- Email: [email protected]
A humanized osteopontin mouse model and its application in immunometabolic obesity studies
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
ORIGINAL ARTICLE Q2 Q1
Q11
A humanized osteopontin mouse model and its application in immunometabolic obesity studies € KARINA ZEYDA, VERONICA MORENO-VIEDMA, KARIN STROHMEIER, NICOLE G. GRUN, € GUNTHER STAFFLER, MAXIMILIAN ZEYDA, and THOMAS M. STULNIG VIENNA, AUSTRIA
Osteopontin (OPN) is a multifunctional protein involved in several inflammatory processes and pathogeneses including obesity-related disorders and cancer. OPN binds to a variety of integrin receptors and CD44 resulting in a proinflammatory stimulus. Therefore, OPN constitutes a novel interesting target to develop new therapeutic strategies, which counteract OPN’s proinflammatory properties. We established a humanized SPP1 (hSPP1) mouse model and evaluated its suitability as a model for obesity and insulin resistance. Unchallenged hSPP1 animals did not significantly differ in body weight and gross behavioral properties compared to wild-type (WT) animals. High-fat diet-challenged hSPP1 similarly developed obesity and inflammation, whereas insulin resistance was markedly changed. However, OPN expression profile in tissues was significantly altered in hSPP1 compared to WT depending on the diet. In conclusion, we developed a versatile humanized model to study the action of OPN in vivo and to develop strategies that target human OPN in a variety of pathologies. (Translational Research 2016;-:1–11) Abbreviations: --- ¼ ---
Q3
INTRODUCTION From the Christian Doppler Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Department of Medicine III, Medical University of Vienna, Vienna, Austria; FH Campus Wien, University of Applied Sciences, Department Health, Section Biomedical Science, Vienna, Austria; AFFiRiS AG, Vienna, Austria; Department of Pediatrics and Adolescent Medicine, Clinical Division of Pediatric Pulmonology, Allergology and Endocrinology, Medical University of Vienna, Vienna Austria. Submitted for publication June 3, 2016; revision submitted July 4, 2016; accepted for publication July 11, 2016. Reprint requests: Thomas M. Stulnig, Christian Doppler Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Department of Medicine III, Medical University of Vienna, W€ahringer G€urtel 18-20, 1090 Vienna, Austria; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2016 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.trsl.2016.07.009
O
steopontin (OPN) is a multifunctional protein, first identified as a T helper type 1 cytokine, which is mainly expressed in various tissues and cells including macrophages, T-cells, dendritic cells, hepatocytes, smooth muscle cells, endothelial, and epithelial cells.1-3 OPN exhibits properties not only of a soluble cytokine that acts in an autocrine or paracrine manner on liver cells but also of an extracellular matrix (ECM)-bound protein.1 OPN plays a prominent role in monocyte migration, adhesion, and differentiation4-6 as well as in the pathophysiology of chronic inflammatory diseases, such as rheumatoid arthritis,7 Crohn’s disease,8 cancer,9 and cardiovascular disease.10,11 OPN is involved in the pathogenesis of Q4 obesity-associated complications, including AT inflammation, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and atherosclerosis.12-16 In vitro 1
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
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 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127
2
128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191
Translational Research - 2016
€ n et al Gru
AT A GLANCE COMMENTARY € n NG, et al. Gru Background
Osteopontin (OPN) is involved in a number of inflammatory processes, including obesity-linked complications, asthma, rheumatoid arthritis as well as cancer progression and metastasis. Proteolytic cleavage of OPN increases OPN’s proinflammatory properties and therefore constitutes an interesting target to develop new strategies to counteract OPN’s actions. To enable research on human OPN, we generated a humanized SPP1 (hSPP1) mouse and evaluated it as a diet-induced obesity model. Translational Significance
Our findings propose the hSPP1 animal model to be applied for a variety of pathologies in which OPN is a key player, including obesity-driven, cardio-metabolic as well as other diseases that can be translated to humans.
studies with adipocytes identified OPN as a modulator of insulin resistance and impaired glucose uptake.17 Obese mice and humans revealed markedly increased messenger RNA (mRNA) levels for OPN in visceral and subcutaneous fat.18 Also, plasma OPN concentrations were markedly elevated in obese human subjects compared with lean controls.19,20 Importantly, high-fat (HF) diet treatment of mice lacking OPN, improved adipose tissue inflammation and insulin sensitivity12 as well as reduced hyperleptinemia and adipocyte hypertrophy.21 OPN acts via integrin binding by different domains. Although the arg-gly-asp (RGD)-containing domain mainly binds integrins avb1, avb3, avb5, and a5b122 on the cell surface, the ser-val-val-tyr-glu-leu-arg (SVVYGLR) motif interacts with integrins a4b1, a4b7, and a9b1.23,24 Importantly, the RGD region is fully conserved between species, whereas the SVVYGLR motif differs between humans (SVVYGLR) and rodents (SLAYGLR).23 The third integrin-binding domain ELVTDFTDLPAT has also been described to bind a4b1.25 Overall, human and murine OPN show only 63% amino acid sequence identity (National Center of Biotechnology Information). OPN’s actions are affected by protease cleavage. The cryptic region SVVYGLR, which is usually hidden in intact OPN, becomes accessible on thrombin and matrix metalloproteinase cleavage. Interestingly, although enzy-
matic thrombin and matrix metalloproteinase cleavage produces a proinflammatory N-terminal OPN fragment, the less described C-terminal OPN form may even attenuate inflammatory processes.26 Moreover, bonding with the hyaluronic acid receptor CD44 leads to the induction of macrophage chemotaxis and the b3-integrin receptors engagement.27 To develop therapeutic strategies against OPN actions, small molecules are not a primary option but sequence-specific approaches are needed such as specific antibodies, RNA aptamers, and antisense oligonucleotides or interfering RNAs. Such approaches targeting OPN have been successfully applied on human tissues transplanted into immunodeficient mice, for example, in cancer studies.28-30 However, human cell transplantation is not feasible for most disease models, particularly inflammatory diseases. To facilitate in vivo studies of blocking human OPN, we developed a humanized mouse model by replacing the mouse gene with human SPP1 sequence. Owing to OPN’s crucial involvement in a variety of inflammatory pathologies and particularly its association with cardiometabolic complications of obesity, we evaluated the humanized SPP1 (hSPP1) mouse model as a dietinduced obesity (DIO) model. We show the functionality of hSPP1 mice as a DIO model by generation of obesity-associated insulin resistance and inflammation following HF diet feeding. Hence, hSPP1 mice are a highly useful model to investigate OPN’s role not only to study prevention and treatment of obesity and its complications but also to facilitate preclinical studies on neutralization of human OPN in a large variety of other inflammatory diseases and cancer. MATERIAL AND METHODS Animals and diets. Heterozygous C57BL/6Spp1tm2737 (SPP1) Arte (hSPP11/2) mice were developed with Taconic (K€oln, Germany). In short, the chimeric SPP1 protein sequence was chosen as depicted in Fig 1, A. Accordingly, exon 2 contains the translation initiation codon and the sequence, which encodes the cleavable signal peptide. The mouse genomic sequence from amino acid 21 (valine) in exon 3 up to the termination codon in exon 7 has been replaced by its human counterpart to leave a single N-terminal mouse-specific amino acid in the mature protein (p.Ile19Leu; Fig 1). Positive selection markers, Q5 including neomycin resistance flanked by FRT and puromycin resistance flanked with F3, were inserted into human intron 3 as well as downstream of the mouse 30 untranslated region, respectively. BAC Q6 clones from the mouse C57BL/6J were used for the targeting vector that was further transfected using
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255
Translational Research Volume -, Number -
3
web 4C=FPO
256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319
€ n et al Gru
Fig 1. Generation of the humanized SPP1 gene for homologous recombination. Description of the constructed chimeric SPP1 in comparison to the murine and human form (A). Stepwise generation of the humanized allele is given in (B).NeoR, neomycin resistance; PuroR, puromycin resistance.
Fig 2. Blood glucose levels are significantly lower in hSPP1 compared to WT animals with similar body weight. Male hSPP1 and WT mice (n 5 20 per group) were kept on normal chow until 9 weeks of age. Body weight (A) and blood glucose levels (B) were determined before LF and HF administration. Data is expressed as mean 6 standard error of the mean. ***P , 0.001. WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1.
electroporation into a Taconic C57BL/6N embryonic stem cell line. After homologous recombination, clones were selected with double positive neomycin resistance–puromycin resistance selection, and the humanized allele was obtained after Flp-mediated
removal of these selection markers. Expression of the human SPP1 gene is controlled by the mouse Spp1 promoter and results in the loss of expression of the mouse Spp1 gene (Fig 1, B). Generation of the transgenic mouse model was according to
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
4
384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447
Translational Research - 2016
€ n et al Gru
Fig 3. Dietary challenge alters body weight and insulin resistance in hSPP1 mice. Male hSPP1 and WT mice (n 5 8–10 per group) were fed a HF and LF diet for 22 weeks. Body weight was determined weekly over 22 weeks (A,B). After 15 weeks on diet, an ITT was performed. Glucose levels were measured over 120 minutes after intraperitoneal insulin application of 0.75-IU/kg body weight for HF and 0.25-IU/kg body weight for LF, respectively, (C) and AUC (D) was calculated. Fasting glucose and insulin levels (E) were measured after 22 weeks, and insulin resistance is given by the HOMA-IR (F). Data is expressed as mean 6 standard error of the mean. *,#,§P , 0.05, **,##,§§P , 0.01, ***,###,§§§P , 0.001. (*) indicates the comparison between WT LF and HF, (#) comparison between hSPP1 LF and WT LF; (§) comparison between hSPP1 LF and HF. AUC, area under the curve; ITT, insulin tolerance test; HOMA-IR, homeostasis model assessment of insulin resistance; WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1.
