Inactivation of a Novel FGF23 Regulator, FAM20C, Leads to Hypophosphatemic Rickets in Mice
Figure 9
Lentiviral shRNA-mediated “knockdown” of FAM20C leads to similar alterations in the expression of DMP1 and FGF23 in human and mouse osteogenic cell lines.
The cells were divided into three groups: uninfected cells, cells infected with control lentivirus expressing the scrambled shRNA and cells infected with FAM20C-shRNA lentivirus containing a mixture of 3 pieces of shRNA targeting different regions of the FAM20C mRNA. The mRNA levels of DMP1 and FGF23 in each group were determined by real-time PCR. The expression levels of the uninfected cells without osteogenic induction were taken as 1, while that of the cells infected with the control virus or FAM20C-shRNA virus were expressed as folds of change to the uninfected cells. (A) The knockdown of FAM20C led to remarkable downregulation of DMP1 in mouse MC3T3-E1 cells. Without the osteogenic induction, the expressional level of DMP1 had no significant difference among the three groups of cells. During the 3-week osteogenesis-induction process, DMP1 was significantly upregulated in the uninfected cells and cells infected with the control lentivirus, while its expression was remarkably reduced in the cells infected with FAM20C-shRNA lentivirus at 1-, 2- and 3-weeks after the start of osteogenic induction. (B) Inactivation of FAM20C led to remarkable downregulation of DMP1 in hMSC cells. The expression level of DMP1 had no significant difference among three groups in the first 2 weeks of culture in the osteogenic medium. After 3 weeks of osteogenic induction, the DMP1 expression was remarkably reduced in the cells infected with FAM20C-shRNA lentivirus when compared with the uninfected cells and cells infected with the control lentivirus. (C) Inactivation of FAM20C led to remarkable downregulation of DMP1 in Saos-2 cells. DMP1 expression was downregulated in human osteoblasts (Saos-2) infected with FAM20C-shRNA lentivirus before the osteogenic induction started, compared with the uninfected cells and cells infected with the control virus. The downregulation of DMP1 in the FAM20C-knockdown cells became more prominent after 1 week of osteogenic induction. (D) Inactivation of FAM20C led to significant upregulation of FGF23 in mouse MC3T3-E1 cells. The expression level of FGF23 had no significant difference among three groups in the first 2 weeks of osteogenic induction. After inducing the cells for osteogenic differentiation for 3 weeks, FGF23 was significantly upregulated in the cells infected with FAM20C-shRNA lentivirus, compared with the uninfected cells and cells infected with the control lentivirus. (E) Inactivation of FAM20C led to significant upregulation of FGF23 in hMSC cells. The FGF23 elevation also occurred in the hMSC cells after 3-week osteogenic induction. Note that upregulation of FGF23 in the hMSC cells was not as remarkable as in the MC3T3-E1 cells at the same time point. (F) Inactivation of FAM20C led to significant upregulation of FGF23 in Saos-2 cells. Without osteogenic induction, FGF23 level had no significant difference among the 3 groups. After 1 week of osteogenic induction, FGF23 was significantly upregulated in the cells infected with FAM20C-shRNA lentivirus, compared with the uninfected cells and cells infected with the control lentivirus. *: P<0.05; **: P<0.01; NI: without osteogenic induction; Control: uninfected control cells; Control virus: cells infected with control lentivirus; FAM20C-shRNA virus: cells infected with FAM20C-shRNA lentivirus.