| Osteoporosis has become an increasingly serious global problem. The pathogenesis of osteoporosis is complicated and there is lack of effective prevention and therapy of osteoporosis. Vitamin D can regulate bone metabolism in an indirect way via controlling calcium and phosphate homeostasis but also via direct effects on osteoblasts. The biologically most active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D] has been shown to stimulate bone formation and mineralization in ex vivo cultures of human osteoblasts. However, the underlying mechanism for 1,25(OH)2D-mediated direct regulation of osteoblasts has yet to be clearly defined. The classic function of heterogeneous nuclear ribonucleoprotein C1/C2( hn RNPC1/C2) is to play a role in post-transcription RNA processing, which including constitutive splicing and alternative splicing. Previous studies from our group have demonstrated a role for hn RNPC1/ C2 as an additional component of the VDR transcription complex, in which hn RNPC1/C2 compete in trans with VDR for occupation of the vitamin D response elements(VDREs) within proximal or distal promoter regions of target genes. Based on the above information, We hypothesize that hn RNPC1/C2 provides a link between the transcriptional activity of 1,25(OH)2D and downstream RNA splicing. In the current study we have utilized primary human osteoblasts(h OBs) and MG63 osteoblastic cell lines as models, in combination with genome wide analyses, to assess the role of hn RNPC1/C2 in coordinating transcriptional and RNA splicing responses to 1,25(OH)2D.Studies have shown the relationship between osteoporosis and cardiovascular diseases in recent years. Both of them occurred in the geriatric population and are frequently o bserved in the same geriatric patient. Moreover, the incidents of both grow with age. Even though age is the common risk factor for both osteoporosis and cardiovascular diseases, most of the studies found that there was still a significant relationship between the two diseases regardless of age. On the one hand, cardiovascular diseases are related with bone loss and increased risk of bone fracture. On the other hand, evidence showed that decreased bone mineral density lead to the increased incidence and mortality of cardiovascular diseases. Further studies revealed a closer and direct relationship between cardiovascular diseases and osteoporosis on the level of pathogenesis. Atherosclerosis is the main pathological basis for cardiovascular diseases and artery calcification is one of its main signs. Therefore, arteries have been regarded as a substantial clinical monitoring factor and a risk factor for cardiovascular diseases. Researches have shown that the mechanism of vascular calcification is the transformation of vascular smooth muscle cells to osteoblasts, as well as that of vascular tissue to bone. Meanwhile vascular calcification is significantly related with bone mineral loss. The current study investigated the relationship between artery calcification, osteoporosis and bone fracture in geriatric population by observing a group of patients over 60 years.MethodsPart I1. We overexpressed hn RNPC1/C2 in h OBs with or without 1,25(OH) 2D treatment, q PCR was used to evaluate the 1,25(OH)2D induced expression of RUNX2,BGLAP,SPP1 and COL1A1.2. We utilized CYP24A1 as a model target gene, chromatin immunoprecipitation(Ch IP) and RNA immunoprecipitation(RIP) were performed to analyze the interaction of VDR and hn RNPC1/C2 with CYP24A1 upstream VDRE as well as CYP24A1 m RNA.3. Two different human hn RNPC1/C2 stealth select si RNA were used to knockdown of hn RNPC1/C2 in MG63 osteoblastic cell line. q PCR and fluorescently labeled PCR were used to assess the functional impact of hn RNPC1/C2 on vitamin D-mediated transcription and prem RNA splicing.4. Further studies were carried out using high throughput RNA sequencing MG63 cells, to explore the genome-wide impact of hn RNPC1/C2 on 1,25(OH)2D-induced transcription and alternative splicing and the potential link between transcription and alternative splicing in response to 1,25(OH)2D.Part II1. Patients over 60 years who visited hospital between 2012.01.01 and 2012.12.31 and met requirements were enrolled in the current study.2. Semi-quantitative measurements of aortic artery calcification: evaluate calcification according to the abdominal aortic calcification deposits at the level of the L1-L4 lumbar vertebrae on the lateral X-Ray lumbar images. Based on the length of deposits and number of involved vertebrae, the aortic calcification score(ACS) varied from 0 to 24 for each patient, with 0 standing for no calcification and 24 for the most severe calcification. Patients were divided into four groups according to ACS scores: G1, ACS = 0; G2, ACS= 1-2; G3, ACS=3-6; G4, ACS > 6.3. Dual-energy X–ray absorptionmetry(DXA) was adopted for the measurement of bone mineral density. Osteoporosis was defined as following: a bone mineral density measured by DXA as 2.5 standard deviations or more below the average peak bone mass of the same gender and race population.4. A multivariate regression analysis model was used to evaluate the relationship between aortic calcification and the risk of osteoporosis.Part III1. This part of study was carried out at the same time with Part II study with postmenopausal women over 60 years who met the requirements.2. Semi-quantitative measurements of aortic calcification were the same as that in Part II.3. Diagnosis of bone fracture: Vertebra fracture was diagnosed by observing the shape of the T4 thoracic vertebrae to L5 lumbar vertebrae on X-Ray(more than 20% reduction in vertebra height).4. A multivariate regression analysis model was used to evaluate the relatio nship between aortic calcification and vertebrae fracture.Part IV1. This part of study was carried out at the same time with Part II study with patients over 60 years who met the requirements as subjects.2. The bone mineral density measurement and the diagnosis of osteoporosis were the same as that in part II.3. Carotid and coronary artery