Follistatin is a potent regulator of the inflammatory response and binds to and inhibits activin A action. Activin A is a member of the TGFβ protein superfamily which has regulatory roles in the inflammatory response and in the fibrotic process. Fibrosis can occur following cell injury and cell death induced by agents such as ionizing radiation (IR). IR is used to treat cancer and marked fibrotic response is a normal tissue (non-tumour) consequence in a fraction of patients under the current dose regimes. The discovery and development of a therapeutic to abate fibrosis in these radiosensitive patients would be a major advance for cancer radiotherapy. Likewise, prediction of which patients are susceptible to fibrosis would enable individualization of treatment and provide an opportunity for pre-emptive fibrosis control and better tumour treatment outcomes. The levels of activin A and follistatin were measured in fibroblasts derived from patients who developed severe radiation-induced fibrosis following radiotherapy and compared to fibroblasts from patients who did not. Both follistatin and activin A gene expression levels were increased following IR and the follistatin gene expression level was lower in the fibroblasts from fibrosis patients compared to controls at both basal levels and after IR. The major follistatin transcript variants were found to have a similar response to IR and both were reduced in fibrosis patients. Levels of follistatin and activin A secreted in the fibroblast culture medium also increased in response to IR and the relative follistatin protein levels were significantly lower in the samples derived from fibrosis patients. The decrease in the follistatin levels can lead to an increased bioactivity of activin A and hence may provide a useful measurement to identify patients at risk of a severe fibrotic response to IR. Additionally, follistatin, by its ability to neutralise the actions of activin A may be of value as an anti-fibrotic for radiation induced fibrosis.
References
[1]
Wynn TA, Ramalingam TR (2012) Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 18: 1028–1040.
[2]
Yarnold J, Brotons MC (2010) Pathogenetic mechanisms in radiation fibrosis. Radiother Oncol 97: 149–161.
[3]
Bentzen SM (2006) Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer 6: 702–713.
[4]
Wynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214: 199–210.
[5]
Hinz B, Gabbiani G (2010) Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives. F1000 Biol Rep 2: 78.
[6]
Hinz B, Phan SH, Thannickal VJ, Prunotto M, Desmouliere A, et al. (2012) Recent developments in myofibroblast biology: paradigms for connective tissue remodeling. Am J Pathol 180: 1340–1355.
[7]
Andreassen CN (2010) Searching for genetic determinants of normal tissue radiosensitivity – are we on the right track? Radiother Oncol 97: 1–8.
[8]
Parliament MB (2012) Radiogenomics: associations in all the wrong places? Lancet Oncol 13: 7–8.
[9]
Alsbeih G, El-Sebaie M, Al-Harbi N, Al-Hadyan K, Shoukri M, et al. (2013) SNPs in genes implicated in radiation response are associated with radiotoxicity and evoke roles as predictive and prognostic biomarkers. Radiat Oncol 8: 125.
[10]
Anscher MS, Thrasher B, Rabbani Z, Teicher B, Vujaskovic Z (2006) Antitransforming growth factor-beta antibody 1D11 ameliorates normal tissue damage caused by high-dose radiation. Int J Radiat Oncol Biol Phys 65: 876–881.
[11]
Anscher MS, Thrasher B, Zgonjanin L, Rabbani ZN, Corbley MJ, et al. (2008) Small molecular inhibitor of transforming growth factor-beta protects against development of radiation-induced lung injury. Int J Radiat Oncol Biol Phys 71: 829–837.
[12]
Nishioka A, Ogawa Y, Mima T, Jin YJ, Sonobe H, et al. (2004) Histopathologic amelioration of fibroproliferative change in rat irradiated lung using soluble transforming growth factor-beta (TGF-beta) receptor mediated by adenoviral vector. Int J Radiat Oncol Biol Phys 58: 1235–1241.
