E XP ER I ME NTAL C E LL RE S E ARCH
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Q1 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways Xinhua Yina, Xiaoyuan Wangb, Xiongke Hua, Yong Chena, Kefeng Zenga, Hongqi Zhanga,n a
Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha, China Department of Nephrology, Xi An Honghui Hospital, Xi an, China
b
article information
abstract
Article Chronology:
Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms
Received 14 February 2015
are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major
Received in revised form
signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway
22 March 2015
and estrogen signaling pathways have been reported in many tissues. In this study, E2
Accepted 28 April 2015
significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-
Keywords:
induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast
17β-estradial (E2)
differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression
Wnt/β-catenin signaling
were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immu-
β-catenin GSK3β
noprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid
Osteoblast
enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest
ERβ
that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. & 2015 Published by Elsevier Inc.
Introduction
in vitro [6,7]. 17β-estradial (E2), a polyphenolic phytoestrogen, on bone cell function has been studied in numerous cell models. E2 has
Estrogen is known to control the processes of bone remodeling during the reproductive life in women [1–3]. Estrogen deficiency is a major determinant of age-related bone loss [4] and it could result in enhanced bone resorption leading to osteoporosis in postmenopausal women [5] and estrogen stimuli promote osteoblastic differentiation
been shown to inhibit bone resorption and enhance bone formation in the tibia and femur in ovariectomized rats [8]. E2 also could protected against bone loss through suppress osteoblasts apoptosis and promote osteoblasts differentiation [9,10]. It has been shown that E2 induced-osteoblast proliferation might be mediated by insulin-like
n
Corresponding author. E-mail address: [email protected] (H. Zhang).
http://dx.doi.org/10.1016/j.yexcr.2015.04.020 0014-4827/& 2015 Published by Elsevier Inc.
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
94 95 96 97 98 99 100 101 102 103 104 105 106 107
2
108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
E XP E RI ME N TAL CE L L R ES E ARC H
growth factor I (IGF-I) [11]. Recent study showed that E2 regulates osteoblastic differentiation in human periodontal ligament cells through oestrogen receptor beta [12]. However, the precise molecular mechanisms underlying the observed the effects of E2 on osteoblastic differentiation are still not fully understood. The Wnt/β-catenin signaling pathway has been demonstrated to be responsible for a variety of biological processes, including tissue homeostasis and cancer. Recently, the role of Wnt/β-catenin signaling pathway in bone formation has been elucidated, and it appears as an important signaling cascade for osteoblast differentiation, bone formation and homeostasis. In the canonical Wnt/ β-catenin signaling pathway, binding of Wnt ligand to its coreceptors Frizzled (Fz) receptor, a seven-transmembrane protein, and a low-density lipoprotein receptor-related protein (LRP) lead to the destruction of a complex that consist of glycogen synthase kinase-3β (GSK-3β), axin and adenomatous polyposis coli (APC) [13]. Disruption of this complex leads to phosphorylation of GSK3β, thus leading to the stabilization of β-catenin through dephosphorylation. Subsequent translocation of β-catenin into the nucleus leads to interaction with Tcf/Lef transcription factors and up-regulation of key osteoblastic genes [14,15]. Accumulating studies demonstrated that the Wnt/β-catenin signaling plays important role in osteogenesis in vitro and in vivo. Mutation with low-density lipoprotein receptor-related protein 5 (LRP5) was shown to cause osteoporosis pseudoglioma syndrome [16], which is characterized by low bone mass. In contrast, mutant mice that overexpressed the constitutive active LRP5 (G171V) in osteoblasts exhibited an enhanced osteoblastic activity, a reduction in osteoblast apoptosis, and high bone mass phenotype [17]. Recently, the interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. Several physiological consequences associated with estrogen signaling, such as neuronal development [18] and bone formation [19], are mediated by the activation of the Wnt/βcatenin signaling pathway. For example, estradiol increased cytosolic β-catenin protein levels and activated the transcriptional activity of Wnt/β-catenin signaling by an inhibition of GSK-3 functions in neuroblastoma cells and primary cortical neurons [18]. Also, in bone cells, the transcriptional activity of Wnt/βcatenin signaling was activated by binding between the TCF and estrogen receptor [20]. Therefore, the regulation of the Wnt/βcatenin signaling pathway and estrogen replacement therapy are considered as the effective treatments for bone loss. In this study, we reported for the first time that E2 induces preosteoblastic cell proliferation and differentiation via ER/GSK3β-dependent Wnt/β-catenin signaling pathway, which subsequently activated the downstream osteogenesis-related gene transcription, CyclinD1, BGP, ALP and OPN.
Materials and methods
] (]]]]) ]]]–]]]
Western blot Protein lysate were separated by 10% SDS-polyacrylamide gel electrophoresis and then electroblotted onto PVDF membranes. The anti-ERβ anti-β-catenin, anti-phospho-β-catenin (Ser33/37/ Thr41), anti-phospho-GSK3 (Ser-9), anti-GSK-3, anti-BGP, antiALP, anti-cyclin D1, anti-TCF and anti-OPN antibodies were purchased from Sigma-Aldrich, with β-actin antibody (ABZOOM) as an internal control. The signal was detected using the enhanced SuperSignal West Pico chemiluminescent.
RNA extraction and qRT-PCR Total RNA was extracted from MC3T3-E1 cells using TRIzol reagent according to the recommendations of the manufacturer with a slight modification. cDNA synthesis was conducted using the iScript TM Select cDNA synthesis kit according to theprotocol of the manufacturer (Bio-Rad). Quantitative real-time PCRs of ERβ were performed on an equal amount of cDNA using KAPA SYBRs FAST quantitative PCR kit according to the instructions of the manufacturer (Kapa Biosystems) with the ABI PRISM 7500 sequence detection system and analysis software (Applied Biosystems). The primers used were as follows: Mus-ERβ, 50 -GAAGCTGGCTGACAAGGAAC-30 and 50 -AACGAGGTCTGGAGCAAAGA-30 ; β-actin, 50 -AGCCATGTACGTAGCCATCC30 and 50 -CTCTCAGCTGTGGTGGTGAA-30 .
Transfection of MC3T3-E1 cells with siRNAs The siRNAs were designed and synthesized by GenePharma. Four siRNAs that were synthesized to target ER2 expression were used in this experiment: siRNA-1 (siRNA-Esr2-992) sense 50 -UCAUUAUGUCCUUGAAUGCTT-30 ; antisense 50 -GCAUUCAAGGACAUAAUGATT-30 . siRNA-2 (siRNA-Esr2-2218) sense 50 -UUAGAACUCAGCAUUCAGCTT; antisense 50 -GCUGAAUGCUGAGUUCUAATT-30 . siRNA-3 (siRNAEsr2-2625) sense 50 -AUAAUCUAGUUAUGUAAGCTT; antisense 50 GCUUACAUAACUAGAUUAUT-30 . Scramble control siRNAs sense 50 UUCUCCGAACGUGUCACGUTT. antisense 50 -ACGUGACACGUUCGGAGAATT-30 . siRNAs transfection was performed using Lipofectamine 2000 (Invitrogen). Total protein assay was prepared 72 h after transfection for protein gel blot analysis.
MTT assay The 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to estimate cell viability [21]. Briefly, cells were plated at a density of 1 104 cells per well in 96-well plates. After exposure to specific treatment, the cells were incubated with MTT at a final concentration of 0.5 mg/ml for 4 h at 37 1C. After the removal of the medium, 150 mM DMSO solutions were added to dissolve the formazan crystals. The absorbance was read at 570 nm using a multi-well scanning spectrophotometer reader. Cells in the control group were considered 100% viable.
Cell culture and transfection Alizarin Red-S staining Mouse preosteoblastic (MC3T3-E1) cells were purchased from Insitute of Biochemistry and Cell Biology (Shanghai, China) and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were cultured at 37 1C in a humidified atmosphere of 5% CO2.
