a protein that in humans is encoded by

260-498: A GAG should qualify as heparin only if its content of N-sulfate groups largely exceeds that of N-acetyl groups and the concentration of O-sulfate groups exceeds those of N-sulfate. Otherwise, it should be classified as HS. Not shown below are the rare disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S) or a free amine group (GlcNH 3 ). Under physiological conditions the ester and amide sulfate groups are deprotonated and attract positively charged counterions to form

390-548: A base material for such a purpose. These gels have exhibited great promises in terms of biocompatibility, wear resistance, shock absorption , friction coefficient, flexibility , and lubrication, and thus are considered superior to polyethylene-based cartilages. A two-year implantation of the PVA hydrogels as artificial meniscus in rabbits showed that the gels remain intact without degradation, fracture, or loss of properties. Several diseases can affect cartilage. Chondrodystrophies are

520-414: A cartilage-like matrix, the best-known being pleomorphic adenoma of the salivary glands . The matrix of cartilage acts as a barrier, preventing the entry of lymphocytes or diffusion of immunoglobulins . This property allows for the transplantation of cartilage from one individual to another without fear of tissue rejection. Cartilage does not absorb X-rays under normal in vivo conditions, but

650-506: A certain developmental point is reached, much like confluence-dependent expression of perlecan in culture. These findings were corroborated by similar results from studies of rat pulmonary artery and lung epithelia. These tissues also were found to begin perlecan production once cell division had ceased, around fetal day 19. The development of the nervous system and extension of axons is precisely directed by cues from extracellular matrix molecules. Olfactory neurite outgrowth in mouse development

780-514: A corresponding fragment was found in the urine of patients suffering end-stage renal failure and in the amniotic fluid of pregnant women who have undergone premature rupture of the membrane. Timing of gene expression during development varies from tissue to tissue. Basement membranes are often the driving force behind separating epithelia from stroma and connective tissue. Perlecan is of particular importance in cardiovascular, neural and cartilaginous development. Pre-implantation blastocyst development

910-585: A critical role in the sequestration and/or delivery of FGF-2 and FGF-18 during endochondral ossification .   Perlecan is also a key component of the vascular extracellular matrix, where it interacts with a variety of other matrix components and helps to maintain the endothelial barrier function. Perlecan is a potent inhibitor of smooth muscle cell proliferation and is thus thought to help maintain vascular homeostasis. Perlecan can also promote growth factor (e.g., FGF-2 ) activity and thus stimulate endothelial growth and re-generation.    Modifications of

1040-447: A dye can be injected into the synovial membrane that will cause the X-rays to be absorbed by the dye. The resulting void on the radiographic film between the bone and meniscus represents the cartilage. For in vitro X-ray scans, the outer soft tissue is most likely removed, so the cartilage and air boundary are enough to contrast the presence of cartilage due to the refraction of

1170-448: A group of diseases, characterized by the disturbance of growth and subsequent ossification of cartilage. Some common diseases that affect the cartilage are listed below. Tumors made up of cartilage tissue, either benign or malignant , can occur. They usually appear in bone, rarely in pre-existing cartilage. The benign tumors are called chondroma , the malignant ones chondrosarcoma . Tumors arising from other tissues may also produce

1300-426: A high collagen content, called cartilage-like matrix, and collagen lacking a highly cellularized core, called osteoid-like matrix. The cartilage-like matrix surrounds the osteoid-like matrix. The amount of the acellular fibrous region is variable. The model organisms used in the study of cartilage in sabellid polychaetes are Potamilla species and Myxicola infundibulum . Vascular plants , particularly seeds , and

1430-517: A large number of extracellular proteins. These are often collectively called the “heparin interactome” or "heparin-binding proteins", because they are isolated by affinity chromatography on the related polysaccharide heparin, though the term “heparan sulfate interactome” is more correct. The functions of heparan sulfate binding proteins ranges from extracellular matrix components, to enzymes and coagulation factors, and most growth factors, cytokines, chemokines and morphogens The laboratory of Mitchell Ho at

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1560-510: A larger number of mineral deposits, which has a similarly undesired stiffening effect. Osteoarthritis has more extreme effects and can entirely wear down cartilage, causing direct bone-to-bone contact. Lubricin , a glycoprotein abundant in cartilage and synovial fluid , plays a major role in bio-lubrication and wear protection of cartilage. Cartilage has limited repair capabilities: Because chondrocytes are bound in lacunae , they cannot migrate to damaged areas. Therefore, cartilage damage

1690-446: A low amount of extra cellular matrix containing collagen. The odontophore contains muscle cells along with the chondrocytes in the case of Lymnaea and other mollusks that graze vegetation. The sabellid polychaetes , or feather duster worms, have cartilage tissue with cellular and matrix specialization supporting their tentacles. They present two distinct extracellular matrix regions. These regions are an acellular fibrous region with

1820-400: A material’s matrix more rapidly, while lower permeability leads to an initial rapid fluid flow and a slow decrease to equilibrium. Typically, the permeability of articular cartilage is in the range of 10^-15 to 10^-16 m^4/Ns. However, permeability is sensitive to loading conditions and testing location. For example, permeability varies throughout articular cartilage and tends to be highest near

1950-694: A matrix consisting of collagen type I and perlecan, as well as several other sulfated matrix glycoproteins. This mimics the in vivo corneal fibroblast's developmental program and response to injury. One of the long-term goals of creating 3D cell culture systems is to engineer tissues that can be used as replacements for patients with many types of disease. In tissue engineered heart valves created by seeding myofibroblasts onto collagen type I followed by endothelial cells, heparan sulfate proteoglycan expression has been verified, although no distinction between syndecan and perlecan has been made in these tissues. Another procedure that could be made possible by tissue engineering

2080-405: A number of possible 3- O -sulfated disaccharides, including GlcA-GlcNS(3S±6S) (modified by HS3ST1 and HS3ST5 ), IdoA(2S)-GlcNH 2 (3S±6S)(modified by HS3ST3A1 , HS3ST3B1 , HS3ST5 and HS3ST6 ) and GlcA/IdoA(2S)-GlcNS(3S) (modified by HS3ST2 and HS3ST4 ). As with all other HS sulfotransferases, the 3OSTs use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfate donor. Despite being

2210-410: A number of regulatory signals. Perlecan in the growth plate of mouse long bones shows glycosylation changes in the chondrocyte progression from the resting zone to the proliferating zone. Although initially the glycosaminoglycan (GAG) chains of perlecan were thought to be exclusively heparan sulfate, chondroitin sulfate chains can be substituted and this may be dependent upon the cell type. By expressing

