The Clinical And Animal Study The Role Of FGFRs In Bone And Mineral Metabolism

BackgroundOsteoporosis is a chronic complex metabolism disease characterized by decreasedbone strength that increases the risk of fracture. Osteoporosis has become an importantpublic health problem with increasing incidence rate as ageing population expanding. Themost harmful aspect of osteoporosis is the increased risk of fracture. About70%ofosteoporotic fractures occur in women, but the fracture-related mortality ratio is higher inmen. The mechanisms under this phenomenon remain unknown. Most of previousresearches focus on postmenopausal osteoporosis in women. Considering the genderdifferences of environmental and hereditary factors on osteoporosis, studying thephenotypes of osteoporosis in men is important for understanding the pathogenesis of maleosteoporosis.Decreased bone strength is the key point for the development of osteoporosis. Bonestrength reflects bone density and quality. Bone mineral density (BMD) is the gold criteriafor diagnosis of osteoporosis. BMD predicts about70%of variation of bone strength and isthe most frequently used trait in studying bone strength. Hip geometry can reflect bonestrength independent of BMD. Femoral geometry has been found being an independent riskfactor for hip fracture in both genders. Bone area is significantly associated with anincidence of osteoporotic fracture. Epidemiologic studies found that the area at femoralneck was an independent risk factor for hip fracture. Quantitative ultrasound (QUS) detectsbone architecture and material characters. QUS parameters can reflect bone quality and aredefined as bone strength traits as well.These bone strength traits are regulated by both environmental and hereditary factorswhile the main contributor is genetic variation. The accumulated effects of environmentalfactors on BMD are relatively weak in peak BMD achieved age stage. Studying geneticvariation of bone strength traits in this age stage helps eliminating the effects of*This study is supported by the National Natural Science Foundation of China (No.31000555). non-hereditary factors on BMD. Many candidate genes of bone strength have beenidentified by using candidate gene approaches, linkage analysis and genome-wideassociation studies (GWAS) along with the accomplishment of Human Genome Projectsand development of bioinformatics and statistics. But the results show more discrepancythan agreement among studies based on different populations or races. This suggests weshould study or validate the association genes of bone strength traits in Chinese population.Previous studies indicated the bone development and metabolism related genes couldbe associated with osteoporosis or bone strength traits. Fibroblast growth factor receptors(FGFRs)1,2,3express in bone tissues and regulate bone development and metabolism.Multiple point mutations in these genes lead to human skeletal hereditary diseases whichinvolved skull, spine, thoracic and limbs. The SNPs of FGFR1and FGFR2were foundassociated with BMD in white elder people. Considering the genetic variation among bonestrength traits, genetic heterogeneity of races and differences of environmental factorsbetween European and Chinese, it’s necessary to indentify the association between FGFRsSNPs and bone strength traits in Chinese young men.Our lab found the three FGFRs affected bone mechanical properties and BMD throughregulating the biologic activity of multiple types of bone cells. FGFRs can also influenceBMD by regulating phosphate metabolism. FGF23, mainly produced by osteocytes, bindsto FGFR1,3,4coupling with Klotho in kidney and depresses renal phosphate reabsorption.FGF23also have effect on intestinal phosphate absorption in a vitamin D receptor (VDR)dependent way. But it’s not clear the role of FGFRs in intestinal phosphate absorption.This study will firstly analyze the association between FGFRs SNPs and bone strengthtraits in Chinese young men, and investigate the effect of FGFR1on intestinal phosphateabsorption and the related mechanisms as well. Furthermore, the effect of alteration ofphosphate metabolism on bone strength will be detected.Main Methods1. Analysis of environmental and hereditary factors affecting bone strength traitsin Chinese young men of Han