Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-κB pathways

Lee,Yonghee; Lee,Dohyun; Koo,Kyotan; Lee,Jay; Song,Yi  Seop; Yoon,Ho  Sang; Choi,Yoo  Mi; Kim,Beom  Joon

doi:10.3892/ijmm.2014.1790

Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-κB pathways

Authors:
- Yonghee Lee
- Dohyun Lee
- Kyotan Koo
- Jay Lee
- Yi Seop Song
- Ho Sang Yoon
- Yoo Mi Choi
- Beom Joon Kim
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Affiliations: Department of Dermatology, Chung-Ang University College of Medicine, Seoul, Republic of Korea, Nutrex Technology R&D Center, Seoul, Republic of Korea, Marketing and Business Division, PacificPharma Corporation, Seoul, Republic of Korea, MB Business Development Team, PacificPharma Corporation, Seoul, Republic of Korea, Cosmeceutical Team, PacificPharma Corporation, Seoul, Republic of Korea
Published online on: May 28, 2014 https://doi.org/10.3892/ijmm.2014.1790
Pages: 624-631

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Abstract

Growth factors are important for regulating a variety of cellular processes and typically act as signaling molecules between cells. In the present study, we examined the mechanisms underlying the inhibitory effects of mixtures of recombinant growth factors (MRGFs) on nitric oxide (NO) and pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. We also examined whether these effects are mediated through the mitogen‑activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signal transduction pathways. NO production was assessed by measuring nitrite acucmulation using the Greiss reaction. Cytokine concentrations were measured using respective ELISA kits for each cytokine. Our results revealed that the MRGFs significantly attenuated the LPS-induced production of pro-inflammatory cytokines and NO in a dose-dependent manner. To elucidate the mechanisms underlying the inhibitory effects of MRGFs, we examined the effects of the LPS-induced phosphorylation of MAPKs and the activation of the NF-κB signaling pathway on the stabilization of NF-κB nuclear translocation and inhibitory factor-κB (IκB) degradation. Western blot analysis was performed to determine the total and phosphorylated levels of ERK, as well as the nuclear translocation of NF-κB, and IκB phosphorylation and degradation. Our results demonstrated that treatment with MRGFs resulted in a reduction in the phosphorylation of the ERK and NF-κB signaling pathways, whereas the phosphorylation of JNK and p38 was not affected. Taken together, our results suggest that MRGFs inhibit the production of pro-inflammatory cytokines and NO by downregulating inducible NO synthase gene expression and blocking the phosphorylation of the ERK and NF-κB signaling pathways. These findings may provide direct evidence of the potential application of MRGFs in the prevention and treatment of inflammatory diseases.

