S100A11, an dual mediator for growth regulation of human keratinocytes

Abstract

We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca(++) or transforming growth factor beta. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21(WAF1). In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; 2) extracellular S100A11 acts on NHK to enhance the production of epidermal growth factor family proteins, resulting in growth stimulation; 3) receptor for advanced glycation end products, nuclear factor-kappaB, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in growth regulation of epithelial cells.

Figure 1.

Secretion of and growth stimulation by S100A11 in NHK. (A) Production and secretion of S100A11. NHK were cultured in EpiLife medium without HKGS but with designated additives for 24 h. Equivalent aliquots of whole protein preparations from the cell extract, and the culture medium was applied for Western blot analysis. Tubulin was used as a control for the applied amount of protein preparations. (B) Screening for cytokines induced by S100A11 (10 ng/ml; 24 h) by using a human cytokine antibody array. Dotted squares, EGF; solid squares, positive and negative controls. (C) Induction of EGF by S100A11 in NHK determined by Northern blot analysis. NHK were cultured in HKGS-free EpiLife 24 h before addition of S100A11. (D) Growth stimulation of NHK by exogenous S100A11. NHK were cultured in HKGS-free EpiLife 24 h before addition of S100A11 and incubated for a further 24 h with the designated cytokines. [³H]Thymidine (1 μCi/ml) was added to the medium 1 h before harvest, and radioactivity in an insoluble fraction was counted. GST was used as a control. Epidermal growth factor receptor (EGFR) I, 10 μg/ml an inhibitor for the EGF receptor AG1478.

Figure 2.

Activation of Akt by S100A11 in NHK. (A) Induction of phosphorylation of Akt. NHK were cultured in HKGS-free EpiLife 24 h before addition of S100A11 protein and incubated for a further 24 h before harvest. Cell extracts were analyzed by Western blot analysis. (B) Time course of activation of Akt and related proteins by S100A11. The experiment was performed under conditions similar to those described in A except for incubation times after addition of S100A11. (C) Specificity in Akt activation among S100 family members. The experiment was performed under conditions similar to those described in A. The recombinant proteins were added at 10 ng/ml. S100A11 was heated at 100°C for 20 min. (D) Inhibition of EGF induction by S100A11 by various agents demonstrated by Northern blot analysis. The experiment was performed under conditions similar to those described in A except for incubation time of 6 h after addition of S100A11 or GST at 10 ng/ml.

Figure 3.

Involvement of RAGE in signal transduction for S100A11. (A) Expression of RAGE in NHK as detected by Western blot analysis. Top, NHK were precultured in HKGS-free EpiLife for 24 h. Cells were harvested 6 h after addition of S100A11 or GST (10 ng/ml). Bottom, after being precultured as described above, NHK were incubated with an inhibitor of NF-κB (0.5 μg/ml) followed by exposure to S100A11 at 10 ng/ml for 16 h. (B) Abrogation of S100A11-induced Akt activation by functional blocking of RAGE. EGFR I, 10 μg/ml AG1478; sRAGE, 1 μg/ml soluble RAGE; antibody (Ab), 20 μg/ml antibody. NHK were pre-cultured in HKGS-free EpiLife for 24 h. The agents were added 1 h before the addition of S100A11 and harvested 6 h later. (C) Preparation of monomer S100A11 and dimer S100A11 (see Materials and Methods). The membrane was stained with Coomassie Brilliant Blue. MM, molecular markers. White arrowhead, monomer; black arrowhead, dimer. (D) Binding of S100A11 monomer and S100A11 dimer to RAGE. Biotinylated proteins (0.1 nmol) were incubated with 1 mg of membrane preparation of NHK, pulled down with a streptavidin agarose, and analyzed for RAGE by Western blot analysis. (E) Phosphorylation of Akt by S100A11 monomer and S100A11 dimer (10 μg/ml). The experiment was performed under conditions similar to those described in B.

Figure 4.

Transcriptional activation of EGF gene by S100A11. (A) Transcription factors bound to the EGF promoter identified by a newly developed method. NHK were precultured in HKGS-free EpiLife for 24 h. An Akt inhibitor and anti-RAGE antibody were added 1 h before the addition of S100A11 or GST and harvested 6 h later. Cell extracts were incubated with a biotinylated EGF promoter fragment (2085 base pairs), and proteins pulled down with streptavidin beads were analyzed by Western blot analysis. (B) Copresence of activated Akt and phosphorylated CREB in nuclei of NHK exposed to S100A11. Histone H1 (H1) and tubulin were used as indicators for nuclei and cytoplasm, respectively. (C) Competitive inhibition of binding of CREB to the EGF promoter by transcription factor-binding elements. Wild-type (W) or mutant (M) oligomers were added to the incubation mixture under conditions similar to those described in A. (D) A luciferase assay for the seventh AP-1 site of the EGF promoter (see Supplemental Figure S7). The seventh AP-1 site was responsive to S100A11 (10 ng/ml), and this was abrogated by blocking of the function of S100A11 (with an antibody, 20 μg/ml), RAGE (with soluble RAGE at 1 μg/ml), and Akt (with an inhibitor at 10 μM). (E) Chromatin immunoprecipitation assay for proteins bound to the seventh AP-1 site described in D. (F) Effect of down-regulation of CREB by siRNA on induction of the EGF gene examined by Northern blot analysis.

Figure 5.

S100A11 in SSC cell lines. (A) Production and secretion of S100A11 in SCCs (A431, BSCC-93, and HSC-5) determined under conditions similar to those described in the legend to Figure 1A. (B) Higher constitutive expression levels of EGF, HB-EGF, and epiregulin in BSCC-93 cells than in NHK (cultivated with Ca⁺⁺ concentration of 0.03 or 1.5 mM) as determined by Northern blot analysis. (C) Down-regulation of constitutively active Akt in BSCC-93 cells by a neutralizing antibody against S100A11 (20 μg/ml; 24 h) as shown by Western blot analysis. (D) Down-regulation of constitutive EGF family mRNA levels by an anti-S100A11 antibody (20 μg/ml; 24 h). (E) Compromise of the growth of NHK and SCCs by an anti-S100A11 antibody. Increase in cell number over a 4-d culture period with or without the antibody (20 μg/ml added at 0 and 2 culture days) was determined. (F) Schematic presentation of the ambivalent role of S100A11 in growth regulation of NHK.