Abrogation of Fam20c altered cell behaviors and BMP signaling of immortalized dental mesenchymal cells

Abstract

FAM20C mutations compromise the mineralization of skeleton and tooth in both human and mouse. Putatively, the mineralization disorder is attributed to the elevated fibroblast growth factor 23 (FGF23), which reduced the serum phosphorus by suppressing the reabsorption of phosphorus in kidney. Besides the regulation on systemic phosphorus homeostasis, FAM20C was also implicated to regulate cell behaviors and gene expression through a cell-autonomous manner. To identify the primary effects of Fam20c on dental mesenchymal cells, mouse Fam20c-deficient dental mesenchymal cells were generated by removing the floxed alleles from the immortalized mouse Fam20c^f/f dental mesenchymal cells with Cre-expressing lentivirus. The removal of Fam20c exerted no impact on cell morphology, but suppressed the proliferation and mobility of the dental mesenchymal cells. Fam20c deficiency also significantly reduced the expression of Osterix, Runx2, type I Collagen a 1 (Col1a1), Alkaline phosphatase (Alpl) and the members of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, but increased Fgf23 expression. Consistently, the in vitro mineralization of Fam20c-deficient dental mesenchymal cells was severely disabled. However, supplements of the non-collagenous proteins from wild type rat dentin failed to rescue the compromised mineralization, suggesting that the roles of FAM20C in tooth mineralization are more than phosphorylating local matrices and regulating systemic phosphorus metabolism. Moreover, the down-regulated BMP signaling pathways in the Fam20c deficient dental mesenchymal cells revealed that the kinase activity of FAM20C might be required to maintain BMP signaling. In summary, our study discloses that Fam20c indeed regulates cell behaviors and cell signaling pathway in a cell-autonomous manner.

Keywords: Extracellular matrix; FAM20C; Mineralization; Phosphorylation; Secretory proteins.

Fig. 1. Generation and identification of DM Fam20c^KO

(A) After infected by CMV-Cre-IRES-EGFP lentivirus, the immortalized DM Fam20c^f/f formed single cell-derived clone and was passaged for 10 generations; All cells from the selected colony were positive for DAPI (blue) and EGFP (green), indicating that all the cells expressed the Cre transgene. (B) Genotyping PCR for Cre transgen, the floxed and recombined Fam20c allele. The genotyping for DM Fam20c^f/f (f/f) and DM Fam20c^KO (KO) with specific primers for the floxed Fam20c allele (Knock-in allele), recombined Fam20c allele (Recom) and Cre transgene. The tail lystae from an identified Fam20c^f/+ mouse was employed as a positive control (f/+) with the primers for the floxed Fam20c allele (Knock-in allele). (C) RT-PCR for the floxed and recombined Fam20c allele. The Set 1 primers amplifying exon 5–8 produced a 388-bp band in DM Fam20c^f/f (f/f), and no band in DM Fam20c^KO (KO), while the Set 2 primers for exon 5–11 gave an 820-bp band in DM Fam20c^f/f (f/f), and a 421-bp band in DM Fam20c^KO (KO). (Std: DNA ladder; Scale bar:100 um)

Fig. 2. Cell morphology and proliferation of the DM Fam20c^f/f and DM Fam20c^KO

(A) The micrographs of DM Fam20c^f/f and DM Fam20c^KO under the Olympus reverse phase-contrast microscope. (B) BrdU-labeling images of DM Fam20c^f/f and DM Fam20c^KO were taken under the Olympus reverse phase-contrast microscope. (C) The statistical assay showed that the difference in the proliferation rate of DM Fam20c^f/f (Mean=52.84%, SD=4.347%) was significantly higher than DM Fam20c^KO (Mean=39.98%, SD=5.423%) (p=0.0107).

Fig. 3. Cell migration of the DM Fam20c^f/f and DM Fam20c^KO

(A) Many DM Fam20c^f/f migrated into the scratches after 24 hours of culture; In contrast, there were only a few DM Fam20c^KO in the scratches. (B–C) After 24 hours of culture, the average diameter of DM Fam20c^f/f colonies from the suspension containing 1×10⁷ of cells was 5.025 mm (Mean=5.025mm, SD=0.359mm), which was significantly larger than those of DM Fam20c^KO (Mean=3.98mm, SD=0.37mm) (***: p<0.001). Scale bar in (A) and (B) equals to100 µm).

Fig. 4. Q-PCR for the transcription of bone-related genes in DM Fam20c^f/f and DM Fam20c^KO

(A) The comparison of the relative transcription of Atf4, Dlx3, Osx/Sp7 and Runx2 between DM Fam20c^f/f and DM Fam20c^KO. (B) The comparison of the relative transcription of Dmp1, Dspp, Bsp, Mepe and Opn/Spp1 between DM Fam20c^f/f and DM Fam20c^KO. (C) The comparison of the relative transcription of Alpl, Fgf23, Col1a1, Ocn/Bglap and Osn/Sparc between DM Fam20c^f/f and DM Fam20c^KO. (*: p<0.05; **:p<0.01; ***: p<0.001.)

Fig. 5. Immunocytochemistry and Western blot for the bone-related genes in DM Fam20c^f/f and DM Fam20c^KO

(A) Immunocytochemistry with the antibodies against DMP1, DSP, BSP, MEPE and OPN/SPP1 for their expression in DM Fam20c^f/f and DM Fam20c^KO. (B) Western blotting with anti-FGF23 antibody for FGF23 concentration in the medium of DM Fam20c^f/f and DM Fam20c^KO. (Scale bar: 100 um)

Fig. 6. Induced mineralized nodules in DM Fam20c^f/f and DM Fam20c^KO

(A) The ALPL activity was assessed by in situ histochemistry assay. (B) After 3 weeks of osteogenic induction, the formation of mineralized nodules in DM Fam20c^f/f and DM Fam20c^KO was evaluated by Alizarin red staining. (C) After 1 week of osteogenic induction, the non-collagenous extract from rat dentin enhanced the formation of mineralized nodules in DM Fam20c^f/f. (D) The addition of non-collagenous extract from rat dentin failed to improve the formation of mineralized nodules in DM Fam20c^KO.

Fig. 7. Western blots for BMP signaling pathways in DM Fam20c^f/f and DM Fam20c^KO

(A) Western blotting with antibodies against pan-Smad1/5/8 and p-Smad1/5/8 for canonical BMP signaling in DM Fam20c^f/f and DM Fam20c^KO. (B) Western blotting with antibodies against pan-p38 and p-p38 for non-canonical BMP/p38 signaling in DM Fam20c^f/f and DM Fam20c^KO. (C) Western blotting with antibodies against pan-Erk and p-Erk for non-canonical BMP/Erk signaling in DM Fam20c^f/f and DM Fam20c^KO. (D) Western blot with antibody against β-actin was used as the internal control to normalize the amount of DM Fam20c^f/f and DM Fam20c^KO. (E) Q-PCR revealed the relative expression of Bmp2, Bmp4 and Bmp7 between DM Fam20c^f/f and DM Fam20c^KO. (*: p<0.05; **:p<0.01; ***: p<0.001.)