After incubation in the differentiation culture for the designated time, the differentiation of MC3T3-E1 cells was monitored by Alizarin Red-S staining. MC3T3-E1 samples were washed with PBS three times and then fixed with ice-cold 70% ethanol for 1 h
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227
E X PE R IM EN TA L C ELL R E S EA RC H
228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287
on ice. Subsequently, the cells were washed with distilled H2O and stained with Alizarin Red-S for 20 min at room temperature. To quantify the relative differentiation, their absorbance at 520 nm was measured in a spectrometer (Tecan infinite M200) after several washes with distilled H2O.
Immunofluorescence staining Primary rat osteoblasts were grown on glass coverslips and incubated with fluoride for 72 h. The cells were washed with cold PBS twice, fixed in ice-cold methanol and permeabilized with 0.5% Triton X-100 for 15 min. Cells were then blocked with 0.5% bovine serum albumin for 1 h at room temperature. Samples were then incubated with mouse monoclonal anti-dephosphorylated βcatenin antibody (1:500) or anti-TCF4 antibody overnight at 4 1C followed by incubation with TRITC-conjugated secondary antibody and DAPI. The signal was visualized by confocal fluorescence microscopy (Olympus, Japan).
Immunoprecipitation Cell lysates were incubated with the indicated antibodies in the presence of 15 ml of protein G-agarose beads for 3 h at 4 1C. After washed five times, the immunoprecipitates were subjected to electrophoresis. Protein expression was examined by probing Western blots of total cell lysates or immunoprecipitates with the appropriate antibodies as noted in the figure legends.
Statistical analysis Statistical significance was determined by a two-tailed unpaired Student's t test or one-way ANOVA followed by post hoc (Bonferroni) multiple comparisons between treatment groups using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, ca.). po0.05 was considered statistically significant.
Results E2 affects the activities of GSK-3β and β-catenin in preosteoblastic cells Estradiol (E2) has been reported to activate Wnt/β-catenin activity in neuronal cells [22] and bone cells [17]. In this study, we examined the effects of E2 on endogenous Wnt/β-catenin signaling in preosteoblastic cells (MC3T3-E1). MC3T3-E1 were treated with 0.1 μM E2 for 0, 30, 60, 90 and 120 min. Western blot analysis demonstrated the expression of β-catenin and p-β-catenin. As shown in Fig. 1, E2 (0.1 mM) exhibited time-dependent activations of Wnt/β-catenin signaling. E2 induced a significant increase in the β-catenin protein level, which peaked at 90 min and declined substantially by 120 min after treatment. On the contrary, the expression level of p-β-catenin was gradually downregulated and reached bottom at 90 min after E2 (0.1 mM) treatment. Because GSK-3β phosphorylates β-catenin and prevents TCF-activated transcription activity, we next determined whether the activation of Wnt/β-catenin signaling mediated by E2 involves GSK-3β. As shown in Fig. 1, E2 greatly promoted GSK-3β phosphorylation at serine 9, but it failed to affect GSK-3β expression. Collectively, these
] (]]]]) ]]]–]]]
3
results suggested that E2 mediates Wnt/β-catenin signaling by phosphorylating GSK-3β and promoting the accumulation of intraQ3 cellular levels of the β-catenin protein in preosteoblastic cells. GSK-3β, p-GSK-3β.
ERβ involves E2-mediated activation of GSK-3β and βcatenin in preosteoblastic cells Because previous study showed that ER involves in Wnt/β-catenin signaling in osteoblastic cells [19] and E2 binds to both ERα and β with high affinity [23], we next determined whether the activation of Wnt/β-catenin signaling mediated by E2 involves ERβ. In this work, we transfected MC3T3-E1 cells with ERβ siRNA and with negative control siRNA containing scrambled random sequence prior to treatment with E2 for 90 min. We examined four siRNAs to ERβ as described above. qRT-PCR analysis showed that siRNA-2 and siRNA-3 effectively blocked the expression of ERβ mRNA. qRT-PCR revealed dramatic reduction of 84.1% with siRNA-2, 78.1% with siRNA-3, and 21.2% with siRNA-1 in the levels of ERβ mRNA after transfection of siRNA in MC3T3-E1 cells, compared with MC3T3-E1 negative control siRNA or the control (untransfected) group (po0.05; Fig. 2A). As shown in Fig. 2B, ERβ siRNA-2 also significantly decreased the level of β-catenin protein. Thus, the cells transfected with ERβ siRNA-2 were used for further experiments. Then, we determine the effect on E2-mediated Wnt/ β-catenin signaling. As shown in Fig. 2C, ERβ siRNA reversed ERinduced upregulation of β-catenin and downregulation of p-βcatenin. Moreover, the induction of GSK-3β phosphorylations mediated by E2 was abolished in the presence of siRNA treatment. These data strongly suggested that ERβ is required for the activation of Wnt/β-catenin signaling by E2 in MC3T3-E1 cells.
Induction of preosteoblastic cell proliferation and differentiation by E2 requires ERβ Because Wnt/β-catenin signaling regulates cell proliferation and differentiation of multiple tissues, including bone cells, we then assessed the proliferative effects of E2 and ERβ on preosteoblastic cells using an MTT assay after treatment with E2 for 90 min. As shown in Fig. 3A, E2 significantly increased cell proliferation, but the cell proliferation was decreased by pretreatment with siRNA. To investigate the effect of E2 on osteoblastic differentiation of MC3T3-E1 cells, mineralization was determined by Alizarin Red S staining. As shown in Fig. 3B, E2 treatment alone significantly promoted mineralized nodule formation, which was reversed by siRNA treatment. Next, we detected the effect of E2 on cyclin D1, bone GLa protein (BGP), alkaline phosphatase (ALP), T-cell factor (TCF) and osteopontin (OPN ) by western blot. As shown in Fig. 3C, E2 and ERβ siRNA failed to affect the expression of TCF. In contrast, BGP, ALP, cyclin D1 and OPN expression levels were significantly increased by E2 treatment, but the expression were reversed by siRNA. Collectively, our results strongly suggested that E2 increases preosteoblastic cell proliferation and differentiation via ERβ.
E2 induces the formation of β-catenin/Tcf protein complex in MC3T3-E1 cells The accumulation of β-catenin in the nucleus is an important step in the activation of the Wnt/β-catenin signaling pathway and
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347
4
348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407
E XP E RI ME N TAL CE L L R ES E ARC H
] (]]]]) ]]]–]]]
Fig. 1 – E2 affects the activities of GSK-3β and β-catenin in preosteoblastic cells. MC3T3-E1 were treated with 0.1 μm E2 for 0, 30, 60, 90 and 120 min. Western blot analysis demonstrated the expression levels of β-catenin, p-β-catenin, GSK-3β and p-GSK-3β. Data are represented as mean7S.E. (n¼ 4). *po0.001 and **po0.05 *po0.01 vs. 0 min group.
Fig. 2 – ERβ involves E2-mediated activation of GSK-3β and β-catenin in preosteoblastic cells. (A) qRT-PCR analyzed the expression of ERβ after transfection with different ERβ siRNAs in MC3T3-E1 cells. (B) The western blot analysis showed the expression of βcatenin protein after siRNA interference. (C) Western blot analysis demonstrated the effect of ERβ on the expression of GSK-3β, pGSK-3β, β-catenin and p-β-catenin. MC3T3-E1 cells were transfected with ERβ siRNA prior to treatment with 0.1 lM E2. Data are the mean7SD of duplicates from a representative experiment of three independent experiments. *po0.01 vs. control group and ** po0.01 vs. E2 group or E2þNC-siRNA group.
