Biomedical subjects

R V Iozzo

Publications and source records attributed to R V Iozzo.

At least 19 recordsLinked to original sources

PubMed2006

A soluble ectodomain of LRIG1 inhibits cancer cell growth by attenuating basal and ligand-dependent EGFR activity.

Leucine-rich repeats and immunoglobulin-like domains-1 (LRIG1) is a transmembrane protein with an ectodomain containing 15 leucine-rich repeats (LRRs) homologous to mammalian decorin and the Drosophila kekkon1 gene. In this study, we demonstrate that a soluble ectodomain of LRIG1, containing only the LRRs, inhibits ligand-independent epidermal growth factor receptor (EGFR) activation and causes growth inhibition of A431, HeLa and MDA-468 carcinoma cells. In contrast, cells that do not express detectable levels of EGFR fail to respond to soluble LRIG1. However, when a functional EGFR gene is introduced in these cells, they become growth-inhibited by soluble LRIG1 protein. Furthermore, we demonstrate the existence of high-affinity (K(d)=10 nM) binding sites on the A431 cells that can be competitively displaced (up to 75%) by molar excess of EGF. Even more powerful effects are obtained with a chimeric proteoglycan harboring the N-terminus of decorin, substituted with a single glycosaminoglycan chain, fused to the LRIG1 ectodomain. Both proteins also inhibit ligand-dependent activation of the EGFR and extracellular signal-regulated protein kinase 1/2 signaling in a rapid and dose-dependent manner. These results suggest a novel mechanism of action evoked by a soluble ectodomain of LRIG1 protein that could modulate EGFR signaling and its growth-promoting activity. Attenuation of EGFR activity without physical downregulation of the receptor could represent a novel therapeutic approach toward malignancies in which EGFR plays a primary role in tumor growth and survival.

Amino Acid Sequence↗

PubMed2005

Targeted disruption of two small leucine-rich proteoglycans, biglycan and decorin, excerpts divergent effects on enamel and dentin formation.

Small leucine-rich proteoglycans have been suggested to affect mineralization of dental hard tissues. To determine the functions of two of these small proteoglycans during the early stages of tooth formation, we characterized the dental phenotypes of biglycan (BGN KO) and decorin deficient (DCN KO) mice and compared them to that of wild type mice. Each targeted gene disruption resulted in specific effects on dentin and enamel formation. Dentin was hypomineralized in both knock out mice, although the effect was more prominent in the absence of decorin. Enamel formation was dramatically increased in newborn biglycan knockout mice but delayed in absence of decorin. Increased enamel formation in the former case resulted from an upregulation of amelogenin synthesis whereas delayed enamel formation in the later case was most probably an indirect consequence of the high porosity of the underlying dentin. Enamelin expression was unchanged in BGN KO, and reduced in DCN KO. Dentin sialoprotein (DSP), a member of the family of phosphorylated extracellular matrix proteins that play a role in dentinogenesis, was overexpressed in BGN-KO odontoblasts and in the sub-odontoblastic layer. In contrast, a decreased expression of DSP was detected in DCN KO. Dentin matrix protein-1 (DMP-1), bone sialoprotein (BSP) and osteopontin (OPN) were upregulated in BGN KO and downregulated in the DCN KO. Despite the strong effects induced by these deficiencies in newborn mice, no significant difference was detected between the three genotypes in adult mice, suggesting that the effects reported here in newborn mice are transient and subjected to self-repair.

Amelogenin↗

PubMed2002

Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues.

Decorin (dcn) and biglycan (bgn), two members of the family of small leucine-rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a "subclinical" cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn- and dcn-deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double-knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double-KO animals in which progeroid changes are observed also in the skin. Ultrastructural analysis of bone collagen fibrils in bone of double-KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked "serrated fibril" morphology. The phenotype of the double-KO animal mimics directly the rare progeroid variant of human Ehlers-Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and den deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.

Animals↗

PubMed2001

A role for the perlecan protein core in the activation of the keratinocyte growth factor receptor.

Perlecan, a widespread heparan sulphate (HS) proteoglycan, is directly involved in the storing of angiogenic growth factors, mostly members of the fibroblast growth factor (FGF) gene family. We have previously shown that antisense targeting of the perlecan gene causes a reduced growth and responsiveness to FGF7 [also known as keratinocyte growth factor (KGF)] in human cancer cells, and that the perlecan protein core interacts specifically with FGF7. In the present paper, we have investigated human colon carcinoma cells in which the perlecan gene was disrupted by targeted homologous recombination. After screening over 1000 clones, we obtained two clones heterozygous for the null mutation with no detectable perlecan, indicating that the other allele was non-functioning. The perlecan-deficient cells grew more slowly, did not respond to FGF7 with or without the addition of heparin, and were less tumorigenic than control cells. Paradoxically, the perlecan-deficient cells displayed increased FGF7 surface binding. However, the perlecan protein core was required for functional activation of the KGF receptor and downstream signalling. Because heparin could not substitute for perlecan, the HS chains are not critical for FGF7-mediated signalling in this cell system. These results provide the first genetic evidence that the perlecan protein core is a molecular entity implicated in FGF7 binding and activation of its receptor.