2340-503: A physiologically abnormal increase in expression of the cytokine it could interfere with implantation. Mechanical and chemical stress can damage basement membranes or the cells they support. This could influence the gene expression profile of the cells, especially in their extracellular matrix, which often provides physical support and a chemical barrier for the cells. Hypoxia, inflammation, mechanical and chemical stress have been examined as to how they relate to perlecan expression. Hypoxia

2470-515: A recombinant form of the N-terminal domain I of the protein and demonstrating that digestion of the peptide with either heparanase or chondroitinase did not lead to complete loss of the peptide's activity, it was shown that chondroitin sulfate chains can be added to human perlecan. This was in agreement with previous data showing chondroitin sulfate GAG chains attached to bovine perlecan produced by chondrocytes and that recombinant human domain I protein

2600-443: A salt. It is in this form that HS is thought to exist at the cell surface. Many different cell types produce HS chains with many different primary structures. Therefore, there is a great deal of variability in the way HS chains are synthesised, producing structural diversity encompassed by the term "heparanome" - which defines the full range of primary structures produced by a particular cell, tissue or organism. However, essential to

2730-463: A sequence repeated in domain IV of the perlecan protein. These cells reproduce acini-like structures similar to those found in the native gland and tight junctions, along with complete basement membranes in culture. While Perlecan suppression causes substantial inhibition of tumor growth and neovascularization in null mice, in contrast, when perlecan-null cells are injected into nude mice enhanced tumor growth

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2860-427: A serious misregulation of extracellular matrix components including perlecan with TGF-β1 over expression. Corneal opacification occurred in both transgenic lines early in development due to greatly increased expression of perlecan, fibronectin and thrombospondin-1 in the corneal mesenchyme. The effect was more pronounced in the βB-1 Crystallin promoter-driven line. The IL family of inflammatory cytokines also upregulates

2990-465: A significant increase in sulfation of the heparan sulfate chains. This is not in contrast to the data shown where perlecan expression is constant beyond e19 in rat VSMC, which suggested that perlecan plays an antiproliferative role for VSMCs. In this case, it seems that the molecule's signaling function is the operative upregulated factor, especially due to the increase in sulfation of the heparan sulfate chains. Chemical damage to organs can affect not only

3120-531: A similar cell stretching mechanism to mimic arterial pressure, this investigation showed that perlecan production increased in response to mechanical strain. This is contingent upon TGF-β autocrine signaling in a positive feedback loop with p38 and ERK. This endothelial cell increase in production of VSMC growth inhibitors (i.e. heparin) is reversed in VSMCs, where mechanical stress induces proliferation. Deformation of VSMC cells in culture leads to perlecan upregulation, with

3250-619: A variably sulfated repeating disaccharide unit. The main disaccharide units that occur in heparan sulfate and heparin are shown below. The most common disaccharide unit within heparan sulfate is composed of a glucuronic acid (GlcA) linked to N -acetylglucosamine (GlcNAc), typically making up around 50% of the total disaccharide units. Compare this to heparin, where IdoA(2S)-GlcNS(6S) makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa. Problems arise when defining hybrid GAGs that contain both 'heparin-like' and 'HS-like' structures. It has been suggested that

3380-483: Is a condition found in disease states and during injury and often results in a lack of endothelial cell proliferation. This and perlecan's role as endorepellin prompted one study into the nature of perlecan expression regulation by endothelial cells during hypoxic conditions. Under hypoxic conditions, this study found that perlecan expression by rat cardiac microvascular endothelial cells was decreased sixty-one percent compared to normal controls. The contention of this paper

3510-457: Is a controlled cascade of gene regulation and intercellular signaling. Extracellular perlecan has been observed at the blastocyst stage of mouse embryonic development, specifically upregulated at the point when the embryo reaches “attachment competence”. This finding was upheld at both the mRNA level and the protein level, shown by RT-PCR and immunostaining. Later embryonic development is just as precisely regulated as pre-implantation development, and

3640-470: Is a hereditary disease with mutations on the EXT1 and EXT2 genes that affect biosynthesis of heparan sulfate. Cartilage Cartilage is a resilient and smooth type of connective tissue . Semi-transparent and non-porous, it is usually covered by a tough and fibrous membrane called perichondrium . In tetrapods, it covers and protects the ends of long bones at the joints as articular cartilage , and

3770-682: Is a key component of the extracellular matrix of cartilage where it is essential for normal growth plate development and long bone growth. The dwarfism exhibited by the perlecan null mouse resembles the phenotype produced by activating mutations in the gene for FGFR3 , a receptor for fibroblast growth factors . Perlecan binds to growth factors involved in growth plate development. Perlecan isolated from developing growth plates has been shown to bind to FGF-2 via its heparan sulfate side chains, and to FGF-18 via domain III of its core protein and mediates their action on FGF receptors. Perlecan likely plays

3900-787: Is a prerequisite for all subsequent modification reactions, and is carried out by one or more members of a family of four GlcNAc N-deacetylase/N-sulfotransferase enzymes (NDSTs). In early studies, it was shown that modifying enzymes could recognize and act on any N-acetylated residue in the forming polymer. Therefore, the modification of GlcNAc residues should occur randomly throughout the chain. However, in HS, N-sulfated residues are mainly grouped together and separated by regions of N-acetylation where GlcNAc remains unmodified. There are four isoforms of NDST (NDST1–4). Both N-deacetylase and N-sulfotransferase activities are present in all NDST-isoforms but they differ significantly in their enzymatic activities. Due to

4030-563: Is a structural component of many body parts including the rib cage , the neck and the bronchial tubes, and the intervertebral discs . In other taxa, such as chondrichthyans and cyclostomes , it constitutes a much greater proportion of the skeleton. It is not as hard and rigid as bone , but it is much stiffer and much less flexible than muscle . The matrix of cartilage is made up of glycosaminoglycans , proteoglycans , collagen fibers and, sometimes, elastin . It usually grows quicker than bone. Because of its rigidity, cartilage often serves

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4160-474: Is a vesicular cell-rich cartilage due to the large, spherical and vacuolated chondrocytes with no homologies in other arthropods. Other type of cartilage found in L. polyphemus is the endosternite cartilage, a fibrous-hyaline cartilage with chondrocytes of typical morphology in a fibrous component, much more fibrous than vertebrate hyaline cartilage, with mucopolysaccharides immunoreactive against chondroitin sulfate antibodies. There are homologous tissues to

