Extracellular matrix: forum introduction.
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Extracellular Matrix↗
Biomedical subjects
Daniel D Carson
Publications and source records attributed to Daniel D Carson.
MUC1: a multifunctional cell surface component of reproductive tissue epithelia.
MUC1 is a large, transmembrane mucin glycoprotein expressed at the apical surface of a variety of reproductive tract epithelia. Functions attributed to MUC1 include those generally associated with mucins such as lubrication and hydration of cell surfaces as well as protection from microorganisms and degradative enzymes. In addition, MUC1 is an effective inhibitor of both cell-cell and cell-extracellular matrix interactions in both normal and malignant contexts. Moreover, a series of recent studies has shown that the highly conserved cytoplasmic tail of MUC1 interacts specifically with a series of important signal transducing molecules including beta-catenin, Grb2 and erbB family members. MUC1 expression in normal epithelia can be quite dynamic, varying in response to steroid hormone or cytokine influences. Following malignant transformation, MUC1 often becomes highly overexpressed, loses its apical restriction, and displays aberrant glycosylation and altered mRNA splice variants. Regulation of MUC1 expression can occur at the transcriptional level. In addition, post-translational regulation of cell surface expression occurs via the activity of cell surface proteases or "sheddases" that release soluble forms of the large ectodomains. This review will briefly summarize studies of MUC1 expression and function in reproductive tissues with particular emphasis on the uterus. In addition, current knowledge of the mechanisms of MUC1 gene regulation, metabolic processing and potential signal transducing functions will be presented.
Animals↗
Heparan sulfate proteoglycans: coordinators of multiple signaling pathways during chondrogenesis.
Heparan sulfate proteoglycans are abundantly expressed in the pericellular matrix of both developing and mature cartilage. Increasing evidence indicates that the action of numerous chondroregulatory molecules depends on these proteoglycans. This review summarizes the current understanding of the interactions of heparan sulfate chains of cartilage proteoglycans with both soluble and nonsoluble ligands during the process of chondrogenesis. In addition, the consequences of mutating genes encoding heparan sulfate biosynthetic enzymes or heparan sulfate proteoglycan core proteins on cartilage development are discussed.
Animals↗
Perlecan functions in chondrogenesis: insights from in vitro and in vivo models.
Perlecan is a large heparan sulfate proteoglycan that is typically found in basal lamina of adult and embryonic tissues. Recent studies have demonstrated that perlecan accumulates impressively during cartilage development and is maintained as the major heparan sulfate proteoglycan of adult cartilage. In vertebrates, perlecan mutations result in skeletal defects. Moreover, in vitro studies indicate that perlecan can stimulate early stages of cartilage differentiation and cooperate with chondrogenic growth factors to promote this process. This short article will summarize these results and propose a model for perlecan function that incorporates these genetic and cell biological findings.
Aggrecans↗
Heparanase degrades syndecan-1 and perlecan heparan sulfate: functional implications for tumor cell invasion.
Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.
Adenocarcinoma↗
A heparin binding synthetic peptide from human HIP / RPL29 fails to specifically differentiate between anticoagulantly active and inactive species of heparin.
Despite extensive progress in determining structures within heparin and heparan sulfate (Hp/HS) and the discovery of numerous proteinaceous binding partners for Hp/HS so far; the only detailed characterization of a specific protein-glycosaminoglycan interaction is antithrombin III (ATIII) binding to a Hp pentasaccharide containing a unique 3-O-sulfated glucosamine residue. Previously, it was reported from our laboratories that a 16 amino acid synthetic peptide derived from the C-terminus of human HIP/RPL29 (HIP peptide-1) enriched for ATIII-dependent anticoagulant activity, presumably by specifically binding the ATIII pentasaccharide. Herein, we demonstrate that HIP peptide-1 cannot enrich ATIII-dependent anticoagulant activity from a starting pool of porcine intestinal mucosa Hp through a bio-specific interaction. However, a HIP peptide-1 column can be used to enrich for anticoagulantly active Hp from a diverse pool of glycosaminoglycans known as Hp byproducts by a mechanism of nonspecific charge interactions. Thus, HIP peptide-1 cannot recognize Hp via bio-specific interactions but binds glycosaminoglycans by non-specific charge interactions.
