Influence Of Endogenous Ligands Of PPARγ2 On MRNA Expression Of Bone Metabolism Related Genes Of Osteoblastic Cells And Bone Marrow Cells

The bone is a dynamic tissue that is continuously being remodeled following two opposite and coordinated processes. Under normal conditions, specialized cells called osteoclasts transiently break down old bone (resorption process) at multiple sites as other cells known as osteoblasts are replacing it with new tissue (bone formation). With aging, alteration of osteoblastic and osteoclastic numbers or functions is thought to compromise the maintenance of bone remodeling equilibrium, and even to be also responsible for senile osteopenia or osteoporosis. Mesenchymal and hematopoetic stem cells (MSCs and HSCs) progenitors which live in bone marrow give rise to bone-forming osteoblasts and bone-resorbing osteoclasts, respectively. Besides, marrow adipocyte is derived from MSCs. Aging inhibits osteogenic differentiation of mesenchymal stem cells and preosteoblasts in favor of adipogenic differentiation, which relates with the high expression of peroxisome proliferator activated receptorγ2 (PPARγ2) gene. It indicates that PPARγ2 gene affects bone metabolism directly. It is supposed that PPARγ2 endogenous ligands level increasing and other inhibiting transcription factors function weakening contributes to high expression of PPARγ2 gene. With aging, some PPARγ2 endogenous ligands, such as polyunsaturated fatty acid, oxidized low-density lipoproteins (Ox-LDL) and so on, increase in vivo, while whether they promote senile osteoporosis or not remains unclear. The aim of the current study is to observe the influence of Ox-LDL, 15d-PGJ2, LTB4, c9,t11-CLA and t10,c12-CLA on mRNA expression of bone metabolism related genes of osteoblastic cells and bone marrow cells, and tentatively to define the mechanism of senile osteoporosis.PartⅠ: Influence of PPARγ2 endogenous ligands on mRNA expression of PPARγ2 and bone metabolism related markers of osteoblastic cellsObjective To observe the effect of oxidized low-density lipoproteins (Ox-LDL), 15-Deoxy-delta(12, 14)-prostaglandinJ2 (15d-PGJ2), leukotrienes B4 (LTB4) and conjugated linoleic acid (c9,t11-CLA and t10,c12-CLA) on mRNA expression of peroxisome proliferator activated receptorγ2 (PPARγ2) , receptor activator of NF-κB ligand (RANKL) , alkaline phosphatase (ALP) and osteoprotegerin (OPG) of osteoblastic cells of rats, and investigate the influence of PPARγ2 endogenous ligands above on bone metabolism.Methods Osteoblastic cells of rats were cultured in vitro and were then cultured for 24h in medium with different PPARγ2 endogenous ligands at different concentrations (the final concentrations of Ox-LDL are 0, 12.5, 25, 50μg/ml; the final concentrations of 15d-PGJ2 are 0, 10, 20, 30μmol/L; the final concentrations of LTB4 are 0, 0.1, 1.0, 10μmol/L; the final concentrations of CLA are 0, 12.5, 25, 50μmol/L). RT-PCR was performed to semi-determine the mRNA expression of PPARγ2, RANKL, ALP, OPG of osteoblastic cells.Results (1) Semi-quantitative RT-PCR analysis showed that Ox-LDL, 15d-PGJ2 and LTB4 all down-regulated the mRNA expression of RANKL, ALP and OPG, while they up-regulated the mRNA expression of PPARγ2 of osteoblastic cells in a dose-dependent manner. Statistical significance was found in interclass comparison (P<0.05, P<0.01). (2) The result also showed that c9,t11-CLA up-regulated the mRNA expression of RANKL and OPG in a dose-dependent manner and statistical significance was found in interclass comparison (P<0.05, P<0.01), but it up-regulated the mRNA expression of ALP, and there was no statistical significance in interclass comparison, while it had no effect on the mRNA expression of PPARγ2. It also showed that t10,c12-CLA up-regulated the mRNA expression of RANKL and OPG in a dose-dependent manner and statistical significance was found