Such a difference may be attributable to [Ca
2+], which is present at 0.07 mM in KSFM, much lower than the 1.8 and 0.9 mM in DMEM and DMEM/F12, respectively. Extracellular [Ca
2+] can affect the balance between proliferation and differentiation in many cell types. For example, the growth of cultured dermal fibroblasts is inhibited at low but promoted in high extracellular [Ca
2+].
43 An increase of extracellular [Ca
2+] stimulates DNA synthesis of 3T3 fibroblasts by activating mitogen activated protein (MAP) kinase.
43 Furthermore, the growth potential is promoted by high extracellular [Ca
2+] (>1.0 mM), even without growth factors.
43 A medium containing a high [Ca
2+] concentration has been used to culture fibroblasts, because it can prevent apoptosis and promote their growth by activating MAP kinase.
44 It remains unknown whether keratocytes (i.e., neural crest-derived mesenchymal cells) prefer a high-[Ca
2+] medium in a similar manner to fibroblasts. In our study, we noted that an increase of [Ca
2+] to 1.8 mM alone in KSFM was not sufficient to alter the dendritic morphology and expression of keratocan and CD34
(Fig. 5) , nor did it upregulate the keratocan promoter activity and the promoter activities of TGF-β1 and -β RII
(Figs 5 6) . However, an increase of [Ca
2+] to 1.8 mM in KSFM with simultaneous addition of 10% FBS synergistically upregulated the promoter activities of TGF-β1 and -β RII, and nuclear translocation of Smad2 and Smad4 to the same extent as did DMEM/10% FBS
(Figs. 6 7) . These results indicate that high [Ca
2+] and FBS synergistically upregulates Smad-mediated TGF-β signaling. In other words, FBS’s detrimental effect against the keratocyte phenotype is contingent on high [Ca
2+]. This finding indicates that keratocytes responded to [Ca
2+] similar to epidermal keratinocytes, in which TGF-β stimulates differentiation in high [Ca
2+],
45 46 47 48 but has no effect or inhibits differentiation in low [Ca
2+].
45 46 49 In epidermal keratinocytes, the synergism between high [Ca
2+] and TGF-β action can further be explained by the fact that TGF-β mRNA can be induced by high [Ca
2+].
41 In fibroblasts, TGF-β signaling depends on extracellular [Ca
2+].
26 These data collectively indicate that a low-[Ca
2+] medium such as in KSFM plays a major role in downregulating TGF-β signaling.