Animals↗
Perlecan-stimulated nodules undergo chondrogenic maturation in response to rhBMP-2 treatment in vitro.
The heparan sulfate proteoglycan, perlecan, is localized to hypertrophic chondrocytes in the growth plates of long bones. Mice mutants for perlecan display severe cartilage and skeletal defects. Previously, we demonstrated that C3H10T1/2 fibroblasts cultured on perlecan stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stain intensely with Alcian blue, and antibodies specific for collagen type II and aggrecan; however, nodules do not express collagen type X, a marker of chondrogenic maturation. In this investigation, we tested the hypothesis that addition of rhBMP-2 to perlecan-induced nodules would promote chondrogenic maturation in vitro. C3H10T1/2 fibroblasts were seeded in Lab-Tek chambered "Permanox" slides uncoated or coated with perlecan (B&D, 5 microg/well), at a density of 2 x 10(5) cells/well. The cells were maintained in CMRL-1066 media supplemented with ascorbic acid, citrate, and pyruvate (50 ng/ml). C3H10T1/2 fibroblasts seeded on perlecan-coated wells began to condense and form cell aggregates within 15 min. On the third day postplating, the media was replaced and supplemented with or without rhBMP-2 (50 ng/ml, Genetics Institute). On day 6 of culture, microscopy revealed that rhBMP-2-treated cultures had significantly proliferated; however, untreated cultures had not. By day 12 of culture, confocal microscopy revealed that perlecan-stimulated nodules treated with rhBMP-2 express a late stage marker of chondrogenesis (collagen type X). Morphologically, cells expressing collagen type X in rhBMP-2-treated nodules appear larger in diameter, relative to cells not expressing collagen type X. Cells cultured on plastic and treated with rhBMP-2 did not form nodules, but attached and spread, yielding a high-density monolayer. In response to rhBMP-2 treatment, these cells also express collagen type X. However, the appearance of collagen type X occurs at a later time point relative to the appearance of collagen type X in perlecan-stimulated nodules. Thus, perlecan-stimulated nodules do mature at a faster rate when treated with rhBMP-2 relative to monolayer cells.
Animals↗
Tumor necrosis factor-alpha converting enzyme/ADAM 17 mediates MUC1 shedding.
MUC1 clearance from the uterine epithelial cell surface is a prerequisite for the creation of an environment conducive to embryo implantation. In some species, reduced mRNA levels along with metabolic turnover account for loss of MUC1 during the receptive phase throughout the uterine epithelium. In other species, MUC1 is rapidly lost solely at the site of blastocyst attachment, suggesting the action of a protease. Correlative studies also indicate the presence of soluble forms of MUC1 in cell culture supernatants in vitro and in bodily fluids in vivo. To characterize the proteolytic activity mediating MUC1 release, shedding of MUC1 was analyzed in a human uterine epithelial cell line (HES) that abundantly expresses and readily sheds MUC1. MUC1 release was stimulated by phorbol 12-myristate 13-acetate and was markedly inhibited by the synthetic peptide hydroxamate metalloprotease inhibitor, tumor necrosis factor-alpha protease inhibitor (TAPI), as well as by an endogenous inhibitor of matrix metalloproteases, tissue inhibitor of metalloproteases (TIMP)-3. These characteristics along with studies conducted with cell lines genetically deficient in various ADAMs (for a disintegrin and metalloprotease) identified tumor necrosis factor-alpha converting enzyme (TACE)/ADAM 17 as a MUC1 sheddase. Furthermore, both TACE and MUC1 were expressed in human uterine epithelia during the receptive phase, and co-immunoprecipitation experiments revealed a physical interaction between TACE and MUC1 in HES cells. These studies establish a proteolytic mechanism for MUC1 clearance from a human uterine epithelial cell line and identify TACE as a MUC1 sheddase.
ADAM Proteins↗
The glycobiology of implantation.