well-described instructions.31 Heterozygous hSPP11/2 animals were crossbred to homozygosity in house. At 9 weeks of age, 10 male hSPP1 and 10 C57BL/6JRj (WT) mice (Janvier Labs, Saint Berthevin, France) were set on high-fat diet (HF, 60 kcal%, D12492; Research
Diets, New Brunswisk, NJ) for 22 weeks to induce obesity, whereas 10 male hSPP1 and 10 WT mice were fed a low-fat diet (LF, 10 kcal%, D12450 B; Research Diets) to serve as negative controls for DIO. All mice were kept on normal chow until 9 weeks of age.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
Translational Research Volume -, Number 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575
€ n et al Gru
5
Fig 4. Obesity-induced adipose tissue inflammation in hSPP1 mice is similar to WT animals. Male hSPP1 and WT mice (n 5 8–10 per group) were set on indicated diets for 22 weeks. GWAT and SWAT weight (A) was determined after sacrification; RNA was isolated and transcribed to complementary DNA. Gene expression of insulin sensitivity markers Adipoq (B) and Pparg (C), macrophage marker Emr1 (F4/80) (D) as well as of cytokines Ccl2 (E) and Tnf-a (F) was assessed by quantitative real-time PCR. Ubc was used as house-keeping gene, and data are given after normalization to GWAT WT LF as mean 6 standard error of the mean. *P , 0.05, **P , 0.01, ***P , 0.001. WT, wild-type; PCR, polymerase chain reaction; Ubc, ubiquitin C; LF, low-fat diet; HF, high-fat diet; mRNA, messenger RNA; hSPP1, humanized SPP1; GWAT, gonadal white AT; SWAT, Subcutaneous white AT.
All mice were housed in a specific pathogen-free facility, maintaining a 12-hour light/dark cycle and had free access to water and food. Food intake was measured 3 times per week. Weight was determined weekly, blood was drawn every third week and immediately before mice were sacrificed. Subcutaneous white AT (SWAT)
and gonadal white AT (GWAT) as well as liver were collected, weighed, and stored for further experiments. The protocol was approved by the local ethics committee for animal studies and followed the guidelines on accommodation and care of animals formulated by the European Convention for the Protection of Vertebrate
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639
640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703
Translational Research - 2016
€ n et al Gru
web 4C=FPO
6
Fig 5. Dietary challenge induces adipocyte hypertrophy and crown-like structure formation in both genotypes. Male hSPP1 and WT mice (n 5 8–10 per group) were fed a HF and LF diet for 22 weeks. Paraffin-embedded GWAT sections were stained for Mac-2 and visualized with light microscopy (A). Formation of crown-like structures (CLS) normalized to total number of adipocytes (B) and adipocyte size (C) was assessed with ImageJ software. Representative pictures are shown after 10-fold magnification. Data are expressed as mean 6 standard error of the mean. *P , 0.05, ***P , 0.001. WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1; GWAT, gonadal white AT; SWAT, Subcutaneous white AT.
Animals Used for Experimental and Other Scientific Purposes. Polymerase chain reaction (PCR) for genotyping analysis. Genomic DNA was isolated from ear tissue af-
ter clipping to identify homozygous hSPP1 mice using 2xKAPPA 2G Fast HS Kit. PCR was performed according to manufacturer’s instructions (KAPA Biosystems, Wilmington, Mass). To amplify the constitutive humanized and WT allele, 2 different primer pairs were used: for the humanized SPP1 allele forward primer
50 -CTGTTTGGCATTGCCTCC-30 and reverse primer 30 -TATACTGTGCTTGGTACTGGCC-50 , for the murine WT Spp1 allele forward primer 50 -TAG CAACCTTTGCGACGC-30 and reverse primer 30 -TATACTGTGCTTGGTACTGGCC-5’. In addition, forward primer 50 -CGAATGTAAGGAGTCTGGTGG-30 and reverse primer 30 -CAAGTTTCAAGCT 0 TTTGCTCG-5 were used to detect both, the humanized and WT allele. To assess DNA size, 100 base pair (bp) DNA ladder was used (Invitrogen, Carlsbad, Calif).
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767
Translational Research Volume -, Number 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831
Q7
€ n et al Gru
The humanized allele was displayed at 362 bp, whereas the WT allele showed a 254 bp band. Genomic DNA isolated from WT and heterozygous mice was employed as negative controls. Serum metabolic parameters. Serum insulin (Mercodia, Uppsala, Sweden) and OPN (R&D Systems, Minneapolis, USA) were analyzed with commercially available enzyme-linked immunosorbent assays. After a 5-hour fasting period, insulin sensitivity was assessed after 15 weeks on indicated diets by measuring glucose levels before, 30, 60, 90, and 120 minutes after an intraperitoneal injection of recombinant human insulin (0.75 IU/kg body weight NovoRapid for HF and 0.25 IU/kg body weight for LF, respectively; Novo Nordisk, Bagsværd, Denmark). Blood glucose levels in mice on normal chow diet were measured without fasting using OneTouch Ultra test strips (LifeScan, Milpitas, USA). Homeostasis model assessment of insulin resistance was calculated as an index for insulin resistance. Reverse transcription and gene expression. One part of SWAT, GWAT, and liver was stored in RNAlater (Qiagen, Hilden, Germany) overnight at 4 C. RNAlater was removed, tissues were snap frozen and stored at 280 C. RNA isolation was performed with peqGOLD TriFastTM (Peqlab, Erlangen, Germany) by using an automated homogenizer (Precellys 24, Erlangen, Germany), and one microgram RNA was transcribed to complementary DNA with M-MLV Reverse Transcriptase (Promega, Madison, USA). Gene expression of F4/80 (Emr1, Mm00802529_m1), Mcp1 (Ccl2, Mm00441242_m1), tumor necrosis factor-a (Tnf, Mm00443258_m1), adiponectin (Adipoq, Mm004564 25_m1), peroxisome proliferator-activated receptor gamma (Pparg, Mm01184322_m1), integrin alpha x (Itgax; Cd11c, Mm00498698_m1), murine OPN (mSpp1; Spp1, Mm00436767_m1), human OPN (hSPP1; SPP1, Hs00959010_m1), and reference gene ubiquitin C (Ubc; Ubc, Mm01201237_m1) was analyzed by quantitative real-time RT-PCR on an ABI Prism 7000 cycler with commercially available assays-on demand kits (TaqMan Gene Expression Assay, Applied Biosystems, Foster City, Calif) and normalized to Ubc. The comparative threshold cycle method was used to calculate relative expression.32 Immunohistochemistry. Murine GWAT samples were fixed with neutral buffered 4% paraformaldehyde at 4 C and were paraffin-embedded. After dewaxation and rehydration, sections were immunoh istochemically stained for Mac-2 (Cedarlane, Ontario, Canada) using the ABC kit (Vector Laboratories) according to manufacturer’s protocol. Counterstaining was performed by using hematoxylin (Merck,
7
Darmstadt, Germany), and sections were analyzed with standard light microscopy using a 10-fold magnification. Crown-like structures and density of adipocytes were quantified with ImageJ software, thereby assessing 5 independent fields per sample. Crown-like structures were normalized to total adipocyte number. Statistical analysis. Data are given as mean and standard error of the mean (mean 6 standard error of the mean). Comparisons between hSPP1 and WT were assessed by 1-way analysis of variance using Tukey’s multiple comparisons test. For body weight gain and insulin tolerance test results, a 2-way analysis of variance with Tukey’s multiple comparisons test was performed. Impact of dietary treatment on OPN expression within a genotype was analyzed by 2-tailed independent Student’s t-test. A P value of #0.05 was considered statistically significant. RESULTS Generation and characterization of the humanized 1/2 hSPP1 mouse. Heterozygous hSPP1 animals were
generated, bred to homozygosity, and subsequently evaluated with respect to its functionality as a DIO model. All hSPP1 mice used were genotyped at 3 weeks of age and displayed a distinct 362 bp DNA band indicating the humanized allele. Moreover, heterozygous hSPP11/2 animals showed a double-band at 362 bp and 254 bp, the latter identifying the mouse allele, whereas genotyping analysis in WT mice revealed a single band at 254 bp (17 hSPP1 animals of 20 are given in Fig S1). A pilot study using 3 unchallenged homozygous hSPP1 mice and WT control animals confirmed the selective expression of human OPN and the complete loss of murine OPN in our humanized mouse model (data not shown). In addition, serum Q8 human OPN levels (96.41 6 18.16 ng/ml; Fig 6, E) were comparable to serum murine OPN concentrations (71.30 6 9.74 ng/ml; data not shown) in lean hSPP1 and WT animals. Breeding efficacy, litter size, and sex distribution of littermates of hSPP1 mice was comparable to WT animals. Unchallenged homozygous hSPP1 animals did not differ in size or gross behavior. Moreover, no apparent abnormalities were observed in hSPP1 mice. Body weight was measured in unchallenged hSPP1 and WT mice at 9 weeks of age giving comparable results (Fig 2, A). However, blood glucose levels were significantly lower in nonfasting hSPP1 compared with WT mice (Fig 2, B). Evaluation of the hSPP1 mouse as a DIO model. To evaluate whether dietary treatment leads to the development of obesity in homozygous hSPP1 comparable to that of
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895
8
896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959
Translational Research - 2016
€ n et al Gru
Fig 6. Insertion of human OPN results in differential OPN expression patterns. Male hSPP1 and WT mice (n 5 8– 10 per group) were fed a HF and LF diet for 22 weeks. Gene expression of mSpp1 and hSPP1 was assessed in GWAT, SWAT (A, B), and liver (C, D). Fasting serum hOPN was determined in hSPP1 mice (E). Data are expressed as mean 6 standard error of the mean. *P , 0.05, **P , 0.01, ***P , 0.001. OPN, osteopontin; WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1; GWAT, gonadal white AT; SWAT, Subcutaneous white AT.
