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Biomedical subjects

R V Iozzo

Publications and source records attributed to R V Iozzo.

At least 37 records · Page 2Linked to original sources
PubMed2000

Decorin binds near the C terminus of type I collagen.

Decorin belongs to a family of small leucine-rich proteoglycans that are directly involved in the control of matrix organization and cell growth. Genetic evidence indicates that decorin is required for the proper assembly of collagenous matrices. Here, we sought to establish the precise binding site of decorin on type I collagen. Using rotary shadowing electron microscopy and photoaffinity labeling, we mapped the binding site of decorin protein core to a narrow region near the C terminus of type I collagen. This region is located within the cyanogen bromide peptide fragment alpha1(I) CB6 and is approximately 25 nm from the C terminus, in a zone that coincides with the c(1) band of the collagen fibril d-period. This location is very close to one of the major intermolecular cross-linking sites of collagen heterotrimers. Thus, decorin protein core possesses a unique binding specificity that could potentially regulate collagen fibril stability.

Amino Acid Sequence↗
PubMed2000

Overexpression of bamacan/SMC3 causes transformation.

Bamacan can occur in certain cell types as either a secreted proteoglycan assembled into basement membranes or as an intracellular protein known as structural maintenance of chromosome 3 (SMC3). To assess the role of this protein in tumorigenesis, we investigated whether induced overexpression of bamacan/SMC3 could transform normal fibroblasts. We generated a full-length cDNA encoding the entire mouse bamacan/SMC3 and demonstrated appropriate transcription and translation into a 146-kDa protein. All the NIH and Balb/c 3T3 murine fibroblasts overexpressing this bamacan/SMC3 transgene generated foci of transformation and acquired anchorage-independent growth. The increased levels of bamacan/SMC3 expression achieved in the transfected fibroblasts were the same as those detected in a series of spontaneously transformed murine and human colon carcinoma cells. Moreover, a 3-4-fold overexpression of bamacan/SMC3 was detected in approximately 70% of human colon carcinoma specimens from matched pairs (n = 19, p < 0.0002) and in a cohort of intestinal tumors from Apc-deficient Min/+ mice. These results support the concept that deregulated expression of bamacan/SMC3 is involved in cell transformation.

Animals↗
PubMed2000

Activation of extracellular signal-regulated protein kinase1,2 results in down-regulation of decorin expression in fibroblasts.

Decorin is a small leucine-rich extracellular matrix proteoglycan, the expression of which is down-regulated in proliferating and malignantly transformed cells. In the present study we show that the expression of decorin in fibroblasts is suppressed by epidermal growth factor (EGF) and PMA, and that the effect of both is potently inhibited by blocking the extracellular signal-regulated protein kinase (ERK)1,2 signalling pathway (Raf/MEK1,2/ERK1,2) with the specific MAPK/ERK kinase (MEK)1,2 inhibitor, PD98059. In addition, specific activation of ERK1,2 by adenovirus-mediated expression of constitutively active MEK1 in dermal fibroblasts results in marked reduction in decorin mRNA abundance and production. Co-transfection of NIH-3T3 fibroblasts with human decorin promoter/chloramphenicol acetyltransferase (CAT) construct (pDEC--879/CAT) in combination with the expression vectors for constitutively active Raf-1 and MEK1 markedly suppressed decorin promoter activity. Co-transfections of human decorin promoter 5'-deletion constructs with constitutively active MEK1 expression vector identified the region -278 to -188 as essential for ERK1,2 mediated down-regulation of decorin promoter activity. These results show that activation of the ERK1,2 signalling pathway by a mitogenic growth factor, a tumour promoter or transformation suppresses decorin gene expression in fibroblasts, which in turn may promote proliferation and migration of normal and malignant cells.

3T3 Cells↗
PubMed2000

Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia.

Perlecan, a heparan sulfate proteoglycan, has been suggested to be critical for regulation of vascular repair. We generated clones of endothelial cells expressing an antisense vector targeting domain III of perlecan. Transfected cells produced significantly less perlecan than parent cells and showed a reduced ability to inhibit the binding and mitogenic activity of fibroblast growth factor-2 in vascular smooth muscle cells. Endothelial cells were seeded onto three-dimensional polymeric matrices and implanted adjacent to porcine carotid arteries subjected to deep injury. Although the parent endothelial cells prevented occlusive thrombosis, perlecan-deficient cells were completely ineffective. The ability of endothelial cells to inhibit intimal hyperplasia, however, was abrogated only in part by perlecan suppression. The differential regulation by perlecan of these different aspects of vascular repair may explain why control of clinical clot formation does not lead to full control of intimal hyperplasia. Thus the use of genetically modified tissue-engineered cells provides a new approach for dissecting the role of specific factors within the complex environment of the blood vessel wall.