ethnicity1.1Subjects and body measurementA total of812Chinese male army recruits of Han ethnicity were involved in this study.Height (cm), weight (kg), waist circumference (WC)(cm), hip circumference (HC)(cm), BMI was measured. A questionnaire recorded age, daily calcium intake and activity, bonediseases of family members and so on.This study received ethical approval from the Ethics Committee of Daping Hospital,Third Military Medical University, China.1.2Bone strength traits measurementBMD (g/cm2) and bone area (cm2) at lumbar (L1-4), left femoral Neck (FN) and totalhip (TH) were measured using DXA (Prodigy, GE). Left hip geometry: hip axis length(HAL)(mm), neck–shaft angle (Angle)(o), femoral strength index (SI), cross-sectional area(CSA)(mm2) and cross-sectional moment of inertia (CSMI)(mm4) were assessed using HipStructure Analysis (HSA) software (Prodigy, GE). Speed of sound (SOS)(m/s), broadbandultrasound attenuation (BUA)(Db/MHz) and stiffness index (QUS-SI) were measuredusing QUS (SONOST2000) at left calcaneus.1.3Serum bone metabolism markers (Tracp5b, BALP) were quantified using ELISAkit.1.4The genotypes of FGFRs were determined by iMLDR assays.1.5Statistic analysisAll the data was represented as mean±SD. Bartlett tests were used to evaluate thehomogeneity of variance. Shapiro-Wilks tests were applied to analyze the distribution of allthe quantitative parameters. Chi-square tests were performed to evaluate theHardy-Weinberg equilibrium of genotypes. PS v3.0.43software was used to calculatedpower. Haploview4.2software was performed to conduct haplotypes of FGFR1. Pearson’scorrelation tests were performed to analyze the relationship between bone metabolismmarkers and BMD. Stepwise regression analyses were applied to bone strength traits usingage, height, weight, WC, HC, BMI as covariates. ANOVA analyses were used to test thedifferences of age, height, weight, WC, HC and BMI among genotypes. ANCOVA analyseswere performed to evaluate the association between genotypes of FGFRs and bone strengthtraits as well as haplotypes of FGFR1and BMD. P<0.05was considered significant.2. The role of FGFR1in intestinal development and absorption of calcium andphosphate2.1Animals: Conditional knockout of FGFR1in intestinal villus epithelial cells(FGFR1-vil) and the littermate controls (WT). 2.2Measurements of weight, intestinal length and circumference, villus height andcrypt depth after birth.2.3BrdU labeling was used to determine the proliferation of epithelial cells. Thenumbers of goblet cells and Paneth’s cells were counted after AB-PAS staining. ALPstaining was used to evaluate the differentiation of enterocytes. TUNEL assay wasperformed to detect the apoptosis of intestinal villus epithelial cells.2.4Immunohistochemistry or immunofluorescence was used to observe theexpressions of FGFR1, NPT2b, P-ERK and P-JNK in intestine.2.5Safranine O/fast green (SO/FG) staining was used to evaluate the changes of boneremodeling of tibia. The differentiation and activation of osteoclasts were detected withtartrate resistant acid phosphatase (TRAP) staining. micro-CT scanning was performed todetermine the bone content at proximal trabecular bone and midshaft cortical bone offemurs.2.6Serum, urine and fecal phosphate and calcium of mice were measured after beingfed with diet contained different contents of phosphate.2.7Real-time PCR was performed to detect the mRNA expression levels of calciumabsorption related genes (calbindin D9k, TRPV6, VDR), phosphate absorption/reabsorption related genes (NPT2a,2b,2c), FGFR1and IGF1in intestinal villus or kidneycortex tissues.2.8Western bolt was used to quantify the protein contents of P-P38and NPT2b inintestine after being fed with diet contained different contents of phosphate.2.9Luciferase activity was determined in CaCO2cells after cotransfection of FGFR1siRNA and NPT2b promotor plasmids.2.10Statistic analysis: Independent t-tests were performed to analyze the differencebetween FGFR1-vil and WT mice. P<0.05was considered significant.Main results1. Analysis of environmental and hereditary factors affecting bone strength traitsin Chinese young men of Han ethnicity1.1Environment factors affecting BMDAge, weight, WC and HC were significantly correlated with BMD and BMCrespectively (P<0.05). All of the