Increase in extracellular Ca2+ concentration inhibits the growth of NHK and HaCaT cells
At first, we confirmed that an increase in extracellular Ca2+
concentration from 0.03 to 1.5 mM results in inhibition of DNA synthesis of NHK cells in dose- and time-dependent manners (). HaCaT cells, an immortalized human keratinocyte line (Boukamp et al., 1988
), were routinely cultivated in a medium with 1.5 mM Ca2+
. When the concentration of Ca2+
was increased to 5 or 10 mM, DNA synthesis of HaCaT cells was inhibited (). Under similar conditions, Sharpe et al. (1989)
and Gonczi et al. (2002)
reported that there was no remarkable difference in intracellular Ca2+
level (70–100 nM) of NHK and HaCaT cells, despite different extracellular Ca2+
concentrations. Intracellular Ca2+
concentrations in NHK and HaCaT cells increased by ~40% within 30 min after increasing the extracellular Ca2+
concentrations from 0.03 to 1.5 mM and from 1.5 to 10 mM, respectively ( C). Overall, the response of HaCaT cells to 10 mM Ca2+
was similar to that of NHK cells to 1.5 mM Ca2+
. No immediate cytotoxicity was noted in HaCaT cells in a medium with 10 mM Ca2+
during the observation period. Therefore, throughout the present work, 0.03 and 1.5 mM Ca2+
was used as low Ca2+
media and 1.5 and 10 mM Ca2+
was used as high Ca2+
media for NHK and HaCaT cells, respectively.
Figure 1. Growth arrest and increased intracellular Ca2+ levels of NHK and HaCaT cells exposed to high Ca2+ media. (A) Dose-dependent inhibition of [3H]TdR incorporation into an insoluble fraction in NHK (left) and HaCaT (right) cells exposed to high Ca2+ for 6 (more ...)
S100C/A11 inhibits the growth of human keratinocytes
To examine the possible involvement of S100C/A11 in growth regulation of human keratinocytes, we transfected various constructs of S100C/A11 flanking SV40-derived NLS to exponentially growing HaCaT cells. As shown in A, incorporation of BrdU was specifically inhibited in the cells transfected with wild-type S100C/A11 (indicated by arrows), whereas GFP alone did not show any effects. DNA synthesis was inhibited by ~25% by the wild-type S100C/A11 ( B). This reduction is substantial, considering that the transfection efficiency was at most 40%. Introduction of recombinant S100C/A11 protein into HaCaT cells by the aid of the protein transduction domain of TAT protein (YGRKKRRQRRR; Schwarze et al., 1999
) efficiently inhibited DNA synthesis ( C). The TAT-flanked protein was transferred to cell nuclei with an efficiency of nearly 100%.
Figure 2. Growth inhibition of human epidermal keratinocytes by S100C/A11. (A) Various constructs of S100C/A11-NLS-Myc (top row) were transfected to HaCaT cells 48 h before the addition of 1 μM BrdU. The cells were further incubated for 12 h before fixation (more ...)
Increase in extracellular Ca2+ concentration results in phosphorylation and nuclear translocation of S100C/A11
When extracts of NHK and HaCaT cells labeled with [32
P]phosphate were analyzed by a two-dimensional gel electrophoresis, increased phosphorylation of S100C/A11 (as identified by sequencing) was noted in both cell types exposed to high Ca2+
( A). Exposure to high Ca2+
increased the immunoprecipitable amount of S100C/A11 and more remarkably the level of phosphorylation in both cell types ( A, bottom), whereas the S100C/A11 protein levels only slightly increased by high Ca2+
(Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200304017/DC1
Figure 3. Phosphorylation of S100C/A11 in NHK and HaCaT cells exposed to high Ca2+ concentrations. (A) Top: two-dimensional electrophoresis of [32P]phosphate-labeled cell extracts. Only a part covering pI (isoelectric point) 6.0–7.0, molecular marker of (more ...)