WT mice, 9-week-old male hSPP1 and WT mice were subdivided into 2 groups, involving 8–10 animals per group and were challenged by HF and LF diet for 22 weeks, respectively. Body weight was measured weekly (Fig 3, A) and was significantly increased in hSPP1 and WT mice on HF diet compared with respective lean controls after 22 weeks. Importantly, there was no difference between obese hSPP1 and
WT mice (Fig 3, B). Interestingly, hSPP1 on LF diet significantly increased the body weight compared with WT mice starting at 6 weeks of LF treatment (Fig 3, A and B), whereas food intake was comparable between all groups (Fig S2). Metabolic changes on HF diet in hSPP1 mice. To determine insulin resistance, an insulin tolerance test was performed in diet-challenged mice, and area under the
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023
Translational Research Volume -, Number 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087
€ n et al Gru
curve was calculated. Owing to a strong insulin response in WT mice on LF diet, we discontinued glucose measurements in WT mice after 30 minutes and rescued with oral glucose. hSPP1 were markedly less insulin sensitive compared to WT on LF (Fig 3, C and D). In addition, we observed significantly higher serum insulin levels in both genotypes after 22 weeks on HF diet compared with corresponding lean controls (Fig 3, E), which were mirrored by elevated homeostasis model assessment of insulin resistance (Fig 3, F). Hence, HF diet challenge induced obesity and insulin resistance in hSPP1 mice comparable to WT mice. However, hSPP1 mice on LF diet had significantly higher body weight and diminished insulin sensitivity compared with lean WT mice. HF diet induces adipose tissue hypertrophy as well as adipose tissue and liver inflammation similar to WT. Next,
we investigated the impact of HF diet on adipose tissues (GWAT and SWAT) of hSPP1 and WT mice. GWAT weight was not increased in hSPP1 animals on HF vs those on LF diet, whereas it was elevated in the latter compared to WT mice on LF diet. SWAT weight was significantly higher in hSPP1 after HF diet compared with lean controls, whereas SWAT weight was increased in hSPP1 on LF compared to WT (Fig 4, A). Gene expression of Adipoq and Pparg was significantly lower in hSPP1 on LF diet compared with WT animals, whereas mRNA levels were markedly higher in GWAT of WT mice fed a LF diet compared to obese WT mice (Fig 4, B and C). In both mouse models, gene expression levels of macrophage marker Emr1 and inflammation markers Ccl2 and Tnf-a were significantly elevated after HF diet in both adipose tissues than in respective control mice, indicating comparable inflammatory gene alterations of hSPP1 mice in adipose tissue under HF diet (Fig 4, D–F). To extend our findings regarding AT inflammation, we investigated macrophage infiltration and adipocyte hypertrophy in GWAT. Mac-2 staining of macrophages showed significantly more crown-like structures after HF diet in both mouse genotypes compared to LF controls (Fig 5, A and B). GWAT adipocyte size, which is often related to insulin resistance,33 was increased after HF diet in WT animals but not significantly changed by HF diet in hSPP1. Notably, adipocyte size of hSPP1 mice on LF diet was significantly increased compared with lean WT mice (Fig 5, C). Because it is well-recognized that obesity-linked inflammation also occurs in the liver, we measured liver weight as well as hepatic macrophage and inflammation markers. All mice on HF diet markedly increased liver weight compared to corresponding lean controls (Fig S3, A). As expected, mRNA levels
9
of macrophage markers Emr1 and Itgax as well as of proinflammatory markers Ccl2 and Tnf-a were significantly upregulated to a similar extent in both mouse genotypes after HF diet compared to respective lean controls (Fig S3, B–E). In conclusion, although several differences in fat distribution and GWAT adipocytes could be found under LF diet, adipose tissue properties were very similar in hSPP1 mice compared to WT mice in HF dietinduced obesity. Furthermore, dietary treatment resulted in hepatomegaly and obesity-associated liver inflammation in hSPP1 comparable to WT mice. Regulation of OPN expression in hSPP1 mice. To analyze the regulation of OPN gene expression after insertion of the human SPP1 gene under LF and HF diet, tissuespecific gene expression and serum levels of murine and human OPN were determined. As expected, homozygous hSPP1 mice did not express mSpp1, neither in AT depots nor in the liver and vice versa (Fig 6, A–D). WT mice showed significantly higher mSpp1 mRNA levels in GWAT, SWAT, and liver after HF diet compared to respective lean controls (Fig 6, A and C). In hSPP1 mice, however, SPP1 mRNA expression was not altered by HF in GWAT but significantly upregulated in SWAT of obese hSPP1 mice (Fig 6, B). Assessment of SPP1 mRNA expression in the liver revealed significantly lower SPP1 mRNA levels in obese hSPP1 mice compared to lean controls, whereas serum human OPN levels were not affected after dietary administration in hSPP1 mice (Fig 6, E). Thus, although OPN expression was significantly upregulated in SWAT in hSPP1 mice on HF challenge, no upregulation was found in GWAT and liver due to high expression already on LF diet. DISCUSSION
Humanized animal models were developed for various research fields, including immunology or cancer research.34 Here, we successfully inserted the human SPP1 gene into C57BL/6 mice and demonstrated similarities in body weight and glucose levels of nonfasting hSPP1 and WT animals, indicating no apparent abnormalities after transgenic modification, although the expression of OPN was high in GWAT and liver already under LF diet. Moreover, phenotypical parameters including litter size, sex distribution, and breeding efficacy of hSPP1 mice did not differ when compared to WT animals. Notably, research data on Spp12/2 animals revealed improved insulin sensitivity, tissue inflammation, and hepatic steatosis when challenged with HF diet,12 whereas our hSPP1 animal model developed obesity-induced complications similar to a wellknown DIO model. Therefore, our results indicate the
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151
10
1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215
Translational Research - 2016
€ n et al Gru
functionality of this classical knock-out-knock-in model. Taken together, these characteristics make the developed hSPP1 model a versatile tool for a variety of preclinical in vivo and ex vivo studies to target human OPN. Diet-induced obesity strikingly resembles the human metabolic syndrome including inflammatory and metabolic characteristics. HF diet administration tremendously increased body weight as well as SWAT and liver weight in the hSPP1 mouse model. Moreover, tissue-specific inflammation and macrophage infiltration was significantly upregulated after HF diet. Importantly, infiltrating macrophages phagocyte dying adipocytes, thereby forming crown-like structures, which have been reported to play a functional role in obesity. Here, we demonstrate that obese hSPP1 mice showed significantly more crown-like structures than lean controls and together with changes in inflammatory gene expression this indicates changes in obesityinduced inflammation in hSPP1 similar to WT animals. In addition, obesity-associated insulin resistance was induced by HF diet in both animal models to a comparable extent. Although mice on HF diet were strikingly similar irrespective of the genotype, hSPP1 mice on LF diet in parallel to high OPN expression, revealed altered body weight, GWAT weight, adipocyte hypertrophy, insulin resistance, and SPP1 expression levels. The increased body weight in hSPP1 compared to WT mice on LF diet as reflected by a marked increase in GWAT weight due larger adipocytes is regarded as fundamental to the other observed alterations including hepatic alterations and insulin resistance.33 Also decreased Adipoq and Pparg gene expression levels in GWAT match the diminished insulin sensitivity and increased GWAT weight. Because food intake was similar in hSPP1 and WT mice on LF, we presume lower energy expenditure in lean hSPP1 mice as a basis for increased body weight in hSPP1 compared to WT animals. Further studies including multiparameter monitoring in metabolic cages are warranted for a more detailed characterization of this model for its use in metabolic studies. Moreover, the hSPP1 model in LF diet constitutes an interesting model for obesityindependent inflammatory and infectious diseases. Although changes in GWAT physiology in hSPP1 mice on LF diet could be a basis for the failure to upregulate hSPP1 gene expression in GWAT and liver by HF diet, intrinsic changes in SPP1 gene regulation by the transgenic modification cannot be ruled out. Although murine 50 regulatory sequences including the signal peptide were conserved, intronic regulatory components that were replaced by the genomic human sequence may play a role for the observed alterations
