Heidi Eberspaecher

Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis

To assess the role of the transcription factor Sox9 in cartilage formation we have compared the expression pattern of Sox9 and Col2a1 at various stages of mouse embryonic development. Expression of Col2a1 colocalized with expression of Sox9 in all chondroprogenitor cells. In the sclerotomal compartment of somites the onset of Sox9 expression preceded that of Col2a1. A perfect correlation was also seen between high levels of Sox9 expression and high levels of Col2a1 expression in chondrocytic cells. However, no Sox9 expression was detected in hypertrophic chondrocytes; only low levels of Col2a1 RNA were found in the upper hypertrophic zone. Coexpression of Sox9 and Col2a1 was also seen in the notochord. At E11.5 Sox9 expression in the brain and spinal neural tube was more widespread than that of Col2a1 although at E14.5 Sox9 and Col2a1 transcripts were colocalized in discrete areas of the brain. Distinct differences between Sox9 and Col2a1 expression were observed in the otic vesicle at E11.5. At E8.5, expression of Sox9 but not of Col2a1 was seen in the dorsal tips of the neural folds and after neural tube closure also in presumptive crest cells emigrating from the dorsal pole of the neural tube. No Col2a1 expression was detected in gonadal ridges in which high levels of Sox9 expression were detected. Together with our previous results showing that the chondrocyte‐specific enhancer element of the Col2a1 gene is a direct target for Sox9, these results suggest that Sox9 plays a major role in expression of Col2a1. The correlation between high expression levels of Sox9 and high expression levels of Col2a1 in chondrocytes suggests the hypothesis that high levels of Sox9 are needed for full expression of the chondrocyte phenotype; lower levels of Sox9 such as in neuronal tissues which are also associated with lower expression levels of Col2a1 would be compatible with other cell specifications. Dev. Dyn. 209:377–386, 1997.

A Gene for a Novel Zinc-finger Protein Expressed in Differentiated Epithelial Cells and Transiently in Certain Mesenchymal Cells

We have identified a novel zinc-finger protein whose mRNA is expressed at high levels in the epidermal layer of the skin and in epithelial cells in the tongue, palate, esophagus, stomach, and colon of newborn mice. Expression in epithelial cells is first detected at the time of their differentiation during embryonic development. In addition, during early embryonic development there is expression in mesenchymal cells of the skeletal primordia and the metanephric kidney which is later down-regulated. The expression pattern suggests that the protein could be involved in terminal differentiation of several epithelial cell types and could also be involved in early differentiation of the skeleton and kidney. The carboxyl terminus of the protein contains three zinc fingers with a high degree of homology to erythroid krüppel-like factor and binds to DNA fragments containing CACCC motifs. The amino-terminal portion of the protein is proline and serine-rich and can function as a transcriptional activator. The chromosomal location of the gene was mapped using mouse interspecific backcrosses and was shown to localize to mouse chromosome 4 and to cosegregate with the thioredoxin gene.

Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo.

To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-α1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity. We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1–3) that were essential for its chondrocyte-specific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bpCol2a1 intron 1 sequence in vivo. SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as largerCol2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocyte-specific complex between the 48-bp element, Sox9, and other protein(s). Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex. Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage.

Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression ofasporin partially overlaps with the expression ofdecorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22–9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2,osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.

The gene for the homeodomain-containing protein Cart-1 is expressed in cells that have a chondrogenic potential during embryonic development

We have examined the expression of the gene for Cart-1, a new homeodomain-containing protein, during rat embryonic development. In early embryos, Cart-1 RNA was detected at high levels in head mesenchyme, lateral mesoderm, sclerotomes and limb bud mesenchyme. These tissues contain prechondrocytic mesenchymal cells responsible for the formation of the cartilaginous skeleton. In addition, Cart-1 RNA was also found in lung buds, tendons and mesonephros. Cells in these tissues have the potential of undergoing chondrogenesis either in explants for mesonephros and tendons, or in vivo for tendons and the precursors of bronchi cartilages. No hybridization was observed in brain, spinal cord, heart, spleen, gastrointestinal tract, liver and muscle. Our results support the hypothesis that Cart-1 may play a role in the pathway of chondrogenesis. The gene for Cart-1 was mapped to a segment of mouse chromosome 10 between the genes for phenylalanine hydroxylase and interferon gamma.