peak BMD at three sites was found in the22years age group. Geographic factor contributed to the variation of BMD. The mean L1-4BMD insouthwestern group was significantly lower than that in northern group and southeasterngroup (P<0.05). We also found ethnic difference in Asia countries. The mean peak BMD atL1-4in this group was higher than that in both Korean and Indian men. But the mean peakBMD of Chinese men at hip was lower than of Indian men.Military training increased BMD at three sites. Comparing with baseline, serum levelsof TRACP5b (P<0.05) were significantly decreased while elevated levels of BALP(P<0.001) were detected after training. Serum concentration of TRACP5b and BALP werenegatively related with age (P<0.001). The variation ratio of BALP was positively relatedwith that of BMD at L1-4and TH (P<0.05).1.2Environment factors affecting bone area, hip geometry and QUS parametersAge was the positive regulator of bone area at L1-4(P<0.05). There was no significantdifference of bone area at HN and TH among age groups.Age was the major factor contributed to variations of CSA and CSMI withanthropometric traits as covariants. All the mean CSA and CSMI in21-23years age groupsare significantly higher than that in17or18years age group (P<0.05). Both mean SI in22and23years age group were significantly higher than that in18years age group (P<0.05).There was no significant relationship between age and HAL or Angle.Age was significantly related with variation of three QUS parameters (P<0.05). Thepeak values of SOS, BUA and QUS-SI were found in23years age group. All the mean SOSand BUA in20-23years age groups are significantly higher than that in17-19years agegroups (P<0.05).2. Association between FGFRs SNPs and BMDrs1047111located at exon1of FGFR2was associated with BMD at L1-4(P=0.013)and FN (P=0.0012). A four loci haplotype in FGFR1constructed by rs2956724, rs6983315,rs6474354and rs4733930had no significant association with BMD.2.1Association between FGFRs SNPs and bone areaIn FGFR1gene, rs2288696located at intron4, rs2956724and rs6474354located atintron3were associated with bone area of FN while rs10958704in5’-Flanking wasassociated with that of TH (P=0.006,0.028,0.025,0.043respectively).2.2Association between FGFRs SNPs and hip geometry Two SNPs of FGFR1was associated with hip geometry parameters: rs2288696inintron4with CSA and CSMI while rs10958704in5’-Flanking with SI (P=0.049,0.035,0.027respectively).2.3Association between FGFRs SNPs and QUS traitsAll the associated SNPs of QUS parameters were found in FGFR1. rs4733946inintron2was associated with SOS (P=0.017) and QUS-SI (P=0.025). rs2956724in intron3was associated with BUA (P=0.000) and QUS-SI (P=0.026) while rs10958704in5’-Flanking with SOS (P=0.023).3. The role of FGFR1in intestinal development and absorption of calcium andphosphate3.1Conditional knockout FGFR1in intestinal epithelial cells lead to reducedcircumference of intestineThe efficiency of gene knockout was confirmed using real-time PCR and IFC.Expression levels of FGFR1in intestinal villus of FGFR1-vil mice were decreased about70%compared with that of WT mice. The body weight and intestine length had no statisticdifference between groups.Compared with WT mice, FGFR1-vil mice exhibited decreased intestinecircumference and increased villus height, but no significant change of crypts depth.Depressed mRNA expression levels of IGF1, known as a regulator of intestinal diameter,were detected in intestines of FGFR1-vil mice. The mRNA expression levels of IGF1andFGFR1were positively related which implied that FGFR1may mediate intestinal diameterexpansion through regulating expression of IGF1in subepithelial myofibroblasts.3.2FGFR1enhanced apoptosis but not proliferation or differentiation ofintestinal epithelial cellsNo remarkable difference in location and numbers of BrdU positive cells was observedbetween FGFR1-vil and WT mice which suggested FGFR1inactivation didn’t seem toaffect epithelial cells proliferation. The numbers of goblet cells and Paneth’s cells inFGFR1-vil mice were not significantly different from that in WT mice. ALP activity whichreflected numbers of enterocytes in FGFR1-vil mice was similar to that in WT mice. Thissuggested that intestine-specific FGFR1inactivation may have no effect on goblet