Biomedical subjects

C W Gibson

Publications and source records attributed to C W Gibson.

At least 19 recordsLinked to original sources

PubMed2006

Amelogenin-mediated regulation of osteoclastogenesis, and periodontal cell proliferation and migration.

We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

Amelogenin↗

PubMed2005

Tooth enamel defects in mice with a deletion at the Arhgap 6/Amel X locus.

The amelogenin proteins regulate enamel mineral formation in the developing tooth. The human AMELX gene, which encodes the amelogenin proteins, is located within an intron of the Arhgap 6 gene. ARHGAP 6 encodes a Rho GAP, which regulates activity of Rho A, a small G protein involved in intracellular signal transduction. Mice were generated in which the entire ARHGAP 6 gene was deleted by Cre-mediated recombination, which also removed the nested Amel X gene. Enamel from these mice appeared chalky white, and the molars showed excessive wear. The enamel layer was hypoplastic and non-prismatic, whereas other dental tissues had normal morphology. This phenotype is similar to that reported for Amel X null mice, which have a short deletion that removed the region surrounding the translation initiation site, and resembles some forms of X-linked amelogenesis imperfecta in humans. Analysis of the enamel from the Arhgap 6/Amel X-deleted mice verifies that the Amel X gene is nested within the murine Arhgap 6 gene and shows that removal of the entire Amel X gene leads to a phenotype similar to the earlier Amel X null mouse results, in which no amelogenin protein was detected. However, an unusual layer of aprismatic enamel covers the enamel surface, which may be related to the 1.1-Mb deletion, which included Arhgap 6 in these mice.

Amelogenesis Imperfecta↗

PubMed2003

The small bovine amelogenin LRAP fails to rescue the amelogenin null phenotype.

Amelogenins are the most abundant secreted proteins in developing dental enamel. These evolutionarily-conserved proteins have important roles in enamel mineral formation, as mutations within the amelogenin gene coding region lead to defects in enamel thickness or mineral structure. Because of extensive alternative splicing of the primary RNA transcript and proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. To address the function of one of the amelogenins, we have created a transgenic mouse that expresses bovine leucine-rich amelogenin peptide (LRAP) in the enamel-secreting ameloblast cells of the dental organ. Our strategy was to breed this transgenic mouse with the recently generated amelogenin knockout mouse, which makes none of the amelogenin proteins and has a severe hypoplastic and disorganized enamel phenotype. It was found that LRAP does not rescue the enamel defect in amelogenin null mice, and enamel remains hypoplastic and disorganized in the presence of this small amelogenin. In addition, LRAP overexpression in the transgenic mouse (wildtype background) leads to pitting in the enamel surface, which may result from excess protein production or altered protein processing due to minor differences between the amino acid compositions of murine and bovine LRAP. Since introduction of bovine LRAP into the amelogenin null mouse does not restore normal enamel structure, it is concluded that other amelogenin proteins are essential for normal appearance and function.

Amelogenesis↗

PubMed2003

Relationship of phenotype and genotype in X-linked amelogenesis imperfecta.

X-linked amelogenesis imperfectas (AI) resulting from mutations in the amelogenin gene (AMELX) are phenotypically and genetically diverse. Amelogenin is the predominant matrix protein in developing enamel and is essential for normal enamel formation. To date, 12 allelic AMELX mutations have been described that purportedly result in markedly different expressed amelogenin protein products. We hypothesize that these AMELX gene mutations result in unique and functionally altered amelogenin proteins that are associated with distinct amelogenesis imperfecta phenotypes. The AMELX mutations and associated phenotypes fall generally into three categories. (1) Mutations (e.g., signal peptide mutations) causing a total of loss of amelogenin protein are associated with a primarily hypoplastic phenotype (though mineralization defects also can occur). (2) Missense mutations affecting the N-terminal region, especially those causing changes in the putative lectin-binding domain and TRAP (tyrosine rich amelogenin protein) region of the amelogenin molecule, result in a predominantly hypomineralization/hypomaturation AI phenotype with enamel that is discolored and has retained amelogenin. (3) Mutations causing loss of the amelogenin C terminus result in a phenotype characterized by hypoplasia. The consistent association of similar hypoplastic or hypomineralization/hypomaturation AI phenotypes with specific AMELX mutations may help identify distinct functional domains of the amelogenin molecule. The phenotype-genotype correlations in this study suggest there are important functional domains of the amelogenin molecule that are critical for the development of normal enamel structure, composition, and thickness.