To determine the phosphorylation sites of S100C/A11, endogenous S100C/A11 protein was purified from 32P-labeled HaCaT cells and degradated to peptides. As shown in B (middle and bottom), only NH2-terminal p1-1 and COOH-terminal p4 were phosphorylated in a totally high Ca2+-depending manner. When the synthesized peptides of S100C/A11 were incubated with cell extracts in vitro, NH2-terminal pepA (amino acid residues 1–23) and COOH-terminal pepF (87–105), but not four other peptides covering the middle part of S100C/A11, were significantly phosphorylated by the extracts prepared from NHK and HaCaT cells cultivated in the high Ca2+ media (unpublished data). Thr and Ser residues were phosphorylated in pepA and pepF, respectively ( C). Finally, phosphorylation sites in p1-1 and p4 were determined as 10Thr and 94Ser, respectively, by an amino acid sequencer ( D). In accordance with this, Ala-substituted forms at 10Thr of pepA and at 94Ser of pepF were not phosphorylated by the cell extracts prepared after cultivating in the high Ca2+ media (unpublished data).
Increases in Ca2+ in the media resulted in partial translocation of endogenous S100C/A11 to nuclei of NHK and HaCaT cells as demonstrated by immunostaining ( A) and by Western blotting and autoradiography ( B). Preincubation with a membrane-permeable Ca2+ chelator, BAPTA-AM, inhibited the nuclear translocation of endogenous S100C/A11 by high Ca2+ ( A). An S100C/A11 variant protein lacking Ca2+-binding capacity was not translocated to nuclei by high Ca2+ ( C). Transfection of the wild-type S100C/A11-GFP construct before the shift to high Ca2+ resulted in nuclear translocation of the protein in HaCaT cells ( D). This nuclear translocation was not observed when 10Thr was substituted with Ala ( D) or when the NH2-terminal 23 amino acids were deleted (unpublished data). 10Thr-to-Asp–substituted protein (TD) was observed in nuclei even in the low Ca2+ medium. On the contrary, 94Ser-substituted forms behaved in a manner similar to the wild-type protein. A COOH-terminal truncated form of S100C/A11 lacking the residues from 87 to 105 was transferred to nuclei by the high Ca2+ (unpublished data). These results indicate that the phosphorylation of 10Thr, but not of 94Ser, is critical for the Ca2+-induced nuclear localization of S100C/A11, and that binding of Ca2+ to S100C/A11 is necessary for the translocation.
Figure 4. Nuclear translocation of S100C/A11 in NHK and HaCaT cells by high Ca2+. (A) Translocation of S100C/A11 to nuclei by high Ca2+. NHK and HaCaT cells were immunostained with anti-S100C/A11 antibody. A membrane-permeable Ca2+ chelator, BAPTA-AM, was added (more ...)
Binding of S100C/A11 to nucleolin is essential for nuclear translocation
Because S100C/A11 lacks the canonical NLS sequence, we screened for possible interacting protein(s) using a pepA(TD)-immobilized column (pepA with Asp at the 10th residue, a phosphorylated pepA equivalent). An ~100-kD protein was identified in extracts of HaCaT cells as showing specific binding ( A, left). Sequencing of the eluted protein gave an amino acid sequence of VKLAKAGKANQGD, corresponding to the NH2
terminus of human nucleolin. The binding of S100C/A11 and nucleolin was confirmed in vitro ( A, right; B). The bound amounts of nucleolin depended on the amounts of S100C/A11 protein immobilized, and S100C/A11(TD), a 10
Thr phosphorylation equivalent form, showed higher affinity to nucleolin than did the wild type ( B). Synthesized pepA having phosphothreonine at the 10th residue showed a higher nucleolin-binding capacity similar to pepA(TD) (Fig. S6, available at http://www.jcb.org/cgi/content/full/jcb.200304017/DC1
). Only pepA, and not the other five partial peptides of S100C/A11, binds to nucleolin. Furthermore, neither GST-S100A2 nor GST-S100A6, the other members of S100 family, bound to nucleolin as assayed in vitro (unpublished data).
Figure 5. Binding of phosphorylated S100C/A11 to nucleolin as a prerequisite for its nuclear translocation. (A) Binding of S100C/A11 to nucleolin. Left: HaCaT cell extract was applied onto a pepA(TD) column (a phosphorylated pepA equivalent), and the bound fraction (more ...)
