Cultured incisors display major modifications in basal lamina deposition without further effect on odontoblast differentiation.

Matrix-mediated epithelio-mesenchymal interactions play a crucial role in the control of dental cytodifferentiations. Ultrastructural observation of the epithelio-mesenchymal junction in cultured embryonic mouse molars showed discrete zones with duplicated or multilayered basal laminae. The use of synthetic peptides demonstrated that the process was RGD*-independent, did not involve the YIGSR* sequence present on laminin and could occur spontaneously. Cultured incisors showed a similar but much more dramatic multiplication of the basal laminae. Furthermore, the deposition of multilayered basal laminae was specific for the labial aspect of the tooth and could be detected after 6 h of culture. Despite these alterations, preodontoblasts differentiated and gradients of differentiation were maintained, suggesting that among basement membrane constituents, the basal lamina itself does not play a critical role. More important is the inner dental epithelium which may still control odontoblast differentiation by means of diffusible molecules able to reach surface receptors expressed by preodontoblasts or matrix receptors underlying the basal lamina. Gradients of odontoblast differentiation could result from a progressive acquisition of competence by preodontoblasts.

Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions.

Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.

Changes in the distribution of actin-associated proteins during epidermal wound healing.

We have examined the distribution of actin filaments and a number of actin-associated proteins during human epidermal wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. Filamentous actin was found in all the living epidermal layers before, during and after wound healing. alpha-actinin was also present in all the living layers of normal epidermis, but diffuse cytoplasmic staining was observed at the leading edge of migrating epidermis. Vinculin and talin were concentrated at the basement membrane prior to wounding, but were absent from the leading edge during wound healing. In normal epidermis, filamin and gelsolin showed a complementary distribution, with filamin most abundant in the basal layer and gelsolin most abundant suprabasally. The abundance of both proteins was reduced at the leading edge of migrating epidermis. All of the changes were transient, as the expression patterns returned to normal by 1 week after wounding, when the epidermis had reformed. The relevance of these changes to the process of keratinocyte migration is discussed.

Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.

We have examined integrin expression during the remodeling of the epidermis that takes place during wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. By immunofluorescence microscopy, we found that the same integrin subunits were expressed during wound healing as in normal epidermis with very little change in the relative intensity or distribution of staining at the leading edge of the migrating epidermis. However, at the time of wound closure, when the epidermis is still hyperproliferative, alpha 2, alpha 3, alpha 6, and beta 1 were no longer confined to the basal layer, as in normal epidermis, but were also found in all the living suprabasal cell layers, coexpressed with the terminal differentiation markers involucrin, keratin 10, and keratin 16. Strong suprabasal staining for alpha v was also found in one specimen. beta 4, which normally forms a heterodimer with alpha 6, and alpha 5 remained predominantly basal. Three of the integrin ligands, fibronectin, type IV collagen, and laminin, remained largely confined to the basement membrane zone and dermis. By 14 d after wounding, the integrins were once more restricted to the basal layer. Suprabasal integrin expression was also observed in involved psoriatic lesions. Thus, in two situations in which the epidermis is hyperproliferative, there is a failure to downregulate integrin expression on initiation of terminal differentiation. The functional consequences of this aberrant integrin expression remain to be explored.

The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation.

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.

Changes in the abundance and distribution of actin and associated proteins during terminal differentiation of human epidermal keratinocytes.

We have examined the abundance and distribution of actin and several actin-associated proteins in human epidermal keratinocytes before and after initiation of terminal differentiation. Keratinocytes were placed in suspension in methylcellulose for 1 h or 24 h and then extracted for immunoblotting. At 24 h, when the proportion of cells expressing the terminal differentiation marker, involucrin, had increased approximately 3-fold, there were marked decreases in the levels of vinculin, talin, filamin and gelsolin. The level of actin was unchanged and the level of alpha-actinin decreased only slightly. To complement the immunoblot analysis, we also examined the distribution of each protein in basal (involucrin-negative) and suprabasal (involucrin-positive) cells in stratified colonies, using confocal microscopy. Gelsolin, filamin, vinculin, talin, alpha-actinin and filamentous actin were all less abundant in suprabasal cells than in basal cells. There were also differences in the distribution of all the proteins in the basal compared to the suprabasal layers. In addition to the changes associated with terminal differentiation, there was variation in the distribution of focal contacts and stress fibres and in gelsolin levels between basal cells at the periphery of colonies and those in the centre. These results are discussed in the context of the known association of the actin cytoskeleton with receptors of the integrin family, the loss of integrins that occurs during keratinocyte terminal differentiation, and the possible role of the cytoskeleton in signalling between integrins and the nucleus.