Animals↗
PubMed2000

The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7.

Perlecan is a multifaceted heparan sulfate proteoglycan that is expressed not only as an intrinsic constituent of basement membranes but also as a cell-surface and pericellular proteoglycan. Perlecan functions as a ligand reservoir for various growth factors that become stabilized against misfolding or proteolysis and acts as a co-receptor for basic fibroblast growth factor by augmenting high affinity binding and receptor activation. These biological properties are mediated by the heparan sulfate moiety. Rather little is known about the protein core's mediation of functions. We have recently discovered that fibroblast growth factor-7 (FGF7) binds to perlecan protein core and that exogenous perlecan efficiently reconstitutes FGF7 mitogenic activity in perlecan-deficient cells. In this report we examined the specific binding of FGF7 to various domains and subdomains of perlecan protein core. Using several experimental approaches including overlay protein assays, radioligand binding experiments, and the yeast two-hybrid system, we demonstrate that FGF7 binds specifically to the N-terminal half of domain III and to a lesser extent to domain V, with affinity constants in the range of 60 nM. Thus, perlecan protein core should be considered a novel biological ligand for FGF7, an interaction that could influence cancer growth and tissue remodeling.

Cells, Cultured↗
PubMed2000

Fibroblast growth factor-2.

Fibroblast growth factor-2 (FGF-2) is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 stimulates the growth and development of new blood vessels (angiogenesis) that contribute to the pathogenesis of several diseases (i.e. cancer, atherosclerosis), normal wound healing and tissue development. FGF-2 contains a number of basic residues (pI 9.6) and consists of 12 anti-parallel beta-sheets organized into a trigonal pyrimidal structure. FGF-2 binds to four cell surface receptors expressed as a number of splice variants. Many of the biological activities of FGF-2 have been found to depend on its receptor's intrinsic tyrosine kinase activity and second messengers such as the mitogen activated protein kinases. However, considerable evidence suggest that intracellular FGF-2 might have a direct biological role particularly within the nucleus. In addition, heparan sulfate proteoglycans have been demonstrated to enhance and inhibit FGF-2 activity. The possibility that FGF-2 activity can be manipulated through alterations in heparan sulfate-binding is currently being exploited in the development of clinical applications aimed at modulating either endogenous or administered FGF-2 activity.

Animals↗
PubMed1999

Spontaneous and mutagen-induced transformation of primary cultures of Msh2-/- p53-/- colonocytes.

Loss of function of mismatch repair (MMR) genes underlies hereditary nonpolyposis colorectal cancer (HNPCC). However, the inability to maintain primary colon epithelial cells in culture has limited the analysis of the contribution of MMR gene defects to colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of Msh2-/- p53-/- double-knockout mice. These cells undergo spontaneous transformation (soft agar colonies and s.c. tumor formation), with a progressively shorter latency as a function of increasing passages in culture. Treatment of early passage cells with the mutagen methylmethane thiosulfonate (MMS) further decreases the transformation latency of Msh2-/- p53-/- cells. Spontaneous transformation of p53-/- colonocytes is only observed using late passage cells, and methylmethane thiosulfonate-treated early passage p53-/- colonocytes do not form tumors when injected into immunodeficient mice. Together, these findings support the pathogenic role of MMR gene inactivation in colorectal tumorigenesis and provide an experimental model for the serial assessment of the molecular phenotype associated with Msh2 deficiency.

Animals↗
PubMed1999

Complete cDNA cloning, genomic organization, chromosomal assignment, functional characterization of the promoter, and expression of the murine Bamacan gene.

