Bifurcated converging pathways for high Ca2+- and TGFβ-induced inhibition of growth of normal human keratinocytes

  1. Masakiyo Sakaguchi*,
  2. Hiroyuki Sonegawa*,
  3. Takamasa Nukui*,
  4. Yoshihiko Sakaguchi†,
  5. Masahiro Miyazaki*,
  6. Masayoshi Namba‡, and
  7. Nam-ho Huh*,§
  1. *Departments of Cell Biology and †Bacteriology, Okayama University Graduate School of Medicine and Dentistry, Shikata-chou, Okayama 700-8558, Japan; and ‡Niimi College, Niimi 718-8585, Japan

  1. Edited by Gerald R. Crabtree, Stanford University School of Medicine, Stanford, CA, and approved August 5, 2005 (received for review January 27, 2005)

 
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Abstract

Growth suppression of normal human keratinocytes by high Ca2+ or TGFβ was shown to be mediated by p21WAF1/CIP1 and Sp1 [Pardali, K., et al. (2000) J. Biol. Chem. 275, 29244–29256; Santini, M. P., Talora, C., Seki, T., Bolgan, L. & Dotto, G. P. (2001) Proc. Nat. Acad. Sci. USA 98, 9575–9580; Al-Daraji, W. I., Grant, K. R., Ryan, K., Saxton, A., & Reynolds, N. J. (2002) J. Invest. Dermatol. 118, 779–788]. We previously demonstrated that S100C/A11 is a key mediator for growth inhibition of normal human epidermal keratinocytes (NHK) triggered by high Ca2+ or TGFβ [Sakaguchi, M., et al. (2003) J. Cell Biol. 163, 825–835; Sakaguchi, M., et al. (2004) 164, 979–984]. On exposure of NHK cells to either agent, S100C/A11 is transferred to nuclei, where it induces p21WAF1/CIP1 through activation of Sp1/Sp3. In the present study, we found that high Ca2+ activated NFAT1 through calcineurin-dependent dephosphorylation. In growing NHK cells, Krueppel-like factor (KLF)16, a member of the Sp/KLF family, bound to the p21WAF1/CIP1 promoter and, thereby, inhibited the transcription of p21WAF1/CIP1. Sp1 complexed with NFAT1 in high Ca2+-treated cells or with Smad3 in TGFβ1-treated cells, but not Sp1 alone, replaced KLF16 from the p21WAF1/CIP1 promoter and transcriptionally activated the p21WAF1/CIP1 gene. Thus, high Ca2+ and TGFβ1 have a common S100C/A11-mediated pathway in addition to a unique pathway (NFAT1-mediated pathway for high Ca2+ and Smad-mediated pathway for TGFβ1) for exhibiting a growth inhibitory effect on NHK cells, and both pathways were shown to be indispensable for growth inhibition.

Normal human epidermal keratinocytes (NHK) provide a rare opportunity for studying mechanisms of growth regulation of normal human cells of epithelial origin. Contrary to many other normal human epithelial cells, NHK cells can be cultivated to grow stably and reproducibly (1, 2). In addition, the cells can be readily induced to stop growing and follow a differentiation pathway under defined conditions, including exposure to high Ca2+ (2, 3) or to TGFβ (4, 5). In the body, proliferating cells are present only in the basal layer of the epidermis. Mechanisms of the cessation of growth of epidermal keratinocytes during transition from the basal layer to the upper layer have not been elucidated, but evidence indicates that Ca2+ and TGFβ play some roles in the cessation of growth (6, 7).

For transduction of signals triggered by TGFβ, the activated TGFβ receptor phosphorylates Smad2 and Smad3, which, in turn, transcriptionally activates or suppresses target genes by binding to a Smad binding region (SBR) located in an upstream region of target genes (8–10). The mode of regulation, however, seems to be more complicated. Transcriptional regulation by the Smad proteins is affected by many different transacting factors and cofactors in cell type- and target gene-dependent manners (11). In the promoter of p21WAF1/CIP1, a principal effector of TGFβ-induced growth suppression, at least three critical regions have been identified, i.e., the distal SBR, the proximal region for Myc-Miz binding, and the intermediate Sp1 sites (12–15). Down-regulation of Myc is a prerequisite for the induction of p21WAF1/CIP1 by TGFβ (13, 14). Seoane et al. (16) showed that the FoxO subfamily of forkhead factors functions as specific Smad partners for binding to SBR.