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1	Goustin AS, Leof EB, Shipley GD and Moses HL: Growth factors and cancer. Cancer Res. 46:1015–1029. 1986.
2	Childs CB, Proper JA, Tucker RF and Moses HL: Serum contains a platelet-derived transforming growth factor. Proc Natl Acad Sci USA. 79:5312–5316. 1982. View Article : Google Scholar : PubMed/NCBI
3	Taguchi M, Moran SL, Zobitz ME, et al: Wound-healing properties of transforming growth factor β (TGF-β) inducible early gene 1 (TIEG1) knockout mice. J Musculoskelet Res. 11:63–69. 2008.
4	Hollwy RW and Kiernan JA: Control of the initiation of DNA synthesis in 3T3 cells: serum factors. Proc Natl Acad Sci USA. 71:2908–2911. 1974. View Article : Google Scholar : PubMed/NCBI
5	Martin P: Wound healing - aiming for perfect skin regeneration. Science. 276:75–81. 1997. View Article : Google Scholar : PubMed/NCBI
6	Matt P, Schoenhoff F, Habashi J, et al: Circulating transforming growth factor-beta in Marfan syndrome. Circulation. 120:526–532. 2009. View Article : Google Scholar : PubMed/NCBI
7	Kim HS: Assignment1 of the human basic fibroblast growth factor gene FGF2 to chromosome 4 band q26 by radiation hybrid mapping. Cytogenet Cell Genet. 83:731998. View Article : Google Scholar : PubMed/NCBI
8	Barrientos S, Stojadinovic O, Golinko MS, Brem H and Tomic-Canic M: Growth factors and cytokines in wound healing. Wound Repair Regen. 16:585–601. 2008. View Article : Google Scholar : PubMed/NCBI
9	Rotolo S, Ceccarelli S, Romano F, Frati L, Marchese C and Angeloni A: Silencing of keratinocyte growth factor receptor restores 5-fluorouracil and tamoxifen efficacy on responsive cancer cells. PLoS One. 3:e25282008. View Article : Google Scholar : PubMed/NCBI
10	Schultz G, Rotatori DS and Clark W: EGF and TGF-alpha in wound healing and repair. J Cell Biochem. 45:346–352. 1991. View Article : Google Scholar : PubMed/NCBI
11	Lee JA, Song HY, Ju SM, et al: Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells. Exp Mol Med. 41:629–637. 2009. View Article : Google Scholar : PubMed/NCBI
12	Andres C, Hasenauer J, Ahn HS, et al: Wound-healing growth factor, basic FGF, induces Erk1/2-dependent mechanical hyperalgesia. Pain. 154:2216–2226. 2013. View Article : Google Scholar : PubMed/NCBI
13	Weston CR, Lambright DG and Davis RJ: Signal transduction. MAP kinase signaling specificity. Science. 296:2345–2347. 2002. View Article : Google Scholar : PubMed/NCBI
14	Su B and Karin M: Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol. 8:402–411. 1996. View Article : Google Scholar : PubMed/NCBI
15	Herlaar E and Brown Z: p38 MAPK signalling cascades in inflammatory disease. Mol Med Today. 5:439–447. 1999. View Article : Google Scholar : PubMed/NCBI
16	Chan-Hui PY and Weaver R: Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades. Biochem J. 336:599–609. 1998.PubMed/NCBI
17	Carter AB, Knudtson KL, Monick MM and Hunninghake GW: The p38 mitogen-activated protein kinase is required for NF-kappaB-dependent gene expression. The role of TATA-binding protein (TBP). J Biol Chem. 274:30858–30863. 1999. View Article : Google Scholar : PubMed/NCBI
18	Pearson G, Robinson F, Beers Gibson T, et al: Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 22:153–183. 2001.PubMed/NCBI
19	Francisco V, Costa G, Figueirinha A, et al: Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-κB pathway inhibition: contribution of chlorogenic acid. J Ethnopharmacol. 148:126–134. 2013.
20	Kim HG, Shrestha B, Lim SY, et al: Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells. Eur J Pharmacol. 545:192–199. 2006. View Article : Google Scholar : PubMed/NCBI
21	O’Connor JC, McCusker RH, Strle K, Johnson RW, Dantzer R and Kelley KW: Regulation of IGF-I function by proinflammatory cytokines: at the interface of immunology and endocrinology. Cell Immunol. 252:91–110. 2008.
22	Twentyman PR and Luscombe M: A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer. 56:279–285. 1987. View Article : Google Scholar : PubMed/NCBI
23	Reddy DB and Reddanna P: Chebulagic acid (CA) attenuates LPS-induced inflammation by suppressing NF-kappaB and MAPK activation in RAW 264.7 macrophages. Biochem Biophys Res Commun. 381:112–117. 2009. View Article : Google Scholar : PubMed/NCBI
24	Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72:248–254. 1976. View Article : Google Scholar : PubMed/NCBI
25	Jeong Y and Mangelsdorf DJ: Nuclear receptor regulation of stemness and stem cell differentiation. Exp Mol Med. 41:525–537. 2009. View Article : Google Scholar : PubMed/NCBI
26	Tilg H, Wilmer A, Vogel W, et al: Serum levels of cytokines in chronic liver diseases. Gastroenterology. 103:264–274. 1992.PubMed/NCBI
27	ter Steege JC, van de Ven MW, Forget PP, Brouckaert P and Buurman WA: The role of endogenous IFN-gamma, TNF-alpha and IL-10 in LPS-induced nitric oxide release in a mouse model. Cytokine. 10:115–123. 1998.
28	Wu KK: Inducible cyclooxygenase and nitric oxide synthase. Adv Pharmacol. 33:179–207. 1995. View Article : Google Scholar : PubMed/NCBI
29	Albini A and Sporn MB: The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer. 7:139–147. 2007. View Article : Google Scholar : PubMed/NCBI
30	Barnes PJ and Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med. 336:1066–1071. 1997. View Article : Google Scholar : PubMed/NCBI
31	Kim SH, Park HS, Lee MS, et al: Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells. Biochem Biophys Res Commun. 372:108–113. 2008. View Article : Google Scholar : PubMed/NCBI
32	Kao SJ, Lei HC, Kuo CT, et al: Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages. Immunology. 115:366–374. 2005. View Article : Google Scholar : PubMed/NCBI
33	Heinrich PC, Castell JV and Andus T: Interleukin-6 and the acute phase response. Biochem J. 265:621–636. 1990.PubMed/NCBI
34	Flamand L, Gosselin J, D’Addario M, et al: Human herpesvirus 6 induces interleukin-1 beta and tumor necrosis factor alpha, but not interleukin-6, in peripheral blood mononuclear cell cultures. J Virol. 65:5105–5110. 1991.PubMed/NCBI
35	Yuk SS, Lim EM, Lee JY, et al: Antiinflammatory effects of Epimedium brevicornum water extract on lipopolysaccharide-activated RAW264.7 macrophages. Phytother Res. 24:1781–1787. 2010. View Article : Google Scholar : PubMed/NCBI
36	Mocellin S, Marincola F, Rossi CR, Nitti D and Lise M: The multifaceted relationship between IL-10 and adaptive immunity: putting together the pieces of a puzzle. Cytokine Growth Factor Rev. 15:61–76. 2004. View Article : Google Scholar : PubMed/NCBI
37	Jana M, Dasgupta S, Saha RN, Liu X and Pahan K: Induction of tumor necrosis factor-alpha (TNF-alpha) by interleukin-12 p40 monomer and homodimer in microglia and macrophages. J Neurochem. 86:519–528. 2003. View Article : Google Scholar : PubMed/NCBI