4290-422: Is absent and is thus resistant to enzyme degration. Also the function of the heparan sulfate analogues is the same as heparan sulfate, protecting a variety of protein ligands such as growth factors and cytokines. By holding them in place, the tissue can then use the different protein ligands for proliferation. Hereditary multiple exostoses (also known as multiple hereditary exostoses or multiple osteochondromas

4420-451: Is carried out by EXT family proteins with glycosyltransferase activities. EXT family genes are tumor suppressors. Mutations at the EXT1-3 gene loci in humans lead to an inability of cells to produce HS and to the development of the disease Multiple Hereditary Exostoses (MHE). MHE is characterized by cartilage-capped tumours, known as osteochondromas or exostoses, which develop primarily on

4550-419: Is concomitant with the role of perlecan's C-terminus as endorepellin. Spatio-temporal specificity in trans-activation of the perlecan gene during development is key to the maturation of basement membranes and thus to the complete separation of epithelia from endothelia and stroma. A thorough study of perlecan expression during chick embryo development has shown that perlecan is present at the morula stage and for

4680-487: Is dependent on the molecular composition of the extracellular matrix (ECM). The ECM consists mainly of proteoglycan and collagens . The main proteoglycan in cartilage is aggrecan, which, as its name suggests, forms large aggregates with hyaluronan and with itself. These aggregates are negatively charged and hold water in the tissue. The collagen, mostly collagen type II, constrains the proteoglycans. The ECM responds to tensile and compressive forces that are experienced by

4810-441: Is difficult to heal. Also, because hyaline cartilage does not have a blood supply, the deposition of new matrix is slow. Over the last years, surgeons and scientists have elaborated a series of cartilage repair procedures that help to postpone the need for joint replacement. A tear of the meniscus of the knee cartilage can often be surgically trimmed to reduce problems. Complete healing of cartilage after injury or repair procedures

4940-415: Is documented to repair at only a very slow rate relative to other tissues. In embryogenesis , the skeletal system is derived from the mesoderm germ layer. Chondrification (also known as chondrogenesis) is the process by which cartilage is formed from condensed mesenchyme tissue, which differentiates into chondroblasts and begins secreting the molecules ( aggrecan and collagen type II) that form

5070-430: Is exerted on the promoter of genes, which can include elements upstream or downstream of the transcriptional start site, some of which can exist inside of the transcribed gene itself. A number of signaling molecules can effect changes in perlecan expression including the transforming growth factor-Beta (TGF-β), interleukin(IL) and vascular endothelial growth factor (VEGF) families of molecules. The upstream 2.5 kilobases of

5200-415: Is free-moving, it makes the material difficult to test. One of the tests commonly used to overcome this obstacle is a confined compression test, which can be used in either a 'creep' or 'relaxation' mode. In creep mode, the tissue displacement is measured as a function of time under a constant load, and in relaxation mode, the force is measured as a function of time under constant displacement. In creep mode,

5330-405: Is guided at least in part by an ECM laid down by olfactory epithelial cells (OECs). Perlecan and laminin-1 appear to be important in this guidance pathway, although perlecan induction occurs slightly later than laminin-1. This data is supported by earlier data showing that OECs express FGF-1 during olfactory development, and that perlecan can stimulate olfactory sensory neurite outgrowth in culture in

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5460-405: Is hindered by cartilage-specific inflammation caused by the involvement of M1/M2 macrophages , mast cells , and their intercellular interactions. Biological engineering techniques are being developed to generate new cartilage, using a cellular "scaffolding" material and cultured cells to grow artificial cartilage. Extensive researches have been conducted on freeze-thawed PVA hydrogels as

5590-416: Is implanted into the cornea of mice and in normal mice angiogenesis is induced. In the knockout mice this angiogenic effect was impaired, although not completely. A full-length perlecan construct, under control of the collagen type II promoter, was used to make a perlecan transgenic mouse. The collagen type II promoter allowed perlecan expression in the extracellular matrix made by chondrocytes only but not in

5720-433: Is insensitive. However, some fibrocartilage such as the meniscus of the knee has partial blood supply. Nutrition is supplied to the chondrocytes by diffusion . The compression of the articular cartilage or flexion of the elastic cartilage generates fluid flow, which assists the diffusion of nutrients to the chondrocytes. Compared to other connective tissues, cartilage has a very slow turnover of its extracellular matrix and

5850-413: Is keratoepithelioplasty. Transplanted tissue must remain intact, which requires a pre-formed basement membrane. Collagen gels have promoted formation of a complete basement membrane by corneal epithelial cells in culture. Perlecan also holds promise to serve as a scaffold for plating cells in culture. Human salivary gland ductal and acinar cells have been successfully grown on a bioactive peptide containing

5980-441: Is more complicated due to differentiation of all tissues. The first study of perlecan expression during embryonal development found that the protein was first expressed during development of the cardiovascular system, and later correlates with maturation of the majority of tissues in the body, i.e. separation of epithelial layers from endothelia and stroma by basement membranes. Again, this upregulation during cardiovascular development

6110-468: Is observed when compared to controls. Cancer progression and pathogenesis is intimately linked to extracellular matrix composition and the role of perlecan and other ECM molecules in cancer is being studied by a large number of laboratories. Since the basement membrane is the first obstacle in the way of extravasating carcinoma cells, the functions of perlecan in this process are multiple. One model system used to study perlecan expression in carcinoma cell lines

6240-433: Is rich in proteoglycans (which dispel and reabsorb water to soften impacts) and thin collagen oriented parallel to the joint surface which have excellent shear resistant properties. Osteoarthritis and natural aging both have negative effects on cartilage as a whole as well as the proper function of the materials gradient within. The earliest changes are often in the superficial zone, the softest and most lubricating part of

6370-467: Is simply a part of the ECM of developing chondrocytes, in addition to collagen II and other cartilage markers that are expressed starting on day 12. Taken with the data, that mice lacking the pln gene cannot maintain stable cartilage, it is apparent that perlecan is essential to the maturation and stability of cartilaginous structure. This is supported by a study showing that knockdown of perlecan production inhibits

6500-752: Is synthesized by both vascular endothelial and smooth muscle cells and deposited in the extracellular matrix of parahoxozoans . Perlecan is highly conserved across species and the available data indicate that it has evolved from ancient ancestors by gene duplication and exon shuffling. Perlecan consists of a core protein of molecular weight 469 kDa to which three long chains (each approximately 70-100 kDa) of glycosaminoglycans (often heparan sulfate , HS, but can be chondroitin sulfate , CS) are attached. The core protein consists of five distinct structural domains . The N-terminal domain I ( aa ~1-195) contains attachment sites for HS chains. Although HS chains are not required for correct folding and secretion of

