J Periodontal Implant Sci.  2017 Apr;47(2):66-76. 10.5051/jpis.2017.47.2.66.

Inhibitors of DNA methylation support TGF-β1-induced IL11 expression in gingival fibroblasts

• Sufaru IG
• Beikircher G
• Weinhaeusel A
• Gruber R

• KMID: 2377741
• DOI: http://doi.org/10.5051/jpis.2017.47.2.66

Abstract

PURPOSE
Oral wound healing requires gingival fibroblasts to respond to local growth factors. Epigenetic silencing through DNA methylation can potentially decrease the responsiveness of gingival fibroblasts to local growth factors. In this study, our aim was to determine whether the inhibition of DNA methylation sensitized gingival fibroblasts to transforming growth factor-β1 (TGF-β1).
METHODS
Gingival fibroblasts were exposed to 5-aza-2'-deoxycytidine (5-aza), a clinically approved demethylating agent, before stimulation with TGF-β1. Gene expression changes were evaluated using quantitative polymerase chain reaction (PCR) analysis. DNA methylation was detected by methylation-sensitive restriction enzymes and PCR amplification.
RESULTS
We found that 5-aza enhanced TGF-β1-induced interleukin-11 (IL11) expression in gingival fibroblasts 2.37-fold (P=0.008). 5-aza had no significant effects on the expression of proteoglycan 4 (PRG4) and NADPH oxidase 4 (NOX4). Consistent with this, 5-aza caused demethylation of the IL11 gene commonly next to a guanosine (CpG) island in gingival fibroblasts. The TGF-β type I receptor kinase inhibitor SB431542 impeded the changes in IL11 expression, indicating that the effects of 5-aza require TGF-β signaling. 5-aza moderately increased the expression of TGF-β type II receptor (1.40-fold; P=0.009), possibly enhancing the responsiveness of fibroblasts to TGF-β1. As part of the feedback response, 5-aza increased the expression of the DNA methyltransferases 1 (DNMT1) (P=0.005) and DNMT3B (P=0.002), which are enzymes responsible for gene methylation.
CONCLUSIONS
These in vitro data suggest that the inhibition of DNA methylation by 5-aza supports TGF-β-induced IL11 expression in gingival fibroblasts.

Keyword

Decitabine; Epigenomics; Fibroblasts; Interleukin-11; Transforming growth factor beta1

MeSH Terms

DNA Methylation*
DNA*
Epigenomics
Fibroblasts*
Gene Expression
Guanosine
In Vitro Techniques
Intercellular Signaling Peptides and Proteins
Interleukin-11*
Methylation
Methyltransferases
NADPH Oxidase
Phosphotransferases
Polymerase Chain Reaction
Proteoglycans
Transforming Growth Factor beta1
Wound Healing
DNA
Guanosine
Intercellular Signaling Peptides and Proteins
Interleukin-11
Methyltransferases
NADPH Oxidase
Phosphotransferases
Proteoglycans
Transforming Growth Factor beta1

Figure

• 

  Figure 1 TGF-β1 increased the respective target genes with and without 5-aza. Human gingival fibroblasts were exposed to 5-aza or left untreated for 72 hours, before cells were stimulated with recombinant human TGF-β1. After 24 hours, the gene expression analysis was performed. (A) IL11 (n=10), (B) PRG4 (n=7), (C) NOX4 (n=11); data from individual experiments, denoted with separate colors, indicate the x-fold changes of gene expression in response to TGF-β1 compared to unstimulated cells. P values were obtained from the paired t-test. Statistical analyses were based on fold-change values of single cell donors and experiments. The colors represent data from different cell donors and experiments. TGF-β1: transforming growth factor-β1, 5-aza: 5-aza-2'-deoxycytidine, IL11: interleukin-11, PRG4: proteoglycan 4, NOX4: NADPH oxidase 4, w/o: without.

• 

  Figure 2 5-aza sensitizes cells to TGF-β1 as indicated by IL11 expression. Human gingival fibroblasts were exposed to 5-aza or left untreated for 72 hours, before cells were stimulated with recombinant human TGF-β1. After 24 hours, gene expression analysis was performed. (A) IL11 (n=10), (B) PRG4 (n=7), (C) NOX4 (n=13), with data from different experiments marked with separate colors. Data indicate the x-fold changes in response to 5-aza in untreated and TGF-β1 treated cells, respectively. P values were obtained from the paired t-test. Statistical analyses were based on fold-change values of single experiments. The colors represent data from different cell donors and experiments. 5-aza: 5-aza-2'-deoxycytidine, TGF-β1: transforming growth factor-β1, IL11: interleukin-11, PRG4: proteoglycan 4, NOX4: NADPH oxidase 4, w/o: without.

