J Bone Metab.  2014 Aug;21(3):169-188. 10.11005/jbm.2014.21.3.169.

Microarray Analysis of Gene Expression Reveals that Cyclo-oxygenase-2 Gene Therapy Up-regulates Hematopoiesis and Down-regulates Inflammation During Endochondral Bone Fracture Healing

• Lau KH
• Popa NL
• Rundle CH

• KMID: 2286287
• DOI: http://doi.org/10.11005/jbm.2014.21.3.169

Abstract

BACKGROUND
Cyclo-oxygenase-2 (Cox-2) is an inflammatory mediator that is necessary for the tissue repair, including bone fracture healing. Although the application of Cox-2 gene therapy to a murine closed femoral fracture has accelerated bony union, but the beneficial effect was not observed until the endochondral stage of bone repair that is well after the inflammatory stage normally subsides.
METHODS
To identify the molecular pathways through which Cox-2 regulates fracture healing, we examined gene expression profile in fracture tissues in response to Cox-2 gene therapy during the endochondral bone repair phase. Cox-2 gene therapy was applied to the closed murine femur fracture model. Microarray analysis was performed at 10 days post-fracture to examine global gene expression profile in the fracture tissues during the endochondral bone repair phase. The entire repertoire of significantly expressed genes was examined by gene set enrichment analysis, and the most up-regulated individual genes were evaluated further.
RESULTS
The genes that normally promote inflammation were under-represented in the microarray analysis, and the expression of several inflammatory chemokines was significantly down-regulated. There was an up-regulation of two key transcription factor genes that regulate hematopoiesis and erythropoiesis. More surprisingly, there was no significant up-regulation in the genes that are normally involved in angiogenesis or bone formation. However, the expression of two tissue remodeling genes was up-regulated.
CONCLUSIONS
The down-regulation of the inflammatory genes in response to Cox-2 gene therapy was unexpected, given the pro-inflammatory role of prostaglandins. Cox-2 gene therapy could promote bony union through hematopoietic precursor proliferation during endochondral bone repair and thereby enhances subsequently fracture callus remodeling that leads to bony union of the fracture gap.

Keyword

Cyclooxygenase 2; Fracture healing; Gene expression; Genetic therapy; Microarray analysis

MeSH Terms

Bony Callus
Chemokines
Cyclooxygenase 2
Down-Regulation
Erythropoiesis
Femoral Fractures
Femur
Fracture Healing
Fractures, Bone*
Gene Expression*
Genetic Therapy*
Hematopoiesis*
Inflammation*
Microarray Analysis*
Osteogenesis
Prostaglandins
Transcription Factors
Transcriptome
Up-Regulation
Chemokines
Cyclooxygenase 2
Prostaglandins
Transcription Factors

Figure

• 

  Fig. 1 Real-time reverse transcription-polymerase chain reaction determination of prostaglandin E receptor (PTGER) gene expression in response to cyclo-oxygenase-2 (Cox-2) transgene at 10 days post-fracture. The three samples in each group size were from the same individuals that underwent microarray analysis. Statistics were performed by t-Test. PTGER, prostaglandin E2 receptors.

• 

  Fig. 2 Correlation of microarray and quantitative real-time reverse transcription-polymerase chain reaction measurements of gene expression for selected genes. The list of genes is shown in Table 2. The correlation coefficient was determined to be r=0.8. The genes below the 1-fold expression boundary displayed negatively regulated expression by each approach. RT-PCR, reverse transcription-polymerase chain reaction.

• 

  Fig. 3 A model proposed for the functions of the genes expressed during fracture callus development in response to cyclo-oxygenase-2 (Cox-2) gene therapy (box). The stages of fracture callus healing normally present at 10 days post-fracture are indicated. IM, intramembranous; Cox-2, cyclo-oxygenase-2; PGE2, prostaglandin E2.

