An 18-Base-Pair Sequence in the Mouse Proα1(II) Collagen Gene Is Sufficient for Expression in Cartilage and Binds Nuclear Proteins That Are Selectively Expressed in Chondrocytes

Abstract

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the pro␣1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3 repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465-and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.Acquisition of the chondrocyte phenotype by mesenchymal cells is one of the major pathways of differentiation of these cells. Chondrocytes form several types of cartilages including the growth plate cartilages essential to skeletal formation and cartilages that have supporting roles and persist throughout adult life such as the articular cartilages and the cartilages of the nose, ear, and trachea. Chondrocyte differentiation presumably involves first the commitment of undifferentiated mesenchymal cells to the chondrocyte lineage (1). Cell condensation and further maturation lead to a fully differentiated phenotype characterized by the synthesis of cartilage extracellular matrix proteins, including collagen types II, IX, and XI, the large proteoglycan aggrecan, the link protein, and the cartilage oligomeric protein (24). Recent molecular and biochemical studies with cell culture, gene inactivation experiments with mice, and the identification of genes responsible for mouse and human skeletal abnormalities have documented the importance of growth and differentiation factors, extracellular matrix proteins, signaling mediators, and tr...

Wnt/β-catenin signaling is sufficient and necessary for synovial joint formation

“…5DЈ,FЈ). As the Col2a1 promoter and enhancer are directly activated by Sox9, which is expressed in differentiating chondrogenic mesenchymal condensations (Wright et al 1995;Lefebvre et al 1996), our results indicate that Wnt14 signals though ␤-catenin in differentiating chondrocytes in vivo to reverse chondrocyte differentiation, the first step in synovial joint formation.…”

Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation

“…The cDNA encoding human SOX9 was cloned into the pGL3-control plasmid (Promega) to replace the luciferase gene. The 4 ϫ 48-p89 luciferase construct and its MA6 mutant were described previously (44,45). For some experiments, cells were incubated with 10 M of one of the p38 inhibitors, SB202190, SB203580, and SB220025 or the inhibitor of MAPK-activated protein kinase 2 (Calbiochem) for the last 12 h. Cell culture and transient transfections were described previously (13).…”

The Transcription Factor δEF1 Is Inversely Expressed with Type II Collagen mRNA and Can Repress Col2a1 Promoter Activity in Transfected Chondrocytes

“…In addition, Sp1 binding sites in the proximal promoter are believed to be important for efficient enhancer-mediated transcription (17) and Sp1 binding activity differs between differentiated and dedifferentiated chondrocytes (18). Although it has been reported that a fragment from the first intron enhancer can direct chondrocyte-specific expression through a heterologous promoter (12), it is likely that in vivo, the endogenous Col2a1 promoter also participates in gene regulation during certain developmental stages and in response to specific regulatory signals. In fact it has been reported recently that both positive and negative elements are located in the 5Ј-flanking region of the human COL2A1 gene and participate in developmental stage-and tissue-specific expression in transgenic mice (19).…”

“…Previously, an enhancer element was discovered in the first intron of the rat gene that was required for chondrocyte-specific expression in vitro (10,11). Other recent studies have identified additional cis-acting regulatory sequences in the first intron of the gene and identified specific transcription factors involved in regulating Col2a1 transcription (12)(13)(14)(15). However, relatively little work has been reported on characterizing cis-acting DNA elements or regulatory proteins operating in the 5Ј-flanking region of the gene.…”