translocation of β-catenin into the nucleus leads to interaction with Tcf/Lef transcription factors and up-regulation of key osteoblastic genes that are Wnt targets. The effect of E2 on subcellular localization of β-catenin and its interaction with Tcf/Lef transcription factors were further explored by immunofluorescence and immunoprecipitation in MC3T3-E1 cells. Consistent with Western blot result in Fig. 3C, immunofluorescence analysis showed that E2 and ERβ siRNA failed to affect the expression of TCF (Fig. 4A), and β-catenin immunostaining was increased significantly after
treatment with 0.1 mM E2 for 90 min (Fig. 4A). Interestingly, the staining was more intense and localized in the nucleus after treated with E2 compared with control group (Fig. 4A). However, accumulation ofβ-catenin in the nucleus were greatly attenuated with ERβ siRNA prior to treatment with E2. PKF118-310, a natural compound of microbial origin, is capable of disrupting the formation of β-catenin/Tcf protein complex. To further confirm that E2 involved the interaction between β-catenin and TCF protein, immunofluorescence was performed in MC3T3-E1 cells
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467
E X PE R IM EN TA L C ELL R E S EA RC H
468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527
] (]]]]) ]]]–]]]
5
Fig. 3 – Induction of preosteoblastic cell proliferation and differentiation by E2 requires ERβ. MC3T3-E1 cells were transfected with ERβ siRNA prior to treatment with 0.1 lM E2. (A) E2 increases MC3T3-E1 cell proliferation. The proliferation of MC3T3-E1 cells was observed by MTT assay. (B) E2-induced preosteoblastic cell differentiation. Mineralization was determined by Alizarin Red S staining. (C) E2 and ERβ affect osteoblastic differentiation-related gene expression. Data are the mean7SD of duplicates from a representative experiment of three independent experiments. *po0.01 vs. control group and **po0.01 vs. E2 group or E2þNCsiRNA group.
using PKF118-310. As shown in Fig. 4A, PKF118-310 successfully abolished E2-induced accumulation of β-catenin in the nucleus. Consistent with immunofluorescence results, immunoprecipitation analysis showed that E2 induced the formation of β-catenin/ Tcf protein complex which was attenuated and abolished by ERβ siRNA and PKF118-310, respectively. These data demonstrated that E2 activate Wnt/β-catenin signaling by promoting nuclear localization of β-catenin protein and formation of β-catenin/Tcf protein complex.
Discussion Both Wnt/β-catenin and estrogen receptor signaling have currently been recognized as important regulators of bone mass and bone cell differentiation. Estrogen is a key regulator of bone formation. 17β-estradial (E2), a polyphenolic phytoestrogen, acts as a stimulator of bone formation in vivo [24]. However, the underlying mechanisms are not fully understood. In this study, our results strongly suggested that E2 promotes preosteoblastic cell proliferation and differentiation through the activation of ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. The Wnt/β-catenin signaling pathway has been demonstrated to be responsible for a variety of biological processes, including
tissue homeostasis and cancer. Cytosolic β-catenin protein is the principal mediator of Wnt signaling [25]. In the activation of Wnt/ β-catenin signaling pathway, β-catenin is dephosphorylated and accumulated in the cytoplasm. In this study, we found that E2 significantly increased the β-catenin protein level and decreased the p-β-catenin expression level. Serine/threonine protein kinase GSK3β is a key negative regulator of β-catenin in the Wnt signaling pathway, it phosphorylates and promotes the degradation of β-catenin in quiescent cells [26]. It has been shown that phosphorylation of GSK-3β leads to inhibition of GSK-3β thus resulting in the stimulation of the Wnt pathway [13]. E2 induced the phosphorylation of Akt at serine 473 and subsequently activated the phosphorylation of GSK-3β at serine 9, leading to inhibition of GSK-3β activity and, hence, activation of Wnt/βcatenin signaling [27]. Consistent with these studies, E2 could also induce phosphorylation of GSK-3β at the serine 9 residue, whereas no significant change in GSK-3β protein expression in preosteoblastic MC3T3-E1 cells. Non-phosphorylated β-catenin accumulated in the cytoplasm and translocates into the nucleus, binds with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF), and activates target gene expression. The immunofluorescence and immunoprecipitation analysis showed that E2 significantly induced translocation of β-catenin into nucleus and formation of β-catenin/Tcf protein complex. These
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587
6
588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647
E XP E RI ME N TAL CE L L R ES E ARC H
results indicated that E2 involved the activation of Wnt/β-catenin signaling by inducing phosphorylation of GSK-3β. Although a previous study reported that, in the absence of mechanical strain, E2 failed to induce β-catenin protein expression and transcription activity in ROS17/2.8 cells [19]. This discrepancy could be due to the difference in the cell type used. Estrogen receptor (ER) signaling pathway has been shown to involve in osteoblastic differentiation, the activation of the ER signaling pathway can occur in a ligand-dependent way by binding of estradiol (E2) to intracellular ERs as well as independently of E2-binding by cross-talk with peptide growth factors [28]. Recently, the interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. Several physiological consequences associated with estrogen signaling, such as neuronal development [18] and bone formation[19], are mediated by the activation of the Wnt/βcatenin signaling pathway. In our study, the siRNA technique was used to inhibit ERβ expression. ERβ siRNA significantly suppressed phosphorylation of GSK-3β, the activate GSK-3β phosphorylated β-catenin and attenuated the accumulation of β-
] (]]]]) ]]]–]]]
catenin. This result indicated that ERβ involves E2-mediated activation of Wnt/β-catenin signaling in preosteoblastic cells. In this study, E2 significantly induced preosteoblastic cell proliferation and differentiation. Osteoblast differentiation is accompanied by the increased expression of bone matrix proteins, such as ALP, BGP and OPN. These osteoblast differentiation factors stimulate mineralization and lead to bone formation. In this study, it was clearly shown that BGP, ALP and OPN expressions were significantly stimulated by E2 in preosteoblastic cells. On the other hand, it was evident that E2-mediated increase on BGP, ALP and OPN expressions were diminished in ERβ siRNA transfected cells prior E2 treatment. BGP is a specific marker for bone formation. As BGP synthesized from bone tissue, half of it deposited in the bone matrix and half in blood circulation, so serum osteocalcin measurements can provide a noninvasive specific marker of bone metabolism [29]. ALP and OPN, as other bone formation marker, observed several cell types including preosteoblastic cells [30]. Studies have proved that ASPP 049 can induce osteoblast differentiation, ALP activity and increase bone mineralization [27]. These osteoblast differentiation factors stimulate mineralization and lead to bone formation. CyclinD1 is up-regulated
Fig. 4 – E2 induces the formation ofβ-catenin/Tcf protein complex in preosteoblastic (MC3T3-E1) cells. (A) subcellular localization of β-catenin and its interaction with Tcf/Lef transcription factors were further explored by immunofluorescence. (B) β-catenin/Tcf association was detected by immunoprecipitate. MC3T3-E1 cells were transfected with ERβ siRNA prior to treatment with 0.1 lM E2. Lysates were immunoprecipitated with anti-β-catenin and immunoblotted with anti-TCF4 antibody. Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707
E X PE R IM EN TA L C ELL R E S EA RC H
708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767
as an important promoter of cell cycle [31], and upregulation of cyclinD1 insulin is involved in osteoclast proliferation [32]. E2 significantly induced preosteoblastic cell proliferation by increasing cyclinD1 expression. Overall, E2 promoted osteoblast proliferation and differentiation by regulating proliferation factors (CyclinD1) and osteoblast differentiation factors (BGP, ALP and OPN) by activating the ER/ GSK-3β-dependent Wnt/β-catenin signaling pathways. Our findings provide novel molecular mechanism by which estrogen may promote bone formation.
[12]
[13]
[14]
Conflict of interest We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.