Bamacan is a chondroitin sulfate proteoglycan that abounds in basement membranes. To gain insights into the bamacan gene regulation and transcriptional control, we examined the genomic organization and identified the promoter region of the mouse bamacan gene. Secondary structure analysis of the protein reveals a sequential organization of three globular regions interconnected by two alpha-helix coiled-coils. The N- and the C-terminal ends carry a P-loop and a DA box motif that can act cooperatively to bind ATP. These features as well as the high sequence homology with members of the SMC (structural maintenance of chromosome) protein family led us to conclude that bamacan is a member of this protein family. The gene comprises 31 exons and is driven by a promoter that is highly enriched in GC sequences and lacks TATA and CAAT boxes. The promoter is highly functional in transient cell transfection assays, and step-wise 5' deletions identify a strong enhancer element between -659 and -481 base pairs that includes Jun/Fos proto-oncogene-binding elements. Using backcrossing experiments we mapped the Bam gene to distal chromosome 19, a locus syntenic to human chromosome 10q25. Bamacan is differentially expressed in mouse tissues with the highest levels in testes and brain. Notably, bamacan mRNA levels are low in normal cells and markedly reduced during quiescence but are highly increased when cells resume growth upon serum stimulation. In contrast, in all transformed cells tested, bamacan is constitutively overexpressed, and its levels do not change with cell cycle progression. These results suggest that bamacan is involved in the control of cell growth and transformation.

Amino Acid Sequence↗
PubMed1999

Cooperative action of germ-line mutations in decorin and p53 accelerates lymphoma tumorigenesis.

Ectopic expression of decorin in a wide variety of transformed cells results in growth arrest and the inability to generate tumors in nude mice. This process is caused by a decorin-mediated activation of the epidermal growth factor receptor, which leads to a sustained induction of endogenous p21(WAF1/CIP1) (the cyclin-dependent kinase inhibitor p21) and growth arrest. However, mice harboring a targeted disruption of the decorin gene do not develop spontaneous tumors. To test the role of decorin in tumorigenesis, we generated mice lacking both decorin and p53, an established tumor-suppressor gene. Mice lacking both genes showed a faster rate of tumor development and succumbed almost uniformly to thymic lymphomas within 6 months [mean survival age (T50) approximately 4 months]. Mice harboring one decorin allele and no p53 gene developed the same spectrum of tumors as the double knockout animals, but had a survival rate similar to the p53 null animals (T50 approximately 6 months). Ectopic expression of decorin in thymic lymphoma cells isolated from double mutant animals markedly suppressed their colony-forming ability. When these lymphoma cells were cocultured with fibroblasts derived from either wild-type or decorin null embryos, the cells grew faster in the absence of decorin. Moreover, exogenous decorin proteoglycan or its protein core significantly retarded their growth in vitro. These results indicate that the lack of decorin is permissive for lymphoma tumorigenesis in a mouse model predisposed to cancer and suggest that germ-line mutations in decorin and p53 may cooperate in the transformation of lymphocytes and ultimately lead to a more aggressive phenotype by shortening the tumor latency.

Animals↗
PubMed1999

Decorin is a biological ligand for the epidermal growth factor receptor.

Ectopic expression of decorin induces profound cytostatic effects in transformed cells with diverse histogenetic backgrounds. The mechanism of action has only recently begun to be elucidated. Exogenous decorin activates the epidermal growth factor (EGF) receptor, thereby triggering a signaling cascade that leads to phosphorylation of mitogen-activated protein (MAP) kinase, induction of p21, and growth suppression. In this study we demonstrate a direct interaction of decorin with the EGF receptor. Binding of decorin induces dimerization of the EGF receptor and rapid and sustained phosphorylation of MAP kinase in squamous carcinoma cells. In a cell-free system, decorin induces autophosphorylation of purified EGF receptor by activating the receptor tyrosine kinase and can also act as a substrate for the EGF receptor kinase itself. Using radioligand binding assays we show that both immobilized and soluble decorin bind to the EGF receptor ectodomain or to purified EGF receptor. The binding is mediated by the protein core and has relatively low affinity (Kd approximately 87 nM). Thus, decorin should be considered as a novel biological ligand for the EGF receptor, an interaction that could regulate cell growth during remodeling and cancer growth.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Transcriptional and posttranscriptional regulation of proteoglycan gene expression.

Proteoglycans are among the most complex and sophisticated molecules of mammalian systems in terms of their protein and carbohydrate moieties. These macromolecules are in a continuous interplay with each other and the cell surface signal-transducing pathways, some of which are beginning to be elucidated. Because of their domain structure, catalytic potential, and diversity, these molecules appear to be designed for integrating numerous signaling events. For example, some proteoglycans interact with hyaluronan and lectins, thereby linking cell surfaces and distant matrix molecules. Some interact with collagen during the complex process of fibrillogenesis and regulate this biological process fundamental to animal life. Others interact with growth factors and serve as depot available during growth or tissue remodeling. In this review, we center on the most recent developments of proteoglycan biology, focusing primarily on genomic organization and transcriptional and posttranscriptional control. We discuss only those proteoglycans whose gene and promoter elements have been characterized and proved to be functional. When possible, we correlate the effects of growth factors and cytokines on proteoglycan gene expression with the topology of cis-acting elements in their genomic control regions. The analysis leads to a comprehensive critical appraisal of the principles that underlie the regulation of proteoglycan gene expression and to the delineation of common regulatory mechanisms.