Reference

1. Bolander ME. Regulation of fracture repair by growth factors. Proc Soc Exp Biol Med. 1992; 200:165–170.

2. Axelrad TW, Kakar S, Einhorn TA. New technologies for the enhancement of skeletal repair. Injury. 2007; 38:Suppl 1. S49–S62.

3. Hadjiargyrou M, Lombardo F, Zhao S, et al. Transcriptional profiling of bone regeneration. Insight into the molecular complexity of wound repair. J Biol Chem. 2002; 277:30177–30182.

4. Li X, Quigg RJ, Zhou J, et al. Early signals for fracture healing. J Cell Biochem. 2005; 95:189–205.

5. Simon AM, Manigrasso MB, O'Connor JP. Cyclo-oxygenase 2 function is essential for bone fracture healing. J Bone Miner Res. 2002; 17:963–976.

6. Zhang X, Schwarz EM, Young DA, et al. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest. 2002; 109:1405–1415.

7. Naik AA, Xie C, Zuscik MJ, et al. Reduced COX-2 expression in aged mice is associated with impaired fracture healing. J Bone Miner Res. 2009; 24:251–264.

8. Tilley SL, Coffman TM, Koller BH. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. J Clin Invest. 2001; 108:15–23.

9. Zhang M, Ho HC, Sheu TJ, et al. EP1(-/-) mice have enhanced osteoblast differentiation and accelerated fracture repair. J Bone Miner Res. 2011; 26:792–802.

10. Paralkar VM, Borovecki F, Ke HZ, et al. An EP2 receptor-selective prostaglandin E2 agonist induces bone healing. Proc Natl Acad Sci U S A. 2003; 100:6736–6740.

11. Xie C, Liang B, Xue M, et al. Rescue of impaired fracture healing in COX-2-/- mice via activation of prostaglandin E2 receptor subtype 4. Am J Pathol. 2009; 175:772–785.

12. Rundle CH, Strong DD, Chen ST, et al. Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat. J Gene Med. 2008; 10:229–241.

13. Xie C, Ming X, Wang Q, et al. COX-2 from the injury milieu is critical for the initiation of periosteal progenitor cell mediated bone healing. Bone. 2008; 43:1075–1083.

14. Amano H, Hayashi I, Endo H, et al. Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth. J Exp Med. 2003; 197:221–232.

15. Katoh H, Hosono K, Ito Y, et al. COX-2 and prostaglandin EP3/EP4 signaling regulate the tumor stromal proangiogenic microenvironment via CXCL12-CXCR4 chemokine systems. Am J Pathol. 2010; 176:1469–1483.

16. Mark H, Penington A, Nannmark U, et al. Microvascular invasion during endochondral ossification in experimental fractures in rats. Bone. 2004; 35:535–542.

17. Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Res. 1984; 2:97–101.

18. Robertson G, Xie C, Chen D, et al. Alteration of femoral bone morphology and density in COX-2-/- mice. Bone. 2006; 39:767–772.

19. Lau KH, Kothari V, Das A, et al. Cellular and molecular mechanisms of accelerated fracture healing by COX2 gene therapy: studies in a mouse model of multiple fractures. Bone. 2013; 53:369–381.

20. Gerstenfeld LC, Thiede M, Seibert K, et al. Differential inhibition of fracture healing by non-selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs. J Orthop Res. 2003; 21:670–675.

21. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102:15545–15550.

22. Ma S, Pathak S, Mandal M, et al. Ikaros and Aiolos inhibit pre-B-cell proliferation by directly suppressing c-Myc expression. Mol Cell Biol. 2010; 30:4149–4158.

23. Fujita H, Hinoi E, Watanabe T, et al. Prevention of bone loss after ovariectomy in mice with preferential overexpression of the transcription factor paired box-5 in osteoblasts. Biol Pharm Bull. 2013; 36:481–484.

24. Pang CJ, Lemsaddek W, Alhashem YN, et al. Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis. Mol Cell Biol. 2012; 32:2628–2644.

