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

Abstract

Growth suppression of normal human keratinocytes by high Ca2+ or TGFbeta 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 TGFbeta [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 p21(WAF1/CIP1). Sp1 complexed with NFAT1 in high Ca2+-treated cells or with Smad3 in TGFbeta1-treated cells, but not Sp1 alone, replaced KLF16 from the p21WAF1/CIP1 promoter and transcriptionally activated the p21WAF1/CIP1 gene. Thus, high Ca2+ and TGFbeta1 have a common S100C/A11-mediated pathway in addition to a unique pathway (NFAT1-mediated pathway for high Ca2+ and Smad-mediated pathway for TGFbeta1) for exhibiting a growth inhibitory effect on NHK cells, and both pathways were shown to be indispensable for growth inhibition.

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 p21^WAF1/CIP1 promoter. p21^WAF1/CIP1 and p15^INK4B was induced by high Ca²⁺ and TGFβ1 (Upper). Under the conditions, NFAT1 and Smad3 bound to the p21^WAF1/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 p21^WAF1/CIP1 (Upper) and dephosphorylation of NFAT1 (Lower) after 6 h of incubation. (g) Abrogation of induction of p21^WAF1/CIP1 and p15^INK4B by depleting PKCα with siRNA, which was applied by lipofection 48 h before the exposure of cells to high Ca²⁺ or TGFβ1.

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 p21^WAF1/CIP1 and p15^INK4B by high Ca²⁺ was examined after 6 h of incubation with the inducer. (b) Induction of p21^WAF1/CIP1 and p15^INK4B by TGFβ1 was assayed under the same conditions as those described in a. (c) Chromatin immunoprecipitation assay for proteins binding to p21^WAF1/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.

Fig. 3.

Competitive binding of KLF16 and Sp1 complexes to the p21^WAF1/CIP1 promoter in vitro. (a) Binding of the p21^WAF1/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 p21^WAF1/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 p21^WAF1/CIP1 promoter by cellular Sp1 complexes but not by Sp1 of baculovirus origin. A region covering –122 to –47 of the p21^WAF1/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.

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 p21^WAF1/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 p21^WAF1/CIP1 promoter. Overexpression of KLF16 inhibited the binding of Sp1/Smad3 complex and Sp1/NFAT1 complex to the p21^WAF1/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 p21^WAF1/CIP1 promoter in high Ca²⁺-treated NHK cells, restoring the binding of KLF16, as assayed by a chromatin immunoprecipitation assay.

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.