• 

  Figure 3 5-aza treatment causes demethylation of IL11 CpG island methylation. UCSC genome browser (hg19) view indicating the location of the IL11 CpG island and PCR amplicon investigated. (A) IL11 gene region (UCSC genome browser view; hg19) presenting the targeted CpG island (intron 1 within IL11: dark green) and the PCR product (indicated as black bar denoted “YourSeq”). DNA methylation tracks (methyl reduced representation bisulfite sequencing; light green indicated 0% and red indicated 100% methylated CpGs). Only Jurkat and HeLa show methylation of the IL11 CpG island. (B) detailed location of the IL11 CpG island and PCR amplicon (primers, bold black) and location of methylation-sensitive restriction sites (denoted as “Rest Enzymes”). (C) IL11-PCR DNA methylation testing results. 1, leukocyte DNA from a normal healthy individual (not 5-aza-treated); 2 and 3, fibroblast DNA: 5-aza-treated vs. non-treated as indicated. 5-aza: 5-aza-2'-deoxycytidine, CpG: commonly next to a guanosine, UCSC: University of California, Santa Cruz, IL11: interleukin-11, PCR: polymerase chain reaction, U: undigested, D: digested by methylation-sensitive restriction enzymes, S: 50 bp ladder, NTC: non-template PCR control 1.

Reference

1. Sculean A, Gruber R, Bosshardt DD. Soft tissue wound healing around teeth and dental implants. J Clin Periodontol. 2014; 41:Suppl 15. S6–S22.

2. Szpaderska AM, Zuckerman JD, DiPietro LA. Differential injury responses in oral mucosal and cutaneous wounds. J Dent Res. 2003; 82:621–626.

3. Finnson KW, Arany PR, Philip A. Transforming growth factor beta signaling in cutaneous wound healing: lessons learned from animal studies. Adv Wound Care (New Rochelle). 2013; 2:225–237.

4. Finnson KW, McLean S, Di Guglielmo GM, Philip A. Dynamics of transforming growth factor beta signaling in wound healing and scarring. Adv Wound Care (New Rochelle). 2013; 2:195–214.

5. Moustakas A, Heldin CH. The regulation of TGFbeta signal transduction. Development. 2009; 136:3699–3714.

6. Elias JA, Zheng T, Whiting NL, Trow TK, Merrill WW, Zitnik R, et al. IL-1 and transforming growth factor-beta regulation of fibroblast-derived IL-11. J Immunol. 1994; 152:2421–2429.

7. Cuellar A, Reddi AH. Stimulation of superficial zone protein/lubricin/PRG4 by transforming growth factor-β in superficial zone articular chondrocytes and modulation by glycosaminoglycans. Tissue Eng Part A. 2015; 21:1973–1981.

8. Sturrock A, Cahill B, Norman K, Huecksteadt TP, Hill K, Sanders K, et al. Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2006; 290:L661–L673.

9. Zimmermann M, Caballé-Serrano J, Bosshardt DD, Ankersmit HJ, Buser D, Gruber R. Bone-conditioned medium changes gene expression in bone-derived fibroblasts. Int J Oral Maxillofac Implants. 2015; 30:953–958.

10. van der Kraan PM. Age-related alterations in TGF beta signaling as a causal factor of cartilage degeneration in osteoarthritis. Biomed Mater Eng. 2014; 24:75–80.

11. Tichauer JE, Flores B, Soler B, Eugenín-von Bernhardi L, Ramírez G, von Bernhardi R. Age-dependent changes on TGFβ1 Smad3 pathway modify the pattern of microglial cell activation. Brain Behav Immun. 2014; 37:187–196.

12. Yan J, Zhang H, Yin Y, Li J, Tang Y, Purkayastha S, et al. Obesity- and aging-induced excess of central transforming growth factor-β potentiates diabetic development via an RNA stress response. Nat Med. 2014; 20:1001–1008.

13. Jinno K, Takahashi T, Tsuchida K, Tanaka E, Moriyama K. Acceleration of palatal wound healing in Smad3-deficient mice. J Dent Res. 2009; 88:757–761.

14. Jones MJ, Goodman SJ, Kobor MS. DNA methylation and healthy human aging. Aging Cell. 2015; 14:924–932.

15. Keating ST, El-Osta A. Epigenetics and metabolism. Circ Res. 2015; 116:715–736.

16. Holroyd C, Harvey N, Dennison E, Cooper C. Epigenetic influences in the developmental origins of osteoporosis. Osteoporos Int. 2012; 23:401–410.

17. Ren J, Singh BN, Huang Q, Li Z, Gao Y, Mishra P, et al. DNA hypermethylation as a chemotherapy target. Cell Signal. 2011; 23:1082–1093.

18. Powell TR, Smith RG, Hackinger S, Schalkwyk LC, Uher R, McGuffin P, et al. DNA methylation in interleukin-11 predicts clinical response to antidepressants in GENDEP. Transl Psychiatry. 2013; 3:e300.