in GWAT and liver. More data in other and more pronounced inflammatory disease models have to be studied to evaluate the comparability of changes in hSPP1 compared to WT mice. Because there is a slight difference between the genotypes used in this study, comparisons were primarily made within a genotype, and similarities or important differences were highlighted between hSPP1 and WT animals. To date, there is a high prevalence of obesityassociated disorders including AT inflammation, insulin resistance, and cardio-metabolic disease. In 2014, the World Health Organization stated a number of approximately 1.9 billion overweight adults, of which over 600 million were recognized of being obese.35 According to this background and the crucial role of OPN in obesity-associated diseases, hSPP1 mice represent a new interesting model to evolve novel strategies to combat OPN-induced inflammation and metabolic complications, for example, by immunotherapy. Moreover, this model is now available to target human SPP1/ OPN in a variety of other inflammatory, infectious, and neoplastic disease models in which OPN plays a key role. ACKNOWLEDGMENTS
Conflicts of Interest: The authors have read the journal’s authorship statement and policy on conflicts of interest and declare no commercial or financial conflict of interest. This work was supported by the Federal Ministry of Economy, Family and Youth and the National Founda- Q9 tion for Research, Technology and Development (to T. M. Stulnig). The authors gratefully thank Melina Amor for technical support. The article has been reviewed and approved by all authors. REFERENCES
1. Lund SA, Giachelli CM, Scatena M. The role of osteopontin in inflammatory processes. J Cell Commun Signal 2009;3:311–22. 2. Tokairin T, Nishikawa Y, Watanabe H, et al. Osteopontin expression in the liver with severe perisinusoidal fibrosis: autopsy case of Down syndrome with transient myeloproliferative disorder. Pathol Int 2008;58:64–8. 3. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J. Osteopontin–a molecule for all seasons. QJM 2002;95: 3–13. 4. Giachelli CM, Lombardi D, Johnson RJ, Murry CE, Almeida M. Evidence for a role of osteopontin in macrophage infiltration in response to pathological stimuli in vivo. Am J Pathol 1998;152: 353–8. 5. Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. J Leukoc Biol 2002;72:752–61.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279
Translational Research Volume -, Number 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343
€ n et al Gru
6. Crawford HC, Matrisian LM, Liaw L. Distinct roles of osteopontin in host defense activity and tumor survival during squamous cell carcinoma progression in vivo. Cancer Res 1998;58:5206–15. 7. Yamamoto N, Sakai F, Kon S, et al. Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis. J Clin Invest 2003;112:181–8. 8. Agnholt J, Kelsen J, Schack L, Hvas CL, Dahlerup JF, Sørensen ES. Osteopontin, a protein with cytokine-like properties, is associated with inflammation in Crohn’s disease. Scand J Immunol 2007;65:453–60. 9. Leitner L, J€ urets A, Itariu BK, et al. Osteopontin promotes aromatase expression and estradiol production in human adipocytes. Breast Cancer Res Treat 2015;154:63–9. 10. Waller AH, Sanchez-Ross M, Kaluski E, Klapholz M. Osteopontin in cardiovascular disease: a potential therapeutic target. Cardiol Rev 2010;18:125–31. 11. Bruemmer D, Collins AR, Noh G, et al. Angiotensin IIaccelerated atherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice. J Clin Invest 2003;112:1318–31. 12. Kiefer FW, Neschen S, Pfau B, et al. Osteopontin deficiency protects against obesity-induced hepatic steatosis and attenuates glucose production in mice. Diabetologia 2011;54:2132–42. 13. Takemoto M, Yokote K, Nishimura M, et al. Enhanced expression of osteopontin in human diabetic artery and analysis of its functional role in accelerated atherogenesis. Arterioscler Thromb Vasc Biol 2000;20:624–8. 14. Sahai A, Malladi P, Melin-Aldana H, Green RM, Whitington PF. Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model. Am J Physiol Gastrointest Liver Physiol 2004;287:G264–73. 15. Isoda K, Kamezawa Y, Ayaori M, Kusuhara M, Tada N, Ohsuzu F. Osteopontin transgenic mice fed a high-cholesterol diet develop early fatty-streak lesions. Circulation 2003;107:679–81. 16. Bertola A, Deveaux V, Bonnafous S, et al. Elevated expression of osteopontin may be related to adipose tissue macrophage accumulation and liver steatosis in morbid obesity. Diabetes 2009;58: 125–33. 17. Zeyda M, Gollinger K, Todoric J, et al. Osteopontin is an activator of human adipose tissue macrophages and directly affects adipocyte function. Endocrinology 2011;152:2219–27. 18. Nomiyama T, Perez-Tilve D, Ogawa D, et al. Osteopontin mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance in mice. J Clin Invest 2007;117:2877–88. 19. Kiefer FW, Zeyda M, Todoric J, et al. Osteopontin expression in human and murine obesity: extensive local up-regulation in adipose tissue but minimal systemic alterations. Endocrinology 2008;149:1350–7. 20. G omez-Ambrosi J, Catalan V, Ramırez B, et al. Plasma osteopontin levels and expression in adipose tissue are increased in obesity. J Clin Endocrinol Metab 2007;92:3719–27.
11
21. Chapman J, Miles PD, Ofrecio JM, et al. Osteopontin is required for the early onset of high fat diet-induced insulin resistance in mice. PLoS One 2010;5:e13959. 22. Yokosaki Y, Tanaka K, Higashikawa F, Yamashita K, Eboshida A. Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin. Matrix Biol J Int Soc Matrix Biol 2005; 24:418–27. 23. Scatena M, Liaw L, Giachelli CM. Osteopontin: a multifunctional molecule regulating chronic inflammation and vascular disease. Arterioscler Thromb Vasc Biol 2007;27:2302–9. 24. Ito K, Kon S, Nakayama Y, et al. The differential amino acid requirement within osteopontin in alpha4 and alpha9 integrinmediated cell binding and migration. Matrix Biol J Int Soc Matrix Biol 2009;28:11–9. 25. Bayless KJ, Davis GE. Identification of dual a4b1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin. J Biol Chem 2001; 276:13483–9. 26. Takahashi K, Takahashi F, Tanabe KK, Takahashi H, Fukuchi Y. The carboxyl-terminal fragment of osteopontin suppresses arginine-glycine-asparatic acid-dependent cell adhesion. Biochem Mol Biol Int 1998;46:1081–92. 27. Okamoto H. Osteopontin and cardiovascular system. Mol Cell Biochem 2006;300:1–7. 28. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis B viruspositive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med 2003;9: 416–23. 29. Zhao J, Dong L, Lu B, et al. Down-regulation of osteopontin suppresses growth and metastasis of hepatocellular carcinoma via induction of apoptosis. Gastroenterology 2008;135:956–68. 30. Bhattacharya SD, Mi Z, Kim VM, Guo H, Talbot LJ, Kuo PC. Osteopontin regulates epithelial mesenchymal transition-associated growth of hepatocellular cancer in a mouse xenograft model. Ann Surg 2012;255:319–25. 31. Doyle A, McGarry MP, Lee NA, Lee JJ. The construction of transgenic and gene knockout/knockin mouse models of human disease. Transgenic Res 2012;21:327–49. 32. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods San Diego Calif 2001;25:402–8. 33. Kim JI, Huh JY, Sohn JH, et al. Lipid-overloaded enlarged adipocytes provoke insulin resistance independent of inflammation. Mol Cell Biol 2015;35:1686–99. 34. Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nat Rev Immunol 2007;7:118–30. 35. WHO. Obesity and overweight [Internet]. WHO. Available at: http://www.who.int/mediacentre/factsheets/fs311/en/. Accessed August 20, 2015.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407
11.e1
1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471
Translational Research - 2016
€ n et al Gru
APPENDIX
Fig S1. PCR analysis confirms homozygosity of hSPP1 mice. Three-week-old male hSPP1 mice (n 5 20; genotyping of 17 animals are given as an example) were ear-clipped, and ear tissue was used for DNA isolation. Genotyping was performed using the 2xKAPPA 2G Fast kit and indicated primer pairs. Electrophoresis revealed the humanized allele at 362 bp and the WT allele at 254 bp. PCR, polymerase chain reaction; WT, wild-type; hSPP1, humanized SPP1.