Animals↗
PubMed1999

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.

Animals↗
PubMed1998

There is binding of collagen IV to beta 1 integrin during early skin basement membrane assembly.

This study is concerned with the mechanism of basement membrane assembly in an in vitro 3-dimensional skin-culture system. Dermal fibroblasts alone can synthesize collagen IV, perlecan, and nidogen, but cannot assemble them into a basement membrane. When keratinocytes are added to the culture, however, linear assembly of collagen IV, perlecan, and nidogen is noted at the epidermo-dermal interface. Northern blots and in situ hybridization showed that perlecan and nidogen mRNAs derive exclusively from fibroblasts, while the alpha 2 (IV) collagen chain is expressed by both keratinocytes and fibroblasts, although the major source is in the mesenchyma (80%). Prior to the development of the lamina densa, collagen IV colocalizes with beta 1 integrins, most likely alpha 1 beta 1 and alpha 2 beta 1, which are known receptors for this collagen. Blocking experiments with the AIIB2 mAb (anti-beta 1 integrin subunit) and by peptide inhibition with the CB3(IV) collagen fragment disrupted the assembly of collagen IV. This study suggests that the initiation of basement-membrane formation involves binding of collagen IV molecules to keratinocyte cell-matrix integrins. These complexes act as nucleation sites for further polymerization of collagen IV molecules mostly derived from fibroblasts, by a process of self-assembly.

Basement Membrane↗
PubMed1998

Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Animals↗
PubMed1998

Tumorigenic conversion of p53-deficient colon epithelial cells by an activated Ki-ras gene.

Distinct genetic abnormalities (loss-of-function mutations of APC and p53 and oncogenic activation of Ki-ras) are associated with specific stages of the sporadic, most common types of colorectal tumors. However, the inability to maintain primary colon epithelial cells in culture has hindered the analysis of the pathogenetic role of these abnormalities in colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of p53-deficient mice; these cells are nontumorigenic as indicated by their failure to form colonies in soft agar and to grow as tumors in immunodeficient SCID mice and in immunocompetent syngeneic hosts. Upon ectopic expression of an activated Ki-ras gene, p53-deficient colon epithelial cells form colonies in soft agar and highly invasive subcutaneous tumors in both immunodeficient and immunocompetent mice. Ectopic expression of wild-type p53, but not of a DNA-binding-deficient mutant, markedly suppressed the colony-forming ability of the Ki-ras-transformed p53-deficient epithelial cells. Together, these findings establish a functional synergism in colorectal tumorigenesis dependent on the effects of an oncogenic Ki-ras in a p53-deficient background. This model of tumorigenic conversion of colon epithelial cells might be useful to identify genetic changes associated with disease progression and to evaluate the therapeutic response to conventional and novel anticancer drugs.

Animals↗
PubMed1998

Transcriptional silencing of perlecan gene expression by interferon-gamma.

Perlecan, a heparan sulfate proteoglycan of basement membranes and cell surfaces, has been implicated in the control of tumor cell growth and metastasis because of its ability to bind and store growth factors and its activity as an inducer of angiogenesis. Because interferon-gamma (IFN-gamma), a cytokine with known antiproliferative and antitumoral activity, binds with high affinity to the heparan sulfate side chains of perlecan, we investigated the activity of IFN-gamma on perlecan gene expression and cell growth in colon carcinoma cells. We found that IFN-gamma rapidly and efficiently blocked perlecan gene expression with concurrent growth suppression, a phenomenon that was independent of a functional p21(WAF1/CIP1). These effects were transcriptionally mediated, did not require new protein synthesis, and were fully reversible. Moreover, we found these IFN-gamma-induced effects to be generalizable because they could be reproduced in a variety of cells with various histogenetic backgrounds. The transcriptional repression of the perlecan gene required intact Stat1 protein, and these effects were likely mediated by Stat1-binding sites in the distal promoter region. Thus, the IFN-gamma-mediated transcriptional repression of perlecan may represent a novel antitumoral effect of this cytokine through which it eliminates a powerful angiogenic stimulus.

Cell Cycle↗