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Background: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls., Methods: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels., Results: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice., Conclusions: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system., ((c) 2007 Wiley-Liss, Inc.)

Abstract: Osteoarthritis is a highly prevalent and disabling disease for which we do not have a cure. The identification of suitable molecular targets is hindered by the lack of standardized, reproducible and convenient screening assays. Following extensive comparisons of a number of chondrocytic cell lines, culture conditions, and readouts, we have optimized an assay utilizing C-28/I2, a chondrocytic cell line cultured in high-density micromasses. Utilizing molecules with known effects on cartilage (e.g. IL-1β, TGFβ1, BMP-2), we have exploited this improved protocol to (i) evoke responses characteristic of primary chondrocytes; (ii) assess the pharmacodynamics of gene over-expression using non-viral expression vectors; (iii) establish the response profiles of known pharmacological treatments; and (iv) investigate their mechanisms of action. These data indicate that we have established a medium-throughput methodology for studying chondrocyte-specific cellular and molecular responses (from gene expression to rapid quantitative measurement of sulfated glycosaminoglycans by Alcian blue staining) that may enable the discovery of novel therapeutics for pharmacological modulation of chondrocyte activation in osteoarthritis. [Copyright &y& Elsevier]