6630-723: Is that of the MeWo/70W melanoma metastatic progression cell lines. MeWo cells are characteristically less invasive than their clonal variant cell line 70W. One lab studied perlecan expression in 27 invasive melanomas and 26 of the 27 samples showed a significant increase in perlecan message when compared to normal tissue from the same patients. They then used the MeWo and 70W cell lines to study if perlecan expression changed during treatment with neurotrophins, which can stimulate cell invasion through matrigel in vitro. The more invasive 70W cells began expressing perlecan message ten minutes after stimulation with

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6760-516: Is that perlecan downregulation leads to a loss of FAK activation and thus less ERK signaling, leading to decreased cell proliferation. It does seem counterintuitive that endothelial cells would proliferate less quickly due to loss of perlecan and its endorepellin subunit. It could be that these endothelial cells merely downregulated transcription of many genes in response to hypoxic conditions. In another study, hypoxia led to induction of genes associated with apoptosis and cell death, but repression of genes

6890-519: Is the VEGF pathway. VEGF165 treatment of human brain microvascular endothelial cells in culture stimulates increased pln transcription. This molecule is a ligand of VEGF Receptor-2 (VGFR2), and it seems that this VEGF165 response is specific for perlecan upregulation, leading to a positive feedback loop involving fibroblastic growth factor (FGF), FGF Receptor (FGFR) and VEGFR2 in response to endothelial damage. This microvascular-specific regulation by VEGF165 raises

7020-446: Is typically 0.45 to 0.80 MPa. The aggregate modulus is “a measure of the stiffness of the tissue at equilibrium when all fluid flow has ceased”, and Young’s modulus is a measure of how much a material strains (changes length) under a given stress. The confined compression test can also be used to measure permeability, which is defined as the resistance to fluid flow through a material. Higher permeability allows for fluid to flow out of

7150-410: Is usually not based on an increase in size or mass of the cartilage itself. It has been identified that non-coding RNAs (e.g. miRNAs and long non-coding RNAs) as the most important epigenetic modulators can affect the chondrogenesis. This also justifies the non-coding RNAs' contribution in various cartilage-dependent pathological conditions such as arthritis, and so on. The articular cartilage function

7280-595: The HSPG2 gene . The HSPG2 gene codes for a 4,391 amino acid protein with a molecular weight of 468,829. It is one of the largest known proteins. The name perlecan comes from its appearance as a "string of pearls" in rotary shadowed images. Perlecan was originally isolated from a tumor cell line and shown to be present in all native basement membranes. Perlecan is a large multidomain (five domains, labeled I-V) proteoglycan that binds to and cross-links many extracellular matrix (ECM) components and cell-surface molecules . Perlecan

7410-472: The X-ray . Cartilaginous fish ( Chondrichthyes ) or sharks , rays and chimaeras have a skeleton composed entirely of cartilage. Cartilage tissue can also be found among some arthropods such as horseshoe crabs , some mollusks such as marine snails and cephalopods , and some annelids like sabellid polychaetes. The most studied cartilage in arthropods is the branchial cartilage of Limulus polyphemus . It

7540-433: The heparan sulfate chains on C- and N-terminal domains are the best-studied differences in the secretory pathway of perlecan. Chondroitin sulfate can be substituted for heparan sulfate, and sulfate incorporation or the sugar composition of the chains can change. Loss of enzymes involved in the heparan sulfate synthetic pathway lead to a number of conditions. Differential heparan sulfate chain modification can occur through

7670-509: The 3-O-sulfation in GlcNS6S3S enhances the binding of Wnt to the glypican. The HS-binding properties of a number of other proteins are also being studied: Heparan sulfate analogues are thought to display identical properties as heparan sulfate with exception of being stable in a proteolytic environment like a wound. Because heparan sulfate is broken down in chronic wounds by heparanase, the analogues only bind sites where natural heparan sulfate

7800-430: The 3OSTs are the largest family of HS modification enzymes and their actions are rate-limiting, substrate specific and produce rare modifications, it has been hypothesized that 3OST modified HS plays an important regulatory role in biological processes. It has been demonstrated that 3- O -sulfation can enhance the binding of Wnt to the glypican and may play a role in regulating Wnt in cancer. Heparan sulfate binds with

7930-410: The ECM, and serve as co-ligands or ligand enhancers when bound to receptors. Another study showed that release of HS-bound basic FGF in culture could be achieved through treatment with stromelysin, heparitinase I, rat collagenase and plasmin, and these proteolysis sites are illustrated in figure 1. This was proposed as a non-exhaustive list of the proteases that could mediate release of growth factors from

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8060-562: The GlcN residue linked to the non-reducing side of a potential GlcA target be N-sulfated. Uronosyl-2-O-sulfotransferase (2OST) sulfates the resulting IdoA residues. Three glucosaminyl 6-O-transferases (6OSTs) have been identified that result in the formation of GlcNS(6S) adjacent to sulfated or non-sulfated IdoA. GlcNAc(6S) is also found in mature HS chains. Currently seven glucosaminyl 3- O -sulfotransferases (3OSTs, HS3STs) are known to exist in mammals (eight in zebrafish). The 3OST enzymes create

8190-408: The HS binding region, near the protein's C-terminal. Binding of HS blocks the receptor binding site and as a result, protein-HS complexes are inactive. Glypican-3 (GPC3) interacts with both Wnt and Frizzled to form a complex and triggers downstream signaling. It has been experimentally established that Wnt recognizes a heparan sulfate motif on GPC3, which contains IdoA2S and GlcNS6S, and that

8320-595: The N-deacetylase and N-sulfotransferase being carried out by the same enzyme N-sulfation is normally tightly coupled to N-acetylation. GlcNH 2 residues resulting from apparent uncoupling of the two activities have been found in heparin and some species of HS. Epimerisation is catalysed by one enzyme, the GlcA C5 epimerase or heparosan-N-sulfate-glucuronate 5-epimerase ( EC 5.1.3.17 ). This enzyme epimerises GlcA to iduronic acid (IdoA). Substrate recognition requires that

8450-644: The NCI isolated the HS20 human monoclonal antibody with high affinity for heparan sulfate by phage display. The antibody binds heparan sulfate, not chondroitin sulfate. The binding of HS20 to heparan sulfate requires sulfation at both the C2 position and C6 position. HS20 blocks the Wnt binding on heparan sulfate and also inhibits infectious entry of pathogenic JC polyomavirus. The cell surface receptor binding region of Interferon-γ overlaps with