Amelogenesis Imperfecta↗

PubMed2003

Over- and ectopic expression of Wnt3 causes progressive loss of ameloblasts in postnatal mouse incisor teeth.

Intercellular signaling is essential for the development of teeth during embryogenesis and in maintenance of the continuously growing incisor teeth in postnatal rodents. WNT intercellular signaling molecules have been implicated in the regulation of tooth development, and the Wnt3 gene shows specific expression in the enamel knot at the cap stage. We demonstrate here that Wnt3 also is expressed in specific epithelial cell layers in postnatal incisor teeth. To begin to delineate the functions of Wnt3 in developing and postnatal teeth, we determined the effects of over- and ectopic expression of Wnt3 in the tooth epithelium of mice carrying a keratin 14-Wnt3 transgene. Expression of the transgene caused a progressive loss of ameloblasts from postnatal lower incisor teeth. Loss of ameloblasts may be due to defective proliferation or differentiation of ameloblast precursors, progressive apoptosis of ameloblasts, or loss of ameloblast stem cells.

Ameloblasts↗

PubMed2002

A new frameshift mutation encoding a truncated amelogenin leads to X-linked amelogenesis imperfecta.

The amelogenin proteins are the most abundant organic components of developing dental enamel. Their importance for the proper mineralization of enamel is evident from the association between previously identified mutations in the X-chromosomal gene that encodes them and the enamel defect amelogenesis imperfecta. In this investigation, an adult male presenting with a severe hypoplastic enamel phenotype was found to have a single base deletion at the codon for amino acid 110 of the X-chromosomal 175-amino acid amelogenin protein. The proband's mother, who also has affected enamel, carries the identical deletion on one of her X-chromosomes, while the father has both normal enamel and DNA sequence. This frameshift mutation deletes part of the coding region for the repetitive portion of amelogenin as well as the hydrophilic tail, replacing them with a 47-amino acid segment containing nine cysteine residues. While greater than 60% of the protein is predicted to be intact, the severity of this phenotype illustrates the importance of the C-terminal region of the amelogenin protein for the formation of enamel with normal thickness.

Adult↗

PubMed2001

Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.

Amelogenesis Imperfecta↗

PubMed2001

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.

Amelogenesis Imperfecta↗

PubMed2001

Model system for evaluation of alternative splicing: exon skipping.

Alternative splicing of the primary RNA transcript is a common mechanism for generating protein diversity. A model system was developed to study this process in vitro that is useful for evaluation of splicing of transcripts expressed in cells that do not grow well in culture. The system was used to analyze skipping of exon 4 of the amelogenin message, normally expressed in ameloblast cells for a short interval during tooth enamel development. Amelogenins are highly conserved proteins resulting from extensive alternative splicing, with domains involved in a range of functions, including mineral formation and intercellular signaling. In the bovine gene, the very short intron 4 was predicted to inhibit inclusion of exon 4, because in murine ameloblasts, exon 4 is detectably included in mRNA, and intron 4 is longer than the bovine counterpart. Bovine intron 4 was lengthened, and this size increase enhanced exon 4 inclusion sixfold to eightfold, although splice site selection was inaccurate. Intron length, therefore, is not the sole determinant controlling amelogenin exon 4 inclusion, and cis-acting inhibitory elements may also be involved in exon skipping. This vector system allows evaluation of splicing of a tissue-specific RNA by focusing on exons of interest through transfection of heterologous cultured cells without complications attributable to background transcription of the gene being evaluated.

Alternative Splicing↗

PubMed1999

Regulation of amelogenin gene expression.

The X-chromosomal amelogenin gene is expressed at a high level by ameloblast cells within the enamel organ for a short time during tooth development. Therefore, expression is both tooth specific and developmentally regulated. A Y-chromosomal amelogenin gene is also active in human and cow, but has not been detected in mouse. Genes and/or cDNAs have been cloned for mouse, human, cow, rat, pig, opossum, and hamster, and analyses have indicated that coding and upstream regions are conserved across species. Alternative splicing is extensive and produces as many as 9 mRNAs from the 7 exon murine gene, resulting from single and multiple exon skipping and alternate 3' site selection within exon 6. The pattern of alternative splicing varies both between species and during development, which is expected to result in some diversity through varying complements of amelogenin proteins associated with this highly conserved gene.

