Growth suppression of normal human keratinocytes by high Ca2+ or TGF beta was shown to be mediated by p2l(WAF1/C1P1) 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. Dermato/. 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 TGF beta [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/ All is transferred to nuclei, where it induces p21(WAF1/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 p21(WAF1/CIP1) promoter and, thereby, inhibited the transcription of p21(WAF1/CIP1). Sp1 complexed with NFAT1 in high Ca2+ -treated cells or with Smad3 in TGF beta 1-treated cells, but not Sp1 alone, replaced KLF16 from the p21(WAF1/CIP1) promoter and transcriptionally activated the p21(WAF1/CIP1) gene. Thus, high Ca2+ and TGF beta 1 have a common S1OOC/A11-mediated pathway in addition to a unique pathway (NFAT1-mediated pathway for high Ca2+ and Smad-mediated pathway for TGF beta 1) for exhibiting a growth inhibitory effect on NHK cells, and both pathways were shown to be indispensable for growth inhibition.