8580-526: The V-3 isoform of CD44 present on keratinocytes and activated monocytes . In the extracellular matrix, especially basement membranes and fractones , the multi-domain perlecan , agrin and collagen XVIII core proteins are the main HS-bearing species. Heparan sulfate is a member of the glycosaminoglycan (GAG) family of carbohydrates and is very closely related in structure to heparin . Both consist of

8710-525: The addition of exogenous VEGF-A. The importance of perlecan to mammalian development is demonstrated by perlecan gene knockout experiments. Nearly half of all mice in which the perlecan gene has been knocked out (perlecan null mice) die at embryonic day 10.5, when the perlecan gene normally starts to be expressed. Others die just after birth with severe defects such as abnormal basement membrane formation, defective cephalic and long bone development and achondroplasia . The knockout strategy employed for one of

8840-694: The aggregate modulus, Poisson's ratio, and permeability of the tissue. Initially, there was a misconception that due to its predominantly water-based composition, cartilage had a Poisson's ratio of 0.5 and should be modeled as an incompressible material. However, subsequent research has disproven this belief. The Poisson’s ratio of articular cartilage has been measured to be around 0.4 or lower in humans and ranges from 0.46–0.5 in bovine subjects. The mechanical properties of articular cartilage are largely anisotropic, test-dependent, and can be age-dependent. These properties also depend on collagen-proteoglycan interactions and therefore can increase/decrease depending on

8970-806: The basement membranes made by endothelial, epithelial or muscle cells. The perlecan transgene in the perlecan null mouse eliminated lethality and restored long bone growth to normal. This suggests that perlecan plays a critical role in cartilage development. The perlecan transgenic mice, however, exhibited muscle hypertrophy, indicating a role for perlecan in muscle development as well as in cartilage growth plate mediated long bone growth. Studies from gene knockout mice and human diseases have also revealed critical in vivo roles for perlecan in cartilage development and neuromuscular junction activity. Signaling pathways function to elevate or decrease levels of transcription of genes, which in turn cause cells to change their gene expression profile. The end effect of signaling pathways

9100-448: The c-terminal endorepellin domain of the perlecan core protein. The laminin-like globular domain contains the active motif of endorepellin, and is unable to be cleaved by cells expressing mutant and inactive forms of the BMP-1 proteins. Furthermore, the critical residue necessary for this cleavage to take place was localized to Asp4197. This proteolytic process may have significance in disease as

9230-406: The cartilage. Cartilage growth thus refers to the matrix deposition, but can also refer to both the growth and remodeling of the extracellular matrix. Due to the great stress on the patellofemoral joint during resisted knee extension, the articular cartilage of the patella is among the thickest in the human body. The ECM of articular cartilage is classified into three regions: the pericellular matrix,

9360-449: The cell's genetic and mechanical integrity but the extracellular matrix of the tissue. To study the effect of chemical damage on liver cells, Wistar rats were treated with carbon tetrachloride for 48 hours prior to sacrificing. Prior to treatment with CCl 4 , perlecan staining was limited to the bile duct and sinusoidal blood vessels of the liver. After treatment, perlecan staining was intense in areas of necrosis. This could have been due to

9490-448: The cells in the organism. This problem is being dealt with by developing 3D cell cultures using a wide variety of substrates as the scaffolds or environments for the cells. In this kind of setting the expression of ECM genes has the potential to more closely resemble that of the native expression profile. 3D scaffolds, the structures on which the cultured cells grow, can be composed of other cells, i.e. cocultures, synthetic polymers mimicking

9620-426: The cells natural environment or purified ECM such as matrigel, and any mixture of these three components. One such system has been developed to study skin development and basal membrane formation between keratinocytes and the stroma. This system is used to delineate the development of basement membrane between fibroblasts in the stroma (in this case fibroblasts in a type-I collagen gel) and keratinocytes grown on top of

9750-485: The core protein is thought to occur in the endoplasmic reticulum (ER) with further assembly of the linkage region and the remainder of the chain occurring in the Golgi apparatus . After attachment of the first N -acetylglucosamine (GlcNAc) residue, elongation of the tetrasacchride linker is continued by the stepwise addition of GlcA and GlcNAc residues. These are transferred from their respective UDP-sugar nucleotides. This

9880-406: The cornea. In the epidermal injury study, a wound spanning the depth of the epidermis was created in exon 3-negative mice and control mice, and in the knockout mice angiogenesis and the hallmarks of wound healing were slow to develop possibly due to decreased growth factor sequestration by the heparan sulfate-negative perlecan. A similar result was produced in the corneal micropocket assay, where FGF-2

10010-482: The creep mode and the relaxation mode of a confined compression test, a disc of cartilage is placed in an impervious, fluid-filled container and covered with a porous plate that restricts the flow of interstitial fluid to the vertical direction. This test can be used to measure the aggregate modulus of cartilage, which is typically in the range of 0.5 to 0.9 MPa for articular cartilage, and the Young’s Modulus, which

10140-492: The data showing that loss of heparan sulfate at the COJ is a key factor in osteogenesis. It is thought that the driving force behind heparanase and chondroitinase activation of osteogenesis is release of bone morphogenetic protein bound in the heparan sulfate chains. Perlecan knockdown in embryonic zebrafish has been achieved through the use of Morpholinos targeted to the perlecan transcript. Morpholinos were used to block translation of

10270-637: The domain IVa and IVb of laminin . Domain IV consists of a series of IG modules. The structure of only the 3rd immunoglobulin (IG)-like repeat in domain IV is shown in the figure labeled "HSPG2". The C-terminal Domain V, which has homology to the G domain of the long arm of laminin , is responsible for self-assembly and may be important for basement membrane formation in vivo. Domain V also has attachment sites for HS/CS chains. Thus, perlecan core protein and HS chains could modulate matrix assembly, cell proliferation , lipoprotein binding and cell adhesion . Perlecan

10400-465: The dynamic regulation of perlecan and its control by extracellular signaling pathways is critical to our understanding of the protein's role in development. To this end, a transgenic mouse line was created expressing porcine TGF-β1 under the lens-specific αA-crystallin promoter and then another similar line was created but with the gene driven by the βb-crystallin promoter, corresponding to another lens-specific gene. This developmentally dynamic tissue showed

10530-445: The effect of proteoglycan composition on nephritic permselectivity, it was noted that puromycin treatment of human glomerular endothelial cells (HGEC) altered the sulfation level of GAG chains on proteoglycans such as perlecan, which in turn caused a decrease in the stability of the GAG chains. The core protein mRNA levels of proteoglycans were not affected, thus the decrease in GAG chains

