Functional analyses of candidate genes for osteoporosis: runx2 and lrp5 interplay during differentiation of the hfob human osteoblast cell line

Osteoporosis is a general skeletal disorder. It is characterized by a low bone mass and a microarchitectural deterioration of bone tissue, which increase bone fragility and susceptibility to fracture. LRP5 is a widely expressed member of the low-density lipoprotein receptor superfamily, which acts as a co-receptor for Wnt. Wnt/β-catenin signaling plays an important role in the development and maintenance of many organs and tissues, among others bone. In bone, LRP5 is expressed by osteoblasts and not by osteoclasts; however, little is known about its regulation. Genetic studies show that LRP5 has a major influence on BMD. Loss-of-function mutations in the LRP5 gene cause osteoporosis pseudoglioma syndrome (OPPG) and Gain-of-function mutations in the LRP5 gene cause high bone mass phenotype, an autosomal dominant condition of increased BMD. RUNX2 is a member of the Runt family of transcription factors with a major role in the control of osteoblast commitment and differentiation, whose expression is necessary for the regulation of skeletal genes. Mutations in the RUNX2 locus in human cause cleidocranial dysplasia (CCD), which is an autosomal-dominant condition. Skeletons from homozygous Runx2 -/- (knock out) mice showed complete lack of Ossification. The DNA-binding sites of Runx2 in major bone matrix protein genes, including Col1a1; Col1a2; Spp1; Ibsp/BSP; Bglap2; Fn1/fibronectin; Mmp13, and Tnfrsf11b/Opg, have been identified, and Runx2 induced the expression of these genes or activated their promoters in vitro. Until recently, RUNX2 and LRP5 had not been directly connected. Studies have revealed the presence of five RUNX2 binding sites within a 2.9 kb region upstream of LRP5 and documented the binding of RUNX2 to these sites in vitro. To further explore this relationship in vivo, in this thesis, an osteoblast differentiation protocol using the hFOB cell line was employed. Transcriptional levels of RUNX2 and LRP5, together with OCN, SOST and ALP were evaluated along 21 days of differentiation in 5 stages. RUNX2 and LRP5 proteins were evaluated along 21 days of differentiation. RUNX2 occupancy of the 5 binding sites on the LRP5 promoter, chromatin immunoprecipitation assays were performed at 3 time-points during hFOB differentiation. Osteoblast differentiation model based on the hFOB displayed characteristic osteoblastic markers of mineralization and alkaline phosphatase activity. \nExpression of LRP5, RUNX2 and also ALP, SOST and OCN was observed in hFOB cell line. RUNX2 showed steady increase reaching a maximum of 4-fold at day 14. All the other genes analyzed showed a peak at day 3, more than 5-fold for SOST and above 4-fold for ALP. OCN and SOST showed a clear decrease after day 3, while ALP showed a slow decrease, and LRP5 maintained relatively similar mRNA levels. Both RUNX2 and LRP5 proteins were detected in hFOB at day 0, 3 and 7. Low levels of LRP5 were also observed at day 14. Overall, the protein levels of both RUNX2 and LRP5 decrease during differentiation of hFOB cell lines. Binding of RUNX2 transcription factor to the promoters of the CDKN1A and SERPINE1 genes was observed in day 7. And RUNX2 binding to CDKN1A and SERPINE1 promoters in hFOB cells was described for the first time. Subsequently, binding of RUNX2 to the five RUNX2 elements in the LRP5 promoter was assessed in chromatin from undifferentiated (day 0) and differentiated (days 7 and 21) hFOB cells. Binding at all five sites was observed at day 7 (above 3-fold enrichment in all cases), while it was negligible at days 0 and 21. No acceptable correlation was observed between RUNX2 binding and LRP5 expression, which leaves the functional effect of RUNX2 binding to the LRP5 promoter as an unsolved question.