In the Ca2+-stimulated HaCaT cells, S100C/A11 protein that was phosphorylated and bound to nucleolin was predominantly found in the nuclei ( C). Among various GFP-conjugated 10Thr- or 94Ser-substituted forms of S100C/A11 transfected to HaCaT cells, only 10Thr-to-Asp–substituted forms were coprecipitated with nucleolin in the low Ca2+ medium ( D). In the high Ca2+ medium, phosphorylation of S100C/A11 was observed when either 10Thr or 94Ser was intact. Nucleolin was coprecipitated with 10Thr-intact forms in addition to 10Thr-to-Asp–substituted forms. BAPTA-AM inhibited the phosphorylation and interaction of wild-type S100C/A11 with nucleolin by high Ca2+ ( E). The S100C/A11 variant protein lacking Ca2+-binding capacity was neither phosphorylated nor coprecipitated with nucleolin by high Ca2+ ( E).
When small interfering RNA (siRNA) against nucleolin was applied to HaCaT cells, the protein levels were substantially reduced ( F, top), whereas the control siRNA against GAPDH showed no effects. Increase in Ca2+
concentration translocated endogenous S100C/A11 into nuclei in the siRNA against GAPDH-treated cells, but not in the siRNA against nucleolin-treated cells ( F, bottom). The necessity of nucleolin for the Ca2+
-induced nuclear translocation of S100C/A11 was also confirmed in an in vitro translocation assay using permeabilized HaCaT cells by depleting nucleolin with a specific antibody (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200304017/DC1
). These results indicate that (1) high Ca2+
treatment of HaCaT cells induces endogenous activity to phosphorylate S100C/A11 at 10
Thr and 94
Ser, and eventually enhances the binding of S100C/A11 to nucleolin; (2) S100C/A11 binds to nucleolin through its NH2
-terminal region in a 10
Thr phosphorylation–dependent manner; (3) the binding of S100C/A11 to nucleolin is essential for the translocation of S100C/A11 to nuclei; and (4) the binding of Ca2+
to S100C/A11 is necessary for the phosphorylation, binding to nucleolin, and nuclear translocation of S100C/A11.
The effector protein for growth inhibition by S100C/A11 is p21CIP1/WAF1
The next question is how nuclear S100C/A11 exerts its inhibitory action on the growth of keratinocytes. We screened a number of possible candidates and found that p21CIP1/WAF1 mRNA was remarkably induced in NHK cells either cultivated at 1.5 mM Ca2+ or exposed to TAT-S100C/A11 ( A). p16INK4a was also induced, but to a moderate extent. Similar induction was observed in HaCaT cells in a time-dependent manner ( B).
Figure 6. Induction of Cdk inhibitors and concomitant inhibition of Cdk2 by S100C/A11. (A) Induction of p21CIP1/WAF1 and p16INK4a by TAT-S100C/A11 or high Ca2+ applied to NHK cells for 6 h as assayed by Northern blot analysis. (B) Time-dependent induction of p21 (more ...)
To confirm the role of S100C/A11 on the induction of p21CIP1/WAF1
, anti-S100C/A11 antibody was introduced into HaCaT cells by the aid of Chariot or polyethyleneimine (PEI). The polyclonal antibody that we used was specific as shown in C, and was demonstrated to recognize the NH2
-terminal pepA and COOH-terminal pepF of S100C/A11 (unpublished data). Binding of the introduced antibody to endogenous S100C/A11 was demonstrated by immunoprecipitation ( D). Introduction of the antibody inhibited the high Ca2+
-induced phosphorylation of S100C/A11 ( E). Fab fragments of the anti-S100C/A11 antibody also reduced the phosphorylation of S100C/A11 in HaCaT cells (Fig. S8, available at http://www.jcb.org/cgi/content/full/jcb.200304017/DC1
). The induction of p21CIP1/WAF1
by TAT-S100C/A11 was dose-dependently abrogated by the antibody introduced into the cells ( F). The decrease in the amount of p21CIP1/WAF1
protein due to the action of anti-S100C/A11 antibody was associated with the recovery of cdk2 activity as assayed in vitro ( F). Inhibition of cdk2 activity by S100C/A11 introduced into HaCaT cells was dose- and time-dependent. Finally, anti-p21CIP1/WAF1
antibody (but not anti-p16INK4a
antibody) completely abrogated the growth inhibition by S100C/A11 as assayed for [3
H]TdR incorporation or for cdk2 activity (), indicating that p21CIP1/WAF1
is the principal effector protein of the S100C/A11-mediated growth inhibition of human keratinocytes. Specificity of anti-p21CIP1/WAF1
antibodies was confirmed as shown in C.