in interclass comparison (P<0.05, P<0.01), while it up-regulated the mRNA expression of ALP and down-regulated the expression of PPARγ2, but there is no statistical significance in interclass comparison.Conclusions These findings suggest that Ox-LDL, 15d-PGJ2 and LTB4 suppress the expression of osteogenic genes through activating the transcription activity of PPARγ2, and it may be a plausible mechanism of senile osteoporosis. While c9,t11-CLA and t10,c12-CLA promote the expression of osteoclastogenesis genes, and the result tentatively suggests a possible bene?cial effect on bone formation.PartⅡ: Influence of PPARγ2 endogenous ligands on mRNA expression of PPARγ2 and bone metabolism related markers of bone marrow cellsObjective To observe the effect of oxidized low-density lipoproteins (Ox-LDL), 15-Deoxy-delta(12, 14)-prostaglandinJ2 (15d-PGJ2), leukotrienes B4 (LTB4) and conjugated linoleic acid (c9,t11-CLA and t10,c12-CLA) on mRNA expression of peroxisome proliferator activated receptorγ2 (PPARγ2), receptor activator of NF-κB ligand (RANKL), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of NF-κB (RANK) and tartrate-resistant acid phosphatase(TRAP) of bone marrow cells of rats, and investigate the influence of PPARγ2 endogenous ligands above on bone metabolism.Methods Bone marrow cells of rats were cultured in vitro and were then cultured for 24h in medium with different PPARγ2 endogenous ligands at different concentrations (the final concentrations of Ox-LDL are 0, 25, 50, 100μg/ml; the final concentrations of 15d-PGJ2 are 0, 10, 20, 30μmol/L; the final concentrations of LTB4 are 0, 0.1, 1.0, 10μmol/L; the final concentrations of CLA are 0, 12.5, 25, 50μmol/L). RT-PCR was performed to semi-determine the mRNA expression of PPARγ2, RANKL, ALP, OPG, RANK and TRAP of bone marrow cells.Results (1) Semi-quantitative RT-PCR analysis showed that Ox-LDL,15d-PGJ2 and LTB4 all down-regulated the mRNA expression of RANKL, ALP and OPG, while they up-regulated the mRNA expression of PPARγ2, RANK and TRAP of osteoblastic cells in a dose-dependent manner. Statistical significance was found in interclass comparison (P<0.05, P<0.01). (2)The result also showed that c9,t11-CLA down-regulated the mRNA expression of RANK and TRAP in a dose-dependent manner, and statistical significance was found in interclass comparison (P<0.05, P<0.01), while it had no effect on the mRNA expression of RANKL, ALP, OPG and PPARγ2. It also showed that t10,c12-CLA up-regulated the mRNA expression of RANKL and OPG in a dose-dependent manner and down-regulated the mRNA expression of RANK, TRAP and PPARγ2 in a dose-dependent manner, and statistical significance was found in interclass comparison (P<0.05, P<0.01), while it had no effect on the mRNA expression of ALP.Conclusions Our findings suggest that Ox-LDL, 15d-PGJ2 and LTB4 suppress the expression of osteogenic genes and promote the expression of osteoclastogenesis genes through activating the transcription activity of PPARγ2, and this may be a plausible mechanism of senile osteoporosis. But c9,t11-CLA suppresses the expression of osteoclastogenesis genes, so does t10,c12-CLA, and t10,c12-CLA also promotes the expression of osteogenic genes. The result tentatively suggests a possible bene?cial effect on bone formation.Summary1. Ox-LDL, 15d-PGJ2 and LTB4 suppress the expression of osteogenic genes of osteoblastic cells and bone marrow cells, while they promote the expression of osteoclastogenesis genes through activating the transcription activity of PPARγ2, and this may be a plausible mechanism of senile osteoporosis.2. c9,t11-CLA suppresses the expression of osteoclastogenesis genes, so does t10,c12-CLA, but t10,c12-CLA also promotes the expression of osteogenic genes of osteoblastic cells and bone marrow cells, and the result tentatively suggests a possible bene?cial effect on bone formation.