Biomedical subjects

Bernhard Ganss

Publications and source records attributed to Bernhard Ganss.

12 recordsLinked to original sources

PubMed2006

Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation.

Endochondral bone formation is orchestrated by mesenchymal cell condensation to form cartilage anlagen, which act as a template for bone formation and eventual mineralization. The current study performed gene expression analysis to examine pre- and post-mineralization stages (E15 and E19) of endochondral bone formation, using fetal metatarsal long bones as a model. An extensive number of genes were differentially expressed, with 543 transcripts found to have at least 2-fold up-regulation and 742 with a greater than 2-fold down-regulation. A bioinformatics approach was adopted based on gene ontology groups, and this identified genes associated with the regulation of signaling and skeletal development, cartilage replacement by bone, and matrix degradation and turnover. Transcripts linked to skeletal patterning, including Hoxd genes 10-12, Gli2 and Noggin were considerably down-regulated at E19. Whereas genes associated with bone matrix formation and turnover, ACP5, MMP-13, bone sialoprotein, osteopontin, dentin matrix protein-1 and MMP-9 all were distinctly up-regulated at this later time point. This approach to studying the formation of the primary ossification center provides a unique picture of the developmental dynamics involved in the molecular and biochemical processes during this intricately regulated process.

Animals↗

PubMed2006

Stereochemical analysis of the functional significance of the conserved inverted CCAAT and TATA elements in the rat bone sialoprotein gene promoter.

Basal transcription of the bone sialoprotein gene is mediated by highly conserved inverted CCAAT (ICE; ATTGG) and TATA elements (TTTATA) separated by precisely 21 nucleotides. Here we studied the importance of the relative position and orientation of the CCAAT and TATA elements in the proximal promoter by measuring the transcriptional activity of a series of mutated reporter constructs in transient transfection assays. Whereas inverting the TTTATA (wild type) to a TATAAA (consensus TATA) sequence increased transcription slightly, transcription was reduced when the flanking dinucleotides were also inverted. In contrast, reversing the ATTGG (wild type; ICE) to a CCAAT (RICE) sequence caused a marked reduction in transcription, whereas both transcription and NF-Y binding were progressively increased with the simultaneous inversion of flanking nucleotides (f-RICE-f). Reducing the distance between the ICE and TATA elements produced cyclical changes in transcriptional activity that correlated with progressive alterations in the relative positions of the CCAAT and TATA elements on the face of the DNA helix. Minimal transcription was observed after 5 nucleotides were deleted (equivalent to approximately one half turn of the helix), whereas transcription was fully restored after deleting 10 nucleotides (approximately one full turn of the DNA helix), transcriptional activity being progressively lost with deletions beyond 10 nucleotides. In comparison, when deletions were made with the ICE in the reversed (f-RICE-f) orientation transcriptional activity was progressively lost with no recovery. These results show that, although transcription can still occur when the CCAAT box is reversed and/or displaced relative to the TATA box, the activity is dependent upon the flexibility of the intervening DNA helix needed to align the NF-Y complex on the CCAAT box with preinitiation complex proteins that bind to the TATA box. Thus, the precise location and orientation of the CCAAT element is necessary for optimizing basal transcription of the bone sialoprotein gene.

