Ann Dermatol.  2015 Feb;27(1):32-39. 10.5021/ad.2015.27.1.32.

Synergistic Inhibition of Tumor Necrosis Factor-Alpha-Stimulated Pro-Inflammatory Cytokine Expression in HaCaT Cells by a Combination of Rapamycin and Mycophenolic Acid

• Kim MY
• Lim YY
• Kim HM
• Park YM
• Kang H
• Kim BJ

• KMID: 2264834
• DOI: http://doi.org/10.5021/ad.2015.27.1.32

Abstract

BACKGROUND
Keratinocytes release various pro-inflammatory cytokines, chemokines, and adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) in response to cytokines such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Rapamycin and mycophenolic acid (MPA) have potent immunosuppressive activity because they inhibit lymphocyte proliferation.
OBJECTIVE
We investigated the effects of rapamycin and MPA on the expression of inflammation-related factors such as ICAM-1 and inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines and chemokines, and related signaling pathways in TNF-alpha-stimulated HaCaT cells.
METHODS
The viability of HaCaT cells treated with rapamycin and MPA was confirmed using MTT assay. The expression of various cytokines such as interleukin (IL)-1beta, IL-6, and IL-8; inflammation-related factors such as ICAM-1 and iNOS; and the activation of mitogen activated protein kinase (MAPK) signaling pathways mediated by extracellular signal-related kinases (ERK), p38, and c-Jun N-terminal kinases (JNK) in TNF-alpha-stimulated HaCaT cells were confirmed using reverse transcription-polymerase chain reaction and western blotting.
RESULTS
Combined treatment of TNF-alpha-induced HaCaT cells with rapamycin and MPA decreased ICAM-1 and iNOS expression and ERK and p38 activation more than treatment with either drug alone. The most significant decrease was observed with a combination of rapamycin (80 nM) and MPA (20 nM). These results show that co-treatment with these agents has a synergistic anti-inflammatory effect by blocking the activation of the ERK/p38 MAPK signaling pathway and thus suppressing the TNF-alpha-induced expression of ICAM-1 and iNOS.
CONCLUSION
The combination of rapamycin and MPA could potentially be used as a therapeutic approach in inflammatory skin diseases.

Keyword

Anti-inflammation; Mycophenolic acid; Sirolimus; Tumor necrosis factor-alpha

MeSH Terms

Blotting, Western
Chemokines
Cytokines
Intercellular Adhesion Molecule-1
Interferons
Interleukin-6
Interleukin-8
Interleukins
Keratinocytes
Lymphocytes
Mycophenolic Acid*
Necrosis*
Nitric Oxide Synthase Type II
Phosphotransferases
Protein Kinases
Sirolimus*
Skin Diseases
Tumor Necrosis Factor-alpha
Chemokines
Cytokines
Intercellular Adhesion Molecule-1
Interferons
Interleukin-6
Interleukin-8
Interleukins
Mycophenolic Acid
Nitric Oxide Synthase Type II
Phosphotransferases
Protein Kinases
Sirolimus
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Effects of rapamycin, mycophenolic acid (MPA), or a combination of the two drugs on the viability of HaCaT cells. (A) Cytotoxic effects of rapamycin and MPA in HaCaT cells. The cells were treated with rapamycin and MPA in a dose-dependent manner for 24 h. (B) Confirmation of the cytotoxic effects of a combination of rapamycin and MPA in HaCaT cells. The cells were treated as indicated in (C). Cell viability was determined using MTT assay as described in the Materials and Methods. The measurements were performed in triplicate. TNF-α: tumor necrosis factor-α.

• 

  Fig. 2 Effects of a combination of rapamycin (RP) and mycophenolic acid (MPA) on tumor necrosis factor (TNF)-α-induced expression of pro-inflammatory genes in HaCaT cells. HaCaT cells were pre-incubated for 24 h and then stimulated with TNF-α (20 ng/ml) after a 1 h pretreatment with RP, MPA, or a combination of the two drugs for 6 h. Pro-inflammatory gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers. The experiment was repeated independently at least three times. IL: interleukin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 3 Inhibitory effects of rapamycin (RP) and mycophenolic acid (MPA) on tumor necrosis factor (TNF)-α-induced activation of nuclear factor-kappa B (NF-κB), extracellular signal-related kinases (ERK), p38, and c-Jun N-terminal kinases (JNK) mitogen activated protein kinases (MAPKs) in HaCaT cells. HaCaT cells were pre-incubated for 24 h. The cells were then pre-treated with RP, MPA, or a combination of the two drugs for 1 h, and then stimulated by TNF-α (20 ng/ml) for 20 min. (A) The phosphorylation of IκB α and the translocation of NF-κB p65 in whole-cell protein lysates were determined by western blot analysis. Cell lysates were prepared and subjected to western blot with the indicated antibodies. (B) The phosphorylation of substrates in the MAPK family (ERK, p38, JNK) was detected by western blot analysis using specific antibodies. The densities of protein expression were expressed as percentages of protein density in the TNF-α-only group. *p<0.05 and **p<0.01 versus the TNF-α-treated group. The densitometric analysis of the western blot was standardized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

• 

  Fig. 4 Inhibitory effects of rapamycin (RP) and mycophenolic acid (MPA) on tumor necrosis factor (TNF)-α-induced intercellular adhesion molecule 1 (ICAM-1) and inducible nitric oxide synthase (iNOS) expression in HaCaT cells. (A) HaCaT cells were pre-incubated for 24 h, and then stimulated with TNF-α (20 ng/ml) in the presence of RP, MPA, or a combination of the two drugs for 3 h (for RNA) or 12 h (for protein). ICAM-1 and iNOS expression were determined after treatment with 100 nM RP, 100 nM MPA, and different combinations of the two drugs (20 nM RP and 80 nM MPA, 50 nM RP and 50 nM MPA, and 80 nM RP and 20 nM MPA) by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. (B) HaCaT cells were pretreated with an extracellular signal-related kinases (ERK) inhibitor (PD98059), p38 inhibitor (SB203580), or c-Jun N-terminal kinases (JNK) inhibitor (SP600125) for 1 h, and then stimulated with TNF-α for 12 h. The band densities are expressed as percentages of the band density of the TNF-α-only group. **p<0.01 compared to treatment with TNF-α alone. The experiment was repeated independently at least three times. The densitometric analysis of the western blot was standardized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control.

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