Q10
Fig S2. Food intake of hSPP1 and WT mice. Male hSPP1 and WT mice (n 5 8–10 per group) were fed a HF and LF diet for 22 weeks. Food intake was measured over 3 weeks and calculated as grams per mouse per day. Data is expressed as mean 6 standard error of the mean. ,0.05, **P , 0.01, ***P , 0.001. WT, wild-type; LF, low fat; HF, high fat; hSPP1, humanized SPP1.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535
Translational Research Volume -, Number 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599
€ n et al Gru
Fig S3. Hepatic gene expression displays inflammation after HF diet. Male hSPP1 and WT mice (n 5 8–10 per group) were set on indicated diets for 22 weeks. Liver weight was determined after sacrification (A). Hepatic gene expression levels of macrophage marker Emr1 (B) and Itgax (C) as well as of inflammation markers Ccl2 (D) and Tnf-a (E) were assessed by quantitative real-time PCR. Gene expression was normalized to WT LF. Data is given as mean 6 standard error of the mean. *P , 0.05, **P , 0.01, and ***P , 0.001. mRNA, messenger RNA; WT, wild-type; LF, low fat; HF, high fat; PCR, polymerase chain reaction; hSPP1, humanized SPP1.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
11.e2
1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663
ORIGINAL ARTICLE Q2 Q1
Q11
A humanized osteopontin mouse model and its application in immunometabolic obesity studies € KARINA ZEYDA, VERONICA MORENO-VIEDMA, KARIN STROHMEIER, NICOLE G. GRUN, € GUNTHER STAFFLER, MAXIMILIAN ZEYDA, and THOMAS M. STULNIG VIENNA, AUSTRIA
Osteopontin (OPN) is a multifunctional protein involved in several inflammatory processes and pathogeneses including obesity-related disorders and cancer. OPN binds to a variety of integrin receptors and CD44 resulting in a proinflammatory stimulus. Therefore, OPN constitutes a novel interesting target to develop new therapeutic strategies, which counteract OPN’s proinflammatory properties. We established a humanized SPP1 (hSPP1) mouse model and evaluated its suitability as a model for obesity and insulin resistance. Unchallenged hSPP1 animals did not significantly differ in body weight and gross behavioral properties compared to wild-type (WT) animals. High-fat diet-challenged hSPP1 similarly developed obesity and inflammation, whereas insulin resistance was markedly changed. However, OPN expression profile in tissues was significantly altered in hSPP1 compared to WT depending on the diet. In conclusion, we developed a versatile humanized model to study the action of OPN in vivo and to develop strategies that target human OPN in a variety of pathologies. (Translational Research 2016;-:1–11) Abbreviations: --- ¼ ---
Q3
INTRODUCTION From the Christian Doppler Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Department of Medicine III, Medical University of Vienna, Vienna, Austria; FH Campus Wien, University of Applied Sciences, Department Health, Section Biomedical Science, Vienna, Austria; AFFiRiS AG, Vienna, Austria; Department of Pediatrics and Adolescent Medicine, Clinical Division of Pediatric Pulmonology, Allergology and Endocrinology, Medical University of Vienna, Vienna Austria. Submitted for publication June 3, 2016; revision submitted July 4, 2016; accepted for publication July 11, 2016. Reprint requests: Thomas M. Stulnig, Christian Doppler Laboratory for Cardio-Metabolic Immunotherapy and Clinical Division of Endocrinology and Metabolism, Department of Medicine III, Medical University of Vienna, W€ahringer G€urtel 18-20, 1090 Vienna, Austria; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2016 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.trsl.2016.07.009
O
steopontin (OPN) is a multifunctional protein, first identified as a T helper type 1 cytokine, which is mainly expressed in various tissues and cells including macrophages, T-cells, dendritic cells, hepatocytes, smooth muscle cells, endothelial, and epithelial cells.1-3 OPN exhibits properties not only of a soluble cytokine that acts in an autocrine or paracrine manner on liver cells but also of an extracellular matrix (ECM)-bound protein.1 OPN plays a prominent role in monocyte migration, adhesion, and differentiation4-6 as well as in the pathophysiology of chronic inflammatory diseases, such as rheumatoid arthritis,7 Crohn’s disease,8 cancer,9 and cardiovascular disease.10,11 OPN is involved in the pathogenesis of Q4 obesity-associated complications, including AT inflammation, insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and atherosclerosis.12-16 In vitro 1
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
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 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127
2
128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191
Translational Research - 2016
€ n et al Gru
AT A GLANCE COMMENTARY € n NG, et al. Gru Background
Osteopontin (OPN) is involved in a number of inflammatory processes, including obesity-linked complications, asthma, rheumatoid arthritis as well as cancer progression and metastasis. Proteolytic cleavage of OPN increases OPN’s proinflammatory properties and therefore constitutes an interesting target to develop new strategies to counteract OPN’s actions. To enable research on human OPN, we generated a humanized SPP1 (hSPP1) mouse and evaluated it as a diet-induced obesity model. Translational Significance
Our findings propose the hSPP1 animal model to be applied for a variety of pathologies in which OPN is a key player, including obesity-driven, cardio-metabolic as well as other diseases that can be translated to humans.
studies with adipocytes identified OPN as a modulator of insulin resistance and impaired glucose uptake.17 Obese mice and humans revealed markedly increased messenger RNA (mRNA) levels for OPN in visceral and subcutaneous fat.18 Also, plasma OPN concentrations were markedly elevated in obese human subjects compared with lean controls.19,20 Importantly, high-fat (HF) diet treatment of mice lacking OPN, improved adipose tissue inflammation and insulin sensitivity12 as well as reduced hyperleptinemia and adipocyte hypertrophy.21 OPN acts via integrin binding by different domains. Although the arg-gly-asp (RGD)-containing domain mainly binds integrins avb1, avb3, avb5, and a5b122 on the cell surface, the ser-val-val-tyr-glu-leu-arg (SVVYGLR) motif interacts with integrins a4b1, a4b7, and a9b1.23,24 Importantly, the RGD region is fully conserved between species, whereas the SVVYGLR motif differs between humans (SVVYGLR) and rodents (SLAYGLR).23 The third integrin-binding domain ELVTDFTDLPAT has also been described to bind a4b1.25 Overall, human and murine OPN show only 63% amino acid sequence identity (National Center of Biotechnology Information). OPN’s actions are affected by protease cleavage. The cryptic region SVVYGLR, which is usually hidden in intact OPN, becomes accessible on thrombin and matrix metalloproteinase cleavage. Interestingly, although enzy-
matic thrombin and matrix metalloproteinase cleavage produces a proinflammatory N-terminal OPN fragment, the less described C-terminal OPN form may even attenuate inflammatory processes.26 Moreover, bonding with the hyaluronic acid receptor CD44 leads to the induction of macrophage chemotaxis and the b3-integrin receptors engagement.27 To develop therapeutic strategies against OPN actions, small molecules are not a primary option but sequence-specific approaches are needed such as specific antibodies, RNA aptamers, and antisense oligonucleotides or interfering RNAs. Such approaches targeting OPN have been successfully applied on human tissues transplanted into immunodeficient mice, for example, in cancer studies.28-30 However, human cell transplantation is not feasible for most disease models, particularly inflammatory diseases. To facilitate in vivo studies of blocking human OPN, we developed a humanized mouse model by replacing the mouse gene with human SPP1 sequence. Owing to OPN’s crucial involvement in a variety of inflammatory pathologies and particularly its association with cardiometabolic complications of obesity, we evaluated the humanized SPP1 (hSPP1) mouse model as a dietinduced obesity (DIO) model. We show the functionality of hSPP1 mice as a DIO model by generation of obesity-associated insulin resistance and inflammation following HF diet feeding. Hence, hSPP1 mice are a highly useful model to investigate OPN’s role not only to study prevention and treatment of obesity and its complications but also to facilitate preclinical studies on neutralization of human OPN in a large variety of other inflammatory diseases and cancer. MATERIAL AND METHODS Animals and diets. Heterozygous C57BL/6Spp1tm2737 (SPP1) Arte (hSPP11/2) mice were developed with Taconic (K€oln, Germany). In short, the chimeric SPP1 protein sequence was chosen as depicted in Fig 1, A. Accordingly, exon 2 contains the translation initiation codon and the sequence, which encodes the cleavable signal peptide. The mouse genomic sequence from amino acid 21 (valine) in exon 3 up to the termination codon in exon 7 has been replaced by its human counterpart to leave a single N-terminal mouse-specific amino acid in the mature protein (p.Ile19Leu; Fig 1). Positive selection markers, Q5 including neomycin resistance flanked by FRT and puromycin resistance flanked with F3, were inserted into human intron 3 as well as downstream of the mouse 30 untranslated region, respectively. BAC Q6 clones from the mouse C57BL/6J were used for the targeting vector that was further transfected using
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255
Translational Research Volume -, Number -
3
web 4C=FPO
256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319
€ n et al Gru
Fig 1. Generation of the humanized SPP1 gene for homologous recombination. Description of the constructed chimeric SPP1 in comparison to the murine and human form (A). Stepwise generation of the humanized allele is given in (B).NeoR, neomycin resistance; PuroR, puromycin resistance.
Fig 2. Blood glucose levels are significantly lower in hSPP1 compared to WT animals with similar body weight. Male hSPP1 and WT mice (n 5 20 per group) were kept on normal chow until 9 weeks of age. Body weight (A) and blood glucose levels (B) were determined before LF and HF administration. Data is expressed as mean 6 standard error of the mean. ***P , 0.001. WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1.
electroporation into a Taconic C57BL/6N embryonic stem cell line. After homologous recombination, clones were selected with double positive neomycin resistance–puromycin resistance selection, and the humanized allele was obtained after Flp-mediated
removal of these selection markers. Expression of the human SPP1 gene is controlled by the mouse Spp1 promoter and results in the loss of expression of the mouse Spp1 gene (Fig 1, B). Generation of the transgenic mouse model was according to
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
4
384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447
Translational Research - 2016
€ n et al Gru
Fig 3. Dietary challenge alters body weight and insulin resistance in hSPP1 mice. Male hSPP1 and WT mice (n 5 8–10 per group) were fed a HF and LF diet for 22 weeks. Body weight was determined weekly over 22 weeks (A,B). After 15 weeks on diet, an ITT was performed. Glucose levels were measured over 120 minutes after intraperitoneal insulin application of 0.75-IU/kg body weight for HF and 0.25-IU/kg body weight for LF, respectively, (C) and AUC (D) was calculated. Fasting glucose and insulin levels (E) were measured after 22 weeks, and insulin resistance is given by the HOMA-IR (F). Data is expressed as mean 6 standard error of the mean. *,#,§P , 0.05, **,##,§§P , 0.01, ***,###,§§§P , 0.001. (*) indicates the comparison between WT LF and HF, (#) comparison between hSPP1 LF and WT LF; (§) comparison between hSPP1 LF and HF. AUC, area under the curve; ITT, insulin tolerance test; HOMA-IR, homeostasis model assessment of insulin resistance; WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1.