In our previous study (17, 18), we found that S100C/A11, a member of the EF-hand Ca2+-binding protein family (19), mediates growth inhibition of NHK cells triggered by high Ca2+ or TGFβ1 in culture. On exposure of NHK cells to high Ca2+ or TGFβ1, S100C/A11 is phosphorylated at 10Thr by PKCα and transferred to nuclei, where it liberates Sp1/Sp3 from nucleolin, which, in turn, transcriptionally activates p21WAF1/CIP1. Activation of S100C/A11 and, therefore, Sp1/Sp3, is indispensable for the growth inhibition of NHK cells induced both by high Ca2+ and by TGFβ1.

Sp1/Sp3 has long been thought to be a ubiquitous transacting factor. However, accumulating evidence, including evidence obtained in our previous study (17, 18), indicates that Sp1 site-dependent transcription is highly regulated by many different factors in a context-dependent manner (ref. 20 and references therein). The amount of Sp1 protein in NHK cells remained unchanged before and after treatment with TGFβ1, but Sp1 could bind to the p21WAF1/CIP1 promoter only when the cells were treated with TGFβ1 (18). Sp1 and Sp3 belong to the Sp/Krueppel-like factor (KLF) family, which comprises at least 20 members (20). The members bind to transcriptional elements designated as Sp1 sites with varying affinities and modulate each other's activity in synergistic or antagonistic manners. Clearly, further studies in defined cell systems are needed to understand the principally important transcriptional regulation on “Sp1 sites.”

The aim of the present study was to determine in a native context why activation of both Smad- and S100C/A11-mediated pathways is necessary for the TGFβ1-triggered growth inhibition of NHK cells and whether any pathways other than the S100C/A11-mediated pathway are present and necessary for high Ca2+-induced growth inhibition. Santini et al. (21) showed that calcineurin and NFAT are involved in transcriptional regulation of p21WAF1/CIP1 in collaboration with Sp1/3 in mouse keratinocytes. Involvement of NFAT in growth regulation was implicated also in human epidermis (22). NFAT is known to be activated through dephosphorylation by calcineurin, and the reaction is blocked by cyclosporin A (23–25). The following results were obtained: (i) High Ca2+ induced calcineurin-dependent dephosphorylation of NFAT1 in NHK cells, (ii) not only the S100C/A11-mediated pathway but also the NFAT1-mediated pathway is indispensable for inhibition of growth of NHK cells by high Ca2+, (iii) in growing NHK cells, KLF16 binds to the p21WAF1/CIP1 promoter and thereby inhibits the transcription of p21WAF1/CIP1, and (iv) Sp1 complexed with NFAT1 or Smad3, but not Sp1 alone, replaces KLF16 from the p21WAF1/CIP1 promoter and transcriptionally activates the p21WAF1/CIP1 gene. Collectively, high Ca2+ and TGFβ1 have a common S100C/A11-mediated pathway in addition to unique pathways (NFAT1-mediated pathway for high Ca2+ and Smad-mediated pathway for TGFβ1) for exhibiting a growth inhibitory effect on NHK cells, and both pathways are indispensable for growth inhibition.

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Materials and Methods

Cells and Chemicals. Neonatal human epidermal keratinocytes (Cascade Biologics, Portland, OR) cultured in an animal-product free medium, EpiLife, with a growth supplement, V2, were used throughout the study. TGFβ1 and cyclosporin A were purchased from Sigma. A myristoylated PKC inhibitor (26) was obtained from Promega.