Animals↗

PubMed2006

3. Protein-protein interactions of the developing enamel matrix.

Extracellular matrix proteins control the formation of the inorganic component of hard tissues including bone, dentin, and enamel. The structural proteins expressed primarily in the enamel matrix are amelogenin, ameloblastin, enamelin, and amelotin. Other proteins, like biglycan, are also present in the enamel matrix as well as in other mineralizing and nonmineralizing tissues of mammals. In addition, the presence of sulfated enamel proteins, and "tuft" proteins has been examined and discussed in relation to enamel formation. The structural proteins of the enamel matrix must have specific protein-protein interactions to produce a matrix capable of directing the highly ordered structure of the enamel crystallites. Protein-protein interactions are also likely to occur between the secreted enamel proteins and the plasma membrane of the enamel producing cells, the ameloblasts. Such protein-protein interactions are hypothesized to influence the secretion of enamel proteins, establish short-term order of the forming matrix, and to mediate feedback signals to the transcriptional machinery of these cells. Membrane-bound proteins identified in ameloblasts, and which interact with the structural enamel proteins, include Cd63 (cluster of differentiation 63 antigen), annexin A2 (Anxa2), and lysosomal-associated glycoprotein 1 (Lamp1). These and related data help explain the molecular and cellular mechanisms responsible for the removal of the organic enamel matrix during the events of enamel mineralization, and how the enamel matrix influences its own fate through signaling initiated at the cell surface. The knowledge gained from enamel developmental studies may lead to better dental and nondental materials, or materials inspired by Nature. These data will be critical to scientists, engineers, and dentists in their pursuits to regenerate an entire tooth. For tooth regeneration to become a reality, the protein-protein interactions involving the key dental proteins must be identified and understood. The scope of this review is to discuss the current understanding of protein-protein interactions of the developing enamel matrix, and relate this knowledge to enamel biomineralization.

Amelogenin↗

PubMed2004

Molecular cloning, structure, expression, and chromosomal localization of the human Osterix (SP7) gene.

We report the isolation of the human orthologue of the mouse Osterix (Osx/Sp7) gene, a C2H2 zinc finger transcription factor of the SP gene family and putative "master" regulator of bone cell differentiation. The human SP7 cDNA encodes a putative 431 amino acid protein that contains three consecutive C2H2 zinc finger repeats. The SP7 protein is highly conserved between mice and humans with an overall sequence identity of 95%. The expression of a SP7 mRNA transcript of approximately 3.2 kb is restricted to bone-derived cell lines in vitro but undetectable in any adult tissues including mandibular bone by Northern blot hybridization. The specific expression of SP7 mRNA in osteoblasts in vivo was further confirmed by in situ hybridization on human embryonic tissues. The highly restricted expression pattern and the divergence of the sequence outside of the zinc finger region distinguish SP7 as a unique member of the SP family. The SP7 gene consists of two exons, with exon 2 containing most of the protein coding sequence. The gene locus was mapped to chromosome 12q13.13 by fluorescent in situ hybridization (FISH). The identification and initial characterization of the SP7 gene will facilitate the study of the molecular regulation of osteoblast differentiation in humans.

Amino Acid Sequence↗

PubMed2004

Zinc finger transcription factors in skeletal development.

Cellular and molecular processes that regulate the development of skeletal tissues resemble those required for regeneration. Given the prevalence of degenerative skeletal disorders in an increasingly aging population, the molecular mechanisms of skeletal development must be understood in detail if novel strategies are to be developed in regenerative medicine. Research in this area over the past decade has revealed that cell differentiation is largely controlled at the level of gene transcription, which in turn is regulated by transcription factors. Transcription factors usually recognize and bind to specific DNA sequences in the promoter of target genes via characteristic DNA-binding domains. Although the gene family containing C2H2 zinc fingers as DNA-binding motifs is the largest family of transciptional regulators, with several hundred individual members in mammals, only a small but increasing number of zinc finger genes have been implicated in bone, cartilage, or tooth development. These zinc finger proteins (ZFPs) contain multiple structural motifs that require zinc to maintain their structural integrity and function. Interestingly, zinc deficiency is known to result in skeletal growth retardation and has been identified as a risk factor in the pathogenesis of osteoporosis. This review attempts to summarize our current state of knowledge regarding the role of ZFPs in the molecular regulation of skeletogenesis.

Animals↗

PubMed2003

Characterization of the 5'-flanking region of the rat AJ18 gene.