25. Hosono K, Sasaki T, Minoshima S, et al. Identification and characterization of a novel gene family YPEL in a wide spectrum of eukaryotic species. Gene. 2004; 340:31–43.

26. Förster R, Mattis AE, Kremmer E, et al. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell. 1996; 87:1037–1047.

27. Ouyang W, Oh SA, Ma Q, et al. TGF-beta cytokine signaling promotes CD8+ T cell development and low-affinity CD4+ T cell homeostasis by regulation of interleukin-7 receptor alpha expression. Immunity. 2013; 39:335–346.

28. Dufour JH, Dziejman M, Liu MT, et al. IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol. 2002; 168:3195–3204.

29. Peperzak V, Veraar EA, Xiao Y, et al. CD8+ T cells produce the chemokine CXCL10 in response to CD27/CD70 costimulation to promote generation of the CD8+ effector T cell pool. J Immunol. 2013; 191:3025–3036.

30. Van Damme J, Proost P, Lenaerts JP, et al. Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family. J Exp Med. 1992; 176:59–65.

31. Bais M, McLean J, Sebastiani P, et al. Transcriptional analysis of fracture healing and the induction of embryonic stem cell-related genes. PLoS One. 2009; 4:e5393.

32. North TE, Goessling W, Walkley CR, et al. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature. 2007; 447:1007–1011.

33. Pelus LM, Hoggatt J. Pleiotropic effects of prostaglandin E2 in hematopoiesis; prostaglandin E2 and other eicosanoids regulate hematopoietic stem and progenitor cell function. Prostaglandins Other Lipid Mediat. 2011; 96:3–9.

34. Salcedo R, Zhang X, Young HA, et al. Angiogenic effects of prostaglandin E2 are mediated by up-regulation of CXCR4 on human microvascular endothelial cells. Blood. 2003; 102:1966–1977.

35. Sinha P, Clements VK, Fulton AM, et al. Prostaglandin E2 promotes tumor progression by inducing myeloid-derived suppressor cells. Cancer Res. 2007; 67:4507–4513.

36. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 2009; 9:162–174.

37. Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A. 2002; 99:9656–9661.

38. Irie A, Sugimoto Y, Namba T, et al. Third isoform of the prostaglandin-E-receptor EP3 subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase. Eur J Biochem. 1993; 217:313–318.

39. Goulet JL, Pace AJ, Key ML, et al. E-prostanoid-3 receptors mediate the proinflammatory actions of prostaglandin E2 in acute cutaneous inflammation. J Immunol. 2004; 173:1321–1326.

40. Jain S, Chakraborty G, Raja R, et al. Prostaglandin E2 regulates tumor angiogenesis in prostate cancer. Cancer Res. 2008; 68:7750–7759.

41. Ueno T, Suzuki T, Oikawa A, et al. Recruited bone marrow cells expressing the EP3 prostaglandin E receptor subtype enhance angiogenesis during chronic inflammation. Biomed Pharmacother. 2010; 64:93–100.

42. Kamoshita E, Ikeda Y, Fujita M, et al. Recruitment of a prostaglandin E receptor subtype, EP3-expressing bone marrow cells is crucial in wound-induced angiogenesis. Am J Pathol. 2006; 169:1458–1472.

43. Arikawa T, Omura K, Morita I. Regulation of bone morphogenetic protein-2 expression by endogenous prostaglandin E2 in human mesenchymal stem cells. J Cell Physiol. 2004; 200:400–406.

44. Colnot C, Thompson Z, Miclau T, et al. Altered fracture repair in the absence of MMP9. Development. 2003; 130:4123–4133.

45. Amano H, Ito Y, Suzuki T, et al. Roles of a prostaglandin E-type receptor, EP3, in upregulation of matrix metalloproteinase-9 and vascular endothelial growth factor during enhancement of tumor metastasis. Cancer Sci. 2009; 100:2318–2324.