Rapamycin and MPA were synthesized at IKSU Co., Ltd. (Seoul, Korea). Dulbecco's modified Eagle medium (DMEM), Dulbecco's phosphate-buffered saline (Dulbecco's PBS), antibiotic (penicillin, streptomycin), fetal bovine serum (FBS), and trypsin-EDTA were purchased from WelGENE Inc. (Daegu, Korea). Recombinant human TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PD98059, and SP600125 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies specific to ICAM-1, phospho-extracellular signal-related kinases (ERK), phospho-IκBα, and lamin B were purchased from Cell Signaling Technology (Beverly, MA, USA). phosphor-c-Jun N-terminal kinases (JNK) and Phosphor-p38, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and inducible nitric oxide synthase (iNOS) antibodies were obtained from BD Bioscience (San Jose, CA, USA). Antibodies specific to NF-κB p65 were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies specific for anti-goat IgG, anti-mouse IgG, and anti-rabbit IgG were purchased from Vector Laboratories (Burlingame, CA, USA). ICAM-1, iNOS, IL-1β, IL-6, IL-8, and GAPDH oligonucleotide primers were obtained from Bioneer (Seoul, Korea).

The human keratinocyte cell line (HaCaT) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured at 37℃ in a humidified incubator containing 5% CO₂ and 95% air in DMEM supplemented with 10% FBS (WelGENE Inc.) and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin).

Total RNA was isolated from cells by using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The RNA (2 µg) was transcribed into cDNA using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Otsu, Japan). The transcribed product was amplified by polymerase chain reaction (PCR) into a final volume of 50 µl by using the following sense and antisense primers (5'→3'): ICAM-1 sense, GGT GAC GCT GAA TGG GGT TCC; ICAM-1 antisense, GTC CTC ATG GTG GGG CTA TGA CTC; iNOS sense, TCC AAC CTG CAG GTC TTC GAT GC; iNOS antisense, GGA CCA GCC AAA TCC AGT CTG C; IL-1β sense, AAA CAG ATG AAG TGG TCC TTC CAG; IL-1β antisense, TGG AGA ACA CCA CTT GTT GCT CCA; IL-6 sense, AGA GTA GTG AGG AAC AAG CC; IL-6 antisense, TAC ATT TGC CGA AGA GCC CT; IL-8 sense, ACA TGA CTT CCA AGC TGG CCG; IL-8 antisense, TTT ATG AAT TCT CAG CCC TC; and GAPDH sense, CAT GGG GAA GGT GAA GGT C; GAPDH antisense, TGG ACT CCA CGA CGT ACT CA. Amplification was performed using Taq polymerase (Takara, Otsu, Japan). The products were electrophoresed for 30 min at 100 V on 1% agarose gel. Gels were visualized using a Molecular Imager Gel Doc XR imaging system (Bio-Rad Laboratories Inc., Hercules, CA, USA).

HaCaT cells were washed twice with ice-cold PBS and then lysed in RIPA buffer. The lysates were clarified by 30 min of centrifugation at 13,000 rpm at 4℃. Total protein in the cell extracts or in the cytoplasmic and nuclear extracts was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% polyacrylamide gel. The separated proteins were transferred to a PVDF membrane and then the membrane was blocked with blocking buffer (5% skim milk in Tris-buffered saline containing 0.5% Tween 20). Western blot analysis was performed by first incubating the membrane in target antibodies overnight at 4℃, and then incubating them with horseradish peroxidase-conjugated secondary antibody. Detection of immunoreactive bands was performed using a SuperSignal West Pico chemiluminescence substrate (PIERCE Biotechnology Inc., IL, USA). The values of the western blot were standardized with an internal control.

Data are presented as means±standard deviations. Data were evaluated using the Student's t-test. A p-value of <0.05 or <0.01 was considered statistically significant. The analysis was performed with Statistical Package for Social Sciences (version 12.0; SPSS Inc., Chicago, IL, USA).

Cell viability was measured at different concentrations of rapamycin, MPA, or a combination of the two drugs by using MTT assay in HaCaT cells. HaCaT cells were incubated with various concentrations (10, 100, 1,000, or 5,000 nM) of rapamycin or MPA for 24 h (Fig. 1A). Viability was not significantly altered by concentrations of rapamycin or MPA up to 1,000 nM. As shown Fig. 1B, the cells were also treated with a combination of different concentrations of rapamycin and MPA (Fig. 1C). The combination of these agents did not show cytotoxicity.

To compare the effect of a combination of rapamycin and MPA to that of either agent alone, the expression levels of TNF-α-induced pro-inflammatory genes such as IL-1β, IL-6, and IL-8 were measured using reverse transcription-PCR (RT-PCR) in HaCaT cells. HaCaT cells were pretreated with rapamycin, MPA, or a combination of rapamycin and MPA for 1 h, and then stimulated with TNF-α for 6 h. The expression of pro-inflammatory genes increased in TNF-α-stimulated HaCaT cells. The treatment of cells with rapamycin (100 nM) or MPA (100 nM) alone either slightly reduced or did not change the levels of IL-1β, IL-6, and IL-8 compared to the corresponding levels in the TNF-α-treated group. The treatment of cells with a combination of rapamycin (20 nM) and MPA (80 nM), or rapamycin (50 nM) and MPA (50 nM) only slightly reduced the level of pro-inflammatory gene expression, whereas rapamycin at 80 nM in combination with MPA at 20 nM strongly inhibited the levels of IL-1β, IL-6, and IL-8. An effect was not seen with any other single or combination treatment. Use of equal amounts of cDNA was confirmed on the basis of GAPDH expression (Fig. 2).

NF-κB is a transcription factor for genes encoding inflammatory cytokines, chemokines, and inflammation-related factors16. To verify the effects of combinations of rapamycin and MPA on TNF-α-induced NF-κB signaling cascades, the phosphorylation of IκBα and the translocation of NF-κB were measured by performing western blot. Cells were pretreated with rapamycin (100 nM), MPA (100 nM), or a combination of both drugs for 1 h, before being stimulated with TNF-α. The activation of NF-κB molecules was analyzed using either nuclear extracts or whole-cell lysates. As shown in Fig. 3A, treatment with rapamycin or MPA slightly inhibited the phosphorylation of IκBα, and similar results were obtained from the combination of rapamycin (20 nM) and MPA (80 nM) or rapamycin (50 nM) and MPA (50 nM). However, in the presence of rapamycin (80 nM) and MPA (20 nM), the inhibition of TNF-α-induced phosphorylation of IκBα was much higher than that with any other treatment. Moreover, a combination of rapamycin (80 nM) and MPA (20 nM) inhibited the TNF-α-induced nuclear localization of NF-κB p65 in HaCaT cells when compared with individual drug treatments and other combinations of the two drugs. These results suggest that the combination of rapamycin (80 nM) and MPA (20 nM) has an inhibitory effect on the signaling pathway that leads to the activation of NF-κB in TNF-α-treated HaCaT cells.

In order to assess the effects of a combination of these drugs on TNF-α-induced MAPK activation in HaCaT cells, we performed western blot analysis. We compared the inhibitory effects of a single drug or a combination of both drugs on the MAPK signaling pathway. As shown in Fig. 3B, in the presence of a combination of 80 nM rapamycin and 20 nM MPA, the inhibition of TNF-α-induced ERK activation was markedly higher than that in the presence of a combination of 50 nM rapamycin and 50 nM MPA. Single-drug treatment and the combination of 20 nM rapamycin and 80 nM MPA did not inhibit TNF-α-induced ERK activation. TNF-α-induced p38 activation decreased in the MPA-only treatment group and in the combination groups but not in the rapamycin-only treatment group.

To investigate the synergistic inhibitory effects of a combination of rapamycin and MPA on TNF-α-induced ICAM-1 and iNOS expression, RT-PCR and western blot analysis were performed. After TNF-α treatment in HaCaT cells, the HaCaT cells were treated with rapamycin and MPA alone, as well as with a combination of the two drugs. Following treatment, the levels of ICAM-1 and iNOS were measured by RT-PCR and western blot. TNF-α-induced ICAM-1 and iNOS expression in HaCaT cells was slightly suppressed by rapamycin and MPA. The co-treatment of rapamycin and MPA resulted in significantly greater inhibition compared to that with single-drug treatments. The greatest inhibitory effect was achieved with a combination of 80 nM rapamycin and 20 nM MPA (Fig. 4A).

In addition, we used pharmacological inhibitors of MAPK to investigate the functional relationship between MAPK activation and ICAM-1 expression. TNF-α-induced ICAM-1 expression was slightly suppressed in HaCaT cells with ERK, p38, and JNK inhibitors. These results suggest that the activation of ERK, p38, and JNK is involved in TNF-α-induced ICAM-1 expression, and the combination of rapamycin (80 nM) and MPA (20 nM) modulates signaling cascades that involve TNF-α-induced activation of ERK and p38 but not JNK (Fig. 4B).