[13]
Rabbani ZN, Anscher MS, Zhang X, Chen L, Samulski TV, et al. (2003) Soluble TGFbeta type II receptor gene therapy ameliorates acute radiation-induced pulmonary injury in rats. Int J Radiat Oncol Biol Phys 57: 563–572.
[14]
Verrecchia F, Mauviel A (2007) Transforming growth factor-beta and fibrosis. World J Gastroenterol 13: 3056–3062.
[15]
Nagashio Y, Ueno H, Imamura M, Asaumi H, Watanabe S, et al. (2004) Inhibition of transforming growth factor beta decreases pancreatic fibrosis and protects the pancreas against chronic injury in mice. Lab Invest 84: 1610–1618.
[16]
Bian Y, Terse A, Du J, Hall B, Molinolo A, et al. (2009) Progressive tumor formation in mice with conditional deletion of TGF-beta signaling in head and neck epithelia is associated with activation of the PI3K/Akt pathway. Cancer Res 69: 5918–5926.
[17]
Tsuchida K, Nakatani M, Uezumi A, Murakami T, Cui X (2008) Signal transduction pathway through activin receptors as a therapeutic target of musculoskeletal diseases and cancer. Endocr J 55: 11–21.
[18]
Nakamura T, Takio K, Eto Y, Shibai H, Titani K, et al. (1990) Activin-binding protein from rat ovary is follistatin. Science 247: 836–838.
[19]
Hedger MP, Winnall WR, Phillips DJ, de Kretser DM (2011) The regulation and functions of activin and follistatin in inflammation and immunity. Vitam Horm 85: 255–297.
[20]
de Kretser DM, O'Hehir RE, Hardy CL, Hedger MP (2012) The roles of activin A and its binding protein, follistatin, in inflammation and tissue repair. Mol Cell Endocrinol 359: 101–106.
[21]
de Kretser DM, Robertson DM (1989) The isolation and physiology of inhibin and related proteins. Biol Reprod 40: 33–47.
[22]
Lin SY, Morrison JR, Phillips DJ, de Kretser DM (2003) Regulation of ovarian function by the TGF-beta superfamily and follistatin. Reproduction 126: 133–148.
[23]
Hedger MP, Drummond AE, Robertson DM, Risbridger GP, de Kretser DM (1989) Inhibin and activin regulate [3H]thymidine uptake by rat thymocytes and 3T3 cells in vitro. Mol Cell Endocrinol 61: 133–138.
[24]
Ohga E, Matsuse T, Teramoto S, Ouchi Y (2000) Activin receptors are expressed on human lung fibroblast and activin A facilitates fibroblast-mediated collagen gel contraction. Life Sci 66: 1603–1613.
[25]
Ohga E, Matsuse T, Teramoto S, Katayama H, Nagase T, et al. (1996) Effects of activin A on proliferation and differentiation of human lung fibroblasts. Biochem Biophys Res Commun 228: 391–396.
[26]
Werner S, Alzheimer C (2006) Roles of activin in tissue repair, fibrosis, and inflammatory disease. Cytokine Growth Factor Rev 17: 157–171.
[27]
Patella S, Phillips DJ, Tchongue J, de Kretser DM, Sievert W (2006) Follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis. Am J Physiol Gastrointest Liver Physiol 290: G137–144.
[28]
Aoki F, Kurabayashi M, Hasegawa Y, Kojima I (2005) Attenuation of bleomycin-induced pulmonary fibrosis by follistatin. Am J Respir Crit Care Med 172: 713–720.
[29]
Phillips DJ, de Kretser DM (1998) Follistatin: a multifunctional regulatory protein. Front Neuroendocrinol 19: 287–322.
[30]
Nakamura T, Sugino K, Titani K, Sugino H (1991) Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells. J Biol Chem 266: 19432–19437.
[31]
Sugino H, Sugino K, Hashimoto O, Shoji H, Nakamura T (1997) Follistatin and its role as an activin-binding protein. J Med Invest 44: 1–14.
