Fig. 1. FGFR3 mediates chondrocytic differentiation in mesenchymal stem cell line C3H10T1/2. (A) RT-PCR analyses of BMP2-dependent expression of FGF- and PTH/PTHrP-receptors in mesenchymal stem cell line C3H10T1/2 incubated for various times in the presence or absence of recombinantly expressed BMP2. (B) Cycloheximide pretreatment of C3H10T1/2 cells does not prevent BMP-induction of the FGFR3 gene. Cells were mock-treated (Control) or treated with BMP2 (50 ng/ml) for various times. Preincubation with cycloheximide (CHX) was for 30 minutes (50 µg/ml). RT-PCR analyses of FGFR3 mRNA levels were as described in Materials and Methods. (C) Western immunoblotting for the detection of BMP2-dependent FGFR3 and FGFR2 expression in cellular extracts of C3H10T1/2 lines. Immunoblotting was performed with polyclonal antibodies SC-123 (FGFR3; Santa Cruz, left) and SC-122 (FGFR2; Santa Cruz, right). (D) (Left) Western immunoblotting to document the forced expression of FGFR3 in mesenchymal progenitors C3H10T1/2. (Right) The recombinant expression of FGFR3 in C3H10T1/2 leads to enhanced levels of activated MAP-kinases pERK-1 and pERK-2 during cultivation (New England Biolabs MAPK Detection Kit 9100). Cell lysates were prepared (at confluence/0) and 4 days post-confluence. (E) The forced expression of FGFR3 in parental C3H10T1/2 cells is sufficient for the induction of the chondrogenic lineage. (Bottom) RT-PCR analyses of expression levels of chondrogenic and ostesteogenic markers were performed as described in Materials and Methods. Collagen 1a1 and osteocalcin are markers predominantly for osteogenic and collagen 2a1 for chondrogenic differentiation, respectively. The PTH/PTHrP-receptor is a marker for early osteogenic and late chondrogenic development. HPRT was used to standardize PCR conditions. (Top) Osteoblast-like cells stably expressing BMP2 or FGFR3 were identified by alkaline phosphatase staining (6 days post-confluence). (Middle) Chondrocyte-like cells secreting proteoglycans were histologically stained with Alcian Blue (12 days post-confluence). Cellular confluence has been arbitrarily termed day 0.