10660-414: The endosternite cartilage in other arthropods. The embryos of Limulus polyphemus express ColA and hyaluronan in the gill cartilage and the endosternite, which indicates that these tissues are fibrillar-collagen-based cartilage. The endosternite cartilage forms close to Hh-expressing ventral nerve cords and expresses ColA and SoxE, a Sox9 analog. This is also seen in gill cartilage tissue. In cephalopods,

10790-583: The exon 3 knockout mice was normal. This suggests that, in the exon 3 knockout, the remaining attachment sites for heparan sulfate on domains I and V available for FGF-2 binding or the site on domain 3 available for FGF-18 binding may be sufficient for normal long bone growth. Changes to the lens in the exon 3 knock out mice are somewhat similar to the TGF-β knockout mouse model. Exon 3 knockout mice also showed decreased wound healing and angiogenesis capabilities when challenged by either epidermal injury or FGF-2 addition to

10920-491: The expression SoxD and SoxE, analogs of the vertebrate Sox5/6 and Sox9, in the developing cartilage. The cartilage growth pattern is the same as in vertebrate cartilage. In gastropods, the interest lies in the odontophore , a cartilaginous structure that supports the radula. The most studied species regarding this particular tissue is Busycotypus canaliculatus . The odontophore is a vesicular cell rich cartilage, consisting of vacuolated cells containing myoglobin, surrounded by

11050-492: The extracellular matrix. In all vertebrates, cartilage is the main skeletal tissue in early ontogenetic stages; in osteichthyans, many cartilaginous elements subsequently ossify through endochondral and perichondral ossification. Following the initial chondrification that occurs during embryogenesis, cartilage growth consists mostly of the maturing of immature cartilage to a more mature state. The division of cells within cartilage occurs very slowly, and thus growth in cartilage

11180-585: The final stages of chondrogenic differentiation in C3H10T1/2 fibroblasts in culture. Bone development, i.e. mineralization of cartilaginous tissue, correlates with loss of perlecan and heparan sulfate at the chondro-osseous junction (COJ). In an effort to understand how heparan sulfate and perlecan direct mesenchymal stem cells into the osteogenic pathway, human mesenchymal stem cells were treated with heparanase and chondroitinase in culture. This led to increased mineralization and expression of osteocyte markers, supporting

11310-500: The formation of HS regardless of primary sequence is a range of biosynthetic enzymes. These enzymes consist of multiple glycosyltransferases , sulfotransferases and an epimerase . These same enzymes also synthesize heparin . In the 1980s, Jeffrey Esko was the first to isolate and characterize animal cell mutants altered in the assembly of heparan sulfate. Many of these enzymes have now been purified, molecularly cloned and their expression patterns studied. From this and early work on

11440-459: The formation of a tetrasaccharide primer O -linked to a serine of the core-protein: βGlcUA-(1→3)-βGal-(1→3)-βGal-(1→4)-βXyl- O -Ser. The pathways for HS/heparin or chondroitin sulfate (CS) and dermatan sulfate (DS) biosynthesis diverge after the formation of this common tetrasaccharide linkage structure. The next enzyme to act, GlcNAcT-I or GalNAcT-I, directs synthesis, either to HS/heparin or CS/DS, respectively. Xylose attachment to

11570-494: The fundamental stages of HS/heparin biosynthesis using a mouse mastocytoma cell free system a lot is known about the order of enzyme reactions and specificity. HS synthesis initiates with the transfer of xylose from UDP -xylose by xylosyltransferase (XT) to specific serine residues within the protein core. Attachment of two galactose (Gal) residues by galactosyltransferases I and II (GalTI and GalTII) and glucuronic acid (GlcA) by glucuronosyltransferase I (GlcATI) completes

11700-402: The gel. Perlecan expression and thus basement membrane maturation is dependent on nidogen crosslinking of collagen IV and laminin γ1 chain in this system. This effect also led to a lack of hemidesmosomes in the developing tissue. Another system using a disorganized hydrated collagen I gel has been used to demonstrate that primary human corneal fibroblasts will eventually invade the gel and create

11830-405: The healing period, along with IL 1-alpha expression. Perlecan expression was traced to microglial cells in the hippocampus and astrocytes. This role for perlecan in beta-amyloid plaque generation is supported by an earlier study showing that perlecan and beta-amyloid treatment of rat brains led to formation of senile plaques, whereas treatment with beta-amyloid alone did not have the same effect. At

11960-429: The heparan sulfate chains of perlecan. Although Whitelock et al. suggested that thrombin cleavage consensus sequences exist in the core protein of perlecan, they also postulate that any thrombin activation of perlecan actually comes from cleavage of other ECM constituents. This article states that heparanase is responsible for cleavage of the heparan sulfate chains of perlecan in matrix. This releases growth factors bound to

12090-419: The heparan sulfate, specifically FGF-10 . Addition of heparanase to cell culture of epithelia in basement membrane caused an increase in epithelial cell proliferation due to FGF-10 release. In a model of explant growth in vitro using corneal epithelium, Matrix Metalloproteinase (MMP) 2 expression correlates with an initial degradation of the original basement membrane. Reformation of basement membrane in culture

12220-424: The increase in capillarization of the liver as an attempt to regenerate damaged tissue. A similar finding was shown in acetamenophin treatment of mice, where perlecan and other matrix components were heavily expressed in necrotic lesions of the liver. One of the resounding arguments against the validity of in vitro results of cell culture on 2D plastic plates is that the environment does not accurately reflect that of

12350-415: The inflammation in the brain context. As previously mentioned, to investigate the effect of brain inflammation on expression levels of perlecan, needle stab wounds were created in mice brains, and after inflammation and variable periods of recovery, mRNA and protein levels were assessed via in situ hybridization and immunostaining. Perlecan levels were increased in the hippocampus but not in the striatum during

12480-493: The joint surface and lowest near the bone (or “deep zone”). Permeability also decreases under increased loading of the tissue. Indentation testing is an additional type of test commonly used to characterize cartilage. Indentation testing involves using an indentor (usually <0.8 mm) to measure the displacement of the tissue under constant load. Similar to confined compression testing, it may take hours to reach equilibrium displacement. This method of testing can be used to measure

12610-438: The lamina cribosa (connective tissue) of the optic nerve head. Their findings were that perlecan and several other proteoglycans were upregulated in response to the stretching stimulus. TGF-β2 and VEGF were induced as well, possibly contributing to the upregulation of the perlecan transcript and protein. It has been shown that autocrine TGF-β signaling is a compensatory result of mechanical stress in vitro in endothelial cells. Using