[15]
[16]
Acknowledgments Q4 This publication was funded in part by the National Natural
Science Foundation of China (81271940) and Key Project of Natural Science Foundation of Hunan Province (12JJ2043).
references [1] R. Rizzoli, J.P. Bonjour, Hormones and bones, Lancet 349 (Suppl 1) (1997) sI20–23. [2] E. Sturm, Liver, hormones, and bones, J. Pediatr. Gastroenterol. Nutr. 54 (2012) 308–309. [3] B.L. Riggs, S. Khosla, L.J. Melton 3rd, A unitary model for involutional osteoporosis: estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men, J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 13 (1998) 763–773. [4] E. Seeman, Estrogen therapy and variable-resistance weight training, J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 7 (1992) 709–711. [5] A. Zallone, Direct and indirect estrogen actions on osteoblasts and osteoclasts, Ann. N. Y. Acad. Sci. 1068 (2006) 173–179. [6] H. Kim, A. Tabata, T. Tomoyasu, T. Ueno, S. Uchiyama, K. Yuasa, A. Tsuji, H. Nagamune, Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells, J. Bone Miner. Metab. (2014). [7] Y. Matsumoto, F. Otsuka, M. Takano, T. Mukai, R. Yamanaka, M. Takeda, T. Miyoshi, K. Inagaki, K.E. Sada, H. Makino, Estrogen and glucocorticoid regulate osteoblast differentiation through the interaction of bone morphogenetic protein-2 and tumor necrosis factor-alpha in C2C12 cells, Mol. Cell Endocrinol. 325 (2010) 118–127. [8] A. Tomkinson, J. Reeve, R.W. Shaw, B.S. Noble, The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone, J. Clin. Endocrinol. Metab. 82 (1997) 3128–3135. [9] Y.H. Yang, K. Chen, B. Li, J.W. Chen, X.F. Zheng, Y.R. Wang, S.D. Jiang, L.S. Jiang, Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway, Apoptosis Int. J. Program. Cell Death 18 (2013) 1363–1375. [10] S.C. Manolagas, S. Kousteni, R.L. Jilka, Sex steroids and bone, Recent Prog. Horm. Res. 57 (2002) 385–409. [11] M. Ernst, J.K. Heath, G.A. Rodan, Estradiol effects on proliferation, messenger ribonucleic acid for collagen and insulin-like growth
[17] [18]
[19]
[20] [21]
[22]
[23]
[24]
[25]
] (]]]]) ]]]–]]]
7
factor-I, and parathyroid hormone-stimulated adenylate cyclase activity in osteoblastic cells from calvariae and long bones, Endocrinology 125 (1989) 825–833. A. Mamalis, C. Markopoulou, A. Lagou, I. Vrotsos, Oestrogen regulates proliferation, osteoblastic differentiation, collagen synthesis and periostin gene expression in human periodontal ligament cells through oestrogen receptor beta, Arch. Oral Biol. 56 (2011) 446–455. J. Mao, J. Wang, B. Liu, W. Pan, G.H. Farr 3rd, C. Flynn, H. Yuan, S. Takada, D. Kimelman, L. Li, D. Wu, Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway, Mol. Cell 7 (2001) 801–809. C.N. Bennett, K.A. Longo, W.S. Wright, L.J. Suva, T.F. Lane, K.D. Hankenson, O.A. MacDougald, Regulation of osteoblastogenesis and bone mass by Wnt10b, Proc. Natl. Acad. Sci. USA 102 (2005) 3324–3329. T. Gaur, C.J. Lengner, H. Hovhannisyan, R.A. Bhat, P.V. Bodine, B.S. Komm, A. Javed, A.J. van Wijnen, J.L. Stein, G.S. Stein, J.B. Lian, Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression, J. Biol. Chem. 280 (2005) 33132–33140. Y. Gong, R.B. Slee, N. Fukai, G. Rawadi, S. Roman-Roman, A.M. Reginato, H. Wang, T. Cundy, F.H. Glorieux, D. Lev, M. Zacharin, K. Oexle, J. Marcelino, W. Suwairi, S. Heeger, G. Sabatakos, S. Apte, W.N. Adkins, J. Allgrove, M. Arslan-Kirchner, J.A. Batch, P. Beighton, G.C. Black, R.G. Boles, L.M. Boon, C. Borrone, H.G. Brunner, G.F. Carle, B. Dallapiccola, A. De Paepe, B. Floege, M.L. Halfhide, B. Hall, R.C. Hennekam, T. Hirose, A. Jans, H. Juppner, C.A. Kim, K. Keppler-Noreuil, A. Kohlschuetter, D. LaCombe, M. Lambert, E. Lemyre, T. Letteboer, L. Peltonen, R.S. Ramesar, M. Romanengo, H. Somer, E. Steichen-Gersdorf, B. Steinmann, B. Sullivan, A. Superti-Furga, W. Swoboda, M.J. van den Boogaard, W. Van Hul, M. Vikkula, M. Votruba, B. Zabel, T. Garcia, R. Baron, B.R. Olsen, M.L. Warman, G. Osteoporosis-Pseudoglioma, Syndrome collaborative, LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development, Cell 107 (2001) 513–523. V. Krishnan, H.U. Bryant, O.A. Macdougald, Regulation of bone mass by Wnt signaling, J. Clin. Invest. 116 (2006) 1202–1209. O. Varea, M.A. Arevalo, J.J. Garrido, L.M. Garcia-Segura, F. Wandosell, P. Mendez, Interaction of estrogen receptors with insulinlike growth factor-I and Wnt signaling in the nervous system, Steroids 75 (2010) 565–569. V.J. Armstrong, M. Muzylak, A. Sunters, G. Zaman, L.K. Saxon, J.S. Price, L.E. Lanyon, Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha, J. Biol. Chem. 282 (2007) 20715–20727. J.J. Westendorf, R.A. Kahler, T.M. Schroeder, Wnt signaling in osteoblasts and bone diseases, Gene 341 (2004) 19–39. B. Liu, W. Che, J. Xue, C. Zheng, K. Tang, J. Zhang, J. Wen, Y. Xu, SIRT4 prevents hypoxia-induced apoptosis in H9c2 cardiomyoblast cells, Cell. Physiol. Biochem. Int. J. Exp. Cell. Physiol. Biochem. Pharmacol. 32 (2013) 655–662. O. Varea, J.J. Garrido, A. Dopazo, P. Mendez, L.M. Garcia-Segura, F. Wandosell, Estradiol activates beta-catenin dependent transcription in neurons, PloS One 4 (2009) e5153. G.G. Kuiper, J.G. Lemmen, B. Carlsson, J.C. Corton, S.H. Safe, P.T. van der Saag, B. van der Burg, J.A. Gustafsson, Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta, Endocrinology 139 (1998) 4252–4263. A. Plant, A. Samuels, M.J. Perry, S. Colley, R. Gibson, J.H. Tobias, Estrogen-induced osteogenesis in mice is associated with the appearance of Cbfa1-expressing bone marrow cells, J. Cell. Biochem. 84 (2002) 285–294. M.D. Gordon, R. Nusse, Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors, J. Biol. Chem. 281 (2006) 22429–22433.
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827
8
828 829 830 831 832 833 834 835 836 837 838 839 840
E XP E RI ME N TAL CE L L R ES E ARC H
[26] D. Wu, W. Pan, GSK3: a multifaceted kinase in Wnt signaling, Trends Biochem. Sci. 35 (2010) 161–168. [27] K. Bhukhai, K. Suksen, N. Bhummaphan, K. Janjorn, N. Thongon, D. Tantikanlayaporn, P. Piyachaturawat, A. Suksamrarn, A. Chairoungdua, A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3beta proteindependent activation of the Wnt/beta-catenin signaling pathway, J. Biol. Chem. 287 (2012) 36168–36178. [28] J.M. Hall, J.F. Couse, K.S. Korach, The multifaceted mechanisms of estradiol and estrogen receptor signaling, J. Biol. Chem. 276 (2001) 36869–36872. [29] W. Zhou, Z. Liu, J. Yao, F. Chi, K. Dong, X. Yue, T. Teng, X. Rausch, The Effects of Exenatide Microsphere on Serum BGP and ALP
] (]]]]) ]]]–]]]
Levels in ZDF Rats after Implantation, Clin. Implant. Dent. Relat. Res. (2013). [30] A.C. Akman, R. Seda Tigli, M. Gumusderelioglu, R.M. Nohutcu, Bone morphogenetic protein-6-loaded chitosan scaffolds enhance the osteoblastic characteristics of MC3T3-E1 cells, Artif. Org. 34 (2010) 65–74. [31] S. Korulu, A. Yildiz-Unal, M. Yuksel, A. Karabay, Protein kinase C activation causes neurite retraction via cyclinD1 and p60-katanin increase in rat hippocampal neurons, Eur. J. Neurosci. 37 (2013) 1610–1619. [32] J.Y. Lee, N.K. Lee, Up-regulation of cyclinD1 and Bcl2A1 by insulin is involved in osteoclast proliferation, Life Sci. 114 (2014) 57–61.