Gel Electrophoresis and Immunological Analyses. Gel electrophoresis, Western blot analysis, and immunoprecipitation were performed under conventional conditions. The antibodies used for Western blot analysis were rabbit anti-mouse NFAT1 antibody (Upstate Biotechnology, Charlottesville, VA), mouse anti-human NFAT2 antibody (Abcam, Inc., Cambridge, MA), rabbit anti-human NFAT3 antibody (Abcam, Inc.), mouse anti-mouse NFAT4 antibody (Santa Cruz Biotechnology), mouse anti-human nucleolin antibody (MBL, Nagoya, Japan), mouse anti-human p21WAF1/CIP1 antibody (BD Biosciences), mouse anti-human p15INK4B antibody (Lab Vision, Fremont, CA), rabbit anti-human Smad3 antibody (Santa Cruz Biotechnology), rabbit anti-human Sp1 antibody (Santa Cruz Biotechnology), and mouse anti-human tubulin antibody (Sigma). Polyclonal rabbit anti-human S100C antibody was prepared in our laboratory (27). For KLF16, rabbit anti-human KLF16 antibody raised by us and goat anti-human KLF16 antibody obtained from Abcam, Inc., were used and gave essentially the same results. For immunoprecipitation, the same antibodies as those used for Western blot analysis were used except for rabbit anti-human Sp1 antibody (Upstate Biotechnology).

Adenovirus Constructs and Short Interfering RNA (siRNA). Adenovirus constructs were prepared by using BD Adeno-X Expression System 2 (BD Biosciences) under the conditions recommended by the manufacturer. siRNAs against Smad3 (5′-aacgtcaacaccaagtgcatc-3′), NFAT1 (5′-aataatgtcacctcgaaccag-3′), Sp1 (5′-aacttgcagcagaattgagtc-3′), and KLF16 (5′-aagtcctcgcacctaaagtcg-3′) were produced by in vitro transcription with a Silencer siRNA construction kit (Ambion, Austin, Texas). The siRNAs were transfected to logarithmically growing NHK cells by using Lipofectamine 2000 (Invitrogen). siRNA for human PKCα was purchased from Santa Cruz Biotechnology.

Introduction of Proteins into Cells. Cationic polyethyleneimine-conjugated S100C antibody (28) was used to introduce antibodies into NHK cells.

Chromatin Immunoprecipitation. Chromatin immunoprecipitation was performed according to the method of Takahashi et al. (29). Briefly, NHK cells were treated with 1% formaldehyde for cross-linking. Cell extracts were immunoprecipitated by using antibodies described above. The primers used were those corresponding to positions –328 to –309 and –56 to –37 of the p21WAF1/CIP1 promoter.

Electrophoretic Mobility Shift Assay. An electrophoretic mobility shift assay was performed as described by Nakano et al. (30). We used a mixture of GC-rich elements located between –122 and –96 bp (A), –95 and –74 bp (B), and –73 and –47 bp (C) of the human p21WAF1/CIP1 promoter as probes (see Fig. 6, which is published as supporting information on the PNAS web site). Recombinant proteins were prepared as GST-fusion proteins by using pGEX-2T (Amersham Pharmacia Biosciences).

Luciferase Assay. A plasmid, pWWP, containing the human p21WAF1/CIP1 promoter and luciferase cDNA (30) was obtained from T. Sakai (Kyoto Prefectural University, Kyoto), and a variant lacking Sp1 sites was made by us. Derivatives of pDNR-CMV (BD Biosciences) expressing individual Sp/KLF family members were cotransfected into NHK cells by using Trans IT-Keratinocyte Transfection Reagent (Mirus, Madison, WI). Any difference in transfection efficiency was compensated by using pTracer-EF/lacZ (Invitrogen).

Acquisition and Processing of Images. To acquire images of cells labeled with tetramethylrhodamine isothiocyanate, a laser-scanning microscope (type, Axioplan 2; objective lens, Plan-Neofluar×40/0.75; Zeiss) was used.

