Barbara J. Gour

A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif

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.

N-Cadherin inhibits Schwann cell migration on astrocytes.

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.

N-Cadherin-Mediated Human Granulosa Cell Adhesion Prevents Apoptosis : A Role in Follicular Atresia and Luteolysis?

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.

N-Cadherin Influences Migration of Oligodendrocytes on Astrocyte Monolayers

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.

N-cadherin is involved in axon-oligodendrocyte contact and myelination.

We have analyzed the influence of the calcium-dependent cell adhesion molecule, N-cadherin, on events leading to CNS myelination. Interactions between axons and oligodendrocyte progenitor (OP) cells and the CG4 OP cell line were examined by video-microscopy. OPs cocultured with dorsal root ganglia explants migrated around the culture and formed numerous contacts with axons. The duration of these contacts depended on the morphology of the OP, with OPs containing four or more processes forming long-lasting contacts and OPs with three or fewer processes forming short-termed contacts. Treatment with N-cadherin function blocking peptides approximately halved the duration of contacts made by cells with four or more processes but contact times for cells with three or less processes were unaffected. The L7 cadherin-blocking antibody and calcium withdrawal had similar effects. Contacts with axons regenerating from explants of adult retina, which do not have N-cadherin on their surface was examined. The contact duration of OPs to adult retina axons was short and similar in length to those formed between OPs and dorsal root ganglion axons in the presence of cadherin blocking reagents. Oligodendrocyte myelination was examined in organotypic rat cerebellar slice cultures, taken before myelination at postnatal day 10 and then allowed to myelinate in vitro over 7 days. When incubated with an N-cadherin function-blocking peptide, myelination of Purkinje cell axons was reduced to about half of control levels, while control peptides were without effect. Cadherin-blockade did not prevent maturation of OPs, since oligodendrocytes showing myelin basic protein immunostaining were still found in these cultures. However, many of the cell processes did not colocalize with calbindin-positive axons. From these data we conclude that N-cadherin is important for the initial contact between a myelinating oligodendrocyte and axons and significantly contributes to the success of myelination.

INP, a novel N-cadherin antagonist targeted to the amino acids that flank the HAV motif

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.

Identification of an occludin cell adhesion recognition sequence.

The molecular mechanisms by which the tight junction integral membrane protein, occludin promotes cell adhesion and establishes an endothelial monolayer permeability barrier have not been elucidated. In particular, the amino acid sequences of the occludin cell adhesion recognition (CAR) sites have not been determined. Here we demonstrate that a cyclic peptide containing the sequence LYHY, which is found in the second extracellular domain of occludins in all mammalian species, inhibits the establishment of endothelial cell barriers in vitro and in vivo. This cyclic peptide also prevents the aggregation of fibroblasts stably transfected with cDNA encoding occludin. The data suggest that the LYHY motif is an occludin CAR sequence.

Tight junction peptide antagonists enhance neutrophil trans-endothelial chemotaxis.

Occludin is an integral membrane protein within tight junctions. Previous studies suggest it functions as a sealing element, which promotes barrier in endothelial and epithelial cell layers. Here, we examine the role of occludin in neutrophil chemotaxis, using cyclic occludin peptide antagonists that incorporate a conserved occludin cell adhesion recognition (CAR) sequence. Human umbilical vein endothelial cells were pre-treated with occludin specific cyclic peptide antagonists to examine effects on neutrophil migration towards a chemotactic gradient of 10(-7) M fMLP. The spatial organization of occludin and VE-cadherin were also assessed in control and occludin peptide-treated monolayers by immunofluorescent staining. The cyclic peptide, peptide B, which contains the CAR sequence of occludin, increased neutrophil chemotaxis in a time and dose dependent manner. Scrambled sequence peptide controls and linear peptides did not. The cyclic occludin antagonist, peptide B, disorganized junctional occludin, but apparently not VE-cadherin as assessed by immunofluorescence. The correlation between diminished occludin organization and increased neutrophil trans-endothelial chemotaxis provides additional support for occludin in the maintenance of the tight junctional barrier.