Wnt/β-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone
2006; Elsevier BV; Volume: 281; Issue: 42 Linguagem: Inglês
10.1016/s0021-9258(19)84086-3
ISSN1083-351X
AutoresJohn A. Robinson, Moitreyee Chatterjee‐Kishore, Paul J. Yaworsky, Diane M. Cullen, Weiguang Zhao, Christine Li, Yogendra P. Kharode, Linda Sauter, Philip Babij, Eugene L. Brown, Andrew A. Hill, Mohammed P. Akhter, Mark L. Johnson, Robert R. Recker, Barry S. Komm, Frederick J. Bex,
Tópico(s)Cancer-related gene regulation
ResumoA preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/β-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/β-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/β-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3β inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3β inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/β-catenin signaling is a normal physiological response to load and that activation of the Wnt/β-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading. A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/β-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/β-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/β-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3β inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3β inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/β-catenin signaling is a normal physiological response to load and that activation of the Wnt/β-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading. Over the past few years the Wnt/β-catenin-signaling pathway has been shown to be an important component of bone mass accrual, regulation, and maintenance. Key to this understanding were the identification of inactivating mutations in LRP5 resulting in an osteoporosis pseudoglioma syndrome (1Gong Y. Slee R.B. Fukai N. Rawadi G. Roman-Roman S. Reginato A.M. Wang H. Cundy T. Glorieux F.H. Lev D. Zacharin M. Oexle K. Marcelino J. Suwairi W. Heeger S. Sabatakos G. Apte S. Adkins W.N. Allgrove J. Arslan-Kirchner M. Batch J.A. Beighton P. Black G.C. Boles R.G. Boon L.M. Borrone C. Brunner H.G. Carle G.F. Dallapiccola B. De Paepe A. Floege B. Halfhide M.L. Hall B. Hennekam R.C. Hirose T. Jans A. Juppner H. Kim C.A. Keppler-Noreuil K. Kohlschuetter A. LaCombe D. Lambert M. Lemyre E. Letteboer T. Peltonen L. Ramesar R.S. Romanengo M. Somer H. Steichen-Gersdorf E. Steinmann B. Sullivan B. Superti-Furga A. Swoboda W. van den Boogaard M.J. Van Hul W. Vikkula M. Votruba M. Zabel B. Garcia T. Baron R. Olsen B.R. Warman M.L. 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We further show that activation of the pathway by treatment with a GSK-3β inhibitor results in an anabolic bone formation response and that use of this inhibitor in combination with mechanical loading produces a synergistic effect on the expression of Wnt/β-catenin pathway target genes. These results strongly implicate the Wnt/β-catenin-signaling pathway as being a critical component of the bone response to mechanical loading. Reagents—Calcein, hematoxylin, and eosin were obtained from Sigma-Aldrich. Fetal bovine serum (FBS), heat-inactivated FBS, α-minimum essential medium, Dulbecco's modified Eagle's medium, penicillin, streptomycin, l-glutamine, glutamax, and Geneticin were purchased from Invitrogen. Bovine serum albumin was purchased from Serologicals Proteins Inc. (Kankakee, IL). In Vivo Loading of Tibiae in LRP5 G171V Transgenic and Non-transgenic Littermates—All animal protocols were conducted with approval of the Wyeth and Creighton University Institutional Animal Care and Use Committees. The heterozygous LRP5 G171V transgenic mice have been described and show a statistically significant increase in bone density (5Babij P. Zhao W. Small C. Kharode Y. Yaworsky P.J. Bouxsein M.L. Reddy P.S. Bodine P.V. Robinson J.A. Bhat B. Marzolf J. Moran R.A. Bex F. J. Bone Miner. Res. 2003; 18: 960-974Crossref PubMed Scopus (443) Google Scholar). Non-transgenic littermates were used as controls. There were a total of 15 animals/sex/genotype in each group. At 17 weeks of age all animals were anesthetized to permit proper leg positioning before loading. Using a 4-point bending device (23Akhter M.P. Wells D.J. Short S.J. Cullen D.M. Johnson M.L. Haynatzki G.R. Babij P. Allen K.M. Yaworsky P.J. Bex F. Recker R.R. Bone (NY). 