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Results

NFAT1 Mediates High Ca2+-Induced Growth Inhibition of NHK Cells. At first, we examined whether NFAT1 is involved in high Ca2+-induced inhibition of growth of NHK cells. Nuclear translocation of NFAT1 is a prerequisite for exerting its transcriptional regulator function (31). When NHK cells usually cultured at a Ca2+ concentration of 0.03 mM were exposed to 1.5 mM Ca2+, NFAT1 was translocated into nuclei (Fig. 1a). In accordance with the results of our previous studies (17, 18), Smad3 and S100C/A11 moved to nuclei in NHK cells exposed to TGFβ1 and to either agent, respectively. NFAT1 that was phosphorylated in the growing NHK cells was readily dephosphorylated when the cells were exposed to high Ca2+ but not when the cells were exposed to TGFβ1 (Fig. 1b). Under a condition of high Ca2+, NFAT1 was coprecipitated with Sp1, which bound to nucleolin in untreated NHK cells and to Smad3 in TGFβ1-treated cells (Fig. 1c). Among NFAT family members, only NFAT1, but not NFAT2, NFAT3, and NFAT4, was coprecipitated with Sp1 on exposure of NHK cells to high Ca2+ (Fig. 1d). p21WAF1/CIP1 and p15INK4B were induced by high Ca2+ and TGFβ1 (Fig. 1e Upper) in accordance with published studies, including that by Feng et al. (32). Chromatin immunoprecipitation assays revealed that not only Sp1 but also NFAT1 bound to the p21WAF1/CIP1 promoter when NHK cells were exposed to high Ca2+ (Fig. 1e Lower). Cyclosporin A is an inhibitor of calcineurin, which activates NFAT1 through dephosphorylation (31). When NHK cells were pretreated with increasing concentrations of cyclosporin A, high Ca2+-induced induction of p21WAF1/CIP1 was abrogated, and phosphorylation of NFAT1 was restored in a dose-dependent manner (Fig. 1f). Finally, we confirmed the involvement of PKCα in the induction of p21WAF1/CIP1 and p15INK4B by high Ca2+ and TGFβ1 by using siRNA (Fig. 1g; see also Fig. 7, which is published as supporting information on the PNAS web site), a more specific method than that used in the previous study (18). These results indicate that on exposure of NHK cells to high Ca2+, calcineurin activates NFAT1 through dephosphorylation, which, in turn, transcriptionally activates the p21WAF1/CIP1 gene in nuclei in collaboration with Sp1.

Fig. 1.
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Fig. 1.

Involvement of NFAT1 in high Ca2+-induced growth inhibition of NHK cells. (a) Nuclear translocation of NFAT1 and Smad3 in NHK cells treated for 6 h with high Ca2+ and TGFβ1, respectively (Scale bars: 20 μm.). (b) Dephosphorylation of NFAT1 in NHK cells treated with high Ca2+ for 6 h. (c) Coprecipitation of various proteins with Sp1. NFAT1 was coprecipitated with Sp1 in NHK cells treated with high Ca2+ for 6 h. Nucl, nucleolin. (d) Binding of NFAT family members to Sp1 on exposure of NHK cells to high Ca2+. (e) Chromatin immunoprecipitation assay for proteins binding to the p21WAF1/CIP1 promoter. p21WAF1/CIP1 and p15INK4B was induced by high Ca2+ and TGFβ1 (Upper). Under the conditions, NFAT1 and Smad3 bound to the p21WAF1/CIP1 promoter in NHK cells treated with high Ca2+ and TGFβ1, respectively (Lower). (f) Cyclosporin A (CsA) added 1 h before abrogated high Ca2+-induced induction of p21WAF1/CIP1 (Upper) and dephosphorylation of NFAT1 (Lower) after 6 h of incubation. (g) Abrogation of induction of p21WAF1/CIP1 and p15INK4B by depleting PKCα with siRNA, which was applied by lipofection 48 h before the exposure of cells to high Ca2+ or TGFβ1.