[32]
Severin DM, Leong T, Cassidy B, Elsaleh H, Peters L, et al. (2001) Novel DNA sequence variants in the hHR21 DNA repair gene in radiosensitive cancer patients. Int J Radiat Oncol Biol Phys 50: 1323–1331.
[33]
Sprung CN, Li J, Hovan D, McKay MJ, Forrester HB (2011) Alternative transcript initiation and splicing as a response to DNA damage. PLoS ONE 6: e25758.
[34]
Forrester HB, Li J, Hovan D, Ivashkevich AN, Sprung CN (2012) DNA repair genes: alternative transcription and gene expression at the exon level in response to the DNA damaging agent, ionizing radiation. PLoS ONE 7: e53358.
[35]
O'Connor AE, McFarlane JR, Hayward S, Yohkaichiya T, Groome NP, et al. (1999) Serum activin A and follistatin concentrations during human pregnancy: a cross-sectional and longitudinal study. Hum Reprod 14: 827–832.
[36]
Knight PG, Muttukrishna S, Groome NP (1996) Development and application of a two-site enzyme immunoassay for the determination of ‘total’ activin-A concentrations in serum and follicular fluid. J Endocrinol 148: 267–279.
[37]
Michel U, Albiston A, Findlay JK (1990) Rat follistatin: gonadal and extragonadal expression and evidence for alternative splicing. Biochem Biophys Res Commun 173: 401–407.
[38]
Shimasaki S, Koga M, Esch F, Cooksey K, Mercado M, et al. (1988) Primary structure of the human follistatin precursor and its genomic organization. Proc Natl Acad Sci U S A 85: 4218–4222.
[39]
Hurwitz C, Tolmach LJ (1969) Time lapse cinemicrographic studies of x-irradiated HeLa S3 cells. I. Cell progression and cell disintegration. Biophys J 9: 607–633.
[40]
Forrester HB, Vidair CA, Albright N, Ling CC, Dewey WC (1999) Using computerized video time lapse for quantifying cell death of X-irradiated rat embryo cells transfected with c-myc or c-Ha-ras. Cancer Res 59: 931–939.
[41]
de Kretser DM, O'Donnell L (2013) Phenotypic assessment of male fertility status in transgenic animal models. Methods Mol Biol 927: 531–548.
[42]
Xavier S, Piek E, Fujii M, Javelaud D, Mauviel A, et al. (2004) Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone. J Biol Chem 279: 15167–15176.
[43]
Ishii H, Choudhuri R, Mathias A, Sowers AL, Flanders KC, et al. (2009) Halofuginone mediated protection against radiation-induced leg contracture. Int J Oncol 35: 315–319.
[44]
Granot I, Halevy O, Hurwitz S, Pines M (1993) Halofuginone: an inhibitor of collagen type I synthesis. Biochim Biophys Acta 1156: 107–112.
[45]
Zion O, Genin O, Kawada N, Yoshizato K, Roffe S, et al. (2009) Inhibition of transforming growth factor beta signaling by halofuginone as a modality for pancreas fibrosis prevention. Pancreas 38: 427–435.
[46]
de Jonge MJ, Dumez H, Verweij J, Yarkoni S, Snyder D, et al. (2006) Phase I and pharmacokinetic study of halofuginone, an oral quinazolinone derivative in patients with advanced solid tumours. Eur J Cancer 42: 1768–1774.
[47]
Oku H, Shimizu T, Kawabata T, Nagira M, Hikita I, et al. (2008) Antifibrotic action of pirfenidone and prednisolone: different effects on pulmonary cytokines and growth factors in bleomycin-induced murine pulmonary fibrosis. Eur J Pharmacol 590: 400–408.
[48]
Azuma A, Nukiwa T, Tsuboi E, Suga M, Abe S, et al. (2005) Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 171: 1040–1047.
[49]
Nagai S, Hamada K, Shigematsu M, Taniyama M, Yamauchi S, et al. (2002) Open-label compassionate use one year-treatment with pirfenidone to patients with chronic pulmonary fibrosis. Intern Med 41: 1118–1123.