atherosclerotic calcification examine: Carotid and coronary CTA(CT angiography) were performed on a 64-row spiral CT scanner. All CTA images were evaluated on a 3D image analysis workstation. The composition and range of artery plaques were evaluated as well.4. A multivariate regression analysis model was used to evaluate the relationship between osteoporosis, bone loss and the risk of calcification plaque in caroti d and coronary arteries.ResultsPart I1. Overexpression of hn RNPC1/C2 in h OBs inhibited 1,25(OH)2D-induced transactivation of RUNX2, BGLAP.2. In MG63 osteoblastic cells, chromatin association of hn RNPC1/C2 was reciprocal with VDR, in apparent competition for VDRE binding; binding of hn RNPC1/C2 to CYP24A1 pre-m RNA or mature m RNA was independent of VDR and VDREs.3. Knockdown of hn RNPC1/C2 in MG63 osteoblastic cells was associated with dysregulated expression of CYP24A1 and aberrant alternative splicing of CYP24A1.4. RNA-seq identified 324 differentially expressed genes(DEG) specifically induced or suppressed by 1,25(OH)2D in MG63 osteoblastic cells: with 187 were unaffected by hn RNPC1/C2 knockdown, whilst 137 were insensitive to 1,25(OH)2D following in hn RNPC1/C2 knockdown. Distinct from these changes, another 168 DEG were exclusively induced by 1,25(OH)2D in combination with hn RNPC1/C2 knockdown.5. RNA-seq identified 310 alternative splicing(AS) with 1,25(OH)2D treatment in MG63 osteoblastic cells. The number of AS events induced by 1,25(OH)2D(310) was enhanced following hn RNPC1/C2 knockdown. Relative to knockdown alone, combined knockdown and 1,25(OH)2D resulted in 390 AS event, with only 3(<1%) of these being common to 1,25(OH)2D alone.6. Of the 324 DEG induced by treatment with 1,25(OH)2D, only 9 genes were also alternative spliced(alternatively spliced genes, ASG). This changed following hn RNPC1/C2 knockdown. Comparison of 1,25(OH)2D-treated cells with cells receiving combined knockdown and 1,25(OH)2D, showed 3572 DEG and 2962 ASG, with 811 genes showing combined altered expression and AS.Part II1. 1448 subjects were enrolled in this study. The mean age was 72.0±4.5. 397 of them(27.4%) developed osteoporosis.2. Compared with the control group, the mean age of the osteoporosis group was older and there were more female subjects(P < 0.001). The bone densities of vertebrae, femoral neck, trochanter, and hip were lower in the osteoporosis group(P < 0.01). The levels of Serum N-terminal propeptide of type I procollagen(PINP), cross-linked C-telopeptide of type I collagen(CTX), total deoxypyridinoline(Total DPD), free deoxypyridinoline(Free DPD) were higher(P<0.05 for PINP and CTX, P<0.01 for Total DPD and Free DPD), while the level of 25-hydroxyvitamin D [25(OH)D] was lower(P<0.01) in the osteoporosis group.3. After adjustment for age and other potential cofounders, ACS(G4), BMD, 25(OH)D and CTX were closely related with the risk of osteoporosis. The risk of osteoporosis increased with the level of ACS. However, this was only significant in the comparison of ACS level of the groups G4 and G1. Higher ACS increased the risk of osteoporosis in both female and male groups.Part III1. Of the 1359 postmenopausal women, 173(12.7%) suffered from fractures with 92 cases of vertebrae fracture and 81 cases of non-vertebrae fracture.2. The prevalence of spine fracture was significantly higher in the aortic calcification group than that in the control group(P<0.01).3. After adjusting for age and other confounders, vertebrae facture was found to be closely related with bone mineral density, age, history of more than 2 falls, 25(OH)D and adiponectin. Compared with ACS group G1, the G3 and G4 groups had higher risk of spine fracture.Part IV1. Of the enrolled 1393 aged patients, 474 had artery calcification plaque, including 158(33.4%) cases of carotid artery calcification plaque, 195(41.1%) cases of coronary artery calcification plaque and 121(25.5%) cases of co-existing carotid and coronary artery calcification plaque.2. After adjusting for age and other confounders, the risk of artery calcification plaque was closely related with osteoporosis, low bone mass and decreased level of 25(OH)D.3. After adjusting for age and other confounders, severe loss of bone mass was significantly related with the increased risk of carotid, coronary and co-existing artery calcification plaques.Conclusion:1. 1,25(OH)2D regulate gene expression by influencing both transcription and m RNA splicing. hn RNPC1/C2 can be involved in and coordinate both transcription and splicing action in response to 1,25(OH)2D. Overexpression of hn RNPC1/C2 significantly inhibited the trans-activation of bone formation marker RUNX2 and BGLAP by 1,25(OH)2D in osteoblasts.2. In the geriatric population, severe aortic calcification increased the risk of osteoporosis. The risk of osteoporosis increased with the level of ACS. The decreased level of bone mineral density and 25(OH)D were both closely related with the risk of osteoporosis.3. In aged postmenopausal women, severe aortic calcification could increase the risk of vertebrae fractures. The decreased bone mineral density and 25(OH) D were closely related with the risk of vertebrae fractures.4. In the geriatric population, osteoporosis, low bone mass and decreased level of 25(OH) D were closely related with artery calcification plaque. Sever bone loss was significantly related with the risk of carotid, coronary and co-existing calcification plaque.The current study revealed the molecular mechanism of the direct regulation of vitamin D on osteoblasts and provided us a novel perspective of the role of vitamin D in bone. The deep exploration of the pathogenesis of osteoporosis possesses substantial significance in looking for new drug treatment targets. Meanwhile, the observational study in a group of geriatric popultion suggested multiple common risk factors and inherent relationships of age related diseases, which is of great importance in the prevention and treatment of osteoporosis and cardiovascular diseases. |
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