Both the Common and Unique Pathways Are Indispensable for the Induction of p21WAF1/CIP1 by High Ca2+ and TGFβ1 in NHK Cells. The above observations, taken together with our previous results (17, 18), indicate that high Ca2+ and TGFβ1 have at least one common pathway and that each has one unique pathway for transducing growth inhibitory signals in NHK cells, i.e., the S100C/A11-mediated and NFAT1-mediated pathways for high Ca2+ and the S100C/A11-mediated and Smad3-mediated pathways for TGFβ1. We next examined the dispensability of each pathway. When the common S100C/A11 pathway was blocked by treatment of cells with a cPKC inhibitor, by introduction of an anti-S100C/A11 antibody into cells or by down-regulation of Sp1 by siRNA, induction of p21WAF1/CIP1 and p15INK4B was abrogated in both high Ca2+- and TGFβ1-treated NHK cells (Fig. 2 a and b). Down-regulation of NFAT1 and Smad3 by siRNA (Fig. 8, which is published as supporting information on the PNAS web site) specifically abrogated the induction of p21WAF1/CIP1 by high Ca2+ and TGFβ1, respectively. Phosphorylation states of S100C/A11, Smad3, and NFAT1 were confirmed as expected (Fig. 9, which is published as supporting information on the PNAS web site). Under these conditions, we performed chromatin immunoprecipitation assays to identify proteins that bind to the p21WAF1/CIP1 promoter. In NHK cells treated with high Ca2+, the amount of Sp1 bound to the p21WAF1/CIP1 promoter decreased in parallel with that of NFAT1 when either the S100C/A11-mediated or NFAT1-mediated pathway was blocked (Fig. 2c). Similarly, the amount of Sp1 bound to the p21WAF1/CIP1 promoter decreased in parallel with that of Smad3 when either the S100C/A11-mediated or Smad3-mediated pathway was blocked (Fig. 2d). Unexpectedly, scrambled siRNA provided by the manufacturer abrogated p21WAF1/CIP1 induction by high Ca2+ (Fig. 2). However, this cancellation was associated with down-regulation of NFAT1 for an unknown reason (Fig. 8). These results clearly show that both the common and unique pathways are indispensable for the induction of p21WAF1/CIP1 by high Ca2+ and TGFβ1 in NHK cells.

Fig. 2.
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Fig. 2.

Essential mediator proteins for high Ca2+- and TGFβ1-induced growth inhibition of NHK cells. (a) NHK cells were subjected to pretreatment with inhibitors (1 h in advance), introduction of anti-S100C/A11 antibody (1 h in advance), or application of siRNAs (48 h in advance) to functionally or physically deplete respective proteins, and then induction of p21WAF1/CIP1 and p15INK4B by high Ca2+ was examined after 6 h of incubation with the inducer. (b) Induction of p21WAF1/CIP1 and p15INK4B by TGFβ1 was assayed under the same conditions as those described in a. (c) Chromatin immunoprecipitation assay for proteins binding to p21WAF1/CIP1 promoter was performed by using NHK cells under the same conditions as those described in a.(d) Chromatin immunoprecipitation assay by using NHK cells under the same conditions as those described in b and c. cPKCI, cPKC inhibitor; CsA, cyclosporin A; Scrm, scrambled control siRNA; –, untreated.


KLF16 Binds to the p21WAF1/CIP1 Promoter and Is Replaced by Sp1/NFAT1 and Sp1/Smad3 on Growth Inhibition of NHK Cells. When the unique pathways, i.e., the NFAT1-mediated and Smad-mediated pathways for high Ca2+ and TGFβ1, respectively, were specifically blocked, Sp1 liberated from nucleolin through the common S100C/A11-mediated pathway (Fig. 10, which is published as supporting information on the PNAS web site) could not efficiently bind to the p21WAF1/CIP1 promoter (Fig. 2 c and d). Uncovering the mechanisms of these events may provide an insight into the reason why the two pathways are indispensable for the inhibition of growth of NHK cells.

