Fig 1.
Schematic representation of the different M-CSF constructs.
(A) WT M-CSF (designated M-CSFWT) constituting a disulfide-bond–linked homodimer. (B) Monospecific M-CSF that can bind αvβ3 integrin (designated M-CSFαvβ3) via an RGD motif but not c-FMS because of mutations in positions 9 and 15. (C) Monospecific M-CSF that can bind only c-FMS (designated M-CSFc-FMS) because of two point mutations that change the RGD motif to RDG, thereby preventing its binding to αvβ3 integrin. (D) Libraries (designated M-CSFRGD) created by changing two loops in the M-CSF dimerization site to an RGD motif with three random amino acids on each side, thereby enabling binding to αvβ3 integrin. M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid; WT, wild type.
Fig 2.
FACS dot plot of M-CSFRGD affinity maturation process.
Yeast-displayed mutant pools were tested for binding to (A) 200 nM c-FMS, (B) 500 nM αvβ3 integrin, (C) 250 nM αvβ3 integrin, (D) 100 nM αvβ3 integrin, (E) 20 nM αvβ3 integrin, and (F) 50 nM c-FMS. High target binders were sorted as indicated in each figure with black square- or polygon-shaped gates. M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid.
Fig 3.
SPR binding sensorgrams of the different purified M-CSF proteins.
Binding of M-CSFαvβ3 (A and F), M-CSFc-FMS (B and G), 4.22 (C and H), 4.24 (D and I), and 5.6 (E and J) to c-FMS (A-E) and αvβ3 integrin (F-J) at concentrations of 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. Source data can be found in S1 Data. M-CSF, macrophage colony-stimulating factor; RUs, response units; SPR, surface plasmon resonance.
Fig 4.
Docking model of the M-CSFRGD variant 4.22/c-FMS-αvβ3 integrin complex.
M-CSFRGD variant 4.22 is shown in pink, c-FMS in cyan, αv in yellow, β3 in green, and QTSRGDSPS mutant residues in red. M-CSF, macrophage colony-stimulating factor; RGD, Arginine-Glycine-Aspartic acid.
Fig 5.
Cell binding, c-FMS, and Akt activation assays.
Purified M-CSFRGD variants 4.22 and 5.6 were tested for binding to (A) MDA-MB-231 breast cancer cell line and (B) murine BMMs at different protein concentrations (1 μM, 2.5 μM, and 7.5 μM). The cellular expression levels of c-FMS and αvβ3 integrin are indicated as superscripts. (C) Cell competition binding assay for variant 5.6 in the presence and absence of the two competitors, namely 10 μM cRGD and 5 μM M-CSFWT. Binding of M-CSFc-FMS and M-CSFαvβ3 to the cells is shown for comparison. (D) Tyrosine phosphorylation of c-FMS and (F) serine phosphorylation of Akt in murine BMMs. Different gel runs are separated by a black line. (E) Relative c-FMS and (G) Akt phosphorylation levels of BMMs following incubation with M-CSFc-FMS, M-CSFαvβ3, and M-CSFRGD variants 4.22, 4.24, and 5.6 in the presence of recombinant M-CSF as a competitor. Chemiluminescence read-outs were quantified by densitometry. Data are means ± SEM of triplicates. *p < 0.05, **p < 0.01, ***p < 0.001. The aspect ratios of the membranes in panels D and F were changed. Source data and analysis can be found in S2 Data. BMM, bone-marrow–derived monocyte; cRGD, cyclic RGD; M-CSF, macrophage colony-stimulating factor; MDA-MB-231, MD Anderson metastatic breast 231; RGD, Arginine-Glycine-Aspartic acid.
Fig 6.
Actin belt formation in mature osteoclasts incubated with M-CSFRGD variants.
(A) Murine BMMs were allowed to differentiate into osteoclasts in the presence of M-CSF and RANKL for 72 h. Then, the cells were incubated for an additional 24 h without (positive control) or with inhibitors (5 μM), followed by fixation and staining for F-actin and nuclei. The cells formed solid actin belts (white arrowheads), actin belts with "scattered" podosomes (white arrows), or "amorphous" actin stain distribution (barbed arrowheads). (B) The actin belts’ formation was quantified by normalizing the numbers of solid actin belt to the number of osteoclasts. Pictures are representatives of 35 images acquired from five different wells per sample. Data are means ± SEM of triplicates. *p < 0.05, **p < 0.01, ***p < 0.001. Source data can be found in S3 Data. BMM, bone-marrow–derived monocyte; cRGD, cyclic RGD; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of the nuclear factor–kappa-B ligand; RGD, Arginine-Glycine-Aspartic Acid.