2004; 35: 162-169Crossref PubMed Scopus (123) Google Scholar), the mechanical loading regimen (∼2500 microstrain) 8Microstrain is defined as a unit of strain that is the percentage of change in length or relative deformation (10,000 microstrain = 0.01 strain = 1% deformation). delivered to the right tibiae (the left tibiae served as the non-loaded controls) was 6N for females and 7N for males (36 cycles, 2 Hz), which ensured that all mice experienced similar levels of maximal compressive and tensile strains during bending loads. RNA from the right tibiae was collected at 4 or 24 h after application of load. Tibiae from 5 mice were pooled to compose a single group. Three replicate groups for each treatment/genotype were analyzed. Cell Culture—MC3T3-E1 osteoblastic cells, used in the in vitro mechanical loading experiments, were cultured in αMEM supplemented with 10% heat inactivated fetal bovine serum, 1% glutamax, and 1% penicillin/streptomycin. Wnt3A-conditioned media was obtained from an overexpressing Wnt3A stable murine L-cell line (ATCC, Manassas, VA) that was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% l-glutamine, and 0.4 mg/ml Geneticin. To obtain Wnt3A-conditioned media, cells were seeded into 100-mm dishes and cultured for 4 days in growth medium without Geneticin, the medium was removed and sterile-filtered, and fresh medium was added to the plates and cultured for an additional 3 days. The medium was then removed and sterile-filtered and combined with the initial batch of cultured media. Control-conditioned medium was obtained in a similar fashion using the parental L-M(TK-) cell line (ATCC, Manassas, VA). The Wnt3A-conditioned media activated canonical Wnt signaling in MC3T3-E1 cells as determined using a T-cell factor-luciferase reporter transiently transfected into these cells (10 μl of conditioned media showed a 10-fold induction of reporter activity compared with control media-treated and untreated cells (data not shown). RNA Isolation—The mouse tibiae were dissected free of soft tissue, and the proximal and distal metaphysis were removed leaving the diaphysis. The tibiae were then cut transversely with bone cutters to expose the trabecular bone and the bone marrow. The trabecular bone and marrow cavities were flushed with ice-cold sterile phosphate-buffered saline to remove the marrow, and the clean bone was placed in liquid nitrogen. A Bessman tissue pulverizer (Fisher) rinsed in 100% ethanol and precooled in liquid nitrogen was used to reduce the tibiae to a powder. Total RNA (2 μg/tibia) was isolated from non-loaded and loaded bones using the ToTALLY RNA kit (Ambion, Austin TX) as per the manufacturer's instructions. Ten μg of total RNA was treated with 4 units of DNase I (Ambion) to remove any genomic DNA contamination. To isolate RNA from MC3T3-E1 cells the cultures were washed twice with 2 ml each of phosphate-buffered saline, and then the RNA was isolated using the QIAshredder and the RNeasy kit (Qiagen, Valencia, CA) as described by the manufacturer. The RNA was treated with 27 units of DNase I (Qiagen) on the RNA isolation column provided in the kit as described by the manufacturer. Quantitative Real-time RT-PCR (TaqMan®)—A two-step TaqMan protocol was used. RNA was first converted to cDNA at 37 °C for 2 h (High Capacity cDNA Archive kit, Applied Biosystems). TaqMan PCR reactions were performed on an ABI Prism 7700 DNA sequence detector (Applied Biosystems) using 20 ng of cDNA/reaction. The conditions for TaqMan PCR were 2 min at 50 °C, 10 min at 95 °C, then 40 cycles each of 15 s at 95 °C and 1 min at 60 °C on MicroAmp Optical 96-well plates covered with MicroAmp Optical caps. Each plate contained triplicates of the test cDNA templates and no-template controls for each reaction mix. The expression for each mouse gene was normalized to murine glyceraldehyde-3-phosphate dehydrogenase. A list of TaqMan probe-primer pairs used in this study can be found in Table 1.TABLE 1TaqMan primer and probe sequencesGeneForward primerReverse