We screened members of the Sp/KLF family for possible involvement in the transcriptional regulation of p21WAF1/CIP1 because they are known to interact either synergistically or antagonistically in very complicated manners (20). A labeled p21WAF1/CIP1 promoter probe containing GC elements specifically bound to recombinant KLF4, 5, 7, 10, and 16 (full-length proteins fused to GST) immobilized onto a membrane (Fig. 3a). When these KLFs were incubated with a recombinant Sp1 protein (the DNA-binding domain of Sp1 fused to GST, produced in Escherichia coli) and the p21WAF1/CIP1 promoter probe in vitro, only KLF16 showed a higher level of affinity to the promoter than that of the Sp1 protein (Fig. 3b). To study the binding of Sp1 and KLF16 to the p21WAF1/CIP1 promoter under more biologically relevant conditions, we prepared Sp1 by immunoprecipitation with antibodies against partner proteins of Sp1, i.e., anti-nucleolin, anti-Smad3, and anti-NFAT1 antibodies for untreated, TGFβ1-treated, and high Ca2+-treated NHK cells (Fig. 3c). Recombinant full-length Sp1 produced by a baculovirus system was of a commercial source (see Materials and Methods). The slightly lower apparent molecular size of the recombinant Sp1 may reflect incomplete modification of Sp1, which is known to be potentially glycosilated and/or phosphorylated (33, 34). When the Sp1 preparations were added to immobilized p21WAF1/CIP1 promoter DNA preincubated with the recombinant KLF16 in vitro, the Sp1 complexed either with Smad3 or NFAT1 bound to the promoter replacing KLF16, whereas nucleolin-bound Sp1 or the recombinant free Sp1 remained in the supernatant (Fig. 3d). KLF16 can specifically bind to each Sp1 site in the p21WAF1/CIP1 promoter (Fig. 11, which is published as supporting information on the PNAS web site). In a luciferase assay, only KLF16 among the Sp/KLF members examined interfered with the activation of the p21WAF1/CIP1 promoter by TGFβ1 in NHK cells (data not shown). KLF16 does not directly associate with Sp1 even in high Ca2+- or TGFβ1-treated cells (Fig. 1c).

Fig. 3.
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Fig. 3.

Competitive binding of KLF16 and Sp1 complexes to the p21WAF1/CIP1 promoter in vitro. (a) Binding of the p21WAF1/CIP1 promoter GC elements (a mixture of A, B, and C shown in Fig. 11) to recombinant KLF family proteins immobilized on a membrane. (b) Replacement of Sp1 from the p21WAF1/CIP1 promoter GC element by KLF16 but not by KLF4, 5, 7 or 10. Filled and open triangles indicate the positions of the probe bound with KLF family proteins and Sp1, respectively. The promoter probe was the same as that used in the experiment for which results are shown in a, and the proteins were produced in E. coli.(c) Preparation of Sp1 protein fractions. Recombinant Sp1 (Rec Sp1) produced in a baculovirus system was obtained from Alexis (Lausen, Switzerland). Cellular Sp1 was immunoprecipitated by using anti-nucleolin (Nucl), anti-Smad3 (Smad), and anti-NFAT (NFAT1) antibodies from NHK cells not treated or treated with TGFβ1(1ng/ml) and high Ca2+ (1.5 mM), respectively. The protein preparations were analyzed by Western blotting. (d) Replacement of KLF16 from the p21WAF1/CIP1 promoter by cellular Sp1 complexes but not by Sp1 of baculovirus origin. A region covering –122 to –47 of the p21WAF1/CIP1 promoter (Fig. 6) preincubated with KLF16 was exposed to various Sp1 preparations (Rec, produced by a Baculovirus system; other Sp1 fractions, prepared by immunoprecipitation with designated antibodies from NHK cells exposed to indicated agents), and the supernatant and bound fractions were analyzed by Western blotting. The DNA fragment was covalently bound to the well surface by using a U-COAT DNA coating kit (TrimGen, Sparks, MD). Nucl, nucleolin.


In untreated NHK cells, KLF16 bound to the p21WAF1/CIP1 promoter and was liberated from the promoter on exposure of NHK cells to high Ca2+ or TGFβ1 (Fig. 1d). Overexpression of KLF16 in NHK cells by an adenovirus vector resulted in abrogation of the induction of p21WAF1/CIP1 by TGFβ1 or high Ca2+ (Fig. 4a) without any effect on the phosphorylation state of Smad3 or NFAT1 (Fig. 4b). The overexpressed KLF16 bound to the p21WAF1/CIP1 promoter at increasing levels in untreated NHK cells (Fig. 4c). Even in the high Ca2+- and TGFβ1-treated cells, the overexpressed KLF16 dose-dependently bound to the promoter by replacing Sp1/NFAT1 and Sp1/Smad3, respectively. When the activation of NFAT1 was inhibited by cyclosporin A in high Ca2+-treated NHK cells, binding of Sp1 and NFAT1 to the p21WAF1/CIP1 promoter was blocked depending on the dose of cyclosporin A, and the binding of KLF16 was recovered (Fig. 4d). These results indicate that in growing NHK cells, p21WAF1/CIP1 is switched off by the negative transcriptional regulator KLF16 and that the KLF16 is replaced with activated Sp1/NFAT1 and Sp1/Smad3 complexes upon exposure to high Ca2+ and TGFβ1, respectively.