12740-526: The largest family of HS modification enzymes, the 3OSTs produce the rarest HS modification, the 3- O -sulfation of specific glucosamine residues at the C3-OH moiety. The 3OSTs are divided into two functional subcategories, those that generate an antithrombin III binding site ( HS3ST1 and HS3ST5 ) and those that generate a herpes simplex virus 1 glycoprotein D (HSV-1 gD) binding site ( HS3ST2 , HS3ST3A1 , HS3ST3B1 , HS3ST4 , HS3ST5 and HS3ST6 ). As

12870-494: The long bones of affected individuals from early childhood until puberty. As an HS chain polymerises, it undergoes a series of modification reactions carried out by four classes of sulfotransferases and an epimerase. The availability of the sulfate donor PAPS is crucial to the activity of the sulfotransferases. The first polymer modification is the N-deacetylation/N-sulfation of GlcNAc residues into GlcNS. This

13000-493: The loss of perlecan expression stems from downregulation of transcription via STAT1 transcription factor activity as shown previously. These in vitro results are not necessarily representative of normal physiological interferon- γ concentrations, nor are the cytokine normally expressed widely but instead at very specific developmental timepoints. Important to note is that perlecan expression can be decreased by treatment with an exogenous cytokine such as interferon- γ, and if there were

13130-679: The millions of loading cycles experienced by human joins over a lifetime, would eventually lead to failure. For example, the elastic modulus of human bone is roughly 20 GPa while the softer regions of cartilage can be about 0.5 to 0.9 MPa. When there is a smooth gradient of materials properties, however, stresses are distributed evenly across the interface, which puts less wear on each individual part. The body solves this problem with stiffer, higher modulus layers near bone, with high concentrations of mineral deposits such as hydroxyapatite. Collagen fibers (which provide mechanical stiffness in cartilage) in this region are anchored directly to bones, reducing

13260-404: The models used for the studies of cartilage are Octopus vulgaris and Sepia officinalis . The cephalopod cranial cartilage is the invertebrate cartilage that shows more resemblance to the vertebrate hyaline cartilage. The growth is thought to take place throughout the movement of cells from the periphery to the center. The chondrocytes present different morphologies related to their position in

13390-502: The neurotrophins, and the MeWo cells did not produce any pln message regardless of treatment. This study took special note of the fact that perlecan upregulation occurred even before that of heparanase, an essential protein involved in the process of extravasation. Heparan sulfate#Proteoglycans The major cell membrane HSPGs are the transmembrane syndecans and the glycosylphosphatidylinositol (GPI) anchored glypicans . Other minor forms of membrane HSPG include betaglycan and

13520-436: The one heparan sulfate attachment site remaining on domain I, the attachment site on domain V would also still be present. The study showed that the exon 3 knockout mice had collapse of lens capsule integrity by postnatal week 3, indicating a role for the amino acids deleted from domain I of perlecan in maintaining lens capsule basement membrane integrity. Unlike the perlecan knockout mice however, viability and long bone growth in

13650-404: The organismic level, mechanical stress has a profound impact on extracellular matrix integrity and probably causes induction of a number of ECM genes for repair and remodeling of ECM in tissue stroma and basement membranes. One study examined the in vitro effects of pressure on global gene transcription using a microarray approach and a cell stretching system meant to simulate intraocular pressure in

13780-420: The perlecan gene was mutated by homologous recombination of the endogenous perlecan gene with a construct containing 2 and 5 kb arms of homology surrounding a deleted exon 3, which codes for 2 of the 3 heparan sulfate attachment sites in domain I of perlecan. Perlecan produced by cultured fibroblasts from exon 3 knockout mice, however, contained 40% Heparan sulfate and 60% chondroitin sulfate because, in addition to

13910-426: The perlecan knockouts was a floxing of exon 6 by insertion of a neomycin cassette, and subsequent CRE expression for removal of exon 6 from the genome. This resulted in the cartilage-compromised phenotype previously discussed and loss of basement membrane integrity in a variety of tissues. The fetal mortality rate is high and the mouse that survive die soon after birth. A separately developed perlecan knockout mouse model

14040-436: The perlecan mRNA in zebrafish embryos, as part of an investigation into perlecan function in skeletal and vascular development. The Morpholino targets the five prime untranslated region of the perlecan mRNA thus blocking translation of the message. Loss of the perlecan protein in these fish led to serious myopathies and circulation problems. As shown in a later study from the same laboratory, this phenotype could be rescued through

14170-738: The perlecan promoter region were studied by CAT activation in cell lines of various histological origins. This study concluded that there existed a TGF-β responsive element in the promoter just 285 base pairs upstream of the transcriptional start site. This result has been corroborated in such tissues as human colon carcinoma cells. and murine uterine epithelium by in vitro addition of the cytokine to cell culture medium. In vitro studies of TGF-β1 signaling and its effects on perlecan expression can have varying results in different cell types. In human coronary smooth muscle cells in culture, TGF-β1 signaling showed no effect on perlecan expression although it did upregulate other matrix constituents. In vivo demonstration of

14300-485: The pln transcript. In a mouse model of Alzheimer's plaque formation, IL-1-alpha effects an increase in perlecan expression in response to brain injury. IL-4 treatment of human gingival fibroblasts in culture led to increased production of various heparan sulfate proteoglycans including perlecan. Treatment of human lung fibroblasts in vitro with IL-1-beta did not lead to any significant increase in perlecan production. Another signaling pathway shown to augment pln transcription

14430-514: The possibility that the anti-coagulant function of perlecan is a part of the damage-control process in brain endothelia. Protein Kinase C signaling is putatively responsible for upregulating transcription and translation of certain proteoglycans including perlecan. When the endocytic pathway of HeLa cells is inhibited by overexpression of a mutant dynamin, Protein Kinase C is activated and perlecan message and protein are subsequently increased. In contrast,

14560-418: The possible deformation. Moving closer to soft tissue into the region known as the tidemark, the density of chondrocytes increases and collagen fibers are rearranged to optimize for stress dissipation and low friction. The outermost layer near the articular surface is known as the superficial zone, which primarily serves as a lubrication region. Here cartilage is characterized by a dense extracellular matrix and

14690-445: The presence of FGF-1. Perlecan also showed nerve adhesive properties in a previous study, further suggesting that it may act in an attractive role in combination with laminin rather than a repulsive one. Cartilage and bone development have proven to be dependent upon perlecan expression. The protein becomes visible by immunostaining on day 15 during mouse development, independently from other basement membrane proteins, suggesting that it