cell,Paneth’s cell and enterocyte differentiation. TUNEL staining found a significant reduction of apoptosis in FGFR1-vil mice which implied FGFR1positively regulated epitheliaapoptosis.3.4Conditional knockout of FGFR1in intestine epithelial cells depressedactivation of MAPK-P38pathwayIFC staining found similar activated signals of P-ERK between two genotypes. Almostno P-JNK positive cells were observed in FGFR1-vil intestine while few ones were foundin intestine of WT mice. WB detected decreased P-P38protein levels in intestinal epitheliaof FGFR1-vil mice than that in WT mice. This suggested MAPK-P38may be the mainpathway contributing to the effects of FGFR1on intestinal development.4. FGFR1inhibited intestinal phosphate absorption through pre-transcriptionalregulating NPT2b expression in the diet phosphate-and VDR-independent way4.1Conditional knockout of FGFR1in intestinal epithelial cells maintainednormal serum phosphate by increasing intestinal absorption and reducing renalreabsorptionCompared with WT mice, serum calcium and phosphate in FGFR1-vil mice had nosignificant change in normal diet condition (0.9%Pi).Higher increased ratio of serumphosphate was detected in FGFR1-vil mice than that in WT mice in a hyperphosphatemiamodel induced by intragastric administration of0.5M NaH2PO4(P<0.05). In long-term highphosphate diet condition (1.25%Pi), elevated serum phosphate was found in FGFR1-vilmice while normal serum phosphate was detected in WT mice (P<0.05). Significantlyincreased urine phosphate and decreased fecal phosphate were detected in FGFR1-vil witheither normal diet or low phosphate diet (P<0.05). This suggested that increased intestinalphosphate absorption in absence of FGFR1lead to reduced renal phosphate resorptionwhich helped normal serum phosphate maintenance.4.2Deletion of FGFR1in intestine upregulated NPT2b expression in intestinalepithelia and downregualted NPT2a expression in renal cortexBoth increased mRNA and protein expression levels of NPT2b were detected inFGFR1-vil mice. Low phosphate diet upregulated protein expression levels of NPT2b inWT mice. High phosphate diet had opposite effects on that in WT mice. The similarresponses were found in FGFR1-vil mice. But the variation ratio of NPT2b in FGFR1-vilmice was significantly lower than that in WT mice. The promoter activity of NPT2b was reduced after transfection of FGFR1siRNA in CaCO2cells. This suggested FGFR1negatively pre-transcriptionally regulatd NPT2b expression in a diet phosphate-independentway.Increased mRNA expression level of NPT2a, known as a phosphate transporter locatedat proximal tubules, was detected in kidney cortical tissues of FGFR1-vil mice. The mRNAexpression level of NPT2c was not significantly different from that in WT mice. Thissuggests abnormal intestinal phosphate absorption may regulate renal phosphatereabsorption by altering NPT2a mRNA expression.No significant difference of mRNA expression levels of calcium absorption relatedgenes (calbindin D9k, VDR and TRPV6) was found between FGFR1-vil and WT mice.This suggested FGFR1may not contribute to intestinal active absorption of calcium.4.3Conditional knockout of FGFR1in intestinal epithelial cells didn’t affect bonephenotypesSO/FG staining found no abnormal pathologic change in FGFR1-vil tibias. TRAPstaining indicated the differentiation and activation of osteoclasts were similar betweenFGFR1-vil and WT mice. micro-CT traits at proximal trabecular bone and midshaft corticalbone of femurs had no significant changes in FGFR1-vil compared with that in WT mice.Conclusions1. BMD of Chinese young men is affected by exercise, geography and race. PeakBMD at three sites (L1-4, FN and TH) are observed in22years age group.2. Age is the major factor influencing bone area, hip geometry and QUS parameters ofChinese men in the period of peak BMD achieved.3. FGFR1is a multiple effect gene associated with bone area, bone geometry and bonequality. FGFR2is the association gene of BMD in Chinese young men.4. FGFR1increases apoptosis of intestinal epithelial cells and positively regulatesintestinal diameter expansion by activating MAPK-P38pathway.5. FGFR1inhibits intestinal phosphate absorption through pre-transcriptionallydepressing NPT2b expression in the diet phosphate-and VDR-independent way.