A 165 kDa membrane antigen mediating fibronectin-vinculin interaction is involved in murine odontoblast differentiation.

Membrane-mediated matrix-microfilament interactions are involved in odontoblast differentiation. In this study, we analyzed the interactions of vinculin and fibronectin with plasma membrane proteins separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene-difluoride (PVDF) paper. Vinculin was found to interact with 58, 63 and 165 kDa plasma membrane proteins. Fibronectin interacted with three high molecular weight (145, 165, and 185 kDa) membrane proteins. Attempts were made to characterize the 165 kDa protein which interacted with vinculin and with fibronectin. The interaction of the 165 kDa protein with fibronectin was not competitively inhibited by synthetic peptides such as GRGDS or GRGDSP, suggesting that the protein was not related to integrins. Antibodies directed against the 165 kDa protein allowed the identification of the precise localization and biological role of this membrane antigen. The data presented in this paper and previous observations indicate that the 165 kDa protein, involved in odontoblast elongation and polarization, mediates a fibronectin-vinculin transmembrane interaction.

Membrane-cytoskeleton interactions: inhibition of odontoblast differentiation by a monoclonal antibody directed against a membrane protein.

It is known that high-molecular-weight (HMW) membrane proteins mediate interactions with constituents of the extracellular matrix and/or with cytoskeletal elements. To study participation of HMW membrane proteins in odontoblast or ameloblast differentiation, an immunological approach has been adopted. Antibodies directed against membrane proteins (Mr, 110-190) from mouse embryos have been produced by the hybridoma technique. Supernatants of hybridoma cultures were screened for their ability to stain dental tissues and also tested for their biological activities on dental cells in primary culture or on developing tooth germs in organ culture. An IgM monoclonal antibody, MC16A16, directed against a 165-kDa antigen present in plasma membrane preparations, reacted strongly with the dental epithelium and weakly with the mesenchyme. MC16A16 also reacted with the cell surface of nonpermeabilized cultured dental cells and could detach epithelial cells cultured on glass, but not mesenchymal cells which maintained vinculin-containing focal contacts. This antibody, which affected the organization of dental-cell microfilaments in primary culture, also inhibited the polarization of odontoblasts, but not that of ameloblasts.

trans-Diamminedichloroplatinum(II), a reversible RNA-protein cross-linking agent. Application to the ribosome and to an aminoacyl-tRNA synthetase/tRNA complex.

A new approach allowing detection of contact points between RNAs and proteins has been developed using trans-diamminedichloroplatinum(II) as the cross-linking reagent. The advantage of the method relies on the fact that the coordination bonds between platinum and the potential acceptors on proteins and nucleic acids (mainly S of cysteine or methionine residues; N of imidazole rings in histidine residues; N7 of guanine, N1 of adenine, and N3 of cytosine residues) can be reversed, so that the cross-linked oligonucleotides or peptides in contact within a complex can be analyzed directly. The method was worked out with the ribosome from Escherichia coli and the tRNAVal/valyl-tRNA synthetase system from the yeast Saccharomyces cerevisiae. In the first system the platinum approach permitted detection of ribosomal proteins cross-linked to 16S rRNA within the 30S subunits (mainly S18 and to a lower extent S3, S4, S11, and S13/S14); in the second system major oligonucleotides of tRNAVal cross-linked to valyl-tRNA synthetase were detected in the anticodon stem and loop, in the variable loop, and in the 3' terminal amino acid accepting region. These results are discussed in light of the current knowledge on ribosome and tRNAs and of potential applications of the methodology.