well-described instructions.31 Heterozygous hSPP11/2 animals were crossbred to homozygosity in house. At 9 weeks of age, 10 male hSPP1 and 10 C57BL/6JRj (WT) mice (Janvier Labs, Saint Berthevin, France) were set on high-fat diet (HF, 60 kcal%, D12492; Research
Diets, New Brunswisk, NJ) for 22 weeks to induce obesity, whereas 10 male hSPP1 and 10 WT mice were fed a low-fat diet (LF, 10 kcal%, D12450 B; Research Diets) to serve as negative controls for DIO. All mice were kept on normal chow until 9 weeks of age.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
Translational Research Volume -, Number 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575
€ n et al Gru
5
Fig 4. Obesity-induced adipose tissue inflammation in hSPP1 mice is similar to WT animals. Male hSPP1 and WT mice (n 5 8–10 per group) were set on indicated diets for 22 weeks. GWAT and SWAT weight (A) was determined after sacrification; RNA was isolated and transcribed to complementary DNA. Gene expression of insulin sensitivity markers Adipoq (B) and Pparg (C), macrophage marker Emr1 (F4/80) (D) as well as of cytokines Ccl2 (E) and Tnf-a (F) was assessed by quantitative real-time PCR. Ubc was used as house-keeping gene, and data are given after normalization to GWAT WT LF as mean 6 standard error of the mean. *P , 0.05, **P , 0.01, ***P , 0.001. WT, wild-type; PCR, polymerase chain reaction; Ubc, ubiquitin C; LF, low-fat diet; HF, high-fat diet; mRNA, messenger RNA; hSPP1, humanized SPP1; GWAT, gonadal white AT; SWAT, Subcutaneous white AT.
All mice were housed in a specific pathogen-free facility, maintaining a 12-hour light/dark cycle and had free access to water and food. Food intake was measured 3 times per week. Weight was determined weekly, blood was drawn every third week and immediately before mice were sacrificed. Subcutaneous white AT (SWAT)
and gonadal white AT (GWAT) as well as liver were collected, weighed, and stored for further experiments. The protocol was approved by the local ethics committee for animal studies and followed the guidelines on accommodation and care of animals formulated by the European Convention for the Protection of Vertebrate
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639
640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703
Translational Research - 2016
€ n et al Gru
web 4C=FPO
6
Fig 5. Dietary challenge induces adipocyte hypertrophy and crown-like structure formation in both genotypes. Male hSPP1 and WT mice (n 5 8–10 per group) were fed a HF and LF diet for 22 weeks. Paraffin-embedded GWAT sections were stained for Mac-2 and visualized with light microscopy (A). Formation of crown-like structures (CLS) normalized to total number of adipocytes (B) and adipocyte size (C) was assessed with ImageJ software. Representative pictures are shown after 10-fold magnification. Data are expressed as mean 6 standard error of the mean. *P , 0.05, ***P , 0.001. WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1; GWAT, gonadal white AT; SWAT, Subcutaneous white AT.
Animals Used for Experimental and Other Scientific Purposes. Polymerase chain reaction (PCR) for genotyping analysis. Genomic DNA was isolated from ear tissue af-
ter clipping to identify homozygous hSPP1 mice using 2xKAPPA 2G Fast HS Kit. PCR was performed according to manufacturer’s instructions (KAPA Biosystems, Wilmington, Mass). To amplify the constitutive humanized and WT allele, 2 different primer pairs were used: for the humanized SPP1 allele forward primer
50 -CTGTTTGGCATTGCCTCC-30 and reverse primer 30 -TATACTGTGCTTGGTACTGGCC-50 , for the murine WT Spp1 allele forward primer 50 -TAG CAACCTTTGCGACGC-30 and reverse primer 30 -TATACTGTGCTTGGTACTGGCC-5’. In addition, forward primer 50 -CGAATGTAAGGAGTCTGGTGG-30 and reverse primer 30 -CAAGTTTCAAGCT 0 TTTGCTCG-5 were used to detect both, the humanized and WT allele. To assess DNA size, 100 base pair (bp) DNA ladder was used (Invitrogen, Carlsbad, Calif).
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767
Translational Research Volume -, Number 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831
Q7
€ n et al Gru
The humanized allele was displayed at 362 bp, whereas the WT allele showed a 254 bp band. Genomic DNA isolated from WT and heterozygous mice was employed as negative controls. Serum metabolic parameters. Serum insulin (Mercodia, Uppsala, Sweden) and OPN (R&D Systems, Minneapolis, USA) were analyzed with commercially available enzyme-linked immunosorbent assays. After a 5-hour fasting period, insulin sensitivity was assessed after 15 weeks on indicated diets by measuring glucose levels before, 30, 60, 90, and 120 minutes after an intraperitoneal injection of recombinant human insulin (0.75 IU/kg body weight NovoRapid for HF and 0.25 IU/kg body weight for LF, respectively; Novo Nordisk, Bagsværd, Denmark). Blood glucose levels in mice on normal chow diet were measured without fasting using OneTouch Ultra test strips (LifeScan, Milpitas, USA). Homeostasis model assessment of insulin resistance was calculated as an index for insulin resistance. Reverse transcription and gene expression. One part of SWAT, GWAT, and liver was stored in RNAlater (Qiagen, Hilden, Germany) overnight at 4 C. RNAlater was removed, tissues were snap frozen and stored at 280 C. RNA isolation was performed with peqGOLD TriFastTM (Peqlab, Erlangen, Germany) by using an automated homogenizer (Precellys 24, Erlangen, Germany), and one microgram RNA was transcribed to complementary DNA with M-MLV Reverse Transcriptase (Promega, Madison, USA). Gene expression of F4/80 (Emr1, Mm00802529_m1), Mcp1 (Ccl2, Mm00441242_m1), tumor necrosis factor-a (Tnf, Mm00443258_m1), adiponectin (Adipoq, Mm004564 25_m1), peroxisome proliferator-activated receptor gamma (Pparg, Mm01184322_m1), integrin alpha x (Itgax; Cd11c, Mm00498698_m1), murine OPN (mSpp1; Spp1, Mm00436767_m1), human OPN (hSPP1; SPP1, Hs00959010_m1), and reference gene ubiquitin C (Ubc; Ubc, Mm01201237_m1) was analyzed by quantitative real-time RT-PCR on an ABI Prism 7000 cycler with commercially available assays-on demand kits (TaqMan Gene Expression Assay, Applied Biosystems, Foster City, Calif) and normalized to Ubc. The comparative threshold cycle method was used to calculate relative expression.32 Immunohistochemistry. Murine GWAT samples were fixed with neutral buffered 4% paraformaldehyde at 4 C and were paraffin-embedded. After dewaxation and rehydration, sections were immunoh istochemically stained for Mac-2 (Cedarlane, Ontario, Canada) using the ABC kit (Vector Laboratories) according to manufacturer’s protocol. Counterstaining was performed by using hematoxylin (Merck,
7
Darmstadt, Germany), and sections were analyzed with standard light microscopy using a 10-fold magnification. Crown-like structures and density of adipocytes were quantified with ImageJ software, thereby assessing 5 independent fields per sample. Crown-like structures were normalized to total adipocyte number. Statistical analysis. Data are given as mean and standard error of the mean (mean 6 standard error of the mean). Comparisons between hSPP1 and WT were assessed by 1-way analysis of variance using Tukey’s multiple comparisons test. For body weight gain and insulin tolerance test results, a 2-way analysis of variance with Tukey’s multiple comparisons test was performed. Impact of dietary treatment on OPN expression within a genotype was analyzed by 2-tailed independent Student’s t-test. A P value of #0.05 was considered statistically significant. RESULTS Generation and characterization of the humanized 1/2 hSPP1 mouse. Heterozygous hSPP1 animals were
generated, bred to homozygosity, and subsequently evaluated with respect to its functionality as a DIO model. All hSPP1 mice used were genotyped at 3 weeks of age and displayed a distinct 362 bp DNA band indicating the humanized allele. Moreover, heterozygous hSPP11/2 animals showed a double-band at 362 bp and 254 bp, the latter identifying the mouse allele, whereas genotyping analysis in WT mice revealed a single band at 254 bp (17 hSPP1 animals of 20 are given in Fig S1). A pilot study using 3 unchallenged homozygous hSPP1 mice and WT control animals confirmed the selective expression of human OPN and the complete loss of murine OPN in our humanized mouse model (data not shown). In addition, serum Q8 human OPN levels (96.41 6 18.16 ng/ml; Fig 6, E) were comparable to serum murine OPN concentrations (71.30 6 9.74 ng/ml; data not shown) in lean hSPP1 and WT animals. Breeding efficacy, litter size, and sex distribution of littermates of hSPP1 mice was comparable to WT animals. Unchallenged homozygous hSPP1 animals did not differ in size or gross behavior. Moreover, no apparent abnormalities were observed in hSPP1 mice. Body weight was measured in unchallenged hSPP1 and WT mice at 9 weeks of age giving comparable results (Fig 2, A). However, blood glucose levels were significantly lower in nonfasting hSPP1 compared with WT mice (Fig 2, B). Evaluation of the hSPP1 mouse as a DIO model. To evaluate whether dietary treatment leads to the development of obesity in homozygous hSPP1 comparable to that of
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895
8
896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959
Translational Research - 2016
€ n et al Gru
Fig 6. Insertion of human OPN results in differential OPN expression patterns. Male hSPP1 and WT mice (n 5 8– 10 per group) were fed a HF and LF diet for 22 weeks. Gene expression of mSpp1 and hSPP1 was assessed in GWAT, SWAT (A, B), and liver (C, D). Fasting serum hOPN was determined in hSPP1 mice (E). Data are expressed as mean 6 standard error of the mean. *P , 0.05, **P , 0.01, ***P , 0.001. OPN, osteopontin; WT, wild-type; LF, low-fat diet; HF, high-fat diet; hSPP1, humanized SPP1; GWAT, gonadal white AT; SWAT, Subcutaneous white AT.
