FAM20C regulates osteoblast behaviors and intracellular signaling pathways in a cell-autonomous manner

Abstract

Recent studies indicate that Family with sequence similarity 20 member C (FAM20C) catalyzes the phosphorylation of secreted proteins, and participates in a variety of biological processes, including cell proliferation, migration, mineralization, and phosphate homeostasis. To explore the local influences of FAM20C on osteoblast, Fam20c-deficient osteoblasts were generated by treating the immortalized Fam20c^f/f osteoblasts with CMV-Cre-IRES-EGFP lentivirus. Compared with the normal Fam20c^f/f osteoblasts, the expression of Bone sialoprotein (Bsp), Osteocalcin (Ocn), Fibroblast growth factor 23 (Fgf23), and transcription factors that promote osteoblast maturation were up-regulated in the Fam20c-deficient osteoblasts. In contrast, the expression of Dental matrix protein 1 (Dmp1), Dentin sialophosphoprotein (Dspp), Osteopontin (Opn), type I Collagen a 1 (Col1a1), and Alkine phosphatase (Alp) were down-regulated in the Fam20c-deficient cells. These alterations disclosed the primary regulation of Fam20c on gene expression. The Fam20c-deficient osteoblasts showed a remarkable reduction in the ability of forming mineralized nodules. However, supplements of extracellular matrix proteins extracted from the normal bone failed to rescue the reduced mineralization, suggesting that FAM20C may affect the biomineralization by the means more than local phosphorylation of extracellular matrix proteins and systemic phosphorus homeostasis. Moreover, although Fam20c deficiency had little impact on cell proliferation, it significantly reduced cell migration and lowered the levels of p-Smad1/5/8, p-Erk and p-p38, suggesting that the kinase activity of FAM20C might be essential to cell mobility and the activity of BMP ligands. In summary, these findings provide evidences that FAM20C may regulate osteoblast maturation, migration, mineralization, and BMP signaling pathways in a cell-autonomous manner.

Keywords: FAM20C; mineralization; osteoblast; phosphorylation; secretory proteins.

Fig 1. Removal of the floxed Fam20c allele by CMV-Cre-IRES-EGFP lentivirus

(A) The immortalized Fam20c^f/f osteoblasts were infected with CMV-Cre-IRES-EGFP lentivirus and cultured after 10 passages; note that all of the cells were positive for DAPI (blue) and EGFP (green), indicating an uniform infection of CMV-Cre-IRES-EGFP lentivirus in these osteoblasts. (B) The genomic DNA from Fam20c^f/+ mouse tail (f/+), OB Fam20c^f/f (f/f; without infection by lentivirus) and OB Fam20c^KO (KO; after treatment with lentivirus) were genotyped with specific primers for the floxed Fam20c allele, recombined Fam20c allele and Cre, respectively. (C) RT-PCR was performed to identify the successful removal of the floxed Fam20c allele. The Set 1 primers for exon 5–8 gave a 388-bp band for OB Fam20c^f/f cells (f/f), but produced no band for OB Fam20c^KO cells (KO); the Set 2 primers for exon 5–11 of Fam20c transcript produced an 820-bp band for OB Fam20c^f/f (f/f), and a truncated 421-bp band for OB Fam20c^KO osteoblasts (KO). (Std: DNA ladder; Scale bar:100 um)

Fig 2. Cell morphology and proliferation of the OB Fam20c^KO cell line

(A) The micrographs OB Fam20c^f/f and OB Fam20c^KO osteoblasts were taken under the Olympus reverse phase-contrast microscope. (B) BrdU-labeling images of OB Fam20c^f/f and OB Fam20c^KO were photographed under the Olympus reverse phase-contrast microscope. (C) The statistical assay showed that there were no significant difference (p>0.05) in the proliferation rate between OB Fam20c^f/f (Mean=45.24%, SD=8.701%) and OB Fam20c^KO (Mean=37.83%, SD=5.814%). Scale bar in (A) and (B) equals to100 µm).

Fig 3. The mRNA levels of bone-related genes in OB Fam20c^f/f and OB Fam20c^KO

Total mRNAs from OB Fam20c^f/f and OB Fam20c^KO were extracted and reversed transcribed into cDNAs. Then, the cDNAs were amplified by Q-PCR to calculate the relative expression levels of bone-related transcription factors: Atf4, Dlx3, Osx and Runx2 (A), the SIBLING proteins: Dmp1, Dspp, Bsp, Mepe and Opn (B), and other bone-related genes: Alp, Fgf23, Col1a1, Ocn and Osn (C).(*: p<0.05; **:p<0.01; ***: p<0.001.)

Fig 4. The levels of bone-related gene expression detected by immunocytochemistry and Western blot

(A) Immunocytochemistry with the antibodies against DMP1, DSP, BSP, MEPE and OPN were performed to assess the cultured OB Fam20c^f/f and Fam20c^KO cells. (B) Western blotting with FGF23 antibody showed that the FGF23 concentration in the serum free medium of the OB Fam20c^KO was higher than in OB Fam20c^f/f.

Fig 5. Impaired mineralization of OB Fam20c^KO

(A) In situ histochemistry assay showed a decrease in ALP activity in OB Fam20c^KO compared with OB Fam20c^f/f. (B) After 3 weeks of induction, Alizarin red staining showed that the formation of mineralized nodules was severely impaired in OB Fam20c^KO, compared to OB Fam20c^f/f. (C) After one week of culture in the osteogenic medium, the addition of non-collagenous proteins extracted from normal bone matrix of rat legs enhanced the formation of mineralization nodules in OB Fam20c^f/f. (D) There was no improvement on the formation of mineralization nodules by the addition of non-collagenous proteins extracted from normal bone matrix into OB Fam20c^KO osteoblasts.

Fig 6. The altered BMP signaling pathways in OB Fam20c^KO

Canonical and non-canonical BMP signaling pathways were analyzed by Western blotting with antibodies against pan-Smad1/5/8 and p-Smad1/5/8 for canonical BMP signaling (A), with antibodies against pan-Erk and p-Erk for BMP/p38 signaling (B), and with antibodies against pan-p38 and p-p38 (C) for BMP/p38 signaling. β-actin was used as the internal control to normalize the loading volume of OB Fam20c^f/f and OB Fam20c^KO cell lysates (D). Q-PCR was applied to compare the relative transcription of Bmp2, Bmp4 and Bmp7 between OB Fam20c^f/f and Fam20c^KO cells (E). (*: p<0.05; **: p<0.01.)

Fig 7. Reduced migration rate of OB Fam20c^KO

(A) After 24 hours of culture, the scratch in OB Fam20c^f/f was nearly fully covered by the cells. In contrast, there were only a few OB Fam20c^KO cells that had migrated into the scratch. (B) After 24 hours of dropping a suspension containing the 1×10⁷ of cells into a culture dish, the diameter of OB Fam20c^KO colony was notably smaller than that of Fam20c^f/f osteoblasts. (C) The statistical assay showed a significant difference between the diameters of OB Fam20c^f/f colonies (Mean=5.025mm, SD=0.359mm) and OB Fam20c^KO colonies (Mean=3.98mm, SD=0.37mm). (***: p<0.001).