Our previous studies clearly demonstrated that S100A11 functions as an essential mediator for growth suppression of NHK triggered by representative growth inhibitors, high Ca++
and TGFβ, in cells (Sakaguchi et al., 2003
). Alternatively, the present study provides evidence that S100A11 enhances growth of NHK through induction of EGF and some other ligands to the EGF receptor when acting on the cells from the outside. Thus, S100A11 plays an ambivalent role with respect to growth regulation of NHK.
Involvement of S100A11 in both growth-suppressive and growth-stimulatory signaling in NHK seems to be paradoxical. We have been learning, however, more and more complex functions of a given protein in multiple biological contexts with recent progress in molecular and cellular biology, and S100A11 is not an exception. S100A11 is constitutively expressed in NHK. Under ordinary culture conditions for NHK, the medium contains EGF. EGF enhanced the production and secretion of S100A11 (A), which in turn induced EGF in an autocrine/paracrine manner (, B and C). Exposure of growing NHK to either Ca++
or TGFβ overrides growth of NHK supported by EGF, and S100A11 cell autonomously functions as an indispensable mediator of the growth suppression via phosphorylation and translocation into nuclei (Sakaguchi et al., 2003
). The mechanistic link between EGF and ambivalent S100A11 demonstrated in the present study may contribute to more exquisite growth regulation and fine tunings.
Secretion of S100A11 was first described in chondrocytes by Cecil et al. (2005)
. S100A11 was markedly overexpressed and secreted into the surrounding matrix by human chondrocytes in osteoarthritis. In the present study, we showed that S100A11 was also secreted from epithelial cells. S100A11 lacks a classical signal peptide sequence for secretion via the endoplasmic reticulum-Golgi pathway. Although several possible mechanisms have been proposed and partly verified for unconventional secretion routes (Nickel, 2005
), the mechanisms by which of S100A11 and other secretory S100 family members are secreted remain to be clarified. In this respect, it is noteworthy that S100A13 was shown to be cosecreted with fibroblast growth factor (FGF)1, both of which lack the conventional signal sequence (Landriscina et al., 2001
). An inhibitor of actin stress fiber formation suppressed secretion of FGF1 and hence of S100A13, which is known to bind to the actin fiber (Prudovsky et al., 2002
). S100A11 also has a capacity to bind to the actin fiber (Sakaguchi et al., 2000
). Phosphorylation of S100A11 does not seem to be involved in the secretion. High Ca++
induced phosphorylation of S100A11 (Sakaguchi et al., 2003
), but it did not affect its secretion (Supplemental Figure S1A), whereas EGF enhanced the secretion (A), but it showed no effect on phosphorylation of S100A11 (data not shown). S100A11 protein was identified in human serum by condensation using affinity chromatography and detection by Western blotting (data not shown), indicating that secretion of S100A11 takes place in vivo and is not merely an in vitro phenomenon.
RAGE is a multiligand receptor that belongs to the immunoglobulin superfamily. RAGE was first identified as a receptor for glycation end products of proteins (Schmidt et al., 1992
), but it was soon found to bind diverse ligands, including amyloid β (Yan et al., 1996
), HMGB1 (amphoterin) (Hori et al., 1995
), and some S100 family members (Donato, 2003
; Hofmann et al., 1999
). Cecil et al. (2005)
showed that exogenous S100A11 acted on articular chondrocytes to increase the production of type X collagen and CXCL8/IL8 in culture and that addition of an anti-RAGE antibody abrogated the action of S100A11. Abrogation of the effect of exogenous S100A11 by a neutralizing antibody was also observed in the present study (B and A). In addition, it was shown that RAGE was coprecipitated with S100A11 from extracts of NHK with a higher efficiency for dimerized S100A11, a functionally more active form than monomeric S100A11 (D). Although binding to the common receptor RAGE, different S100 family members seem to exert distinct biological effects. Heterodimeric S100A8 and S100A9, candidate ligands for RAGE (Ehlermann et al., 2006
), neither induced EGF nor activated Akt, but they induced cytokines such as IL-8, IL-1F9, and TNF-α in NHK (data not shown). Hsieh et al. (2004)
showed that intracellular translocation of S100A4, S100A12, S100A13, and S100B was brought about by application of the respective proteins onto endothelial cells. Although those S100 proteins seemed to share a common receptor, RAGE, intracellular translocation was observed in an individual protein-specific manner. The underlying mechanisms of the differential signal transduction by RAGE remain to be elucidated.