WT mice, 9-week-old male hSPP1 and WT mice were subdivided into 2 groups, involving 8–10 animals per group and were challenged by HF and LF diet for 22 weeks, respectively. Body weight was measured weekly (Fig 3, A) and was significantly increased in hSPP1 and WT mice on HF diet compared with respective lean controls after 22 weeks. Importantly, there was no difference between obese hSPP1 and
WT mice (Fig 3, B). Interestingly, hSPP1 on LF diet significantly increased the body weight compared with WT mice starting at 6 weeks of LF treatment (Fig 3, A and B), whereas food intake was comparable between all groups (Fig S2). Metabolic changes on HF diet in hSPP1 mice. To determine insulin resistance, an insulin tolerance test was performed in diet-challenged mice, and area under the
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023
Translational Research Volume -, Number 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087
€ n et al Gru
curve was calculated. Owing to a strong insulin response in WT mice on LF diet, we discontinued glucose measurements in WT mice after 30 minutes and rescued with oral glucose. hSPP1 were markedly less insulin sensitive compared to WT on LF (Fig 3, C and D). In addition, we observed significantly higher serum insulin levels in both genotypes after 22 weeks on HF diet compared with corresponding lean controls (Fig 3, E), which were mirrored by elevated homeostasis model assessment of insulin resistance (Fig 3, F). Hence, HF diet challenge induced obesity and insulin resistance in hSPP1 mice comparable to WT mice. However, hSPP1 mice on LF diet had significantly higher body weight and diminished insulin sensitivity compared with lean WT mice. HF diet induces adipose tissue hypertrophy as well as adipose tissue and liver inflammation similar to WT. Next,
we investigated the impact of HF diet on adipose tissues (GWAT and SWAT) of hSPP1 and WT mice. GWAT weight was not increased in hSPP1 animals on HF vs those on LF diet, whereas it was elevated in the latter compared to WT mice on LF diet. SWAT weight was significantly higher in hSPP1 after HF diet compared with lean controls, whereas SWAT weight was increased in hSPP1 on LF compared to WT (Fig 4, A). Gene expression of Adipoq and Pparg was significantly lower in hSPP1 on LF diet compared with WT animals, whereas mRNA levels were markedly higher in GWAT of WT mice fed a LF diet compared to obese WT mice (Fig 4, B and C). In both mouse models, gene expression levels of macrophage marker Emr1 and inflammation markers Ccl2 and Tnf-a were significantly elevated after HF diet in both adipose tissues than in respective control mice, indicating comparable inflammatory gene alterations of hSPP1 mice in adipose tissue under HF diet (Fig 4, D–F). To extend our findings regarding AT inflammation, we investigated macrophage infiltration and adipocyte hypertrophy in GWAT. Mac-2 staining of macrophages showed significantly more crown-like structures after HF diet in both mouse genotypes compared to LF controls (Fig 5, A and B). GWAT adipocyte size, which is often related to insulin resistance,33 was increased after HF diet in WT animals but not significantly changed by HF diet in hSPP1. Notably, adipocyte size of hSPP1 mice on LF diet was significantly increased compared with lean WT mice (Fig 5, C). Because it is well-recognized that obesity-linked inflammation also occurs in the liver, we measured liver weight as well as hepatic macrophage and inflammation markers. All mice on HF diet markedly increased liver weight compared to corresponding lean controls (Fig S3, A). As expected, mRNA levels
9
of macrophage markers Emr1 and Itgax as well as of proinflammatory markers Ccl2 and Tnf-a were significantly upregulated to a similar extent in both mouse genotypes after HF diet compared to respective lean controls (Fig S3, B–E). In conclusion, although several differences in fat distribution and GWAT adipocytes could be found under LF diet, adipose tissue properties were very similar in hSPP1 mice compared to WT mice in HF dietinduced obesity. Furthermore, dietary treatment resulted in hepatomegaly and obesity-associated liver inflammation in hSPP1 comparable to WT mice. Regulation of OPN expression in hSPP1 mice. To analyze the regulation of OPN gene expression after insertion of the human SPP1 gene under LF and HF diet, tissuespecific gene expression and serum levels of murine and human OPN were determined. As expected, homozygous hSPP1 mice did not express mSpp1, neither in AT depots nor in the liver and vice versa (Fig 6, A–D). WT mice showed significantly higher mSpp1 mRNA levels in GWAT, SWAT, and liver after HF diet compared to respective lean controls (Fig 6, A and C). In hSPP1 mice, however, SPP1 mRNA expression was not altered by HF in GWAT but significantly upregulated in SWAT of obese hSPP1 mice (Fig 6, B). Assessment of SPP1 mRNA expression in the liver revealed significantly lower SPP1 mRNA levels in obese hSPP1 mice compared to lean controls, whereas serum human OPN levels were not affected after dietary administration in hSPP1 mice (Fig 6, E). Thus, although OPN expression was significantly upregulated in SWAT in hSPP1 mice on HF challenge, no upregulation was found in GWAT and liver due to high expression already on LF diet. DISCUSSION
Humanized animal models were developed for various research fields, including immunology or cancer research.34 Here, we successfully inserted the human SPP1 gene into C57BL/6 mice and demonstrated similarities in body weight and glucose levels of nonfasting hSPP1 and WT animals, indicating no apparent abnormalities after transgenic modification, although the expression of OPN was high in GWAT and liver already under LF diet. Moreover, phenotypical parameters including litter size, sex distribution, and breeding efficacy of hSPP1 mice did not differ when compared to WT animals. Notably, research data on Spp12/2 animals revealed improved insulin sensitivity, tissue inflammation, and hepatic steatosis when challenged with HF diet,12 whereas our hSPP1 animal model developed obesity-induced complications similar to a wellknown DIO model. Therefore, our results indicate the
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151
10
1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215
Translational Research - 2016
€ n et al Gru
functionality of this classical knock-out-knock-in model. Taken together, these characteristics make the developed hSPP1 model a versatile tool for a variety of preclinical in vivo and ex vivo studies to target human OPN. Diet-induced obesity strikingly resembles the human metabolic syndrome including inflammatory and metabolic characteristics. HF diet administration tremendously increased body weight as well as SWAT and liver weight in the hSPP1 mouse model. Moreover, tissue-specific inflammation and macrophage infiltration was significantly upregulated after HF diet. Importantly, infiltrating macrophages phagocyte dying adipocytes, thereby forming crown-like structures, which have been reported to play a functional role in obesity. Here, we demonstrate that obese hSPP1 mice showed significantly more crown-like structures than lean controls and together with changes in inflammatory gene expression this indicates changes in obesityinduced inflammation in hSPP1 similar to WT animals. In addition, obesity-associated insulin resistance was induced by HF diet in both animal models to a comparable extent. Although mice on HF diet were strikingly similar irrespective of the genotype, hSPP1 mice on LF diet in parallel to high OPN expression, revealed altered body weight, GWAT weight, adipocyte hypertrophy, insulin resistance, and SPP1 expression levels. The increased body weight in hSPP1 compared to WT mice on LF diet as reflected by a marked increase in GWAT weight due larger adipocytes is regarded as fundamental to the other observed alterations including hepatic alterations and insulin resistance.33 Also decreased Adipoq and Pparg gene expression levels in GWAT match the diminished insulin sensitivity and increased GWAT weight. Because food intake was similar in hSPP1 and WT mice on LF, we presume lower energy expenditure in lean hSPP1 mice as a basis for increased body weight in hSPP1 compared to WT animals. Further studies including multiparameter monitoring in metabolic cages are warranted for a more detailed characterization of this model for its use in metabolic studies. Moreover, the hSPP1 model in LF diet constitutes an interesting model for obesityindependent inflammatory and infectious diseases. Although changes in GWAT physiology in hSPP1 mice on LF diet could be a basis for the failure to upregulate hSPP1 gene expression in GWAT and liver by HF diet, intrinsic changes in SPP1 gene regulation by the transgenic modification cannot be ruled out. Although murine 50 regulatory sequences including the signal peptide were conserved, intronic regulatory components that were replaced by the genomic human sequence may play a role for the observed alterations
