Identification of CD44 residues important for hyaluronan binding and delineation of the binding site.

CD44 is a widely distributed cell surface protein that plays a role in cell adhesion and migration. As a proteoglycan, CD44 is also implicated in growth factor and chemokine binding and presentation. The extracellular region of CD44 is variably spliced, giving rise to multiple CD44 isoforms. All isoforms contain an amino-terminal domain, which is homologous to cartilage link proteins. The cartilage link protein-like domain of CD44 is important for hyaluronan binding. The structure of the link protein domain of TSG-6 has been determined by NMR. Based on this structure, a molecular model of the link-homologous region of CD44 was constructed. This model was used to select residues for site-specific mutagenesis in an effort to identify residues important for ligand binding and to outline the hyaluronan binding site. Twenty-four point mutants were generated and characterized, and eight residues were identified as critical for binding or to support the interaction. In the model, these residues form a coherent surface the location of which approximately corresponds to the carbohydrate binding sites in two functionally unrelated calcium-dependent lectins, mannose-binding protein and E-selectin (CD62E).

A comprehensive survey of the cadherins expressed in the testes of fetal, immature, and adult mice utilizing the polymerase chain reaction.

The cadherins are a family of calcium-binding membrane glycoproteins. Most cadherins are capable of acting as cell adhesion molecules (CAMs). In order to begin a thorough analysis of the roles of these CAMs in the testis, we employed a reverse transcriptase-polymerase chain reaction (RT-PCR) strategy to identify the cadherins expressed in this tissue at various stages of development. Oligonucleotides encoding amino acid sequences that are conserved among all of the known cadherins were used as primers in the RT-PCR, with cDNA preparations of fetal, newborn, 7-day, 21-day, and adult mouse testes employed as templates. The PCR products were subcloned into a plasmid vector and sequenced. On the basis of the nucleotide sequences of these PCR products, we have determined that five previously characterized cadherins (E-cadherin, N-cadherin, P-cadherin, K-cadherin, and OB-cadherin), as well as two novel cadherins (T1-cadherin and T2-cadherin), are expressed at various stages during testicular development. In order to determine the expression patterns of these cadherins, we ascertained the mRNA levels of each cadherin normalized to the levels of hypoxanthine phosphoribosyltransferase mRNA in fetal, newborn, 7-day, 21-day, and adult mouse testes. We observed that N-cadherin mRNA is expressed at all stages of testicular development, with maximal levels being present in the testes of 21-day-old mice. Furthermore, we found that E-, P-, K-, OB-, and T2-cadherin mRNAs are all expressed in the fetal gonad. The testicular levels of these cadherin mRNAs decreased dramatically after birth. Conversely, T1-cadherin mRNA was not detected in the fetal, newborn, and 7-day-old testes but was present in 21-day-old and adult testes. T1-cadherin levels were 10-fold higher in the testes of adult mice, compared to the levels found in the testes of 21-day-old mice. We speculate that these cadherins will be found to be intimately involved in mediating cell interactions during testicular development.

Remodelling of the nuclear periphery during muscle cell differentiation in vitro.

We have examined the composition and ultrastructure of the nuclear periphery during in vitro myogenesis of the rat myoblast cell line, L6E9. Immunofluorescence labelling and immunoblotting showed that lamins A/C and B were all present in undifferentiated cells, but that they increased significantly before extensive cell fusion had occurred, with lamins A/C increasing proportionately more. Electron microscopic observations were consistent with these results, showing an increase in the prominence of the lamina during differentiation. On the other hand, immunofluorescence labelling suggested that the P1 antigen began to disappear from the nuclear periphery as the cells were fusing, after the increase in lamin quantity, and was no longer detectable in multinucleated cells. Unexpectedly, however, P1 was readily detected in isolated nuclei, whether prepared from myoblast or differentiated cultures, as well as in both myoblast and myotube nuclear matrices. It appears probable, therefore, that the fading of P1 labelling is due to masking of the epitope by a soluble factor recruited to the nuclear periphery as cell differentiate. These data, together with evidence that the genome is substantially rearranged during L6E9 myogenesis [Chaly and Munro, 1996], suggest that L6E9 cells are a useful model system in which to study the interrelationship of nuclear envelope organization, chromatin spatial order, and nuclear function.