[50]
Raghu G, Johnson WC, Lockhart D, Mageto Y (1999) Treatment of idiopathic pulmonary fibrosis with a new antifibrotic agent, pirfenidone: results of a prospective, open-label Phase II study. Am J Respir Crit Care Med 159: 1061–1069.
[51]
Simone NL, Soule BP, Gerber L, Augustine E, Smith S, et al. (2007) Oral Pirfenidone in patients with chronic fibrosis resulting from radiotherapy: a pilot study. Radiat Oncol 2: 19.
Denton CP, Merkel PA, Furst DE, Khanna D, Emery P, et al. (2007) Recombinant human anti-transforming growth factor beta1 antibody therapy in systemic sclerosis: a multicenter, randomized, placebo-controlled phase I/II trial of CAT-192. Arthritis Rheum 56: 323–333.
[54]
Schultze-Mosgau S, Blaese MA, Grabenbauer G, Wehrhan F, Kopp J, et al. (2004) Smad-3 and Smad-7 expression following anti-transforming growth factor beta 1 (TGFbeta1)-treatment in irradiated rat tissue. Radiother Oncol 70: 249–259.
[55]
Inouye S, Guo Y, DePaolo L, Shimonaka M, Ling N, et al. (1991) Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin. Endocrinology 129: 815–822.
[56]
Morrissey J, Hruska K, Guo G, Wang S, Chen Q, et al. (2002) Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function. J Am Soc Nephrol 13 Suppl 1S14–21.
[57]
Lepparanta O, Tikkanen JM, Bespalov MM, Koli K, Myllarniemi M (2012) The BMP-inducer tilorone identified by high-throughput screening is antifibrotic in vivo. Am J Respir Cell Mol Biol.
[58]
Bo Li Z, Zhang J, Wagner KR (2012) Inhibition of myostatin reverses muscle fibrosis through apoptosis. J Cell Sci 125: 3957–3965.
[59]
West CM, Davidson SE, Elyan SA, Swindell R, Roberts SA, et al. (1998) The intrinsic radiosensitivity of normal and tumour cells. Int J Radiat Biol 73: 409–413.
[60]
Distel LV, Neubauer S, Keller U, Sprung CN, Sauer R, et al. (2006) Individual differences in chromosomal aberrations after in vitro irradiation of cells from healthy individuals, cancer and cancer susceptibility syndrome patients. Radiother Oncol 81: 257–263.
[61]
Zhou PK, Sproston AR, Marples B, West CM, Margison GP, et al. (1998) The radiosensitivity of human fibroblast cell lines correlates with residual levels of DNA double-strand breaks. Radiother Oncol 47: 271–276.
[62]
Sprung CN, Chao M, Leong T, McKay MJ (2005) Chromosomal radiosensitivity in two cell lineages derived from clinically radiosensitive cancer patients. Clin Cancer Res 11: 6352–6358.
[63]
Sprung CN, Davey DS, Withana NP, Distel LV, McKay MJ (2008) Telomere length in lymphoblast cell lines derived from clinically radiosensitive cancer patients. Cancer Biol Ther 7.
[64]
Alsner J, Andreassen CN, Overgaard J (2008) Genetic markers for prediction of normal tissue toxicity after radiotherapy. Semin Radiat Oncol 18: 126–135.
[65]
Ismail SM, Buchholz TA, Story M, Brock WA, Stevens CW (2004) Radiosensitivity is predicted by DNA end-binding complex density, but not by nuclear levels of band components. Radiother Oncol 72: 325–332.
[66]
Vasireddy RS, Sprung CN, Cempaka NL, Chao M, McKay MJ (2010) H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype. Br J Cancer 102: 1511–1518.
[67]
Fields SZ, Parshad S, Anne M, Raftopoulos H, Alexander MJ, et al. (2013) Activin receptor antagonists for cancer-related anemia and bone disease. Expert Opin Investig Drugs 22: 87–101.
