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1.
Mol Cell Neurosci ; 15(5): 456-64, 2000 May.
Article in English | MEDLINE | ID: mdl-10833302

ABSTRACT

The classical cadherins are homophilic binding molecules that play fundamental roles in several biological processes, including axonal growth and synaptic plasticity. The structures of the amino-terminal homophilic binding domains of N-cadherin and E-cadherin have been resolved. However, the mechanisms that govern cadherin binding and specificity remain contentious. In the present study we have used a peptide competition approach to probe for small linear determinants of cadherin binding. We demonstrate that a linear peptide mimetic of a short sequence in ECD1 of N-cadherin (INPISGQ) functions as a highly specific and potent antagonist of N-cadherin function with an IC(50) value of approximately 15 microM. Peptide mimetics of the corresponding motif in chick R-cadherin also inhibited N-cadherin function, albeit with lower efficacy. In contrast, peptide mimetics of the corresponding motif in E- or P-cadherin failed to inhibit N-cadherin function. A short cyclic peptide that contained only the INP motif from N-cadherin was also a potent N-cadherin antagonist (IC(50) approximately 15 microM). Analysis of existing crystal structures suggests that the peptides are likely to antagonize N-cadherin function by binding to the region that flanks the HAV motif at the adhesion dimer interface.


Subject(s)
Amino Acid Motifs , Cadherins/genetics , Cadherins/metabolism , Peptide Fragments/pharmacology , Peptides, Cyclic/pharmacology , 3T3 Cells , Animals , Cadherins/chemistry , Cells, Cultured , Chick Embryo , Crystallography , Fibroblast Growth Factors/metabolism , Humans , Leukocyte L1 Antigen Complex , Membrane Glycoproteins/metabolism , Mice , Molecular Conformation , Neural Cell Adhesion Molecules/metabolism , Peptide Fragments/chemistry , Peptides, Cyclic/chemistry , Rats
2.
Mol Cell Neurosci ; 15(3): 288-302, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10736205

ABSTRACT

Oligodendrocyte cell migration is required for the development of the nervous system and the repopulation of demyelinated lesions in the adult central nervous system. We have investigated the role of the calcium-dependent adhesion molecules, the cadherins, in oligodendrocyte-astrocyte interaction and oligodendrocyte progenitor migration. Immunostaining demonstrated the expression of N-cadherin on the surfaces of both oligodendrocytes and astrocytes, and oligodendrocyte-like cells adhered to and spread on N-cadherin substrates. The blocking of cadherin function by antisera or specific peptides reduced adhesion of oligodendroglia to astrocyte monolayers, diminished contact time between oligodendrocyte processes and individual astrocytes, and significantly increased the migration of oligodendrocyte-like cells on astrocyte monolayers. Furthermore, a soluble cadherin molecule without adhesive properties increased oligodendroglial proliferation on various extracellular matrix substrates. These data suggest that cadherins are at least partially responsible for the poor migration-promoting properties of astrocytes and that decreasing cell-cell adhesion might effect repopulation of demyelinated multiple sclerosis lesions by oligodendrocyte progenitors.


Subject(s)
Astrocytes/physiology , Cadherins/physiology , Cell Movement/physiology , Oligodendroglia/physiology , Animals , Cadherins/drug effects , Coculture Techniques , Demyelinating Diseases , Immune Sera , Microscopy, Video , Myelin Sheath/physiology , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Rats , Recombinant Fusion Proteins/physiology
3.
J Biol Chem ; 275(6): 4007-12, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660557

ABSTRACT

The classical cadherins (e.g. N-, E-, and P- cadherin) are well established homophilic adhesion molecules; however, the mechanism that governs cadherin specificity remains contentious. The classical cadherins contain an evolutionarily conserved His-Ala-Val (HAV) sequence, and linear peptides harboring this motif are capable of inhibiting a variety of cadherin-dependent processes. We now demonstrate that short cyclic HAV peptides can inhibit N-cadherin function. Interestingly, the nature of the amino acids that flank the HAV motif determine both the activity and specificity of the peptides. For example, when the HAV motif is flanked by a single aspartic acid, which mimics the natural HAVD sequence of N-cadherin, the peptide becomes a much more effective inhibitor of N-cadherin function. In contrast, when the HAV motif is flanked by a single serine, which mimics the natural HAVS sequence of E-cadherin, it loses its ability to inhibit the N-cadherin response. Our results demonstrate that subtle changes in the amino acids that flank the HAV motif can account for cadherin specificity and that small cyclic peptides can inhibit cadherin function. An emerging role for cadherins in a number of pathological processes suggests that the cyclic peptides reported in this study might be developed as therapeutic agents.