In nuclei, S100C/A11 competitively binds to nucleolin and liberates Sp1 and Sp3, which eventually induce p21CIP1/WAF1
Next, we addressed the question of how S100C/A11 in the nuclei induces the effector p21CIP1/WAF1. Screening using Signal Transduction AntibodyArray™ (Hypromatrix) resulted in identification of Sp1 as a protein that binds to nucleolin, and the binding was inhibited by the addition of S100C/A11(TD) ( A). Anti-Sp2 antibody (but not anti-Sp3 antibody) was also on the membrane, but showed a negative result. Binding of Sp1 to nucleolin was confirmed by applying the extract of HaCaT cells onto GST-nucleolin beads ( B). Sp1 did not bind directly to S100C/A11.
Figure 7. Induction of p21CIP1/WAF1 via Sp1 by S100C/A11. (A) HaCaT cell extract was applied onto Signal Transduction AntibodyArray™, followed by incubation with either GST-nucleolin alone or GST-nucleolin mixed with S100C/A11(TD), and was visualized with (more ...)
To examine whether nucleolin binds to Sp1 in the cells, we performed a coprecipitation assay ( C). Sp1 and S100C/A11 were detected in the precipitates prepared using anti-nucleolin antibody, whereas nucleolin, but not S100C/A11, was detected in the precipitates prepared using anti-Sp1 antibody. These results indicate that binding among the proteins actually takes place in vivo, and that S100C/A11 does not bind to Sp1. In HaCaT cells cultivated in the presence of 1.5 mM Ca2+, nucleolin bound to Sp1, but not to S100C/A11. When the Ca2+ concentration was increased to 10 mM, S100C/A11 instead of Sp1 was detected in the immunoprecipitates prepared using anti-nucleolin antibody, and the bound S100C/A11 was phosphorylated. When the extract was treated with alkaline phosphatase in advance, the amount of S100C/A11 bound to nucleolin was dramatically reduced and the binding of Sp1 was recovered concomitantly. Immunoprecipitation with anti-Sp1 antibody confirmed that the stimulation with high Ca2+ liberated Sp1 from nucleolin.
Regions of nucleolin and Sp1 involved in the interaction were determined using recombinant partial proteins ( E). The COOH-terminal region of Sp1 (segment 3) including a zinc-finger domain was shown to be responsible for the binding to nucleolin ( D, top). In turn, Sp1 in the cell extract bound to segment 4 of nucleolin immobilized on a membrane ( D, middle). Recombinant Sp1 segment 3 also bound to the nucleolin segment 4, indicating direct interaction between the two proteins. S100C/A11 was recovered only from beads conjugated with segment 3 of nucleolin ( D, bottom). When segment 3 of nucleolin was introduced into HaCaT cells, the Ca2+-induced p21CIP1/WAF1 protein level was decreased and the activity of cdk2 was recovered within 8 h, despite continuous cultivation at 10 mM Ca2+ (unpublished data). This is possibly due to a dominant-negative effect of the peptide through sequestering S100C/A11 by binding. The interacting regions of the proteins are depicted in E.
Binding of nucleolin in cell extracts to Sp1 (segment 3) was dose-dependently inhibited by recombinant S100C/A11 ( F). S100C/A11(TD) showed a stronger effect, whereas S100C/A11(TA) did not affect the binding between nucleolin and Sp1 (segment 3).
Because Sp3 is also known to activate the p21CIP1/WAF1
promoter (Prowse et al., 1997
), we examined possible interaction with nucleolin and S100C/A11 and found that (1) Sp3 can bind to nucleolin segment 4 via its COOH-terminal zinc-finger domain; and (2) the binding of Sp3 to nucleolin was inhibited by S100C/A11 (unpublished data).