19. Sanders YY, Liu H, Liu G, Thannickal VJ. Epigenetic mechanisms regulate NADPH oxidase-4 expression in cellular senescence. Free Radic Biol Med. 2015; 79:197–205.

20. Christman JK. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 2002; 21:5483–5495.

21. Varga AE, Stourman NV, Zheng Q, Safina AF, Quan L, Li X, et al. Silencing of the Tropomyosin-1 gene by DNA methylation alters tumor suppressor function of TGF-beta. Oncogene. 2005; 24:5043–5052.

22. Xiao X, Tang W, Yuan Q, Peng L, Yu P. Epigenetic repression of Krüppel-like factor 4 through Dnmt1 contributes to EMT in renal fibrosis. Int J Mol Med. 2015; 35:1596–1602.

23. Neveu WA, Mills ST, Staitieh BS, Sueblinvong V. TGF-β1 epigenetically modifies Thy-1 expression in primary lung fibroblasts. Am J Physiol Cell Physiol. 2015; 309:C616–C626.

24. Bian EB, Huang C, Wang H, Chen XX, Zhang L, Lv XW, et al. Repression of Smad7 mediated by DNMT1 determines hepatic stellate cell activation and liver fibrosis in rats. Toxicol Lett. 2014; 224:175–185.

25. Kang SH, Bang YJ, Im YH, Yang HK, Lee DA, Lee HY, et al. Transcriptional repression of the transforming growth factor-beta type I receptor gene by DNA methylation results in the development of TGF-beta resistance in human gastric cancer. Oncogene. 1999; 18:7280–7286.

26. Zhang Q, Rubenstein JN, Liu VC, Park I, Jang T, Lee C. Restoration of expression of transforming growth factor-beta type II receptor in murine renal cell carcinoma (renca) cells by 5-Aza-2′-deoxycytidine. Life Sci. 2005; 76:1159–1166.

27. Takai R, Uehara O, Harada F, Utsunomiya M, Chujo T, Yoshida K, et al. DNA hypermethylation of extracellular matrix-related genes in human periodontal fibroblasts induced by stimulation for a prolonged period with lipopolysaccharide derived from Porphyromonas gingivalis. J Periodontal Res. 2016; 51:508–517.

28. Zhang S, Barros SP, Moretti AJ, Yu N, Zhou J, Preisser JS, et al. Epigenetic regulation of TNFA expression in periodontal disease. J Periodontol. 2013; 84:1606–1616.

29. Kojima A, Kobayashi T, Ito S, Murasawa A, Nakazono K, Yoshie H. Tumor necrosis factor-alpha gene promoter methylation in Japanese adults with chronic periodontitis and rheumatoid arthritis. J Periodontal Res. 2016; 51:350–358.

30. Schulz S, Immel UD, Just L, Schaller HG, Gläser C, Reichert S. Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis. Hum Immunol. 2016; 77:71–75.

31. Baptista NB, Portinho D, Casarin RC, Vale HF, Casati MZ, De Souza AP, et al. DNA methylation levels of SOCS1 and LINE-1 in oral epithelial cells from aggressive periodontitis patients. Arch Oral Biol. 2014; 59:670–678.

32. Andia DC, Planello AC, Portinho D, da Silva RA, Salmon CR, Sallum EA, et al. DNA methylation analysis of SOCS1, SOCS3, and LINE-1 in microdissected gingival tissue. Clin Oral Investig. 2015; 19:2337–2344.

33. Larsson L, Castilho RM, Giannobile WV. Epigenetics and its role in periodontal diseases: a state-of-the-art review. J Periodontol. 2015; 86:556–568.

34. Barros SP, Offenbacher S. Modifiable risk factors in periodontal disease: epigenetic regulation of gene expression in the inflammatory response. Periodontol 2000. 2014; 64:95–110.

35. Mossman D, Kim KT, Scott RJ. Demethylation by 5-aza-2′-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists. BMC Cancer. 2010; 10:366.

36. Zhu WG, Hileman T, Ke Y, Wang P, Lu S, Duan W, et al. 5-aza-2′-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation. J Biol Chem. 2004; 279:15161–15166.

37. Weinhaeusel A, Thiele S, Hofner M, Hiort O, Noehammer C. PCR-based analysis of differentially methylated regions of GNAS enables convenient diagnostic testing of pseudohypoparathyroidism type Ib. Clin Chem. 2008; 54:1537–1545.

38. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013; 153:1194–1217.

39. Gomes FS, de-Souza GF, Nascimento LF, Arantes EL, Pedro RM, Vitorino DC, et al. Topical 5-azacytidine accelerates skin wound healing in rats. Wound Repair Regen. 2014; 22:640–646.

40. Stähli A, Bosshardt D, Sculean A, Gruber R. Emdogain-regulated gene expression in palatal fibroblasts requires TGF-βRI kinase signaling. PLoS One. 2014; 9:e105672.