Animals↗

PubMed2001

B-Myb overexpression results in activation and increased Fas/Fas ligand-mediated cytotoxicity of T and NK cells.

The human B-myb gene encodes a transcriptional regulator that plays an important role in cell cycle progression, differentiation, and survival. To assess the in vivo role of B-myb, we investigated the phenotype of mouse transgenic lines in which B-Myb expression in lymphoid tissues was driven by the LCK proximal promoter. Overexpression of B-Myb had no measurable effect on the subsets of splenic and thymic lymphocytes, but was associated with increased expression of Fas ligand in NK and T cells. B-Myb-overexpressing splenocytes expressed higher IFN-gamma levels and contained higher percentages of cytokine-producing cells than wild-type (wt) splenocytes, as detected by Western blot analysis and ELISPOT assays, respectively. Ex vivo-cultured transgenic thymocytes and splenocytes had decreased survival compared with the corresponding cells from wt mice, possibly dependent on increased expression of Fas ligand. In addition, Fas ligand-dependent cytotoxicity of transgenic T and NK cells was significantly higher than that mediated by their wt counterparts. Together, these results indicate that B-Myb overexpression results in T and NK cell activation and increased cytotoxicity. Therefore, in addition to its well-established role in proliferation and differentiation, B-myb also appears to be involved in activation of NK and T cells and in their regulation of Fas/Fas ligand-mediated cytotoxicity

Animals↗

PubMed2001

A composite element binding the vitamin D receptor and the retinoic X receptor alpha mediates the transforming growth factor-beta inhibition of decorin gene expression in articular chondrocytes.

Decorin, a small leucine-rich proteoglycan may play an important role in the attempt of cartilage repair initiated by chondrocytes in early stages of osteoarthritis, through its ability to bind collagen fibrils and growth factors such as transforming growth factor-beta (TGF-beta). We previously demonstrated that TGF-beta decreased decorin mRNA steady state levels in articular chondrocytes (Demoor, M., Rédini, F., Boittin, M., and Pujol, J.-P. (1998) Biochim. Biophys. Acta 1398, 179-191). Here, we investigated the effect of TGF-beta on decorin gene expression in both primary cultures of articular chondrocytes and chondrocytes dedifferentiated by serial passages. Transient transfection of cells with plasmid constructs of the decorin promoter linked to the luciferase reporter gene revealed transcriptional repression by TGF-beta, in fully differentiated as well as dedifferentiated chondrocytes. Experiments with 5'-deleted constructs allowed characterization of a TGF-beta-responsive element in the shortest construct (base pairs (bp) -155/+269). DNase I footprinting analysis delineated a negative TGF-beta-responsive region between -140 and -111 bp in the decorin proximal promoter. Gel retardation assays demonstrated that TGF-beta modulates decorin gene expression through transcription factors, the nature and mode of action of which depend on the differentiation state of the chondrocytes; two DNA-protein complexes were formed in the region -144/-127 bp with nuclear extracts from primary chondrocytes, whereas a higher mobility complex was observed in the -127/-111 bp region for dedifferentiated cells. Antibodies against vitamin D and retinoic acid receptors used in supershift experiments showed that these nuclear receptors are involved in the regulation of decorin gene expression in articular chondrocytes.

Animals↗

PubMed2001

Fibroblast growth factor-binding protein is a novel partner for perlecan protein core.

Perlecan, a widespread heparan sulfate proteoglycan, functions as a bioactive reservoir for growth factors by stabilizing them against misfolding or proteolysis. These factors, chiefly members of the fibroblast growth factor (FGF) gene family, are coupled to the N-terminal heparan sulfate chains, which augment high affinity binding and receptor activation. However, rather little is known about biological partners of the protein core. The major goal of this study was to identify novel proteins that interact with the protein core of perlecan. Using the yeast two-hybrid system and domain III of perlecan as bait, we screened approximately 0.5 10(6) cDNA clones from a keratinocyte library and identified a strongly interactive clone. This cDNA corresponded to FGF-binding protein (FGF-BP), a secreted protein previously shown to bind acidic and basic FGF and to modulate their activities. Using a panel of deletion mutants, FGF-BP binding was localized to the second EGF repeat of domain III, a region very close to the binding site for FGF7. FGF-BP could be coimmunoprecipitated with an antibody against perlecan and bound in solution to recombinant domain III-alkaline phosphatase fusion protein. Immunohistochemical analyses revealed colocalization of FGF-BP and perlecan in the pericellular stroma of various squamous cell carcinomas suggesting a potential in vivo interaction. Thus, FGF-BP should be considered a novel biological ligand for perlecan, an interaction that could influence cancer growth and tissue remodeling.

Amino Acid Motifs↗

PubMed2001

Resistance to Lyme disease in decorin-deficient mice.

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Animals↗

PubMed2001