primerProbePtgisTGGCTTCGGTCTGATGCACCCAGGTGAGTCTGCTCCATCCAGAGGAAGACGTGCCCATCCGeNOSTCTGCGGCGATGTCACTATGGCCCTCTGTTGCCAGAATTCAGCGTCCTGCAAACCGTGCACOX-2AGGCTGTTGGAATTTACGCATAACATGCTTGGGTCAGTCAATATTGAGCAGACTGCATAGATc-FosGCCTCTGCACAGCAATTCCTCAGCTTCAGGGTAGGTGAAGACACCATGGTCACAGAGCc-JunTCCACGGCCAACATGCTCGTGGTTCATGACTTTCTGCTTAGGAACAGGTGGCACALrp5CCCCTCTATGACCGGAATCACCGGATATAGTGTGGCCTTTGTGCATCCAGCAGCTCGTLrp6CCACCGGGACATGTAAATACACAGCACCCACCCACTTTATAATGTTAACTGCCTCACTCTTCCxn43GGCCGGAAGCACCATCTTGGCTGTCGTCAGGGAAATCCAACTCCCACGCCCAGCCGTTSFRP4TGGAGCCACCCTTACAGGATGCAAGTGGTATGTGGCCTTCTGAGGCTGTCCCAGGCAGCACCASFRP1CCCTCCAAGGCTTGAGTAAAAGAGCACATGCATAGGCGGTGTATCGTTGACTGCCCAAGGCTGCCCCND1GAGAAATGTACTCTGCTTTGCTGAAGGGCTGTAGGCACTGAGCAAAGGCCCTCAGCCTCACTCCCTGGWISP2GGTGACCTTGTAAGTGTGCCTTTTCCATCTCTTCATGTTCCCAGAATCTGAGAACACCCTGCCCGGCTFzD2GCCCGACTTCACAGTCTACATGGCCGGACCAGATCCAGAACCGACGTGATGCCCACGATGAWnt10BCCTCGGGCTCAGGTTCCTAAAGAGGAGTGGCCAAAAGATAGACTCCCTATCCAAAGGAAG Open table in a new tab Calvariae Treatment and Histochemistry and Immunohistochemistry Analysis—The GSK-3β inhibitor (GSK3βi) (3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione) (36Coghlan M.P. Culbert A.A. Cross D.A. Corcoran S.L. Yates J.W. Pearce N.J. Rausch O.L. Murphy G.J. Carter P.S. Roxbee Cox L. Mills D. Brown M.J. Haigh D. Ward R.W. Smith D.G. Murray K.J. Reith A.D. Holder J.C. Chem. Biol. 2000; 7: 793-803Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar) at 1 mg/kg or vehicle was injected subcutaneously daily for the first 7 days over the right side of the calvaria in 4-week-old male Swiss-Webster mice. For each treatment group that contained 8 animals each, calcein (15 mg/kg) was administered subcutaneously on day 1 and again on day 8 for mineral apposition rate (MAR) determination. On day 10 the calvariae were removed, and the bones were fixed in 70% ethanol for 24 h. The anterior portion of the calvaria was paraffin-embedded, whereas the posterior portion was used for frozen sections. The paraffin sections were stained with hematoxylin and eosin for measurement of calvarial thickness. To calculate mineral apposition rates linear measurements of single label surface (SLS), double label surface (DLS), and bone surface (BS) were taken, and the equation DLS + (1/2 SLS)/BS × 100 was used to calculate percent mineralized surface/bone surface. Measurements were made on unstained 6-μm sections at 20× magnification. All measurements were made using the Bioquant Image Analysis System (Bioquant, Nashville, TN). For immunohistochemical analysis of β-catenin, calvaria were decalcified in Surgipath Decalcifier II (Surgipath, Richmond, IL) for 7-8 h, dehydrated in graded alcohol, and sectioned. Nonphosphorylated β-catenin was detected using a mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). Signal was detected using the avidin-linked AP system (Vector Laboratories, Burlingame, CA). Endogenous alkaline phosphatase was detected histochemically using the Vector Red alkaline phosphatase substrate kit (Vector Laboratories) in 6-μm frozen sections of the mouse parietal bone after fixation in 70% ethanol. Effect of Systemic GSK3βi Administration on the in Vivo Response to Mechanical Load—17-Week old female, wild type C57Bl6 mice were injected with GSK3βi 50 mg/kg/BID or vehicle (control) subcutaneously twice daily for 14 days. There were 15 animals per group. The right tibiae were loaded at 6N for 36 cycles at 2 Hz. The left tibiae served as unloaded controls. The animals were sacrificed at 4 h post-load, and the tibia was isolated and flash-frozen in liquid nitrogen. Tibiae (five) were pooled from each group to provide three replicates per group. The RNA was purified from tibiae (loaded and unloaded). Transcriptional analyses were performed by TaqMan® on samples from the tibiae on selected load- and Wnt-response genes. In Vitro Mechanical Loading—MC3T3-E1 cells were plated at 80,000-100,000 cells per well in a type I collagen-coated Bioflex 6-well plate (Flexcell International Corp., McKeesport, PA) and then cultured for 3-4 days or until confluent. Twenty-four hours before loading the cells were washed twice with 2 ml of basal α-minimum essential medium (αMEM) before adding 2 ml of fresh serum-free media containing αMEM, 0.25% bovine serum albumin, glutamax, and penicillin/streptomycin. Immediately before