Fig. 4.
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Fig. 4.

Involvement of Smad3 in TGFβ1-induced growth inhibition. (a) Overexpression of KLF16 by an adenovirus vector (10 moi; infected 48 h in advance) abrogated induction of p21WAF1/CIP1 in NHK cells treated with high Ca2+ or TGFβ1 for 6 h. –, uninfected; lacZ, adenovirus carrying the lacZ gene. (b) Overexpression of KLF16 did not affect the phosphorylation states of Smad3 and NFAT1 under the same conditions as those described in a.(c) Chromatin immunoprecipitation assay for proteins binding to the p21WAF1/CIP1 promoter. Overexpression of KLF16 inhibited the binding of Sp1/Smad3 complex and Sp1/NFAT1 complex to the p21WAF1/CIP1 promoter in NHK cells treated with TGFβ1 and high Ca2+, respectively. (d) Cyclosporin A (CsA) abrogated the binding of Sp1 and NFAT1 to the p21WAF1/CIP1 promoter in high Ca2+-treated NHK cells, restoring the binding of KLF16, as assayed by a chromatin immunoprecipitation assay.


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Discussion

During the last two decades, much progress has been made in the understanding of intracellular signal transduction pathways through identification of involved molecules and elucidation of mechanisms of their interaction/regulation. The mode of signal transduction, however, appears far more complicated than initially expected. Many molecules still remain to be identified, and cross-talks among different pathways have been only partially elucidated. Activation of a given molecule has often different biological roles in different biological contexts. Because the “biological context” is an integrated form of temporal functional states of individual molecules that can be only partially defined, it is important to study intracellular signaling events in well defined native biological systems. We used NHK cells in early cultures throughout the present study as a representative cell system of human cells of epithelial origin. As shown in Fig. 5, high Ca2+ and TGFβ1 need a common S100C/A11-mediated pathway in addition to individual unique pathways for exhibiting inhibitory effects on the growth of NHK cells. These bifurcated converging signal pathways may provide a better opportunity to cross-talk with other pathways, enabling more sophisticated regulations and finer tunings.

Fig. 5.
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Fig. 5.

Bifurcated converging pathways for high Ca2+- and TGFβ1-induced inhibition of growth of NHK cells. Black arrows, known from the results of studies by others; red arrows, revealed by our previous studies (17, 18) and present study.


Involvement of calcineurin and NFAT in the induction of p21WAF1/CIP1 was first shown by Santini et al. (21) in mouse keratinocytes. A promoter construct covering the Sp1 binding sites of the p21WAF1/CIP1 promoter was activated by high Ca2+ in a calcineurin-dependent manner and NFAT1/2 was coprecipitated with Sp1. Gafter-Gvili et al. (35) showed that cyclosporin A enhanced hair growth in the mouse in vivo and inhibited p21WAF1/CIP1 induction in cultured follicular keratinocytes. In the present study, we showed that upon exposure of NHK cells to high Ca2+, NFAT1 was dephosphorylated, translocated into nuclei, and bound to the Sp1-binding site of the genomic p21WAF1/CIP1 promoter in association with Sp1, replacing KLF16 from it. These results clearly demonstrate the involvement of calcineurin-NFAT1 in transcriptional induction of p21WAF1/CIP1 in NHK cells exposed to high Ca2+ in a native setting.