Alternative Splicing↗

PubMed1998

Comparison of upstream regions of X- and Y-chromosomal amelogenin genes.

The amelogenin genes encode abundant enamel proteins that are required for the development of normal tooth enamel. These genes are active only in enamel-forming ameloblasts within the dental organ of the developing tooth, and are part of a small group of genes that are active on both sex chromosomes. The upstream regions of the bovine X- and Y-chromosomal and the sole murine X-chromosomal amelogenin genes have been cloned and sequenced, and conservation at nearly 60% is found in the 300 bp upstream of exon 1 for the 3 genes. A region of the bovine X-chromosomal gene that has inhibitory activity when assayed by gene transfer into heterologous cells includes motifs that have a silencing activity in other genes, and may be important to the mechanism that represses amelogenin expression in non-ameloblast cells in vivo. A comparison of sequences from three genes has led to the identification of several regions with conserved motifs that are strong candidates for having positive or negative regulatory functions, and these regions can now be tested further for interaction with nuclear proteins, and for their ability to regulate expression in vivo.

Amelogenin↗

PubMed1998

DNA sequences of amelogenin genes provide clues to regulation of expression.

The amelogenins are a heterogeneous group of enamel proteins, which have an important function in enamel formation, as mutations in the amelogenin gene result in the enamel defect amelogenesis imperfecta. The cDNAs that encode murine, bovine, human, porcine, rat and opossum amelogenins have been cloned, and as many as nine alternatively spliced messages can be produced from a single primary transcript, explaining some of the protein heterogeneity. Bovine and human amelogenin genes are found on both X and Y chromosomes, and the sexually dimorphic proteins would have 87-93% identity. A comparison of genes from human, bovine and mouse indicates that they are organized into seven exons, and sequences are highly homologous among species. Bovine, murine and human upstream regions also have similarities, with consensus sequences for potential binding of transcription factors, such as AP1 and CTF/NF1. Transgenic mouse studies have shown that 2300-3500 bp of upstream region are sufficient for expression, while 900 bp are insufficient. Analysis of DNA sequence has identified (a) major homology between species for coding exons with the exception of exon 4, (b) similarities in upstream regions likely involved in tissue specific regulation of expression, and (c) sequences at the RNA splice sites which may determine exon inclusion or skipping.

Alternative Splicing↗

PubMed1997

Regulation of amelogenin gene expression.

The amelogenins are found uniquely in enamel, where they constitute the predominant class of secreted matrix proteins and where they play a fundamental role in normal enamel formation. To better understand the high level of tissue-specific expression, we cloned the bovine X and Y chromosomal amelogenin genes and the murine amelogenin gene and determined the DNA sequences for the regions upstream of the transcription start sites. We observed segments of strong homology among species, and identified consensus sequences for the binding of various transcription factors, including the glucocorticoid receptor, AP1, RXR and p53. Although specific sis-elements conferring enhanced transcription have not yet been identified, elements have been localized that have silencing effect in non-ameloblast cells. Conserved sequences are likely to be involved in tissue-specific expression. Transgenic mouse studies have shown that 3.5 kb of upstream region is sufficient but 900 bp is insufficient for specific expression in vivo. Alternative splicing of the primary transcript is an effective mechanism for generating molecular heterogeneity. Amelogenin genes contain seven exons, and exons 3, 4, 5 and most of 6 can be deleted by alternative splicing. However, the pattern of exon splicing varies according to the species, and skipping of bovine exon 3 appears to be developmentally regulated. It will be important to determine whether the relative amounts of translation products differ among species as do the mRNAs, and to correlate the various protein structures with function. These findings also suggest that the regulation of amelogenin gene expression is complex and takes place at several levels.

Alternative Splicing↗

PubMed1997

An amelogenin gene defect associated with human X-linked amelogenesis imperfecta.

Dental enamel is a product of ameloblast cells, which secrete a mineralizing organic matrix, composed primarily of amelogenin proteins. The amelogenins are thought to be crucial for development of normal, highly mineralized enamel. The X-chromosomal amelogenin gene is a candidate gene for those cases of amelogenesis imperfecta, resulting in defective enamel, in which inheritance is X-linked. In this report, a kindred is described that has a C to A mutation resulting in a pro to thr change in exon 6 of the X-chromosomal amelogenin gene in three affected individuals, a change not found in unaffected members of the kindred. The proline that is changed by the mutation is conserved in amelogenin genes from all species examined to date.