Subject(s)
Cadherins/genetics , Conserved Sequence/genetics , Peptides, Cyclic/pharmacology , 3T3 Cells , Animals , Brain/metabolism , Cadherins/chemistry , Cells, Cultured , Mice , Models, Molecular , Molecular Structure , Structure-Activity Relationship
4.
Mol Cell Neurosci ; 14(1): 66-84, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10433818

ABSTRACT

Astrocytes exclude Schwann cells (SCs) from the central nervous system (CNS) at peripheral nerve entry zones and restrict their migration after transplantation into the CNS. We have modeled the interactions between SCs, astrocytes, and fibroblasts in vitro. Astrocytes and SCs in vitro form separate territories, with sharp boundaries between them. SCs migrate poorly when placed on astrocyte monolayers, but migrate well on various other surfaces such as laminin (LN) and skin fibroblasts. Interactions between individual SCs and astrocytes result in long-lasting adhesive contacts during which the SC is unable to migrate away from the astrocyte. In contrast, SC interactions with fibroblasts are much shorter with less arrest of migration. SCs adhere strongly to astrocytes and other SCs, but less well to substrates that promote migration, such as LN and fibroblasts. SC-astrocyte and SC-SC adhesion is mediated by the calcium-dependent cell adhesion molecule N-cadherin. Inhibition of N-cadherin function by calcium withdrawal, peptides containing the classical cadherin cell adhesion recognition sequence His-Ala-Val, or antibodies directed against this sequence inhibit SC adhesion and increase SC migration on astrocytes. We suggest that N-cadherin-mediated adhesion to astrocytes inhibits the widespread migration of SCs in CNS tissue.


Subject(s)
Astrocytes/physiology , Cadherins/physiology , Schwann Cells/physiology , Sciatic Nerve/physiology , Amino Acid Sequence , Animals , Animals, Newborn , Astrocytes/cytology , Calcium/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Aggregation/drug effects , Cell Aggregation/physiology , Cell Line , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Fibroblasts/drug effects , Fibroblasts/physiology , Microscopy, Video , Oligopeptides/pharmacology , Peptide Fragments/pharmacology , Schwann Cells/cytology , Skin/cytology
5.
Am J Pathol ; 154(5): 1391-406, 1999 May.
Article in English | MEDLINE | ID: mdl-10329592

ABSTRACT

Studies suggest that cell-cell interactions may regulate apoptosis, and in particular, the calcium-dependent cell adhesion molecule N-cadherin has been shown to be capable of modulating this process. Rat granulosa cells (GCs) are known to express N-cadherin whereas cAMP is known to induce apoptosis in human and rat GCs. Based on these observations, we hypothesized that N-cadherin regulates human GC apoptosis via a cAMP-dependent mechanism. N-cadherin expression was evaluated in ovarian follicles and corpora lutea utilizing immunohistochemical techniques and in luteinized GCs in culture using immunoblotting, flow cytometric analysis, immunohistochemistry, and indirect immunofluorescence techniques utilizing anti-N-cadherin antibodies directed against both the extracellular and cytoplasmic domains of the molecule. Apoptosis was assessed by TUNEL and DNA fragmentation analysis and confirmed by flow cytometric cell cycle analysis and electron microscopy. The rate of GC apoptosis was found to be two- to three-fold lower among aggregated cells, as compared with single cells. N-cadherin was found to be expressed by aggregating GCs in vitro and GCs cultured in the presence of either N-cadherin function disrupting antibodies or peptides exhibiting enhanced rates of apoptosis. GCs in situ stained intensely for N-cadherin in preantral and normal growing preovulatory follicles as well as early corpora lutea. N-cadherin was weak in atretic follicles and regressing corpora lutea. Exposure of GCs to cAMP increased apoptosis while decreasing N-cadherin protein expression in a dose-dependent manner. Cell culture under serum-free conditions increased apoptosis and decreased N-cadherin expression, in part through cleavage of the extracellular domain of the molecule. The metalloproteinase inhibitor 1-10-phenanthroline inhibited the cleavage of the extracellular domain of N-cadherin and concomitantly inhibited the serum-deprivation-induced apoptosis of aggregated GCs. Collectively, these observations suggest that down-regulation of N-cadherin or the absence of a functional extracellular domain of the molecule prevents cell aggregation and is associated with GC apoptosis. In addition, cAMP induces apoptosis in a dose-dependent manner, and this process is dependent, at least in part, on regulation of the N-cadherin molecule at the surface of the cells. We conclude that N-cadherin-mediated GC signaling plays a central role in follicular and luteal cell survival.


Subject(s)
Apoptosis/physiology , Cadherins/physiology , Follicular Atresia/physiology , Granulosa Cells/pathology , Luteolytic Agents/metabolism , Adult , Cell Adhesion/physiology , Cell Aggregation/physiology , Cells, Cultured , Cyclic AMP/physiology , Down-Regulation , Female , Humans , In Situ Nick-End Labeling , Protein Structure, Tertiary
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