14820-411: The protein in the extracellular environment when cells have a reason to move or change their surroundings. Cathepsin S is a cysteine protease that moderately attenuates binding of FGF-positive cells to a perlecan-positive substrate. Cathepsin S is a potential protease that acts on the core protein of perlecan in the basement membrane or stroma. The heparan sulfate chains of perlecan bind growth factors in

14950-578: The protein, lack of HS or decreased sulfation can decrease perlecan's ability to interact with matrix proteins . Removal of HS chains may affect matrix organization and endothelial barrier function. Domain II comprises four repeats homologous to the ligand-binding portion of the LDL receptor with six conserved cysteine residues and a pentapeptide, DGSDE, which mediates ligand binding by the LDL receptor. Domain III has homology to

15080-587: The purpose of holding tubes open in the body. Examples include the rings of the trachea, such as the cricoid cartilage and carina . Cartilage is composed of specialized cells called chondrocytes that produce a large amount of collagenous extracellular matrix , abundant ground substance that is rich in proteoglycan and elastin fibers. Cartilage is classified into three types — elastic cartilage , hyaline cartilage , and fibrocartilage — which differ in their relative amounts of collagen and proteoglycan. As cartilage does not contain blood vessels or nerves , it

15210-461: The rest of development, although expression can be transient and precisely timed in certain tissue predecessors. In the rat embryo, perlecan expression has been shown to increase in vascular smooth muscle cells (VSMCs) post e19 in fetal development. This correlates perfectly with the ceasing of proliferation of VSMCs at e18 and a change in their phenotype. The theory put forward in this study is that perlecan plays an anti-proliferative role for VSMCs once

15340-446: The study in 2007 and the epithelial cell studied in these experiments is indicative of how varied the regulatory mechanisms of perlecan may be in different cell types. The development of beta-amyloid plaques on the brain is associated with onset of Alzheimer's disease. These plaques induce a constant state of inflammation in areas of accumulation, leading to expression of certain inflammation-related gene products, some of which perpetuate

15470-451: The territorial matrix, and the interterritorial matrix. The mechanical properties of articular cartilage in load-bearing joints such as the knee and hip have been studied extensively at macro, micro, and nano-scales. These mechanical properties include the response of cartilage in frictional, compressive, shear and tensile loading. Cartilage is resilient and displays viscoelastic properties. Since cartilage has interstitial fluid that

15600-457: The tissue displacement is measured as a function of time under a constant load. During this mode, the deformation of the tissue has two main regions. In the first region, the displacement is rapid due to the initial flow of fluid out of the cartilage, and in the second region, the displacement slows down to an eventual constant equilibrium value. Under the commonly used loading conditions, the equilibrium displacement can take hours to reach. In both

15730-420: The tissue. The embryos of S. officinalis express ColAa, ColAb, and hyaluronan in the cranial cartilages and other regions of chondrogenesis. This implies that the cartilage is fibrillar-collagen-based. The S. officinalis embryo expresses hh, whose presence causes ColAa and ColAb expression and is also able to maintain proliferating cells undiferentiated. It has been observed that this species presents

15860-413: The tissue. Degradation of this layer can put additional stresses on deeper layers which are not designed to support the same deformations. Another common effect of aging is increased crosslinking of collagen fibers. This leads to stiffer cartilage as a whole, which again can lead to early failure as stiffer tissue is more susceptible to fatigue based failure. Aging in calcified regions also generally leads to

15990-586: The total content of water, collagen, glycoproteins, etc. For example, increased glucosaminoglycan content leads to an increase in compressive stiffness, and increased water content leads to a lower aggregate modulus. In addition to its role in load-bearing joints, cartilage serves a crucial function as a gradient material between softer tissues and bone. Mechanical gradients are crucial for your body’s function, and for complex artificial structures including joint implants. Interfaces with mismatched material properties lead to areas of high stress concentration which, over

16120-501: The transcription factor STAT1 was interacting with the Pln promoter in the distal region, localized to 660 base pairs upstream of the transcription start site. Interferon- γ treatment of blastocyst-stage murine embryos leads to a loss of perlecan expression on the trophectoderm, and thus an embryonic morphology and phenotype in cell culture, which is suggestive that these interferon-γ treated blastocysts would be defective in implantation. Presumably

16250-427: The usual downregulation of perlecan in response to hyperglycemia is lost in mice negative for PKC-α. Interferon-γ signaling mediates transcriptional repression of the perlecan gene. This was first shown in colon cancer cell lines, and subsequently in cell lines of other tissue origins, but in each case intact STAT1 transcription factor was required for the signal to take effect. This led the investigators to believe that

16380-480: Was as a result of some other factor, which in this case turned out to be a decrease in expression of sulfate transferase enzymes, which play a key role in GAG biosynthesis. It seems that there may be some overlap in diseases stemming from loss of heparan sulfate proteoglycan expression and loss of enzymes involved in heparan sulfate biosynthesis. Cells can modify their extracellular matrix and basement membranes in response to signals or stress. Specific proteases act on

16510-450: Was created by insertion of a neomycin cassette into exon 7 of the perlecan gene. These knockout mice were also 40% embryonic lethal, with the rest of the mice dying soon after birth due to severe skeletal abnormalities. The perlecan knock out phenotype in both studies were identical and similar to the phenotype produced by activating mutations in the gene for FGFR3, a receptor for fibroblast growth factors. In yet another mouse knockout model,

16640-472: Was dependent on an initial upregulation followed by a downregulation of MMP-9, in contrast to the constant expression of MMP-2. This is not evidence that MMP-2 and MMP-9 directly cleave perlecan protein in vivo but shows that the proteins clearly modulate some factor in maturation of basement membrane. Another family of metalloproteases, the Bone Morphogenetic Protein 1/Tolloid-like family, releases

16770-522: Was glycosylated with both heparan and chondroitin sulfate chains when expressed in Chinese Hamster Ovary cells. The preferential addition of heparan sulfate or chondroitin sulfate chains to domains I and V could have an effect on the differentiation of mesenchymal tissues into cartilage, bone or any number of tissues, but the regulatory mechanism of changing from heparan sulfate to chondroitin sulfate addition are not well understood. While studying

16900-412: Was not limited to proteins associated with a specific pathway. When T84 intestinal epithelial cells are exposed to hypoxic conditions for 24 hours a significant increase in perlecan mRNA and protein production occurs. They relate this to the fact that many genes elevated in response to hypoxia contain a cAMP response element (CRE) in their promoter, as does pln. This difference between endothelial cells from

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