Two haplotypes defined by polymorphisms in the 5' flank of the COLIA1 regulate BMD in a bidirectional manner in women.

Three polymorphisms (-1997G/T; -1663IndelT and +1245G/T) have been identified in the 5' flank of COL1A1 gene that are associated with osteoporosis but the underlying mechanism is unclear. Here we investigated the functional effects of these variants on COL1A1 transcription. Transcription was 2-fold higher with the osteoporosis-associated G-del-T haplotype compared with the common G-Ins-G haplotype. Gel shift assays showed that the region surrounding the -1663IndelT polymorphism recognized a complex of proteins essential for osteoblast differentiation and function including Nmp4 and Osterix, and the osteoporosis-associated -1663delT allele had increased binding affinity for this complex. Chromatin immunoprecipitation assays confirmed that the region flanking -1663insdelT bound a complex of proteins including Osterix and Nmp4 and also showed evidence of recruitment of Nmp4 to the Sp1 binding site in intron 1. Further studies showed that haplotype G-del-T had higher binding affinity for RNA polymerase II, consistent with increased transcription of the G-del-T allele and there was a significant inverse association between carriage of G-del-T and bone mineral density (BMD) in a cohort of 3270 Caucasian women. We conclude that common polymorphic variants in the 5' flank of COLIA1 regulate transcription by affecting DNA-protein interactions and that increased levels of transcription correlate with reduced BMD values in vivo. This is consistent with a model whereby increased COL1A1 transcription predisposes to osteoporosis, probably by increasing production of the alpha 1 chain and disrupting the normal ratio of collagen type 1 alpha 1 and alpha 2 chains.

ObjectiveThe articular cartilage is known to be highly mechanosensitive, and a number of mechanosensing mechanisms have been proposed as mediators of the cellular responses to altered mechanical load. These pathways are likely to be important in tissue homeostasis as well as in the pathogenesis of osteoarthritis. One important injury-activated pathway involves the release of pericellular fibroblast growth factor 2 (FGF-2) from the articular cartilage. Using a novel model of murine cartilage injury and surgically destabilized joints in mice, we examined the extent to which FGF-2 contributes to the cellular gene response to injury.MethodsFemoral epiphyses from 5-week-old wild-type mice were avulsed and cultured in serum-free medium. Explant lysates were Western blotted for phospho-JNK, phospho-p38, and phospho-ERK or were fixed for immunohistochemical analysis of the nuclear translocation of p65 (indicative of NF-κB activation). RNA was extracted from injured explants, rested explants that had been stimulated with recombinant FGF-2 or FGF-18, or whole joints from either wild-type mice or FGF-2−/− mice. Reverse transcription–polymerase chain reaction was performed to examine a number of inflammatory response genes that had previously been identified in a microarray analysis.ResultsMurine cartilage avulsion injury resulted in rapid activation of the 3 MAP kinase pathways as well as NF-κB. Almost all genes identified in murine joints following surgical destabilization were also regulated in cartilage explants upon injury. Many of these genes, including those for activin A (Inhba), tumor necrosis factor–stimulated gene 6 (Tnfaip6), matrix metalloproteinase 19 (Mmp19), tissue inhibitor of metalloproteinases 1 (Timp1), and podoplanin (Pdpn), were significantly FGF-2 dependent following injury to cartilage in vitro and to joint tissues in vivo.ConclusionFGF-2–dependent gene expression occurs in vitro and in vivo in response to cartilage/joint injury in mice.