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
841 842 843 844 845 846 847 848 849 850 851 852
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Q1 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways Xinhua Yina, Xiaoyuan Wangb, Xiongke Hua, Yong Chena, Kefeng Zenga, Hongqi Zhanga,n a
Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha, China Department of Nephrology, Xi An Honghui Hospital, Xi an, China
b
article information
abstract
Article Chronology:
Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms
Received 14 February 2015
are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major
Received in revised form
signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway
22 March 2015
and estrogen signaling pathways have been reported in many tissues. In this study, E2
Accepted 28 April 2015
significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-
Keywords:
induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast
17β-estradial (E2)
differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression
Wnt/β-catenin signaling
were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immu-
β-catenin GSK3β
noprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid
Osteoblast
enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest
ERβ
that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. & 2015 Published by Elsevier Inc.
Introduction
in vitro [6,7]. 17β-estradial (E2), a polyphenolic phytoestrogen, on bone cell function has been studied in numerous cell models. E2 has
Estrogen is known to control the processes of bone remodeling during the reproductive life in women [1–3]. Estrogen deficiency is a major determinant of age-related bone loss [4] and it could result in enhanced bone resorption leading to osteoporosis in postmenopausal women [5] and estrogen stimuli promote osteoblastic differentiation
been shown to inhibit bone resorption and enhance bone formation in the tibia and femur in ovariectomized rats [8]. E2 also could protected against bone loss through suppress osteoblasts apoptosis and promote osteoblasts differentiation [9,10]. It has been shown that E2 induced-osteoblast proliferation might be mediated by insulin-like
n
Corresponding author. E-mail address: [email protected] (H. Zhang).
http://dx.doi.org/10.1016/j.yexcr.2015.04.020 0014-4827/& 2015 Published by Elsevier Inc.
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
94 95 96 97 98 99 100 101 102 103 104 105 106 107
2
108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
E XP E RI ME N TAL CE L L R ES E ARC H
growth factor I (IGF-I) [11]. Recent study showed that E2 regulates osteoblastic differentiation in human periodontal ligament cells through oestrogen receptor beta [12]. However, the precise molecular mechanisms underlying the observed the effects of E2 on osteoblastic differentiation are still not fully understood. The Wnt/β-catenin signaling pathway has been demonstrated to be responsible for a variety of biological processes, including tissue homeostasis and cancer. Recently, the role of Wnt/β-catenin signaling pathway in bone formation has been elucidated, and it appears as an important signaling cascade for osteoblast differentiation, bone formation and homeostasis. In the canonical Wnt/ β-catenin signaling pathway, binding of Wnt ligand to its coreceptors Frizzled (Fz) receptor, a seven-transmembrane protein, and a low-density lipoprotein receptor-related protein (LRP) lead to the destruction of a complex that consist of glycogen synthase kinase-3β (GSK-3β), axin and adenomatous polyposis coli (APC) [13]. Disruption of this complex leads to phosphorylation of GSK3β, thus leading to the stabilization of β-catenin through dephosphorylation. Subsequent translocation of β-catenin into the nucleus leads to interaction with Tcf/Lef transcription factors and up-regulation of key osteoblastic genes [14,15]. Accumulating studies demonstrated that the Wnt/β-catenin signaling plays important role in osteogenesis in vitro and in vivo. Mutation with low-density lipoprotein receptor-related protein 5 (LRP5) was shown to cause osteoporosis pseudoglioma syndrome [16], which is characterized by low bone mass. In contrast, mutant mice that overexpressed the constitutive active LRP5 (G171V) in osteoblasts exhibited an enhanced osteoblastic activity, a reduction in osteoblast apoptosis, and high bone mass phenotype [17]. Recently, the interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. Several physiological consequences associated with estrogen signaling, such as neuronal development [18] and bone formation [19], are mediated by the activation of the Wnt/βcatenin signaling pathway. For example, estradiol increased cytosolic β-catenin protein levels and activated the transcriptional activity of Wnt/β-catenin signaling by an inhibition of GSK-3 functions in neuroblastoma cells and primary cortical neurons [18]. Also, in bone cells, the transcriptional activity of Wnt/βcatenin signaling was activated by binding between the TCF and estrogen receptor [20]. Therefore, the regulation of the Wnt/βcatenin signaling pathway and estrogen replacement therapy are considered as the effective treatments for bone loss. In this study, we reported for the first time that E2 induces preosteoblastic cell proliferation and differentiation via ER/GSK3β-dependent Wnt/β-catenin signaling pathway, which subsequently activated the downstream osteogenesis-related gene transcription, CyclinD1, BGP, ALP and OPN.
Materials and methods
] (]]]]) ]]]–]]]
Western blot Protein lysate were separated by 10% SDS-polyacrylamide gel electrophoresis and then electroblotted onto PVDF membranes. The anti-ERβ anti-β-catenin, anti-phospho-β-catenin (Ser33/37/ Thr41), anti-phospho-GSK3 (Ser-9), anti-GSK-3, anti-BGP, antiALP, anti-cyclin D1, anti-TCF and anti-OPN antibodies were purchased from Sigma-Aldrich, with β-actin antibody (ABZOOM) as an internal control. The signal was detected using the enhanced SuperSignal West Pico chemiluminescent.
RNA extraction and qRT-PCR Total RNA was extracted from MC3T3-E1 cells using TRIzol reagent according to the recommendations of the manufacturer with a slight modification. cDNA synthesis was conducted using the iScript TM Select cDNA synthesis kit according to theprotocol of the manufacturer (Bio-Rad). Quantitative real-time PCRs of ERβ were performed on an equal amount of cDNA using KAPA SYBRs FAST quantitative PCR kit according to the instructions of the manufacturer (Kapa Biosystems) with the ABI PRISM 7500 sequence detection system and analysis software (Applied Biosystems). The primers used were as follows: Mus-ERβ, 50 -GAAGCTGGCTGACAAGGAAC-30 and 50 -AACGAGGTCTGGAGCAAAGA-30 ; β-actin, 50 -AGCCATGTACGTAGCCATCC30 and 50 -CTCTCAGCTGTGGTGGTGAA-30 .
Transfection of MC3T3-E1 cells with siRNAs The siRNAs were designed and synthesized by GenePharma. Four siRNAs that were synthesized to target ER2 expression were used in this experiment: siRNA-1 (siRNA-Esr2-992) sense 50 -UCAUUAUGUCCUUGAAUGCTT-30 ; antisense 50 -GCAUUCAAGGACAUAAUGATT-30 . siRNA-2 (siRNA-Esr2-2218) sense 50 -UUAGAACUCAGCAUUCAGCTT; antisense 50 -GCUGAAUGCUGAGUUCUAATT-30 . siRNA-3 (siRNAEsr2-2625) sense 50 -AUAAUCUAGUUAUGUAAGCTT; antisense 50 GCUUACAUAACUAGAUUAUT-30 . Scramble control siRNAs sense 50 UUCUCCGAACGUGUCACGUTT. antisense 50 -ACGUGACACGUUCGGAGAATT-30 . siRNAs transfection was performed using Lipofectamine 2000 (Invitrogen). Total protein assay was prepared 72 h after transfection for protein gel blot analysis.
MTT assay The 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to estimate cell viability [21]. Briefly, cells were plated at a density of 1 104 cells per well in 96-well plates. After exposure to specific treatment, the cells were incubated with MTT at a final concentration of 0.5 mg/ml for 4 h at 37 1C. After the removal of the medium, 150 mM DMSO solutions were added to dissolve the formazan crystals. The absorbance was read at 570 nm using a multi-well scanning spectrophotometer reader. Cells in the control group were considered 100% viable.
Cell culture and transfection Alizarin Red-S staining Mouse preosteoblastic (MC3T3-E1) cells were purchased from Insitute of Biochemistry and Cell Biology (Shanghai, China) and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were cultured at 37 1C in a humidified atmosphere of 5% CO2.