Characterization of cadherins expressed by murine thymocytes.

Thymocytes develop in close apposition to the stromal cells of the thymus. The ontogeny of thymocytes is dependent on intimate interactions between these cells and the stromal cells. The molecular mechanisms involved in regulating thymocyte-stromal cell interactions remain to be clearly defined. In this study, we utilized a polymerase chain reaction strategy to identify members of the cadherin family of cell adhesion molecules that are expressed by CD4+ CD8+ thymocytes, the major cell type in the thymus. One classical cadherin (E-cadherin), three atypical cadherins (OB-cadherin) K-cadherin, and cadherin-8), and two novel cadherins (T1-cadherin and T2-cadherin) were found to be expressed by the CD4+ CD8+ thymocytes. The discovery that these cells display multiple cadherins opens a new area of investigation concerning the adhesive mechanisms involved in modulating thymocyte-stromal cell interactions. We speculate that cadherins will prove to play an essential role in the ontogeny of thymocytes.

Centromeres reposition to the nuclear periphery during L6E9 myogenesis in vitro.

To test the hypothesis that genome architecture in interphase is related to nuclear function, we have compared the disposition of centromeres in nuclei of undifferentiated rat L6E9 myoblasts with that in nuclei of L6E9 myotubes differentiated in vitro. Immunofluorescence labeling showed that centromeres repositioned to the nuclear periphery during differentiation, and condensed chromatin was more prominent at the myotube nuclear envelope by electron microscopy. These data indicate that, in parallel with considerable changes in gene expression, the spatial order of the genome undergoes substantial rearrangement during myogenesis.

Adhesion or anti-adhesion in cancer: what matters more?

The regulation of adhesion processes between normal epithelial cells is an essential condition for the maintenance of appropriate tissular architecture and differentiation. Quantitative and qualitative alterations in these homotypic adhesions occur during the transformation of normal into malignant epithelium. How these complex alterations in various homotypic adhesions modify the ability of tumor cells to detach from the original neoplastic site, to grow and move as single or clumped cells, and to invade the stroma are current issues in tumor biology. This review contrasts tumor cell adhesion mediated by E-cadherin which is consistently decreased in carcinomas, with adhesion mediated by CD44 and CEA which are increased in the tumors. A model proposing to resolve the apparent paradox of simultaneous adhesion and anti-adhesion mediated by the same protein is proposed.

E-cadherin and OB-cadherin mRNA levels in normal human colon and colon carcinoma.

A polymerase chain reaction strategy was utilized to identify members of the cadherin family of cell adhesion molecules that are expressed in normal human colon and colon carcinoma. E-cadherin (an epithelial cadherin) and OB-cadherin (a mesenchymal cadherin) were found to be present in the colon specimens. Furthermore, a novel semiquantitative polymerase chain reaction (PCR) assay was developed for each of these two cadherins. These assays were used to determine the relative levels of E-cadherin and OB-cadherin in normal human colon and colon carcinoma specimens. E-cadherin levels were found to be reduced approximately twofold in the adenocarcinoma specimens in comparison to matched normal colon specimens. In contrast, OB-cadherin levels did not correlate with malignant transformation. We speculate that this novel PCR method will be widely applicable for assessing E-cadherin mRNA levels in carcinomas.

Cadherins, steroids and cancer.

The cadherins are a family of calcium-dependent cell adhesion molecules which are thought to be key regulators of morphogenesis. This review contains a discussion of the structure, function and regulation of these cell adhesion molecules. In particular, we discuss recent studies that demonstrate the ability of steroids to modulate cadherin levelsin vivo. We speculate that steroids and estrogenic organochlorines exert their diverse morphoregulatory actions on tissues by altering cadherin levels.

E-cadherin, estrogens and cancer: is there a connection?

E-cadherin is a calcium-dependent, epithelial cell adhesion molecule. It has recently been implicated as a tumor suppressor. This review article contains a description of the structure, function, and regulation of E-cadherin and other members comprising the cadherin family. In particular, we discuss studies concerning the ability of estrogens to modulate E-cadherin levels in vivo. Finally, we consider the hypothesis that estrogens may promote breast, uterine and ovarian cancer by down-regulating E-cadherin levels in these tissues.