br Materials and Methods br Results br

Materials and Methods
Results
Discussion We previously demonstrated the importance of the WNK4–OSR1/SPAK–NCC signaling cascade in the kidney for blood pressure regulation (Uchida, 2014). However, the extrarenal roles of WNK4 still required clarification. In this study, we found that WNK4 regulates expression of adipogenic genes and WNK4−/− mice had reduced adiposity. Intriguingly, WNK4 in mouse adipose tissues was expressed predominantly in mouse mature adipocytes, rather than in primary preadipocytes (Fig. 1), and the expression was greatly induced with adipocyte differentiation. These data also were confirmed in the 3T3-L1 Thirty percent of and hMSC-AT cells (Fig. 1 and Fig. S4). Then, WNK4 inhibition impaired the expression of PPARγ and C/EBPα, which are core transcriptional factors to form mature white adipocytes. These results indicated that WNK4 is an important factor for adipocyte differentiation. Among WNKs, WNK1 and WNK4 were expressed in the adipose tissue (Fig. 1 and Fig. S1). In contrast to the dramatic change of WNK4 during adipocyte differentiation, the protein level of WNK1 was not changed through adipogenesis, and si-WNK1 did not affect the expression of adipogenic genes (Fig. 1 and Fig. S5). These results indicated that only WNK4 is involved in adipogenesis. We indicated that the increase of WNK4 protein would be due mainly to the increase of WNK4 transcription, since WNK4 mRNA was, indeed, increased and KLHL2/3 or CUL3 mRNA levels were not decreased during 3T3-L1 differentiation. We also showed that WNK4 expression was induced by Dexa (Fig. S2) in 3T3-L1 cells. However, such induction by Dexa was not observed in different cell types (mpkDCT cells). Since microarray analysis revealed that hundreds of genes in 3T3-L1 cells are differently regulated by Dexa (Burton et al., 2004), the mechanism of WNK4 induction in Dexa could be involved in this complex machinery of adipogenesis, rather than a simple transcriptional activation by Dexa (Fig. S2d). We sought to clarify the regulatory mechanism of adipogenic genes by WNK4. We showed the function of WNK4 in adipocytes that was independent of the WNK-OSR1/SPAK-NKCC1 signaling cascade (Fig. S5). Consistent with this finding, when kinase-dead mutant WNK4 (D318A) was exogenously expressed in 3T3-L1 cells, increased expression of PPARγ and C/EBPα was observed (Fig. S7). It was interesting that si-WNK4 did not affect the expression of C/EBPβ, but did affect the expression of C/EBPα and PPARγ. We also showed that WNK4 inhibition decreased the binding ability of C/EBPβ to the PPARγ2 promoter region, suggesting that WNK4 affected PPARγ transcription by C/EBPβ. Furthermore, we identified significant expression of WNK4 in the nuclei of differentiated 3T3-L1 adipocytes (Fig. 3b). Since WNK1 has been shown previously to be involved in cell division in Hela cells (Tu et al., 2011), we focused our attentions on MCE. Although MCE is considered an essential step in adipogenesis (Guo et al., 2015), its regulatory mechanism is poorly understood. Previously, it has been reported that blocking the cell cycle at the G1-S stage can inhibit MCE and thereby delay subsequent events in adipogenesis (Tang et al., 2003a). Similarly we revealed that si-WNK4 inhibited cell cycle progression during MCE, potentially indicating why the DNA-binding ability of C/EBPβ was affected by WNK4 (Fig. 3c and d), because C/EBPβ is known to obtain its binding ability during MCE (Guo et al., 2015). Thus, we determined that WNK4 in adipocytes had an important positive role in MCE. We believe that these results provide an insight into the regulatory mechanism of adipogenic gene expression by WNK4, and that they also shed light on the regulatory mechanisms of MCE. In WNK4−/− mice fed a HFD, the expression of PPARγ, C/EBPα, and FABP4 in the adipose tissue was significantly depressed. Furthermore, they had reduced insulin resistance and obesity induced by HFD. It has been reported previously that NCC knockout mice did not show lower body weight on a normal diet (Soleimani et al., 2012), and we have shown that administration of HCTZ did not inhibit the gain of body weight by HFD (Fig. S8). These results indicated that the decreased body weight of WNK4−/− mice was not due to low NCC activity or volume depletion. The association between reduced WNK4 expression and decreased expression of core transcriptional factors regulating adipocyte functions, both in 3T3-L1 adipocytes and in WNK4−/− mice, suggested that there may be an explanation for the observed phenotypes of the knockout mice. Although it is well known that PPARγ agonists improve insulin sensitivity, various studies have indicated that moderate reduction of PPARγ activity prevents insulin resistance and HFD-induced obesity (Kubota et al., 1999; Yamauchi et al., 2001; Jones et al., 2005). Intriguingly, adipose-specific PPARγ-knockout mice exhibited insulin sensitivity, reduced fat development, and resistance to HFD-induced obesity, despite their higher TG levels and lower plasma leptin and adiponectin levels (Jones et al., 2005). These phenotypes of the adipose-specific PPARγ-knockout mice were quite similar to those of our WNK4−/− mice. TG levels of WNK4-KO mice also tended to be higher than those of WT mice (Fig. 5). If we could increase the number of mice examined, the difference might reach statistical significance. Thus, most of the phenotypes observed in WNK4−/− mice could be explained by the reduced expression of PPARγ in adipocytes.