Interactions between PIAS Proteins and SOX9 Result in an Increase in the Cellular Concentrations of SOX9

We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -xα, and -xβ as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMO-activating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7 cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions.

Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5

Sox9 is a transcription factor of the SRY family required for several steps of chondrogenesis. It activates the expression of various chondrocyte-specific genes, but the mechanisms and role of cofactors involved in Sox9-regulated gene transcription are not fully understood. Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes.

Coordinate patterns of expression of type I and III collagens during mouse development

The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen mRNAs are systematically examined from 7.5 to 17.5 days of development (E7.5 to E17.5) in the mouse using in situ hybridization with specific riboprobes. Coordinated expression of pro alpha 1(I) and pro alpha 2(I) collagen mRNA was found throughout development in all regions examined. Widespread type I collagen expression starting at E8.5 occurred in embryonic mesoderm, sclerotomes, dermatomes, and in the forming connective tissues. After E14.5, regions of ossification showed highest levels of type I collagen expression. Pro alpha 1(III) collagen expression was specific to and coordinated with patterns of type I collagen expression in many fibroblast-containing tissues. No expression of type III collagen occurred in osteoblasts. This comprehensive study of the transcripts of abundantly expressed structural proteins should provide a basis for comparison of other key extracellular matrix molecules and serve as a reference for studies on the patterns of activities of various promoter/enhancer-reporter gene constructions of type I and III collagen genes in transgenic mice.

Molecular cloning and characterization of a novel rat activin receptor

We report the isolation of a full-length rat cDNA for a new activin receptor. The deduced amino acid sequence of this receptor shows 67 percent overall identity with that of a previously identified mouse activin receptor. As predicted for the mouse activin receptor, the amino acid sequence of the rat receptor is consistent with a polypeptide containing an extracellular ligand binding domain, a hydrophobic transmembrane domain, and a serine/threonine kinase intracellular domain. In an expression assay, this new receptor was found to bind I125 radiolabeled activin.

Downregulation of rheumatoid arthritis-related antigen RA-A47 (HSP47/Colligin-2) in chondrocytic cell lines induces apoptosis and cell-surface expression of RA-A47 in association with CD9

Previously, we showed that gene expression of the rheumatoid arthritis‐related antigen RA‐A47, which is identical to human heat shock protein (HSP)47, was downregulated in chondrocytes by inflammatory cytokines such as TNFα. Associated with this phenomenon, RA‐A47 appeared on the cell surface concomitant with upregulation of metabolic factors related to cartilage destruction. The upregulation of the metabolic factors could be achieved by downregulation of RA‐A47 expression with ra‐a47‐specific anti‐sense oligonucleotide. Here, we show that the enhanced surface expression of RA‐A47 on a chondrocytic cell line, HCS‐2/8 was also a direct result of RA‐A47 downregulation by ra‐a47 anti‐sense oligonucleotide, independent of the cytokine effects. Moreover, cell‐surface expression of CD9, a β1 integrin‐associated transmembrane protein that is involved in cell adhesion and cell motility events, was enhanced in the ra‐a47 anti‐sense oligonucleotide‐treated cells. The CD9 was colocalized with RA‐A47 on the cell surface, where it may have affected integrin signaling. Furthermore, Annexin‐V binding to the cell surface and the level of a number of apoptosis‐related genes including caspase‐9 were increased after ra‐a47 anti‐sense oligonucleotide treatment, suggesting that enhanced surface expression of RA‐A47 and CD9 may be initiating apoptosis. Differential screening using a cDNA gene array showed induction of metallothionein‐III and chemokine receptor CXCR4 and of factors of the Notch signaling pathway by the anti‐sense treatment, but not by TNFα. Thus, here we show for the first time an alternative mechanism of inducing apoptosis by downregulating molecular chaperones, independent of the action of TNFα. The surface‐exposed RA‐A47 may induce autoantibodies and inflammatory reactions in autoimmune disease situations such as rheumatoid arthritis.