Wnts differentially regulate colony growth and differentiation of chondrogenic rat calvaria cells.

The wingless- and int-related proteins (Wnts) have an important role during embryonic development and limb patterning. To investigate their function during chondrocyte differentiation, we used NIH3T3 cells producing seven members of the Wnt family and secreted frizzled-related protein (sFRP-2) for co-culture experiments with the rat chondrogenic cell line pColl(II)-EGFP-5. Pilot experiments showed a negative effect of Wnt-7a on the proliferation of three rodent chondrogenic cell lines, RCJ3.1(C5.18), CFK-2, and C1. To establish a reporter system for chondrogenic differentiation we then produced a stably transfected chondrogenic cell line based on RCJ3.1(C5.18) for further experiments, which expresses green fluorescence protein (EGFP) under the collagen type II promoter (pColl(II)-EGFP-5). This cell line permits convenient observation of green fluorescence as a marker for differentiation in life cultures. The colony size of this cell line in agarose suspension cultures was reduced to 20-40% of control, when exposed to Wnt-1, 3a, 4, 7a, and 7b for 14 days. Similarly, reporter gene expression and the synthesis of cartilage-specific proteoglycans were inhibited by this group of Wnts. In contrast, pColl(II)-EGFP-5 cells exposed to Wnt-5a and Wnt-11 reached 140% of control, and reporter gene expression and proteoglycan synthesis were stimulated. The effects of Wnt-7a and Wnt-5a were additive in pColl(II)-EGFP-5 cells and some but not all Wnt effects were antagonized by the inhibition of proteoglycan sulfation with chlorate, by sFRP-2, which may modulate Wnt receptor binding, or by inhibitors of protein kinase C. These results suggest two functional Wnt subclasses that differentially regulate proliferation and chondrogenic differentiation in vitro which may have implications for cartilage differentiation in vivo. Since some, but not all Wnt effects were sensitive to inhibitors of proteoglycan synthesis or protein kinase C, multiple modes of signal transduction may be involved.

A versatile chondrogenic rat calvaria cell line R-tTA-24 that permits tetracycline-regulated gene expression.

The clonal rat calvaria cell line RCJ3.1C5.18 (RCJ) undergoes chondrogenic differentiation after long-term culture post confluence. To allow flexible genetic manipulation, a tetracycline-regulated gene expression system was established in this cell line. Treatment with tetracycline in operational doses does not affect the differentiation of RCJ cells with respect to the markers tested. After stable transfection with pUHD15.1 containing the tetracycline transactivator (tTA) in the presence of pTK-hyg for hygromycin selection, 28 clones were isolated and characterized for alcian blue staining of cartilage-specific proteoglycans and for collagen type II expression. Clone R-tTA-24 was selected on the basis of phenotype and displayed tetracycline-dependent down-regulation of luciferase activity (tet-OFF system) by two orders of magnitude (57-149-fold) after stable transfection with the reporter gene pBI-EGFP/luc. The novel, chondrogenic cell line R-tTA-24 may be stably transfected with various genes of interest for tetracycline-regulated gene expression using neomycin selection and may be a valuable tool to study the process of chondrogenic differentiation in vitro.