Sustained BMP signaling in osteoblasts stimulates bone formation by promoting angiogenesis and osteoblast differentiation

Abstract

Angiogenesis and bone formation are tightly coupled during the formation of the skeleton. Bone morphogenetic protein (BMP) signaling is required for both bone development and angiogenesis. We recently identified endosome-associated FYVE-domain protein (endofin) as a Smad anchor for BMP receptor activation. Endofin contains a protein-phosphatase pp1c binding domain, which negatively modulates BMP signals through dephosphorylation of the BMP type I receptor. A single point mutation of endofin (F872A) disrupts interaction between the catalytic subunit pp1c and sensitizes BMP signaling in vitro. To study the functional impact of this mutation in vivo, we targeted expression of an endofin (F872A) transgene to osteoblasts. Mice expressing this mutant transgene had increased levels of phosphorylated Smad1 in osteoblasts and showed increased bone formation. Trabecular bone volume was significantly increased in the transgenic mice compared with the wildtype littermates with corresponding increases in trabecular bone thickness and number. Interestingly, the transgenic mice also had a pronounced increase in the density of the bone vasculature measured using contrast-enhanced microCT imaging of Microfil-perfused bones. The vessel surface and volume were both increased in association with elevated levels of vascular endothelial growth factor (VEGF) in osteoblasts. Endothelial sprouting from the endofin (F872A) mutant embryonic metatarsals cultured ex vivo was increased compared with controls and was abolished by an addition of a VEGF neutralizing antibody. In conclusion, osteoblast targeted expression of a mutant endofin protein lacking the pp1c binding activity results in sustained signaling of the BMP type I receptor, which increases bone formation and skeletal angiogenesis.

FIG. 1

Generation of transgenic mice with point mutation in endofin (F872A). (A) Diagram of expression construct of endofin (F872A) driven by 2.3-kb mouse type I collagen promoter (Col1α1) for generation of transgenic mice. (B) Representative genotyping of endofin (F872A) transgenic mice by PCR analysis of expression of endofin (F872A). Lanes 3, 5, and 8 represent the mutant transgene, whereas lanes 1, 2, 4, 6, and 7 represent WT littermates. CO, positive control. (C) Western blot analysis of protein extracted from bone tissue of WT littermates and the mutant for endofin (F872A) expression. The ratio of mutant endofin to endogenous endofin was 2.45. (D) Representative histological sections of distal femurs from WT and endofin (F872A) transgenic mice with immunostaining with an antibody against endofin.

FIG. 2

Increased bone formation in endofin (F872A) mutant mice. (A and B) Increased BMD is shown (A) in radiography and (B) μCT images of femur of endofin (F872A) mutant mice and their WT littermates at 16 wk of age. Two lines were shown. Quantitation of bone structure by μCT shows comparison of endofin mutant mice (gray bars) with their WT littermates (white bars), increased (C) bone volume per tissue volume (BV/TV), (D) trabecular number (Tb.N), (E) trabecular thickness (Tb.Th.), and decreased (F) trabecular separation (Tb.Sp). (G) Dynamic parameter bone formation rate (BFR) was assessed by two sequential doses of calcein injection in mice at 6 wk of age before death. Representative calcein-labeled sections of proximal tibias are visualized by fluorescence micrography. Bone histomorphometric analysis of trabecular bone of the femur, (H) bone surface referent bone formation rate (BFR/BS), (I) mineralizing surface per bone surface (MS/BS), and (J) osteoblast surface per bone surface (Ob.S/BS) were increased in endofin (F872A) mutant mice, but there is no significant difference in osteoclast surface per bone surface (Oc.S/BS) between endofin (F872A) mutant mice and their WT littermates (K). Quantitative data are expressed as means ± SD. *p < 0.05, n = 4.