mechanical loading, the medium was removed, and 1 ml of α-minimum essential medium/bovine serum albumin with or without GSK3βi, Wnt3A-conditioned media or control-conditioned media was added to each well. The cells were subjected to mechanical distortion equivalent to 3400 microstrain (2 Hz, 7200 cycles/h) for 5 h using a FX-3000 Flexercell® strain unit (Flexcell International Corp). RNA was harvested immediately or 24 h post-loading from both the mechanical-strained samples as well as the non-strained controls. Statistical Analysis—The data are represented as the mean ± S.D. For those data comparing non-loaded versus loaded results, unpaired 2-tailed t-tests was performed. To compare strain and GSK-3β inhibitor or Wnt3A, multiple comparisons of 2-factor analysis of variance was performed. A Tukey HSD multiple comparisons was then performed between each dose of the GSK-3β inhibitor or Wnt3A with strain versus the strain only treatment. Application of in Vivo Mechanical Load Induces Transcription of Wnt/β-Catenin Pathway Genes—It has been previously reported that the LRP5 G171V transgenic mice have increased bone formation compared with non-transgenic mice (5Babij P. Zhao W. Small C. Kharode Y. Yaworsky P.J. Bouxsein M.L. Reddy P.S. Bodine P.V. Robinson J.A. Bhat B. Marzolf J. Moran R.A. Bex F. J. Bone Miner. Res. 2003; 18: 960-974Crossref PubMed Scopus (443) Google Scholar), and mechanical loading increases bone formation. Given the fact that this mutation leads to altered regulation of the Wnt/β-catenin pathway, we tested the hypothesis that downstream targets of the pathway would have exaggerated changes in expression in response to loading compared with normal mice. We performed a preliminary analysis of gene expression profiles from tibial osteoblast explant cultures from LRP5 G171V versus non-transgenic mice and identified several differentially expressed genes including Wnt10B, secreted frizzled-related protein 1 (SFRP1), SFRP2, Dickkopf-3 (Dkk3), cyclin D1 (CCND1), and Wnt1-inducible signaling pathway protein 2 (WISP2) associated with the mutation (data not shown). We next examined whether the expression of these Wnt/β-catenin target genes were regulated by mechanical loading. Transcriptional analysis of tibia samples from LRP5 G171V mice after in vivo mechanical loading by four-point bending resulted in expected increases in the mRNA expression of known stress-responsive genes including prostaglandin synthase (COX-2), prostacyclin synthase (Ptgis), and endothelial nitric-oxide synthase (eNOS) (37Pitsillides A.A. Rawlinson S.C. Suswillo R.F. Bourrin S. Zaman G. Lanyon L.E. FASEB J. 1995; 9: 1614-1622Crossref PubMed Scopus (303) Google Scholar, 38Tang L.Y. Cullen D.M. Yee J.A. Jee W.S. Kimmel D.B. J. Bone Miner. Res. 1997; 12: 276-282Crossref PubMed Scopus (55) Google Scholar) (Fig. 1). The expression of these genes was increased 4 h post-loading in wild type and LRP5 G171V transgenic mice tibiae; however, the response was 4-10-fold greater in the transgenics (Fig. 1). This response was attenuated 24 h post-loading (data not shown). In non-transgenic mice, transcription of Wnt10b, SFRP1, CCND1, connexin 43 (Cxn43), and WISP2 genes was significantly increased (∼2-4-fold, p < 0.01) at both 4 and 24 h after in vivo mechanical loading (Fig. 2, A and B). RNA levels of frizzled 2 (FzD2) was increased 2.8-fold (p < 0.01) at 4 h post-load but returned to base line by 24 h. Expression of LRP5 was not affected by load in this model, whereas LRP6 expression was marginally increased in male LRP5 G171V animals at 4 h post-load (Fig. 2, A, and B). In LRP5 G171V transgenic mice, a more significant and sustained increase (3-30-fold, p < 0.01) in the transcription of the Wnt/β-catenin target genes analyzed was observed, including SFRP4, which was not regulated by mechanical loading in wild type mice (Fig. 2, A and B). Unlike the pattern demonstrated in the non-transgenic mice, there was an increased expression of FzD2 at both 4- and 24-h post-load. Thus, application of mechanical load increased expression of Wnt/β-catenin-regulated genes, and the response was significantly greater in the LRP5 G171V tra
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