A gel shift assay was performed using GC-rich regions of the p21CIP1/WAF1
promoter, the well-known Sp1/Sp3-binding sites (see Materials and methods). As shown in G, exposure of NHK and HaCaT cells to high Ca2+
resulted in an increase in the intensity of the shifted bands by Sp1 and Sp3, which were further retarded by the addition of anti-Sp1 or -Sp3 antibody. Under similar experimental conditions in which HaCaT cells were cultivated in a high Ca2+
medium, addition of GST-nucleolin into the incubation mixtures inhibited the extent of the band shift as compared with GST ( G, right). This was possibly due to sequestration of Sp1 by exogenous nucleolin. The inhibition of the band shift by GST-nucleolin was abrogated by the 10
Thr phosphorylation equivalent form S100C/A11(TD), whereas wild-type S100C/A11 and S100A2 showed no effect. We found that the interaction of the proteins were dose-dependent, and addition of an excess amount of wild-type S100C/A11 showed recovering effects similar to S100C/A11(TD) (unpublished data). These results indicate the competitive nature of the binding between S100C/A11 and Sp1/Sp3 to nucleolin. We performed a series of luciferase assays using a plasmid containing the p21CIP1/WAF1
promoter region (Nakano et al., 1997
) and obtained essentially the same results as those obtained by using the gel shift assay (unpublished data). Deletion of the Sp1/Sp3-binding sites in the promoter abrogated the induction of luciferase activity by S100C/A11.
Biological relevance of the S100C/A11 pathway as a mediator of the growth inhibition of keratinocytes
The findings described in the previous paragraphs indicate that S100C/A11 is involved in growth inhibition of human keratinocytes induced by high Ca2+ ( A). Under low Ca2+ conditions, S100C/A11 is present mostly in the cytoplasm. The unphosphorylated S100C/A11 partly binds to actin fiber via its COOH-terminal region (unpublished data). An increase in intracellular Ca2+ results in activation of an as-yet-unidentified protein kinase(s) that phosphorylates S100C/A11 at 10Thr and 94Ser. Phosphorylated S100C/A11 binds to nucleolin via its NH2-terminal region and is translocated to nuclei. In the nuclei, S100C/A11 competes with Sp1 and Sp3 for binding to nucleolin, and the resulting free Sp1 and Sp3 induce p21CIP1/WAF1.
Figure 8. An S100C/A11-mediated pathway for inhibition of cell growth and implication of its biological significance. (A) A schematic diagram of the S100C/A11-mediated pathway revealed in the present paper. (B) Significance of the S100C/A11-mediated pathway in (more ...)
A question that arises is to what extent the pathway is biologically relevant. To address this question, anti-S100C/A11 antibody was introduced into NHK and HaCaT cells by PEI and Chariot, respectively. The introduction efficiency was confirmed to be >70% by immunostaining. The introduced antibody inhibited the high Ca2+-induced phosphorylation of S100C/A11 ( E). High Ca2+ suppressed DNA synthesis to ~17% of the control level, and the antibody recovered the DNA synthesis to 64 and 72% of the control level in NHK and HaCaT cells, respectively, indicating that S100C/A11 is a principal (if not sole) mediator of growth regulation ( B). The incomplete recovery may be either due to incomplete blockage of S100C/A11 function by the introduced antibody or due to the presence of some additional pathway mediating high Ca2+-induced growth inhibition.
S100C/A11 was found to be expressed at various levels in many human tissues ( C), among which skin showed a particularly high level of S100C/A11 expression. When normal human skin tissues were stained with anti-S100C/A11 antibody, S100C/A11 protein was detected in epidermal keratinocytes ( D). Nuclei of cells in the basal layer were consistently negative for S100C/A11 protein, whereas nuclei of cells in the suprabasal layers were positive. p21CIP1/WAF1 protein was not detected in the basal layer, but appeared in suprabasal cell nuclei in which S100C/A11 was present. Among 21 nuclei counted in the basal layer, 20 and 1 nuclei were negative and positive for both S100C/A11 and p21CIP1/WAF1, respectively. In the suprabasal layer, 89 nuclei were positive for S100C/A11 and p21CIP1/WAF1, whereas 11 nuclei were positive for S100C/A11, but negative for p21CIP1/WAF1. No S100C/A11-negative nuclei expressed p21CIP1/WAF1. These results indicate that S100C/A11 not only mediates high Ca2+-induced growth inhibition of human keratinocytes in culture, but most likely plays a key role in the growth regulation in vivo as well.