Fig 7.
Effects of M-CSFRGD variants on murine osteoclast differentiation.
Murine BMMs were cultured for 96 h in a medium containing recombinant mouse M-CSF (20 ng/ml), RANKL (20 ng/ml), and different concentrations of inhibitors. The same medium without inhibitors was used as a positive control. The medium for the negative control was supplemented with recombinant M-CSF. (A) Cells were fixed and stained for TRAP. (B–D) Cells were examined for: (B) number of osteoclasts, (C) number of nuclei within osteoclasts, (D) total surface area, and (E) total TRAP absorbance were normalized to the positive control. The effect of the inhibitors on markers of osteoclast differentiation was assessed using quantitative PCR for (F) NFATc1 and (G) Oscar mRNA expression. (H) The effect of the inhibitor on pre-osteoclast cell survival was assayed by measuring PI incorporation in osteoclasts cultured for 48 h in the presence of 1 μM of each inhibitor. (I) To test whether there is an unspecific toxic effect of the inhibitors, BMSCs were tested for cell viability in the XTT assay in the presence of three different concentrations of inhibitor (50 nM, 1 μM, and 5 μM). Data are means ± SEM of triplicates. A total of 2,340 frames were analyzed for 1,581 osteoclasts and 5,313 nuclei. *p < 0.05, **p < 0.01, ***p < 0.001. Source data and analysis can be found in S4 Data. BMM, bone-marrow–derived monocyte; BMSC, bone-marrow–derived mesenchymal stromal cell; M-CSF, macrophage colony-stimulating factor; PI, propidium iodide; RANKL, receptor activator of the nuclear factor–kappa-B ligand; RGD, Arginine-Glycine-Aspartic acid; TRAP, tartrate-resistant acid phosphatase; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
Fig 8.
Effects of M-CSFRGD variants on human osteoclast differentiation.
Human CD14+ cells were cultured for 96 h in medium containing recombinant human M-CSF (20 ng/ml), murine RANKL (20 ng/ml), and different concentrations of inhibitors. The same medium without inhibitors was used as a positive control. The αMEM medium for the negative control was supplemented with recombinant M-CSF. (A) Cells were fixed and stained for TRAP. (B-D) Cells were examined for (B) number of mature osteoclasts, (C) number of nuclei within osteoclasts, and (D) total surface area. Results were normalized to the positive control. Data are means ± SEM of triplicates. A total of 1,620 frames were analyzed for 477 osteoclasts and 1,897 nuclei. *p < 0.05, **p < 0.01, ***p < 0.001. Source data and analysis can be found in S5 Data. αMEM, alpha Minimum Essential Medium; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of the nuclear factor–kappa-B ligand; RGD, Arginine-Glycine-Aspartic acid; TRAP, tartrate-resistant acid phosphatase.
Fig 9.
M-CSFRGD proteins inhibit bone resorption in ovariectomized mice.
(A) Ex vivo images of 12-weeks-old WT C57BL6 mice organs. The organs were removed and imaged 1.5 h and 3 h after mice were injected s.c. and compared with organs of a mouse that had been injected with unconjugated dye and of a mouse that had not been injected with the proteins (right). To determine whether M-CSFRGD variant 5.6 accumulates in the bones, the epiphysis and diaphysis of the femur of a mouse injected with variant 5.6 were compared with those of the control mice (left). (B) Ten-weeks-old mice were ovariectomized, and starting 2 wk after the surgery, they were injected twice a day with PBS or M-CSFRGD variants 4.22 or 5.6 for 3 d. Thereafter, serum CTX-I levels were determined by ELISA. Data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data and its analysis can be found in S6 Data. CTX-I, carboxy-terminal telopeptide of type I collagen; ELISA, enzyme linked immunosorbent assay; M-CSF, macrophage colony-stimulating factor; OVX, ovariectomy-induced bone loss; PBS, phosphate-buffered saline; RGD, Arginine-Glycine-Aspartic acid; s.c., subcutaneously; WT, wild type.