# Shotkuseki Extract Promotes Keratinocyte Differentiation Even at a Low Calcium Concentration
## 4 Discussion  
In this study, we hypothesized that Shotokuseki extract contained key minerals that affected keratinocyte differentiation. Initially, minerals present in SE were measured by AAS, revealing the presence of Na, Mg, K, Ca, and Zn ions, indicating that SE contains various minerals (Figure 1). The epidermis consists of keratinocytes that differentiate from the basal layer to the stratum corneum. Keratinocytes present in the basal layer proliferate and express keratins 5 and 14 [1,2]. As the expression levels of keratins 5 and 14 decrease, these cells move upwards to the stratum spinosum and express the early differentiation markers keratins 1 and 10 [1,2]. Subsequently, the keratinocytes express late differentiation markers involucrin and loricin [4,5,18]. During the final differentiation process, the cells express transglutaminase, which is essential for the formation of the stratum corneum, and filaggrin, which is a precursor of natural moisturizing factor in keratinocytes [5,19]. Calcium ions are a major regulator of keratinocyte differentiation and proliferation [8], playing important roles in all stages of keratinocyte differentiation, including differentiation in the basal and spinous layers to final differentiation in the stratum granulosum [3,20,21]. The calcium content required for stage-specific expression of differentiation marker proteins varies in each layer of the epidermis [20,21,22]. For example, the calcium concentration required for expression of the late differentiation marker proFLG is high when compared with the extracellular calcium concentration required for expression of keratins 1 and 10 [23]. This suggests that the calcium gradient in the epidermis is important for epidermal differentiation and barrier formation. In general, keratinocytes are promoted to grow at an extracellular calcium concentration of 0.03-0.15 mmol/L. At higher calcium concentrations (1 mmol/L or more), keratinocyte differentiation is induced and proliferative ability is lost [8,24]. In this report, Shotokuseki extract was added to a final concentration of 5% (\(v/v\)). The concentration of calcium at this SE concentration was determined to be 0.01 mmol/L. Because the calcium concentration in the medium was 0.06 mmol/L, this indicates that keratinocyte differentiation was not promoted solely by the calcium level in SE. However, the mRNA expression levels of K5, an undifferentiated marker; K10, an early differentiation marker; proFLG, a late differentiation marker; and TGase1 increased significantly in the presence of  
Figure 5: Effects of Shotokuseki extract on intracellular calcium concentration in NHEKs. Intracellular calcium concentration was evaluated as detailed in the Materials and Methods section using Fura-2 AM. (**a**) intracellular calcium concentration profile intracellular calcium concentration profile (**b**) intracellular calcium concentration after 20 s. Data represent the mean \(\pm\) S.D. of four independent experiments. * \(p\) < 0.05 and **\(p\) < 0.01. Statistical significance was evaluated using the [PERSON]’s post hoc multiple comparison test. SE (Figure 3). Furthermore, protein expression of transglutaminase was measured. The expression level of transglutaminase increased significantly when 5% (\(\tau/v\)) SE was added to the keratinocyte culture (Figure 4). Application of the same concentration of calcium (0.01 mM) as 5% SE slightly increased TGase1 protein expression, which is involved in epidermal final differentiation, and its expression was upregulated more than that in 5% SE. This suggests that the promotion of final differentiation of the epidermis by SE treatment is not solely the action of calcium or calcium-dependent but may involve other minerals. Calcium is an important factor in the differentiation process of keratinocytes. We monitored the changes in intracellular calcium concentration in keratinocytes after SE application. The intracellular calcium concentration in the group to which 5% SE was applied increased about 1.4 times (Figure 5).