Krüppel-associated box (KRAB) domains are present in one-third of all C(2)H(2) zinc finger containing proteins, making the KRAB/C(2)H(2) proteins one of the largest known families of putative transcription repressors. AJ18 has been identified as a novel KRAB/C(2)H(2) gene that is involved in the differentiation of osteogenic cells. To study the regulation of expression of the AJ18 gene, the 5'-flanking region of the AJ18 gene was obtained by screening a rat genomic library. This region was sequenced, and the transcription start site mapped by primer extension. The AJ18 gene consists of at least four exons, the first exon coding for an unusually long 2.3 kb 5'-UTR region. A putative internal ribosome entry site, immediately upstream of the translation initiation site, is indicated from the complementarity of a 12 nucleotide sequence with a region in the rat 18S rRNA. Chimeric constructs encompassing the region surrounding the transcription start site (-77-+171), as well as constructs with additional 1.9 kb upstream from this region revealed strong transcriptional activity when ligated to a luciferase reporter gene and tested in transient transfection assays. This activity was lost on deletion of the 5'-flanking region to -77. In addition, transcriptional activity was progressively lost with the inclusion of downstream sequences extending into the 5'-UTR. Several known response elements for proteins such as Runx2, NFkappaB, Smads, Sp1, and Ets1 are retained within the conserved sequences of rat and mouse AJ18, which was retrieved from mouse genomic libraries. Interestingly, the transcriptional activity was approximately 100-fold higher in the osteocarcinoma cell line ROS 2.8/17 compared to the fibroblast-like C3H10T1/2. Notably, this is the first gene promoter from the large KRAB/C(2)H(2) zinc finger family of proteins to be identified and characterized.

5' Flanking Region↗

PubMed2003

Developmental expression pattern and DNA-binding properties of the zinc finger transcription factor Krox-26.

The development of teeth through epithelial-mesenchymal interactions is mediated on a molecular level through the iterative and reciprocal action of secreted growth factors and responsive transcription factors. Although zinc finger transcription factors constitute by far the largest class of transcriptional regulators in mammals, little is known about their specific role in the regulation of tissue formation. The C2H2 zinc finger transcription factor Krox-26 is transiently expressed during organ formation, most prominently at sites of early tooth development and subsequently in secretory stage ameloblasts. The objective of this study was to determine the developmental expression pattern of Krox-26 during mouse embryogenesis and to determine its preferred DNA-binding site. Krox-26 protein expression could be detected in multiple tissues, most prominently in developing craniofacial structures. A target detection assay using recombinant Krox-26 protein identified the sequence CAATG as the preferred Krox-26 DNA-binding site. These results suggest that Krox-26 may regulate the expression of target genes through CCAAT box-related sequences in multiple tissues.

Animals↗

PubMed2002

An L1 element disrupts human bone sialoprotein promoter: lack of tissue-specific regulation by distalless5 (Dlx5) and runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements.

Bone sialoprotein (BSP) is a phosphorylated and sulphated glycoprotein with hydroxyapatite nucleating properties that is specifically expressed in association with physiological and pathological mineralization. Although previous studies have indicated that tissue-specific expression of murine BSP is regulated through a proximal homeodomain element that binds distalless5 (Dlx5) transcription analysis of the homologous human promoter revealed modest enhancement in osteogenic cells. Moreover, whereas forced expression of an antisense Dlx5 vector increased transcription, Dlx5 expression did not alter transcription significantly. Since extended promoter sequences are required to confer absolute tissue-specific expression of BSP in vivo, we characterized the upstream region of the human BSP gene. In contrast to the rat and mouse promoters, which show conserved sequences extending several kbs upstream, analysis of approximately 3 kb of the human promoter showed no sequence conservation beyond -0.99 kb. Southern blot analysis of genomic DNA from four different BAC clones showed that this sequence was not an aberration in the human genomic library used to isolate the BSP gene. Using clone BAC H-NH0811I08, the human BSP promoter sequence was extended approximately 8 kb upstream from which the non-homologous region was characterized as a 3.48 kb insert coding for an L1 retrotransposon element. Transcriptional analyses of chimeric promoter constructs revealed that the retrotransposon element suppresses transcription <80%. Upstream of the inserted DNA several regions, varying in length from 26 to 161 bps, were conserved within the mouse and human promoters. One of these conserved regions included a runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements consensus element in reverse orientation. Whereas a multimeric form of the element was transcriptionally active in response to Runx2/Cbfa1 when ligated to the BSP basal promoter, the single element in the context of the extended promoter was unresponsive. These studies have characterized the upstream promoter of the human BSP gene, which is interrupted by a unique high-frequency DNA insert that suppresses BSP gene transcription.

Animals↗

PubMed2002

Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.

