Brian Houston

Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes

Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes.

Elevated expression of hypoxia inducible factor-2α in terminally differentiating growth plate chondrocytes

Growth plate chondrocytes exist in a hypoxic environment where it is recognized that hypoxia‐inducible factor‐1α (HIF‐1α) is essential for their survival. Its regulation of chondrocyte viability may be mediated by the increased expression of vascular endothelial growth factor (VEGF) and glycolytic enzymes. However, the full chondrocyte response to hypoxia and the molecular control of VEGF expression in relation to growth plate differentiation and vascularization remains poorly understood. Using Percoll density gradient centrifugation, chick chondrocytes were separated into populations of different maturational phenotype. A differential display analysis of the populations showed highly upregulated expression of HIF‐2α mRNA during chondrocyte differentiation. HIF‐2α is a homologue of the HIF‐1α transcription factor, both of which play a role in the activation of a number of hypoxia responsive genes. HIF‐1α mRNA was also found to be expressed, although levels of expression were found to be similar in all of the chondrocyte fractions. The elevated expression of HIF‐2α during chondrocyte differentiation was accompanied by increased VEGF expression. Analysis of the murine chondrocyte cell line, ATDC5, which undergoes ordered maturation indicated that HIF‐2α, VEGF, placental growth factor, and glucose transporter‐1 expression all increased in parallel with chondrocyte differentiation. This observation was supported by immunohistochemistry on sections of mouse bone which showed staining corresponding to the presence of HIF‐2α in hypertrophic growth plate chondrocytes. The presence of HIF‐2α was also observed in articular chondrocytes but was restricted to the superficial tangential zone. HIF‐2α is, therefore, likely to be involved in the initiation of blood vessel formation and a metabolic shift in the growth plate, processes crucial for endochondral ossification. J. Cell. Physiol. 206: 435–440, 2006.

Expression patterns of chondrocyte genes cloned by differential display in tibial dyschondroplasia

Tibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought.

Cloning differentially regulated genes from chondrocytes using agarose gel differential display

The technique of RNA differential display has been used extensively to clone differentially expressed genes from a wide variety of cells and tissues. Recently, a simplified method of cloning differential display products, separated on agarose gels, was described. Here we report an adaption of this method, using total RNA, to clone differentially expressed genes. The approach is simple and rapid, and requires only small quantities of total RNA. Utilising this approach, we have cloned three differentially regulated genes from chondrocytes stimulated to hypertrophy in vitro, and confirmed their pattern of expression by Northern blotting. These gene fragments were sequenced and found to correspond to known genes, although only one has previously been isolated from chondrocytes.