After incubation in the differentiation culture for the designated time, the differentiation of MC3T3-E1 cells was monitored by Alizarin Red-S staining. MC3T3-E1 samples were washed with PBS three times and then fixed with ice-cold 70% ethanol for 1 h
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227
E X PE R IM EN TA L C ELL R E S EA RC H
228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287
on ice. Subsequently, the cells were washed with distilled H2O and stained with Alizarin Red-S for 20 min at room temperature. To quantify the relative differentiation, their absorbance at 520 nm was measured in a spectrometer (Tecan infinite M200) after several washes with distilled H2O.
Immunofluorescence staining Primary rat osteoblasts were grown on glass coverslips and incubated with fluoride for 72 h. The cells were washed with cold PBS twice, fixed in ice-cold methanol and permeabilized with 0.5% Triton X-100 for 15 min. Cells were then blocked with 0.5% bovine serum albumin for 1 h at room temperature. Samples were then incubated with mouse monoclonal anti-dephosphorylated βcatenin antibody (1:500) or anti-TCF4 antibody overnight at 4 1C followed by incubation with TRITC-conjugated secondary antibody and DAPI. The signal was visualized by confocal fluorescence microscopy (Olympus, Japan).
Immunoprecipitation Cell lysates were incubated with the indicated antibodies in the presence of 15 ml of protein G-agarose beads for 3 h at 4 1C. After washed five times, the immunoprecipitates were subjected to electrophoresis. Protein expression was examined by probing Western blots of total cell lysates or immunoprecipitates with the appropriate antibodies as noted in the figure legends.
Statistical analysis Statistical significance was determined by a two-tailed unpaired Student's t test or one-way ANOVA followed by post hoc (Bonferroni) multiple comparisons between treatment groups using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, ca.). po0.05 was considered statistically significant.
Results E2 affects the activities of GSK-3β and β-catenin in preosteoblastic cells Estradiol (E2) has been reported to activate Wnt/β-catenin activity in neuronal cells [22] and bone cells [17]. In this study, we examined the effects of E2 on endogenous Wnt/β-catenin signaling in preosteoblastic cells (MC3T3-E1). MC3T3-E1 were treated with 0.1 μM E2 for 0, 30, 60, 90 and 120 min. Western blot analysis demonstrated the expression of β-catenin and p-β-catenin. As shown in Fig. 1, E2 (0.1 mM) exhibited time-dependent activations of Wnt/β-catenin signaling. E2 induced a significant increase in the β-catenin protein level, which peaked at 90 min and declined substantially by 120 min after treatment. On the contrary, the expression level of p-β-catenin was gradually downregulated and reached bottom at 90 min after E2 (0.1 mM) treatment. Because GSK-3β phosphorylates β-catenin and prevents TCF-activated transcription activity, we next determined whether the activation of Wnt/β-catenin signaling mediated by E2 involves GSK-3β. As shown in Fig. 1, E2 greatly promoted GSK-3β phosphorylation at serine 9, but it failed to affect GSK-3β expression. Collectively, these
] (]]]]) ]]]–]]]
3
results suggested that E2 mediates Wnt/β-catenin signaling by phosphorylating GSK-3β and promoting the accumulation of intraQ3 cellular levels of the β-catenin protein in preosteoblastic cells. GSK-3β, p-GSK-3β.
ERβ involves E2-mediated activation of GSK-3β and βcatenin in preosteoblastic cells Because previous study showed that ER involves in Wnt/β-catenin signaling in osteoblastic cells [19] and E2 binds to both ERα and β with high affinity [23], we next determined whether the activation of Wnt/β-catenin signaling mediated by E2 involves ERβ. In this work, we transfected MC3T3-E1 cells with ERβ siRNA and with negative control siRNA containing scrambled random sequence prior to treatment with E2 for 90 min. We examined four siRNAs to ERβ as described above. qRT-PCR analysis showed that siRNA-2 and siRNA-3 effectively blocked the expression of ERβ mRNA. qRT-PCR revealed dramatic reduction of 84.1% with siRNA-2, 78.1% with siRNA-3, and 21.2% with siRNA-1 in the levels of ERβ mRNA after transfection of siRNA in MC3T3-E1 cells, compared with MC3T3-E1 negative control siRNA or the control (untransfected) group (po0.05; Fig. 2A). As shown in Fig. 2B, ERβ siRNA-2 also significantly decreased the level of β-catenin protein. Thus, the cells transfected with ERβ siRNA-2 were used for further experiments. Then, we determine the effect on E2-mediated Wnt/ β-catenin signaling. As shown in Fig. 2C, ERβ siRNA reversed ERinduced upregulation of β-catenin and downregulation of p-βcatenin. Moreover, the induction of GSK-3β phosphorylations mediated by E2 was abolished in the presence of siRNA treatment. These data strongly suggested that ERβ is required for the activation of Wnt/β-catenin signaling by E2 in MC3T3-E1 cells.
Induction of preosteoblastic cell proliferation and differentiation by E2 requires ERβ Because Wnt/β-catenin signaling regulates cell proliferation and differentiation of multiple tissues, including bone cells, we then assessed the proliferative effects of E2 and ERβ on preosteoblastic cells using an MTT assay after treatment with E2 for 90 min. As shown in Fig. 3A, E2 significantly increased cell proliferation, but the cell proliferation was decreased by pretreatment with siRNA. To investigate the effect of E2 on osteoblastic differentiation of MC3T3-E1 cells, mineralization was determined by Alizarin Red S staining. As shown in Fig. 3B, E2 treatment alone significantly promoted mineralized nodule formation, which was reversed by siRNA treatment. Next, we detected the effect of E2 on cyclin D1, bone GLa protein (BGP), alkaline phosphatase (ALP), T-cell factor (TCF) and osteopontin (OPN ) by western blot. As shown in Fig. 3C, E2 and ERβ siRNA failed to affect the expression of TCF. In contrast, BGP, ALP, cyclin D1 and OPN expression levels were significantly increased by E2 treatment, but the expression were reversed by siRNA. Collectively, our results strongly suggested that E2 increases preosteoblastic cell proliferation and differentiation via ERβ.
E2 induces the formation of β-catenin/Tcf protein complex in MC3T3-E1 cells The accumulation of β-catenin in the nucleus is an important step in the activation of the Wnt/β-catenin signaling pathway and
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347
4
348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407
E XP E RI ME N TAL CE L L R ES E ARC H
] (]]]]) ]]]–]]]
Fig. 1 – E2 affects the activities of GSK-3β and β-catenin in preosteoblastic cells. MC3T3-E1 were treated with 0.1 μm E2 for 0, 30, 60, 90 and 120 min. Western blot analysis demonstrated the expression levels of β-catenin, p-β-catenin, GSK-3β and p-GSK-3β. Data are represented as mean7S.E. (n¼ 4). *po0.001 and **po0.05 *po0.01 vs. 0 min group.
Fig. 2 – ERβ involves E2-mediated activation of GSK-3β and β-catenin in preosteoblastic cells. (A) qRT-PCR analyzed the expression of ERβ after transfection with different ERβ siRNAs in MC3T3-E1 cells. (B) The western blot analysis showed the expression of βcatenin protein after siRNA interference. (C) Western blot analysis demonstrated the effect of ERβ on the expression of GSK-3β, pGSK-3β, β-catenin and p-β-catenin. MC3T3-E1 cells were transfected with ERβ siRNA prior to treatment with 0.1 lM E2. Data are the mean7SD of duplicates from a representative experiment of three independent experiments. *po0.01 vs. control group and ** po0.01 vs. E2 group or E2þNC-siRNA group.