Animals↗

PubMed2002

Krox-26 is a novel C2H2 zinc finger transcription factor expressed in developing dental and osteogenic tissues.

The development of teeth through epithelial-mesenchymal interactions is mediated on a molecular level by a network of secreted growth factors and responsive transcription factors. Although zinc finger transcription factors constitute by far the largest class of transcriptional regulators with an estimated number of approximately 1000 genes present in mammals [14], little is known about their role in the regulation of mineralized tissue formation. A fragment (Y150) of the novel C2H2 zinc finger transcription factor Krox-26 has initially been isolated from highly proliferative dental pulp tissue in rats [19]. The objective of this study was to clone the full-length cDNA sequence of the murine homologue and to determine its mRNA and protein expression pattern during mouse embryonic development. Mouse Krox-26 contains five C2H2 zinc finger repeats. Its expression was found to be most prominent in the developing craniofacial bones and dental organs. These results suggest Krox-26 as a potential regulator of gene transcription during the development of teeth and the craniofacial skeleton.

Amino Acid Sequence↗

PubMed2002

Temporal and spatial expression of a novel zinc finger transcription factor, AJ18, in developing murine skeletal tissues.

Bone morphogenetic proteins (BMPs) are characterized by their ability to induce osteoblastic differentiation. However, the mechanism of osteo-induction by BMPs has yet to be determined. Using differential display we previously identified AJ18, a zinc finger transcription factor, as an immediate-early response gene to BMP-7. AJ18 was shown to bind to the osteoblast-specific element2 (OSE2) and to modulate transactivation by Runx2, a master gene in osteoblastic differentiation. Here we describe the temporal and spatial expression of AJ18 in developing mouse tissues. AJ18 mRNA expression was observed in most tissues, except liver, and was generally highest early in embryonic development, decreasing markedly after parturition. Consistent with immunohistochemical analysis, AJ18 mRNA expression was highest in the brain, kidney, and bone of 17 dpc (days post coitum) embryos. In endochondral bones of embryonic and 4-week-old mice, immunostaining for AJ18 was strong in the nuclei of proliferating and pre-hypertrophic chondrocytes, and osteoblasts, whereas there was low or no staining in hypertrophic chondrocytes. In teeth of embryonic and 4-week-old mice, nuclear staining was observed in precursor and mature ameloblasts, odontoblasts, and cementoblasts, respectively. In addition, in 4-week-old mice staining of AJ18 was observed within alveolar bone cells and periodontal ligament cells. In general, the spatial expression of AJ18 in skeletal and non-skeletal tissues of mouse embryos showed striking similarity to the expression of BMP-7 mRNA. Therefore, the expression of AJ18 is consistent with its perceived role as a transcriptional factor that regulates developmental processes downstream of BMP-7.

Amino Acid Sequence↗

PubMed2002

The zinc finger transcription factor Zfp60 is a negative regulator of cartilage differentiation.

The differentiation of many mesenchyme-derived cells, including cells that form bone and cartilage, is regulated at the level of gene transcription, but many of the factors involved in this regulation remain to be identified. In this study, a modified RNA fingerprinting technique was used to identify the KRAB domain zinc finger transcription factor Zfp60 as a candidate regulator of cell differentiation in mouse calvaria primary cultures. The highest expression of Zfp60 mRNA in vivo was found between embryonic day 11 (E11) and E15 during mouse embryonic development, coinciding with stages of active organ formation. The expression of Zfp60 mRNA and protein was analyzed further in mouse embryos during skeletal development. The most prominent expression was found in prehypertrophic chondrocytes, where it coincides with the expression of key regulators of chondrocyte maturation, Indian hedgehog (Ihh), and the parathyroid hormone-related peptide (PTHrP) receptor. Zfp60 mRNA was also found transiently expressed during chondrogenesis of C1 cells in vitro, preceding collagen type X expression and cellular hypertrophy. Overexpression of Zfp60 inhibited cartilage differentiation in the chondrogenic ATDC5 cell line. These results suggest a role for Zfp60 as a negative regulator of gene transcription, specifically during the development and/or differentiation of chondrocytes.

Amino Acid Sequence↗