Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation.

Alternative splicing of the type II procollagen gene (COL2A1) is developmentally regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow-derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 nucleotides of exon 2 by the use of an alternative 5' splice site, resulting in a premature termination codon and possible nonsense-mediated decay of IIC mRNA. Low levels of the IIC isoform were detected by RT-PCR and Southern analysis of COL2A1 cDNA amplified from differentiating rabbit and human MSCs. A second novel transcript, named IID, arises by the use of another 5' alternative splice site in intron 2. The IID isoform contains exon 2 plus 3 nucleotides, resulting in the insertion of an additional amino acid. The IID isoform was co-expressed with the IIA isoform during chondrogenesis, and was approximately one-third as abundant. Deletion of the IIC alternative 5' splice site from a COL2A1 mini-gene construct resulted in a significant increase in the IIA:IIB ratio. A mutant mini-gene that inhibited production of the IID isoform, however, had differential effects on the production of the IIA and IIB isoforms: this was apparently related to the differentiation status of the cell type used. These data suggest that COL2A1 mRNA abundance and other aspects of chondrocyte differentiation may be regulated by the use of these previously undetermined alternative splice sites.

Chondrogenic potential of progenitor cells derived from human bone marrow and adipose tissue: a patient-matched comparison.

PURPOSE: Stem cell-based tissue engineering represents a possible alternative for the repair of cartilage defects. Both bone marrow and adipose tissue contain pluripotential cells capable of chondrogenesis. This study was a qualitative and quantitative comparison of the chondrogenic potential of progenitor cells isolated from bone marrow aspirates and adipose tissue. METHODS: Bone marrow aspirates (BM) and matching adipose tissue (AD) overlying the posterior superior iliac crest were obtained from patients undergoing elective spine surgery. Chondrogenesis was induced using an established aggregate culture technique. Qualitative analysis was performed by histology and immunohistochemistry. DNA and glycosaminoglycan (GAG) quantitative assays were performed. Quantitative RT-PCR analysis was performed to compare expression of type II collagen between BM and AD aggregates. Osteogenic and adipogenic assays were also performed to confirm pluripotentiality of both AD-derived progenitor cells (ADPC) and BM-derived progenitor cells (BMPC). RESULTS: Toluidine blue metachromasia and type II collagen immunohistochemical staining were more extensive in the aggregates formed by BMPC. Quantitative RT-PCR showed a 500-5000 fold higher expression of type II collagen in the BMPC aggregates. The DNA content was 68% higher in the AD aggregates (p<0.02) but proteoglycan deposition per cell was 120% greater for BM-derived cell aggregates as measured by GAG assays (p<0.05). CONCLUSIONS: The tissue formed by the aggregate culture of the expanded ADPC population was less cartilaginous. It is unclear whether this is because there are fewer chondroprogenitor cells or if the monolayer expansion culture favors cells with higher proliferative rates but without differentiation potential. Under the conditions described in this study, BMPCs may represent a better choice for progenitor cell-based strategies for cartilage repair.

Ligand dependence in the copper-catalyzed oxidation of hydroquinones.

Transition metal-mediated oxidation of hydroquinones is an important physiologic reaction, and copper(II) effectively catalyzes the reaction in phosphate-buffered saline (PBS). Studies reported herein in phosphate buffer alone demonstrate that copper(II) is an ineffective catalyst in the absence of coordinating ligands, but that 1,10-phenanthroline and histamine facilitate the copper(II)-mediated oxidation of hydroquinone and its 2,5- and 2,6-di-tert-butyl analogs to the corresponding benzoquinones. The high concentration of chloride in PBS is the key element that allows copper(II) to work in this system. Although the bis-bathocuproine disulfonate complex of Cu(II), (BC)(2)Cu(II), is a strong stoichiometric oxidant, stoichiometric amounts of copper(II) in the presence of ligands other than BC oxidize hydroquinones very slowly under anaerobic conditions. Thus, the rapid copper(II)-catalyzed reaction operating aerobically does not involve a simple ping-pong reduction of copper(II) to copper(I) by hydroquinone and reoxidation of copper(I) by O(2).

Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470.

As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys(2)His(2) zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression.