Fig 1.
Differential expression of RAGE, S100A8 and S100A9 on transcriptional level in whole skin and epidermis.
Expression of RAGE, S100A8 and S100A9 on transcriptional level. The expression was assessed by real-time PCR using specific primers for RAGE, S100A8 and S100A9. The expression level was normalized to 36B4 housekeeping gene. a: whole skin: No significant difference of RAGE expression was detected within or among the groups. S100A8 and S100A9 mRNAs are increased only in IC patients with invasive SCC versus normal skin (*p<0.05). b: epidermis: The expression of RAGE, S100A8 and S100A9 transcripts was significantly increased in OTR and IC patients with invasive or in situ SCC versus normal epidermis (***p<0.001). Significant difference in the expression of S100A8 and S100A9 was also detected between the IC and OTR groups with invasive SCC (*p<0.05; **p<0.01).
Fig 2.
Differential expression of RAGE and S100A8/A9 on protein level in whole skin.
The expression level was analysed by immunochistochemistry using specific anti human S100A8/A9 and anti human RAGE antibodies. The expression was tested in 5 patient samples per group (IC and OTR with in situ or invasive SCC). a: Expression of RAGE and S100A8/A9 in IC and OTR patients with in situ SCC in comparison to normal skin. b: Expression of RAGE and S100A8/A9 in IC and OTR patient with invasive SCC in comparison to normal skin.
Fig 3.
Endogenous S100A8/A9 is involved in cellular proliferation.
a: Spontaneous secretion of S100A8/A9 from normal and SCC-derived keratinocytes. Normal and SCC-derived keratinocytes were grown in 96 well plates for 24 hours. Afterwards, supernatant was collected and preceded for the assessment of secreted S100A8/A9 using specific ELISA for S100A8/A9. b: Blockade of RAGE using anti RAGE blocking antibody reduces spontaneous proliferation of keratinocytes. Normal and SCC-derived keratinocytes were incubated with a blocking anti-RAGE antibody (8ug/100μl) for 24 hours. A decrease of the proliferation rate was detected (20–30%) based on the BrdU incorporation (t-test *p = 0.002). All the results are presented as percentage deviation from corresponding control and represent the mean +/- SD of duplicate values.
Fig 4.
Exogenous S100A8/A9 induces keratinocyte proliferation.
a: Assessment of the proliferation by BrdU assay. To assess the role of S100A8/A9 in normal primary, AK and SCC cultures, they were treated with purified S100A8/A9 for 24 hours and proliferation was assessed by BrdU incorporation. The induction of cellular proliferation was between 30–70% (2 way Anova, p<0.0001). b: Assessment of the proliferation by the incorporation of BrdU analysed by flow cytrometry. Normal keratinocytes were treated with S100A8/A9 (10ng/ml) and BrdU for 24 hours. Afterwards cells were fixed in 4% PFA, permeabilized with 0.5% Triton and stained for RAGE and BrdU using the antibodies mentioned above. The differential BrdU incorporation was compared to untreated cell.
Fig 5.
The receptor RAGE critically mediates the cellular response to S100A8/A9.
a: Direct blockade by a specific RAGE blocking antibody reduces cellular proliferation. RAGE dependent proliferation was analyzed in normal primary, AK and SCC cells. Cells were incubated for 1 hour with a blocking anti-RAGE antibody (80μg/ml, as recommended by manufacturer) followed by S100A8/A9 stimulation for additional 24 hours. The differences in the proliferation after the blockade were assessed by BrdU incorporation (1 way Anova, Bonferroni`s Multiple test, **p<0.05, ***p<0.05). b: Knockdown of RAGE using shRNA reduces cellular proliferation. The knockdown studies were performed using specific lentiviral shRNA against RAGE. Primary keratinocytes were infected by shRAGE and sh control viral particles. Selection of positive clones was performed by puromycine selection. Cells were grown to optimal confluence and were stimulated with 10ng/ml S100A8/A9.Cells with RAGE knockdown showed a delay in proliferation in comparison to control as assessed by BrdU (70%) (1 way Anova, Bonferroni`s Multiple test p****<0.0001) and microscope images. Exogenous S100A8/A9 did not induce proliferation of shRAGE keratinocytes, but only in the control. c: Blocking TLR4 using specific blocking antibody (HTA125) does not impair cellular proliferation. AK cells were treated with a specific blocking TLR4 antibody (HTA125). AK cells were grown for 24 hours in the presence of HTA125 antibody (1μg/ml) and S100A8/A9 (10ng/ml). For detection of the cellular proliferation rate BrdU proliferation assay was performed.
Fig 6.
Exogenous S100A8/A9 induces migration of normal and SCC-derived primary keratinocytes.
a, b: Cells were treated with different concentrations of purified S100A8/A9 (0.01–1 μg/ml) for 15h. The assessment of the migratory potential was analyzed by scratch assay where the number of migrated cells was analyzed. The migration of both normal and SCC-derived keratinocytes was increased significantly (between 60–100% depending on cell type) when 0.01 and 0.1μg/ml of S100A8/A9 were used (t-test, PK, *p = 0.003, *p = 0.004; SCC, *p = 0.0014,*p = 0.0015).
Fig 7.
Exogenous S100A8/A9 induces RAGE surface expression and MAPK phosphorylation.
a: Rapid and transient induction of RAGE surface expression after stimulation with S100A8/A9. Normal keratinocytes were induced with an exogenous S100A8/A9 (10ng/ml) in time intervals of 30 and 60 minutes. Afterwards cells were stained with the specific goat polyclonal anti RAGE antibody and a secondary FITC-conjugated antibody for analysis of both surface and total RAGE expression before and after S100A8/A9 treatment. Total expression of RAGE was analyzed prior to cell permeabilization with PFA and followed by blocking step with 1% BSA. As controls isotype control antibody and untreated cells were used. b: Exogenous S100A8/A9 induces phosphorylation of MAPKs. Normal and SCC-derived keratinocytes were seeded in 6cm dishes in 50% confluence. Cells were starved for 18 h and stimulated afterwards with S100A8/A9 (10ng/ml) for different time intervals (0–60min). Cell lysates were collected after each time interval and the alteration of the phosphorylation of ERK1/2, p38 and SAPK/JNK was analyzed by western blot using the indicated antibodies.