Embryo implantation involves complex, coordinated interactions of cell surface components of embryonic (trophectoderm/trophoblast) and uterine cells. Oligosaccharides are common constituents of cell surface molecules. A variety of oligosaccharide structures have been identified at embryonic and uterine cell surfaces and change dynamically during progression of the implantation reaction. In many cases, the oligosaccharides have been shown to have biological activities with great relevance to the implantation process. This review summarizes the available information on glycoconjugates as it relates to the implantation process. It is suggested that carbohydrate bearing molecules at embryonic and uterine cell surfaces play diverse roles during the implantation process that are similar to the roles suggested for these molecules in other biological contexts.
Animals↗
Formation of MUC1 metabolic complex is conserved in tumor-derived and normal epithelial cells.
MUC1 is abundantly expressed at the surface of epithelial cells in many tissues and their carcinomas. In humans, genetic polymorphism and differential splicing produce isoforms that may contribute to MUC1 participation in protection of the cell surface, modulation of cell-cell interactions, signaling, and metastasis. Biosynthetic and processing studies in tumor-derived cell lines indicate that cell surface MUC1 consists of a non-covalently associated heterodimer of separate cytoplasmic tail and extracellular domains. This heterodimer results from a single precursor proteolytically cleaved intracellularly. To determine whether similar processing of this isoform occurs in normal epithelial cells, we have examined cell-associated MUC1 and MUC1 released into medium by normal human uterine, mammary, and prostate epithelial cells. Cell-associated MUC1/REP was extracted as an SDS labile complex which was resistant to dissociation by boiling, urea, sulfhydryl reduction, peroxide, high salt, or low pH and was present in all normal cells examined. Addition of various proteolytic inhibitors during extraction had no effect on the proportion of this complex detected. MUC1 released into the medium was not associated with a particulate fraction (100,000g insoluble) and lacked the cytoplasmic tail. MUC1/REP and the MUC1 isoform lacking the transmembrane/cytoplasmic tail region, MUC1/SEC, mRNA were detected in all normal cells examined indicating that both shed and secreted MUC1 are likely to contribute to soluble forms found in culture media.
Alternative Splicing↗
Changes in the cytologic distribution of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) during in vivo and in vitro mouse mammary epithelial cell expression and differentiation.
HIP/RPL29 is a small, highly basic, heparin/heparan sulfate interacting protein identical to ribosomal protein L29 and present in most adult epithelia. In the present study, we show that mouse HIP/RPL29 is ubiquitously present in adult mammary epithelia and is significantly increased during pregnancy and lactation. We observed for the first time that HIP/RPL29 intracellular expression and distribution varies, depending on the growth/differentiation state of the luminal epithelium. HIP/RPL29 was detected at low levels in mammary glands of virgin animals, increased markedly during lactation, and was lost again during involution. HIP/RPL29, preferentially found in the expanded cytoplasm of mature epithelial cells secreting milk, is present also in the nucleus of proliferating and differentiating ductal and alveolar elements. We used COMMA-D cells as an in vitro model for mammary-specific differentiation and examined similar intracellular redistribution of HIP/RPL29 associated with functional differentiation. However, no changes in HIP/RPL29 expression levels were detected in response to lactogenic hormones. Finally, the cellular distribution of HIP/RPL29 in both nuclear and cytoplasmic compartments was confirmed by transfecting a normal mammary epithelial cell line, NMuMG, with a fusion protein of HIP/RPL29 and EGFP. Collectively, these data support the idea that HIP/RPL29 plays more than one role during adult mammary gland development.
Animals↗
Synergistic stimulation of MUC1 expression in normal breast epithelia and breast cancer cells by interferon-gamma and tumor necrosis factor-alpha.
The MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in human breast cancers. Persistent stimulation by proinflammatory cytokines may contribute to increased MUC1 transcription by tumor cells. We demonstrate that MUC1 expression in T47D breast cancer cells and normal human mammary epithelial cells (HMEC) is enhanced by tumor necrosis factor-alpha (TNF-alpha) in the presence of interferon-gamma (IFN-gamma). MUC1 responsiveness to these cytokines was modest in T47D cells and robustly induced in HMEC. Transient transfection of T47D cells with mutant MUC1 promoter constructs revealed that a kappaB site at -589/-580 and the STAT-binding element at -503/-495 and were required for cooperative stimulation by TNFalpha and IFN-gamma. Binding of NFkappaB p65 to the MUC1 kappaB site was induced by TNF-alpha treatment, as demonstrated by electrophoretic mobility shift assay. Specific mutation of the kappaB site prevented binding of NFkappaB p65 and blocked TNF-alpha stimulation of MUC1 promoter activity. Collectively, these studies demonstrate synergistic stimulation of MUC1 expression by TNF-alpha and IFN-gamma that is mediated by independent actions of NFkappaB p65 and STAT1alpha upon kappaB and STAT sites, respectively, in the MUC1 promoter. Strong induction of MUC1 expression by these proinflammatory cytokines is clearly evident in normal mammary epithelium. In contrast, breast tumor cells appear to override normal regulatory responses via as yet undefined cis-elements.
Binding Sites↗
Mucins and blastocyst attachment.
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Animals↗
A rapid and simple nonradioactive method for in vitro testing of ribozyme activity.
Ribozymes that target specific messenger RNA transcripts are powerful tools in the emerging fields of functional genomics, proteomics, and metabolomics. We have found that successful in vitro testing greatly increases the likelihood of producing ribozymes with good efficacy in living cells. A rapid and simple nonradioactive method for systematic in vitro testing of ribozyme-cleaving activity is reported. Ribozymes are synthesized enzymatically from double-stranded DNA (dsDNA) oligonucleotides without vector cloning. Substrate target DNA template is cloned into a vector flanked with SP6 and T7 promoters at multiple cloning sites that permit colorimetric screening and ampicillin selection, enhancing the efficiency of the cloning procedure. Ribozyme cleavage products are satisfactorily resolved on 2.0% NuSieve 3:1 agarose (FMC Products, Rockland, ME)/formaldehyde gels by electrophoresis. This method avoids the preparation of polyacrylamide gels. Using this procedure, the ribozyme, target substrate RNA, and ribozyme cleavage products are all easily detected by ethidium bromide staining. Resolution and detection are fast and simple, eliminating the need for either polyacrylamide gel analysis or radiolabeling. The use of RNase inhibitors in the assays is also assessed and discussed.
DNA↗
Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.
High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium. Of approximately 12,000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages. Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results. In addition, novel genes were identified. Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes. Several genes were studied further by Northern blot analyses. These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased approximately 4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition. Immunostaining also revealed strong specific staining for claudin-4/CPE, EP(1) and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage. Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans. Some of these gene products, e.g. OPN, may play direct roles in embryo-uterine interactions during the implantation process.
Blotting, Northern↗
Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I.
C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln). To identify the region(s) within the large Pln molecule that provides a differentiation signal, recombinant Pln-sequence-based polypeptides representing distinct structural domains were assayed for their ability to promote chondrogenesis in C3H10T1/2 cells. Five distinct domains, along with structural variations, were tested. The N-terminal domain I was tested in two forms (IA and IB) that contain only heparan sulfate (HS) chains or both HS and chondroitin sulfate (CS) chains, respectively. A mutant form of domain I lacking attachment sites for both HS and CS (Pln I(mut)) was tested also. Other constructs consecutively designated Pln domains II, III(A-C), IV(A,B), and V(A,B) were used to complete the structure-function analysis. Cells plated onto Pln IA or Pln IB but no other domain rapidly assembled into cellular aggregates of 40-120 microm on average. Aggregate formation was dependent on the presence of glycosaminoglycan (GAG) chains, because Pln I-based polypeptides lacking GAG chains either by enzymatic removal or mutation of HS/CS attachment sites were inactive. Aggregates formed on GAG-bearing Pln IA stained with Alcian Blue and were recognized by antibodies to collagen type II and aggrecan but were not recognized by an antibody to collagen type X, a marker of chondrocyte hypertrophy. Collectively, these studies indicate that the GAG-bearing domain I of Pln provides a sufficient signal to trigger C3H10T1/2 cells to enter a chondrogenic differentiation pathway. Thus, this matrix proteoglycan (PG) found at sites of cartilage formation in vivo is likely to enhance early stage differentiation induced by soluble chondrogenic factors.
Animals↗