FIG. 3

Enhanced osteoblastic differentiation in endofin (F872A) mutant mice. Primary pre-osteoblasts isolated from either endofin (F872A) mutant mice or their WT littermates were cultured in osteogenic medium. (A) Western blot analysis shows that endogenous phosphorylated Smad1 (P-Smad1) levels of the primary calvaria osteoblast were increased in endofin (F872A) mutant mice relative to the WT mice. Endogenous β-actin was used as internal control. (B) Representative micrographs of histochemical staining of ALP in calvarial pre-osteoblasts isolated from endofin (F872A) mutant mice or their WT littermates. Before staining, the primary pre-osteoblasts were cultured in osteogenic medium for 14 days. (C) Quantitative densitometric analysis of ALP activity in B using NIH Image J 1.38. (D) Representative micrographs of von Kossa staining for mineralized nodule formation in pre-osteoblasts cultured in osteogenic medium for 21 days. (E) Quantification of von Kossa–stained mineralized nodules is expressed as percent of WT pre-osteoblast staining. Ten randomly selected microscopic fields were examined in each of three independent experiments. Quantitative data represent mean ± SD. *p < 0.05. (F) Western blot analysis shows that endogenous osteocalcin expression of the primary pre-osteoblasts was increased in endofin (F872A) mutant mice relative to the WT mice. The ratio of osteocalcin expressed in the mutant mice to the control mice was 1.47. Endogenous β-actin was used as internal control. (G) Immunostaining of femoral sections for P-Smad1 and (H) quantitative analysis of the nuclear expression of P-Smad1 shows that the level of P-Smad1 was increased in endofin (F872A) mutant mice relative to that of WT mice. Sections were counterstained with hematoxylin. Gray arrows indicate positive staining. Total cells represent the cells covering the bone surface. Quantitative data represent mean ± SD. **p < 0.01. (I) Immunostaining of femoral sections for osteocalcin and (J) quantitative analysis of the expression of osteocalcin in density shows that the level of osteocalcin was higher in endofin (F872A) mutant mice than WT mice. Black arrows indicate weakly positive staining (WP staining). Gray arrows indicate strong positive staining (SP staining). Total cells represent the cells covering the bone surface. Quantitative data represent mean ± SD. *p < 0.05.

FIG. 4

Stimulated angiogenesis in long bones of endofin (F872A) mutant mice. (A) Photograph of hind limbs from endofin (F872A) mutant mice and their WT littermates. (B) Representative μCT images of vasculature in Microfil-perfused femurs from 2-mo-old endofin (F872A) mutant mice and their WT littermates. Quantitative μCT angiography analysis shows increased vessel surface (C), vessel volume (D), and ratio of vessel volume/total volume (VV/TV) (E) within femoral bone of endofin (F872A) mutant mice (gray bars) relative to control mice (white bars). Data represent mean ± SD. *p < 0.05. n = 5. (F) Representative images show increased endothelial sprouting in metatarsals from endofin (F872A) mutant mice in an in vitro angiogenesis assay at 9 and 18 days, respectively. The metatarsals were dissected from E17.5 fetuses of endofin (F872A) mutant mice and their WT littermates. Endothelial sprouting is visualized by immunostaining for CD31. Magnification, ×25.

FIG. 5

Increased expression of VEGF necessary for the enhanced angiogenesis in endofin (F872A) mutant mice. (A) Levels of VEGF mRNA in primary osteoblasts measured by RT-PCR. The primary cells were isolated from endofin (F872A) mutant mice or their WT littermates and cultured under osteogenic medium for 18 days before harvesting of total RNA. (B) Western blot analysis shows increased expression of VEGF protein in osteoblasts as prepared in A. (C) Immunostaining of femoral sections with the anti-VEGF antibody shows increased VEGF expression (gray arrows) in endofin (F872A) mutant mice relative to control mice. Nonimmune serum was used as negative control. Black arrows indicate negative staining in osteoblast. Magnification, ×400. (D) Representative images show endothelial sprouting in metatarsals from endofin (F872A) mutant mice and WT littermates, which were treated with the different serums described below. (I) Metatarsal from WT littermates. (II) Metatarsal from WT littermates treated with recombinant VEGF (10 ng/ml). (III) Metatarsal from endofin (F872A) mutant mice treated with mouse IgG (100 ng/ml). (IV) Metatarsal from endofin (F872A) mutant mice treated with a VEGF-neutralizing antibody (100 ng/ml). Data are representative of three independent experiments.