Adenine↗

PubMed1996

Analysis of amelogenin mRNA during bovine tooth development.

The amelogenins are highly conserved enamel-matrix proteins that are essential for proper mineral formation. Transcriptionally active genes encoding the bovine amelogenin proteins reside on both the X and Y chromosomes. Comparison of relative levels of amelogenin mRNAs at various stages of development indicated that the X-chromosomal amelogenin message is at least six fold more abundant than the Y. Alternative splicing generates at least seven messages, five from the X primary transcript, and two from the Y. The two most abundant X-chromosomal amelogenin messages are approx. 850 and 450 nucleotides long, and nearly 10-fold more 850-nucleotide mRNA can be measured than 450 nucleotide, which has lost most of exon 6 by splicing. The predominant small message encodes leucine-rich amelogenin protein (LRAP), and amounts of LRAP message are relatively constant during development. However, the amelogenin message from which exon 3 has been spliced declines approximately 2.3-fold, when compared to total X chromosomal amelogenin transcripts, suggesting differential regulation of alternative splicing. In addition, a new exon was identified within genomic DNA, which was shown to be expressed by the use of reverse transcriptase-polymerase chain reaction, and the exons were renamed accordingly. This new exon-4 sequence is unusual in that it is not highly conserved between species.

Alternative Splicing↗

PubMed1996

Albumin gene expression during mouse odontogenesis.

Albumin protein is present in developing teeth of several species. Oligomer primers and cRNA probes specific for albumin were designed to perform RT-PCR, and for in situ hybridization, respectively. In situ hybridization failed to reveal albumin expression in any tooth cells, however, albumin PCR products were amplified from tissues adhering to the roots of developing teeth from four-week-old mice. It is concluded that this source is not the primary source of albumin protein found in developing enamel, because of the location and level of expression of albumin mRNA in periodontal tissue.

Albumins↗

PubMed1995

Bovine enamel organ cells express tissue non-specific alkaline phosphatase mRNA.

Alkaline phosphatase (AP) is expressed at high levels in all mineralizing tissues, and the isoform identified in developing enamel has biochemical properties similar to that found in bone. While the bone AP is referred to as the liver/bone/kidney or tissue non-specific (TNS) form, other APs are highly specific for tissue of expression. To determine unequivocally the AP isoform made by enamel organ cells, we constructed a fetal bovine enamel organ cDNA library, which yielded eight AP cDNA clones. In each case, the DNA sequence was homologous to the partial cDNA reported for bovine kidney AP (Garattini et al., 1987). It is concluded that enamel organ cells express the TNS-AP isoform. The extended 3' untranslated region of the cDNA has considerable homology to human TNS-AP, and the conservation of sequence suggests that the 3' end may have a role in post-transcriptional regulation of expression.

Alkaline Phosphatase↗

PubMed1995

Analysis of amelogenin proteins using monospecific antibodies to defined sequences.

Amelogenins are the predominant proteins found in the developing enamel matrix and are believed to play a crucial role in normal mineralization. Although the amelogenin gene is found as a single copy in all species in which it has been examined, multiple amelogenin polypeptides ranging in size from 5 to 25 kDa are obtained upon extraction of developing enamel matrix, making identification and characterization of individual components difficult. This heterogeneity may be ascribed to transcription of divergent genes located on the X and Y chromosomes, alternative splicing of the primary transcripts, physiologic degradative processing, and artefactual degradation. In order to characterize individual components, antibodies were produced to the following peptides: (1) QPLQPMQPMQPLQPLQPL (corresponding to the repeat sequence encoded only in the bovine X chromosome gene), (2) IRHPPLPP (corresponding to a unique sequence generated by alternative splicing found in leucine-rich amelogenin peptide (LRAP), (3) LPDLPLEAWPATDKTKREEVD corresponding to the amelogenin carboxy-terminus. Amelogenin proteins obtained from fetal bovine molars were subjected to SDS PAGE and Western electrotransfer, and immuno-ultrastructural analysis. These analyses demonstrated that: (1) the distribution of amelogenin polypeptides isolated from male fetuses differed appreciably from that of females, (2) the LRAP junctional peptide sequence can be specifically identified, and (3) the LRAP peptide can be immunolocalized in the enamel matrix of both males and females.

Alternative Splicing↗