The culture of human submandibular gland (HSG) cells on laminin-1 induces acinar differentiation. We identified a site on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin ␣1 and ␣2 chains. The ␣1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on laminin-1. Cells cultured with either AG73 or the homologous ␣2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinarlike, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on laminin are required for complete differentiation. The G-domain of laminin-1 contains both integrin and heparin binding sites, and anti-␤ 1 -integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of laminin-1 containing AG73, is specific, since other laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-␤ 1 -integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of laminin-1 and laminin-2. In summary, multiple interactions between laminin and HSG cells contribute to acinar differentiation, involving both ␤ 1 -integrins and syndecan-1.

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Amelogenesis imperfecta is a broad classification of hereditary enamel defects, exhibiting both genetic and clinical diversity. Most amelogenesis imperfecta cases are autosomal dominant disorders, yet only the local hypoplastic form has been mapped to human chromosome 4q between D4S242 1 and the albumin gene. An enamel protein cDNA, termed ameloblastin (also known as amelin and sheathlin), has been isolated from rat, mouse and pig. Its human homolog has been mapped to chromosome 4q21 between markers D4S409 and D4S400, flanking the local hypoplastic amelogenesis imperfecta critical region. Therefore, ameloblastin is a strong candidate gene for this form of amelogenesis imperfecta. To facilitate genetic studies related to this dental disease, we isolated and characterized a human ameloblastin cDNA. A human third molar cDNA library was screened and two ameloblastin clones identified. Nucleotide sequencing of these cDNAs indicated alternative splicing of the putative open reading frame, use of different polyadenylation signals, and a high degree of similarity to reported rat, mouse and porcine cDNAs. Immunohistochemistry studies on embryonic human teeth using an antibody to recombinant ameloblastin indicated ameloblastin expression by ameloblasts with localization in the enamel matrix associated with the sheath structures.

Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared from rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0·1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1·6 kb intron fragment where chondrocytespecific Sry/Sox-consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes chondrocyte differentiation through enhancement of SOX9.

Ameloblastin was first identified as one of the most abundant novel transcripts from a random screening of a rat incisor cDNA library. In situ hybridization experiments have shown ameloblastin expression to be specific to ameloblasts, with highest levels in secretory and maturation stage ameloblasts and cells of the epithelial root sheath. Ameloblastin has been identified as a candidate gene for the local hypoplastic form of autosomal dominant amelogenesis imperfecta, by virtue of it's location within the critical disease locus. The purpose of this study was to isolate a full length mouse ameloblastin cDNA and determine its temporal expression pattern during odontogenesis. A newborn mouse molar cDNA library was screened using a rat ameloblastin cDNA probe. Positive clones were confirmed by PCR analysis with ameloblastin-specific primers, and their size determined with vector-specific primers. Phage clones were rescued to phagemid using Exassist helper phage and the nucleotide sequence determined. We report here the identification of two clones, exhibiting alternative splicing of the putative open reading frame, and use of multiple polyadenylation signals. Nucleotide sequence analysis indicated a high degree of similarity to rat ameloblastin, rat amelin 1 and 2 and porcine sheathlin. Reverse transcriptase-PCR analysis using mouse first and second mandibular molar mRNA indicated initial expression at E-14. This is one day after the initial expression of tuftelin (E-13) and one day prior to that of amelogenin (E-15).

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