translocation of β-catenin into the nucleus leads to interaction with Tcf/Lef transcription factors and up-regulation of key osteoblastic genes that are Wnt targets. The effect of E2 on subcellular localization of β-catenin and its interaction with Tcf/Lef transcription factors were further explored by immunofluorescence and immunoprecipitation in MC3T3-E1 cells. Consistent with Western blot result in Fig. 3C, immunofluorescence analysis showed that E2 and ERβ siRNA failed to affect the expression of TCF (Fig. 4A), and β-catenin immunostaining was increased significantly after
treatment with 0.1 mM E2 for 90 min (Fig. 4A). Interestingly, the staining was more intense and localized in the nucleus after treated with E2 compared with control group (Fig. 4A). However, accumulation ofβ-catenin in the nucleus were greatly attenuated with ERβ siRNA prior to treatment with E2. PKF118-310, a natural compound of microbial origin, is capable of disrupting the formation of β-catenin/Tcf protein complex. To further confirm that E2 involved the interaction between β-catenin and TCF protein, immunofluorescence was performed in MC3T3-E1 cells
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467
E X PE R IM EN TA L C ELL R E S EA RC H
468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527
] (]]]]) ]]]–]]]
5
Fig. 3 – Induction of preosteoblastic cell proliferation and differentiation by E2 requires ERβ. MC3T3-E1 cells were transfected with ERβ siRNA prior to treatment with 0.1 lM E2. (A) E2 increases MC3T3-E1 cell proliferation. The proliferation of MC3T3-E1 cells was observed by MTT assay. (B) E2-induced preosteoblastic cell differentiation. Mineralization was determined by Alizarin Red S staining. (C) E2 and ERβ affect osteoblastic differentiation-related gene expression. Data are the mean7SD of duplicates from a representative experiment of three independent experiments. *po0.01 vs. control group and **po0.01 vs. E2 group or E2þNCsiRNA group.
using PKF118-310. As shown in Fig. 4A, PKF118-310 successfully abolished E2-induced accumulation of β-catenin in the nucleus. Consistent with immunofluorescence results, immunoprecipitation analysis showed that E2 induced the formation of β-catenin/ Tcf protein complex which was attenuated and abolished by ERβ siRNA and PKF118-310, respectively. These data demonstrated that E2 activate Wnt/β-catenin signaling by promoting nuclear localization of β-catenin protein and formation of β-catenin/Tcf protein complex.
Discussion Both Wnt/β-catenin and estrogen receptor signaling have currently been recognized as important regulators of bone mass and bone cell differentiation. Estrogen is a key regulator of bone formation. 17β-estradial (E2), a polyphenolic phytoestrogen, acts as a stimulator of bone formation in vivo [24]. However, the underlying mechanisms are not fully understood. In this study, our results strongly suggested that E2 promotes preosteoblastic cell proliferation and differentiation through the activation of ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. The Wnt/β-catenin signaling pathway has been demonstrated to be responsible for a variety of biological processes, including
tissue homeostasis and cancer. Cytosolic β-catenin protein is the principal mediator of Wnt signaling [25]. In the activation of Wnt/ β-catenin signaling pathway, β-catenin is dephosphorylated and accumulated in the cytoplasm. In this study, we found that E2 significantly increased the β-catenin protein level and decreased the p-β-catenin expression level. Serine/threonine protein kinase GSK3β is a key negative regulator of β-catenin in the Wnt signaling pathway, it phosphorylates and promotes the degradation of β-catenin in quiescent cells [26]. It has been shown that phosphorylation of GSK-3β leads to inhibition of GSK-3β thus resulting in the stimulation of the Wnt pathway [13]. E2 induced the phosphorylation of Akt at serine 473 and subsequently activated the phosphorylation of GSK-3β at serine 9, leading to inhibition of GSK-3β activity and, hence, activation of Wnt/βcatenin signaling [27]. Consistent with these studies, E2 could also induce phosphorylation of GSK-3β at the serine 9 residue, whereas no significant change in GSK-3β protein expression in preosteoblastic MC3T3-E1 cells. Non-phosphorylated β-catenin accumulated in the cytoplasm and translocates into the nucleus, binds with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF), and activates target gene expression. The immunofluorescence and immunoprecipitation analysis showed that E2 significantly induced translocation of β-catenin into nucleus and formation of β-catenin/Tcf protein complex. These
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587
6
588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647
E XP E RI ME N TAL CE L L R ES E ARC H
results indicated that E2 involved the activation of Wnt/β-catenin signaling by inducing phosphorylation of GSK-3β. Although a previous study reported that, in the absence of mechanical strain, E2 failed to induce β-catenin protein expression and transcription activity in ROS17/2.8 cells [19]. This discrepancy could be due to the difference in the cell type used. Estrogen receptor (ER) signaling pathway has been shown to involve in osteoblastic differentiation, the activation of the ER signaling pathway can occur in a ligand-dependent way by binding of estradiol (E2) to intracellular ERs as well as independently of E2-binding by cross-talk with peptide growth factors [28]. Recently, the interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. Several physiological consequences associated with estrogen signaling, such as neuronal development [18] and bone formation[19], are mediated by the activation of the Wnt/βcatenin signaling pathway. In our study, the siRNA technique was used to inhibit ERβ expression. ERβ siRNA significantly suppressed phosphorylation of GSK-3β, the activate GSK-3β phosphorylated β-catenin and attenuated the accumulation of β-
] (]]]]) ]]]–]]]
catenin. This result indicated that ERβ involves E2-mediated activation of Wnt/β-catenin signaling in preosteoblastic cells. In this study, E2 significantly induced preosteoblastic cell proliferation and differentiation. Osteoblast differentiation is accompanied by the increased expression of bone matrix proteins, such as ALP, BGP and OPN. These osteoblast differentiation factors stimulate mineralization and lead to bone formation. In this study, it was clearly shown that BGP, ALP and OPN expressions were significantly stimulated by E2 in preosteoblastic cells. On the other hand, it was evident that E2-mediated increase on BGP, ALP and OPN expressions were diminished in ERβ siRNA transfected cells prior E2 treatment. BGP is a specific marker for bone formation. As BGP synthesized from bone tissue, half of it deposited in the bone matrix and half in blood circulation, so serum osteocalcin measurements can provide a noninvasive specific marker of bone metabolism [29]. ALP and OPN, as other bone formation marker, observed several cell types including preosteoblastic cells [30]. Studies have proved that ASPP 049 can induce osteoblast differentiation, ALP activity and increase bone mineralization [27]. These osteoblast differentiation factors stimulate mineralization and lead to bone formation. CyclinD1 is up-regulated
Fig. 4 – E2 induces the formation ofβ-catenin/Tcf protein complex in preosteoblastic (MC3T3-E1) cells. (A) subcellular localization of β-catenin and its interaction with Tcf/Lef transcription factors were further explored by immunofluorescence. (B) β-catenin/Tcf association was detected by immunoprecipitate. MC3T3-E1 cells were transfected with ERβ siRNA prior to treatment with 0.1 lM E2. Lysates were immunoprecipitated with anti-β-catenin and immunoblotted with anti-TCF4 antibody. Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707
E X PE R IM EN TA L C ELL R E S EA RC H
708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767
as an important promoter of cell cycle [31], and upregulation of cyclinD1 insulin is involved in osteoclast proliferation [32]. E2 significantly induced preosteoblastic cell proliferation by increasing cyclinD1 expression. Overall, E2 promoted osteoblast proliferation and differentiation by regulating proliferation factors (CyclinD1) and osteoblast differentiation factors (BGP, ALP and OPN) by activating the ER/ GSK-3β-dependent Wnt/β-catenin signaling pathways. Our findings provide novel molecular mechanism by which estrogen may promote bone formation.
[12]
[13]
[14]
Conflict of interest We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.