Thus far, three critical elements have been identified for transcriptional regulation of the p21WAF1/CIP1 gene by TGFβ, i.e., the distal SBR, the proximal Myc-Miz-binding region, and the Sp1 sites in between (12–15). FoxO, a subfamily of Folkhead transcriptional factors, recruits Smad3 onto SBR of the p21WAF1/CIP1 promoter in cells exposed to TGFβ (16). Because the function of FoxO was shown to be regulated by the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway (36), the SBR functions as a scaffold for converging the Smads and PI3-K/Akt pathways. Activation of the p21WAF1/CIP1 promoter through SBR by interaction of Smad and FoxO proteins needs relief of Myc-Miz-induced transcriptional repression (16). The relief may be accomplished by down-regulation of Myc transcription mediated by Smads (37, 38).

In NHK cells exposed to TGFβ or high Ca2+, S100C/A11 phosphorylated by PKCα is transferred to nuclei and liberates Sp1 (17, 18). Without the partner molecules activated by the unique pathways, however, Sp1 cannot bind to and transcriptionally activate the p21WAF1/CIP1 promoter (Fig. 2 c and d). In growing NHK cells, GC-rich elements of the p21WAF1/CIP1 promoter are not opened for free binding of irrelevant proteins but are tightly covered by KLF16. Bifurcation and conversion of the high Ca2+- and TGFβ1-triggered growth inhibitory signals demonstrated in the present study confer on the resulting Sp1/NFAT1 and Sp1/Smad3 complexes an affinity to the promoter strong enough to replace KLF16. As a similar line of work, KLF4/GKLF was shown to inhibit Sp1-mediated activation of the CYP1A1 promoter and the cyclin D1 promoter through competitive binding (39, 40). Pardali et al. (41) showed that Smad proteins functionally cooperate with Sp1 in an Sp1 site-dependent manner in HepG2 cells exposed to TGFβ. The activated Smad proteins increased affinity of Sp1 to the proximal p21WAF1/CIP1 promoter without binding of Smad proteins to the DNA region. These results are consistent with the results of the present study by using NHK cells. Physical interaction of endogenous Sp1 with Smad3 was demonstrated in cells exposed to TGFβ1 (Fig. 1c). The question of whether binding of Smad3 to SBR of the p21WAF1/CIP1 promoter is needed for interaction with Sp1 on the Sp1 sites in a native setting remains to be answered.

Events that take place in nuclei of high Ca2+- and TGFβ1-treated NHK cells seem even more complicated. We found that p300 bound to the p21WAF1/CIP1 promoter in NHK cells exposed to both growth inhibitory agents. Similarly, c-Jun bound to the p21WAF1/CIP1 promoter in cells exposed to high Ca2+ (data not shown). Xiao et al. (42) reported that p300 collaborates with Sp1 in activation of the p21WAF1/CIP1 promoter. c-Jun was also shown to transactivate the p21WAF1/CIP1 promoter by enhancing the function of Sp1 (43). Although further study is needed to obtain a better understanding of the growth control mechanisms in NHK cells, our observations provide some clues for artificial intervening in cell growth and for developing measures against human diseases due to altered cell growth/differentiation.

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Acknowledgments

We thank Dr. H. Yamada (Okayama University, Okayama, Japan) for conjugation of anti-S100C antibody with cationic polyethyleneimine. This work was supported by Ministry of Education, Culture, Sports, Science, and Technology of Japan Grants 14370260 and 17014065 (to N.H.) and grants by the Okayama Medical Foundation (to M.S.).

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Footnotes

  • ↵ § To whom correspondence should be addressed. E-mail: namu{at}md.okayama-u.ac.jp.

  • Author contributions: M.S., M.N., and N.-h.H. designed research; M.S., H.S., T.N., and Y.S. performed research; M.S. and Y.S. contributed new reagents/analytic tools; M.S., M.M., and N.-h.H. analyzed data; and M.S. and N.-h.H. wrote the paper.

  • This paper was submitted directly (Track II) to the PNAS office.

  • Abbreviations: KLF, Krueppel-like factor; NHK, normal human epidermal keratinocytes; SBR, Smad binding region; siRNA, short interfering RNA.

  • Freely available online through the PNAS open access option.

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  1. Published online before print September 19, 2005, doi: 10.1073/pnas.0500630102 PNAS September 27, 2005 vol. 102 no. 39 13921-13926
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