BMP regulation of the mouse connexin43 promoter in osteoblastic cells and embryos

Abstract

We examined BMP regulation of the gap junction gene Gjal (Cx43α1) using a series of lacZ reporter constructs containing up to 6.7 kbs of mouse Cx43α1 promoter sequence. Using transient transfection assays, we showed that BMP2, BMP4, and GDF5, but not BMP6 or BMP7, can modulate Cx43α1 promoter activity in the osteosarcoma cell line ROS17/2.8. Positive regulatory elements were found at the proximal and distal ends of the 6.7 kb promoter fragment, while negative regulatory elements were found in the intervening region. Comparison of Cx43α1 promoter sequences from the human vs. mouse showed five regions with significant sequence conservation, two of which contained Smad binding elements in conjunction with a BMP response element. Analysis of a transgenic mouse line containing a Cx43α1 promoter driven lacZ reporter construct revealed lacZ expression in the developing joints, an expression pattern similar to that previously reported for Gdf5. LacZ expression was also observed in axial regions of the skeletal anlage, which in situ hybridization analysis confirmed as sites of Gdf5 transcript expression. When the Cx43α1 promoter driven lacZ transgene was bred into the brachypodism mouse Gdf5bpJ (bp) harboring a Gdf5 loss of function mutation, lacZ expression was extinguished. This was observed in homozygous and heterozygous bp animals, suggesting that Cx43α1 promoter regulation by GDF5 is subject to haploinsufficiency. Overall, these observations are consistent with recent studies by others indicating a role for Cx43α1 in osteogenesis and osteoblastic function during mouse development.