[15]
[16]
Acknowledgments Q4 This publication was funded in part by the National Natural
Science Foundation of China (81271940) and Key Project of Natural Science Foundation of Hunan Province (12JJ2043).
references [1] R. Rizzoli, J.P. Bonjour, Hormones and bones, Lancet 349 (Suppl 1) (1997) sI20–23. [2] E. Sturm, Liver, hormones, and bones, J. Pediatr. Gastroenterol. Nutr. 54 (2012) 308–309. [3] B.L. Riggs, S. Khosla, L.J. Melton 3rd, A unitary model for involutional osteoporosis: estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men, J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 13 (1998) 763–773. [4] E. Seeman, Estrogen therapy and variable-resistance weight training, J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 7 (1992) 709–711. [5] A. Zallone, Direct and indirect estrogen actions on osteoblasts and osteoclasts, Ann. N. Y. Acad. Sci. 1068 (2006) 173–179. [6] H. Kim, A. Tabata, T. Tomoyasu, T. Ueno, S. Uchiyama, K. Yuasa, A. Tsuji, H. Nagamune, Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells, J. Bone Miner. Metab. (2014). [7] Y. Matsumoto, F. Otsuka, M. Takano, T. Mukai, R. Yamanaka, M. Takeda, T. Miyoshi, K. Inagaki, K.E. Sada, H. Makino, Estrogen and glucocorticoid regulate osteoblast differentiation through the interaction of bone morphogenetic protein-2 and tumor necrosis factor-alpha in C2C12 cells, Mol. Cell Endocrinol. 325 (2010) 118–127. [8] A. Tomkinson, J. Reeve, R.W. Shaw, B.S. Noble, The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone, J. Clin. Endocrinol. Metab. 82 (1997) 3128–3135. [9] Y.H. Yang, K. Chen, B. Li, J.W. Chen, X.F. Zheng, Y.R. Wang, S.D. Jiang, L.S. Jiang, Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway, Apoptosis Int. J. Program. Cell Death 18 (2013) 1363–1375. [10] S.C. Manolagas, S. Kousteni, R.L. Jilka, Sex steroids and bone, Recent Prog. Horm. Res. 57 (2002) 385–409. [11] M. Ernst, J.K. Heath, G.A. Rodan, Estradiol effects on proliferation, messenger ribonucleic acid for collagen and insulin-like growth
[17] [18]
[19]
[20] [21]
[22]
[23]
[24]
[25]
] (]]]]) ]]]–]]]
7
factor-I, and parathyroid hormone-stimulated adenylate cyclase activity in osteoblastic cells from calvariae and long bones, Endocrinology 125 (1989) 825–833. A. Mamalis, C. Markopoulou, A. Lagou, I. Vrotsos, Oestrogen regulates proliferation, osteoblastic differentiation, collagen synthesis and periostin gene expression in human periodontal ligament cells through oestrogen receptor beta, Arch. Oral Biol. 56 (2011) 446–455. J. Mao, J. Wang, B. Liu, W. Pan, G.H. Farr 3rd, C. Flynn, H. Yuan, S. Takada, D. Kimelman, L. Li, D. Wu, Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway, Mol. Cell 7 (2001) 801–809. C.N. Bennett, K.A. Longo, W.S. Wright, L.J. Suva, T.F. Lane, K.D. Hankenson, O.A. MacDougald, Regulation of osteoblastogenesis and bone mass by Wnt10b, Proc. Natl. Acad. Sci. USA 102 (2005) 3324–3329. T. Gaur, C.J. Lengner, H. Hovhannisyan, R.A. Bhat, P.V. Bodine, B.S. Komm, A. Javed, A.J. van Wijnen, J.L. Stein, G.S. Stein, J.B. Lian, Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression, J. Biol. Chem. 280 (2005) 33132–33140. Y. Gong, R.B. Slee, N. Fukai, G. Rawadi, S. Roman-Roman, A.M. Reginato, H. Wang, T. Cundy, F.H. Glorieux, D. Lev, M. Zacharin, K. Oexle, J. Marcelino, W. Suwairi, S. Heeger, G. Sabatakos, S. Apte, W.N. Adkins, J. Allgrove, M. Arslan-Kirchner, J.A. Batch, P. Beighton, G.C. Black, R.G. Boles, L.M. Boon, C. Borrone, H.G. Brunner, G.F. Carle, B. Dallapiccola, A. De Paepe, B. Floege, M.L. Halfhide, B. Hall, R.C. Hennekam, T. Hirose, A. Jans, H. Juppner, C.A. Kim, K. Keppler-Noreuil, A. Kohlschuetter, D. LaCombe, M. Lambert, E. Lemyre, T. Letteboer, L. Peltonen, R.S. Ramesar, M. Romanengo, H. Somer, E. Steichen-Gersdorf, B. Steinmann, B. Sullivan, A. Superti-Furga, W. Swoboda, M.J. van den Boogaard, W. Van Hul, M. Vikkula, M. Votruba, B. Zabel, T. Garcia, R. Baron, B.R. Olsen, M.L. Warman, G. Osteoporosis-Pseudoglioma, Syndrome collaborative, LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development, Cell 107 (2001) 513–523. V. Krishnan, H.U. Bryant, O.A. Macdougald, Regulation of bone mass by Wnt signaling, J. Clin. Invest. 116 (2006) 1202–1209. O. Varea, M.A. Arevalo, J.J. Garrido, L.M. Garcia-Segura, F. Wandosell, P. Mendez, Interaction of estrogen receptors with insulinlike growth factor-I and Wnt signaling in the nervous system, Steroids 75 (2010) 565–569. V.J. Armstrong, M. Muzylak, A. Sunters, G. Zaman, L.K. Saxon, J.S. Price, L.E. Lanyon, Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha, J. Biol. Chem. 282 (2007) 20715–20727. J.J. Westendorf, R.A. Kahler, T.M. Schroeder, Wnt signaling in osteoblasts and bone diseases, Gene 341 (2004) 19–39. B. Liu, W. Che, J. Xue, C. Zheng, K. Tang, J. Zhang, J. Wen, Y. Xu, SIRT4 prevents hypoxia-induced apoptosis in H9c2 cardiomyoblast cells, Cell. Physiol. Biochem. Int. J. Exp. Cell. Physiol. Biochem. Pharmacol. 32 (2013) 655–662. O. Varea, J.J. Garrido, A. Dopazo, P. Mendez, L.M. Garcia-Segura, F. Wandosell, Estradiol activates beta-catenin dependent transcription in neurons, PloS One 4 (2009) e5153. G.G. Kuiper, J.G. Lemmen, B. Carlsson, J.C. Corton, S.H. Safe, P.T. van der Saag, B. van der Burg, J.A. Gustafsson, Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta, Endocrinology 139 (1998) 4252–4263. A. Plant, A. Samuels, M.J. Perry, S. Colley, R. Gibson, J.H. Tobias, Estrogen-induced osteogenesis in mice is associated with the appearance of Cbfa1-expressing bone marrow cells, J. Cell. Biochem. 84 (2002) 285–294. M.D. Gordon, R. Nusse, Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors, J. Biol. Chem. 281 (2006) 22429–22433.
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827
8
828 829 830 831 832 833 834 835 836 837 838 839 840
E XP E RI ME N TAL CE L L R ES E ARC H
[26] D. Wu, W. Pan, GSK3: a multifaceted kinase in Wnt signaling, Trends Biochem. Sci. 35 (2010) 161–168. [27] K. Bhukhai, K. Suksen, N. Bhummaphan, K. Janjorn, N. Thongon, D. Tantikanlayaporn, P. Piyachaturawat, A. Suksamrarn, A. Chairoungdua, A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3beta proteindependent activation of the Wnt/beta-catenin signaling pathway, J. Biol. Chem. 287 (2012) 36168–36178. [28] J.M. Hall, J.F. Couse, K.S. Korach, The multifaceted mechanisms of estradiol and estrogen receptor signaling, J. Biol. Chem. 276 (2001) 36869–36872. [29] W. Zhou, Z. Liu, J. Yao, F. Chi, K. Dong, X. Yue, T. Teng, X. Rausch, The Effects of Exenatide Microsphere on Serum BGP and ALP
] (]]]]) ]]]–]]]
Levels in ZDF Rats after Implantation, Clin. Implant. Dent. Relat. Res. (2013). [30] A.C. Akman, R. Seda Tigli, M. Gumusderelioglu, R.M. Nohutcu, Bone morphogenetic protein-6-loaded chitosan scaffolds enhance the osteoblastic characteristics of MC3T3-E1 cells, Artif. Org. 34 (2010) 65–74. [31] S. Korulu, A. Yildiz-Unal, M. Yuksel, A. Karabay, Protein kinase C activation causes neurite retraction via cyclinD1 and p60-katanin increase in rat hippocampal neurons, Eur. J. Neurosci. 37 (2013) 1610–1619. [32] J.Y. Lee, N.K. Lee, Up-regulation of cyclinD1 and Bcl2A1 by insulin is involved in osteoclast proliferation, Life Sci. 114 (2014) 57–61.
Please cite this article as: X. Yin, et al., ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.04.020
841 842 843 844 845 846 847 848 849 850 851 852