NF-κB is a well-known immediate mediator for RAGE-triggered signaling (Huttunen et al., 1999
). In accordance with this, phosphorylation and subsequent degradation of IκB were observed within 1 h in NHK exposed to S100A11 (B). Activation of Akt by phosphorylation was essential for the induction of EGF (D and A). The phosphorylation level of Akt was appreciably elevated at 1 h and further elevated at 3 h after exposure of NHK to S100A11 (B). The enhanced phosphorylation state of Akt was sustained up to 24 h. Transient and sustained modes of Akt activation have been shown to often lead to distinct outcomes in various biological contexts (Foulstone et al., 1999
). The products of phosphatidylinositol 3-kinase (PI3K) are absolutely necessary for activation of Akt (Alessi et al., 1996
), and PI3K is activated by RAGE via generation of reactive oxygen species (Alessi et al., 1996
; Xu and Kyriakis, 2003
To identify transcription factors involved in induction of the EGF gene, we developed a new method to fish proteins binding in vitro to a long region of the EGF promoter (Supplemental Figure S5). The method is facile and costless, and it can be applied for any promoters. Although the length of the target promoter region is restricted by size amplifiable by PCR, usually 2~3 kb because of the necessity of a substantial amount of DNA, longer regions (>10 kb) may be analyzed by mixing flanking segments amplified separately. It is also possible to screen unknown promoter-binding proteins by combination with mass spectrometry. The electrophoretic mobility shift assay, another analytical method for in vitro protein-promoter binding, is usually used for a specific short segment of the promoter. The chromatin immunoprecipitation assay is a strong tool for analyzing in vivo binding of a given protein to a defined region of the promoter. It is also possible to screen unknown binding regions in genomic DNA when combined with a microarray (chromatin immunoprecipitation on chip; Wu et al., 2006
S100A11 was produced and secreted at higher levels in squamous cell carcinoma cells than in NHK (A). As expected, higher mRNA levels of EGF, HB-EGF, and epiregulin were observed in BSCC-93, A431, and HSC-5 cells than in NHK (B; data not shown). If the S100A11-EGF family member axis constitutively functions in squamous carcinoma cells, sequestering of endogenous S100A11 would result in compromise of the pathway. When a neutralizing anti-S100A11 antibody was added to the culture medium, the constitutively higher phosphorylation state of Akt in BSCC-93 cells was almost completely suppressed (C), and mRNA levels of EGF, betacellulin, epiregulin and HB-EGF were markedly reduced (D). Addition of the anti-S100A11 antibody resulted in a decrease in the number of NHK and squamous carcinoma cells after 4 d of culture by 30~40% (E). The observed partial growth suppression was probably due to the fact that, among the EGF family members, TGFα and amphiregulin were hardly affected by S100A11 (D) and the fact that some growth-stimulating pathways other than the S100A11-EGF family member axis also play significant roles. The advantageous effect for cancer cells of overexpressed S100A11 and hence activation of RAGE, however, is not necessarily restricted to growth stimulation. S100A11 induced expression of Bcl-2, a representative antiapoptotic protein (B). RAGE-mediated signaling has been shown to confer cells resistance to induction of apoptosis in different biological systems (Huttunen et al., 2000
; Arumugam et al., 2004
). S100P, another ligand for RAGE, was shown to be overexpressed in pancreatic cancer (Crnogorac-Jurcevic et al., 2003
; Sato et al., 2004
) and to promote growth, survival, and invasion of pancreatic cancer cells (Arumugam et al., 2005
). Angiogenesis, which is essential for growth and metastasis of cancer cells, has been shown to be promoted by ligands for the EGF receptor directly and/or via induction of vascular endothelial growth factor and its receptor (Goldman et al., 1993
; Tsai et al., 1995
). Recently, Gupta et al. (2007)
showed that epiregulin is one of the genes that facilitate the assembly of new tumor blood vessels and thus promote lung metastasis. Together, the results suggest that overexpression of S100A11 and eventual activation of EGF family members contribute to facilitation of growth, survival, invasion, and metastasis because S100A11 and EGF family members compose a positive feedback loop. This process could be a target for exploitation of new therapeutic measures.