in GWAT and liver. More data in other and more pronounced inflammatory disease models have to be studied to evaluate the comparability of changes in hSPP1 compared to WT mice. Because there is a slight difference between the genotypes used in this study, comparisons were primarily made within a genotype, and similarities or important differences were highlighted between hSPP1 and WT animals. To date, there is a high prevalence of obesityassociated disorders including AT inflammation, insulin resistance, and cardio-metabolic disease. In 2014, the World Health Organization stated a number of approximately 1.9 billion overweight adults, of which over 600 million were recognized of being obese.35 According to this background and the crucial role of OPN in obesity-associated diseases, hSPP1 mice represent a new interesting model to evolve novel strategies to combat OPN-induced inflammation and metabolic complications, for example, by immunotherapy. Moreover, this model is now available to target human SPP1/ OPN in a variety of other inflammatory, infectious, and neoplastic disease models in which OPN plays a key role. ACKNOWLEDGMENTS
Conflicts of Interest: The authors have read the journal’s authorship statement and policy on conflicts of interest and declare no commercial or financial conflict of interest. This work was supported by the Federal Ministry of Economy, Family and Youth and the National Founda- Q9 tion for Research, Technology and Development (to T. M. Stulnig). The authors gratefully thank Melina Amor for technical support. The article has been reviewed and approved by all authors. REFERENCES
1. Lund SA, Giachelli CM, Scatena M. The role of osteopontin in inflammatory processes. J Cell Commun Signal 2009;3:311–22. 2. Tokairin T, Nishikawa Y, Watanabe H, et al. Osteopontin expression in the liver with severe perisinusoidal fibrosis: autopsy case of Down syndrome with transient myeloproliferative disorder. Pathol Int 2008;58:64–8. 3. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, Johnson R, Hughes J. Osteopontin–a molecule for all seasons. QJM 2002;95: 3–13. 4. Giachelli CM, Lombardi D, Johnson RJ, Murry CE, Almeida M. Evidence for a role of osteopontin in macrophage infiltration in response to pathological stimuli in vivo. Am J Pathol 1998;152: 353–8. 5. Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. J Leukoc Biol 2002;72:752–61.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279
Translational Research Volume -, Number 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343
€ n et al Gru
6. Crawford HC, Matrisian LM, Liaw L. Distinct roles of osteopontin in host defense activity and tumor survival during squamous cell carcinoma progression in vivo. Cancer Res 1998;58:5206–15. 7. Yamamoto N, Sakai F, Kon S, et al. Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis. J Clin Invest 2003;112:181–8. 8. Agnholt J, Kelsen J, Schack L, Hvas CL, Dahlerup JF, Sørensen ES. Osteopontin, a protein with cytokine-like properties, is associated with inflammation in Crohn’s disease. Scand J Immunol 2007;65:453–60. 9. Leitner L, J€ urets A, Itariu BK, et al. Osteopontin promotes aromatase expression and estradiol production in human adipocytes. Breast Cancer Res Treat 2015;154:63–9. 10. Waller AH, Sanchez-Ross M, Kaluski E, Klapholz M. Osteopontin in cardiovascular disease: a potential therapeutic target. Cardiol Rev 2010;18:125–31. 11. Bruemmer D, Collins AR, Noh G, et al. Angiotensin IIaccelerated atherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice. J Clin Invest 2003;112:1318–31. 12. Kiefer FW, Neschen S, Pfau B, et al. Osteopontin deficiency protects against obesity-induced hepatic steatosis and attenuates glucose production in mice. Diabetologia 2011;54:2132–42. 13. Takemoto M, Yokote K, Nishimura M, et al. Enhanced expression of osteopontin in human diabetic artery and analysis of its functional role in accelerated atherogenesis. Arterioscler Thromb Vasc Biol 2000;20:624–8. 14. Sahai A, Malladi P, Melin-Aldana H, Green RM, Whitington PF. Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model. Am J Physiol Gastrointest Liver Physiol 2004;287:G264–73. 15. Isoda K, Kamezawa Y, Ayaori M, Kusuhara M, Tada N, Ohsuzu F. Osteopontin transgenic mice fed a high-cholesterol diet develop early fatty-streak lesions. Circulation 2003;107:679–81. 16. Bertola A, Deveaux V, Bonnafous S, et al. Elevated expression of osteopontin may be related to adipose tissue macrophage accumulation and liver steatosis in morbid obesity. Diabetes 2009;58: 125–33. 17. Zeyda M, Gollinger K, Todoric J, et al. Osteopontin is an activator of human adipose tissue macrophages and directly affects adipocyte function. Endocrinology 2011;152:2219–27. 18. Nomiyama T, Perez-Tilve D, Ogawa D, et al. Osteopontin mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance in mice. J Clin Invest 2007;117:2877–88. 19. Kiefer FW, Zeyda M, Todoric J, et al. Osteopontin expression in human and murine obesity: extensive local up-regulation in adipose tissue but minimal systemic alterations. Endocrinology 2008;149:1350–7. 20. G omez-Ambrosi J, Catalan V, Ramırez B, et al. Plasma osteopontin levels and expression in adipose tissue are increased in obesity. J Clin Endocrinol Metab 2007;92:3719–27.
11
21. Chapman J, Miles PD, Ofrecio JM, et al. Osteopontin is required for the early onset of high fat diet-induced insulin resistance in mice. PLoS One 2010;5:e13959. 22. Yokosaki Y, Tanaka K, Higashikawa F, Yamashita K, Eboshida A. Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin. Matrix Biol J Int Soc Matrix Biol 2005; 24:418–27. 23. Scatena M, Liaw L, Giachelli CM. Osteopontin: a multifunctional molecule regulating chronic inflammation and vascular disease. Arterioscler Thromb Vasc Biol 2007;27:2302–9. 24. Ito K, Kon S, Nakayama Y, et al. The differential amino acid requirement within osteopontin in alpha4 and alpha9 integrinmediated cell binding and migration. Matrix Biol J Int Soc Matrix Biol 2009;28:11–9. 25. Bayless KJ, Davis GE. Identification of dual a4b1 integrin binding sites within a 38 amino acid domain in the N-terminal thrombin fragment of human osteopontin. J Biol Chem 2001; 276:13483–9. 26. Takahashi K, Takahashi F, Tanabe KK, Takahashi H, Fukuchi Y. The carboxyl-terminal fragment of osteopontin suppresses arginine-glycine-asparatic acid-dependent cell adhesion. Biochem Mol Biol Int 1998;46:1081–92. 27. Okamoto H. Osteopontin and cardiovascular system. Mol Cell Biochem 2006;300:1–7. 28. Ye QH, Qin LX, Forgues M, et al. Predicting hepatitis B viruspositive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med 2003;9: 416–23. 29. Zhao J, Dong L, Lu B, et al. Down-regulation of osteopontin suppresses growth and metastasis of hepatocellular carcinoma via induction of apoptosis. Gastroenterology 2008;135:956–68. 30. Bhattacharya SD, Mi Z, Kim VM, Guo H, Talbot LJ, Kuo PC. Osteopontin regulates epithelial mesenchymal transition-associated growth of hepatocellular cancer in a mouse xenograft model. Ann Surg 2012;255:319–25. 31. Doyle A, McGarry MP, Lee NA, Lee JJ. The construction of transgenic and gene knockout/knockin mouse models of human disease. Transgenic Res 2012;21:327–49. 32. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods San Diego Calif 2001;25:402–8. 33. Kim JI, Huh JY, Sohn JH, et al. Lipid-overloaded enlarged adipocytes provoke insulin resistance independent of inflammation. Mol Cell Biol 2015;35:1686–99. 34. Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nat Rev Immunol 2007;7:118–30. 35. WHO. Obesity and overweight [Internet]. WHO. Available at: http://www.who.int/mediacentre/factsheets/fs311/en/. Accessed August 20, 2015.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407
11.e1
1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471
Translational Research - 2016
€ n et al Gru
APPENDIX
Fig S1. PCR analysis confirms homozygosity of hSPP1 mice. Three-week-old male hSPP1 mice (n 5 20; genotyping of 17 animals are given as an example) were ear-clipped, and ear tissue was used for DNA isolation. Genotyping was performed using the 2xKAPPA 2G Fast kit and indicated primer pairs. Electrophoresis revealed the humanized allele at 362 bp and the WT allele at 254 bp. PCR, polymerase chain reaction; WT, wild-type; hSPP1, humanized SPP1.
Q10
Fig S2. Food intake of hSPP1 and WT mice. Male hSPP1 and WT mice (n 5 8–10 per group) were fed a HF and LF diet for 22 weeks. Food intake was measured over 3 weeks and calculated as grams per mouse per day. Data is expressed as mean 6 standard error of the mean. ,0.05, **P , 0.01, ***P , 0.001. WT, wild-type; LF, low fat; HF, high fat; hSPP1, humanized SPP1.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535
Translational Research Volume -, Number 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599
€ n et al Gru
Fig S3. Hepatic gene expression displays inflammation after HF diet. Male hSPP1 and WT mice (n 5 8–10 per group) were set on indicated diets for 22 weeks. Liver weight was determined after sacrification (A). Hepatic gene expression levels of macrophage marker Emr1 (B) and Itgax (C) as well as of inflammation markers Ccl2 (D) and Tnf-a (E) were assessed by quantitative real-time PCR. Gene expression was normalized to WT LF. Data is given as mean 6 standard error of the mean. *P , 0.05, **P , 0.01, and ***P , 0.001. mRNA, messenger RNA; WT, wild-type; LF, low fat; HF, high fat; PCR, polymerase chain reaction; hSPP1, humanized SPP1.
FLA 5.4.0 DTD TRSL1074_proof 30 July 2016 11:50 am ce
11.e2
1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624 1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663