Selective deletion of the membrane-bound colony stimulating factor 1 isoform leads to high bone mass but does not protect against estrogen-deficiency bone loss

Abstract

To better define the biologic function of membrane-bound CSF1 (mCSF1) in vivo, we have generated mCSF1 knockout (k/o) mice. Spinal bone density (BMD) was 15.9% higher in k/o mice compared to wild-type (wt) controls (P < 0.01) and total BMD was increased by 6.8% (P < 0.05). A higher mean femur BMD was also observed but did not reach statistical significance (6.9% P = NS). The osteoclastogenic potential of bone marrow isolated from mCSF1 k/o mice was reduced compared to wt marrow. There were no defects in osteoblast number or function suggesting that the basis for the high bone mass phenotype was reduced resorption. In addition to a skeletal phenotype, k/o mice had significantly elevated serum triglyceride levels (123 ± 7 vs. 88 ± 3.2 mg/dl; k/o vs. wt, P < 0.001), while serum cholesterol levels were similar (122 ± 6 vs. 116 ± 6 mg/dl; k/o vs. wt, P = NS). One month after surgery, 5-month-old k/o and wt female mice experienced the same degree of bone loss following ovariectomy (OVX). OVX induced a significant fourfold increase in the expression of the soluble CSF1 isoform (sCSF1) in the bones of wt mice while expression of mCSF1 was unchanged. These findings indicate that mCSF1 is essential for normal bone remodeling since, in its absence, BMD is increased. Membrane-bound CSF1 does not appear to be required for estrogen-deficiency bone loss while in contrast; our data suggest that sCSF1 could play a key role in this pathologic process. The reasons why mCSF1 k/o mice have hypertriglyceridemia are currently under study.

Fig. 1

Strategy for generating conditional mCSF1 knock out mice. The targeting construct for deletion of mCSF1 introduces a stop codon followed by a lox-p site into exon 6 at a position 5′ to the splice acceptor site for mCSF1. The stop codon is designed to include all three reading frames. A Neo cassette with flanking frt sites (frt-Neo-frt-lox-p) is introduced into intron 6 (a). Excision of the Neo cassette with flp recombinase leads to a CSF1 allele that contains two lox-p sites, one in intron 6 and one, which is upstream of the splice acceptor site for mCSF1 (d). In animals bearing two of these floxed alleles (i.e. before cre-mediated recombination) both isoforms of CSF1 will be generated. Splicing to the mCSF1 acceptor site will yield a normal mCSF1 transcript (d). Splicing to the sCSF1 acceptor site will lead to generation of an mRNA that includes the sequences for the proteolyitc cleavage sites for sCSF1 followed by a stop codon. The protein product of this mRNA will yield normal mature sCSF1 since it contains the appropriate signals for proteolytic cleavage with the surrounding sequences (e). Recombination with cre leads to selective deletion of the splice acceptor site for the mCSF1 isoform as well as removal of the transmembrane domain (e). This leaves a modified exon 6 that includes the splice acceptor site for sCSF1 followed by the proteolytic sites for sCSF1, followed by a stop codon. Thus a mature functional sCSF-1 will be generated but no mCSF1 (e). b The wt allele. c The engineered allele before removal of the Neo cassette in vitro

Fig. 2

Isoform specific RT-PCR of bone mRNA demonstrates the absence of mCSF1 transcripts in mCSF1 k/o mice. A 2% agarose gel was used for this analysis. MW markers are shown in the far right lane

Fig. 3

Western blot for sCSF1 using media conditioned by osteoblasts from either wt or k/o mice. Media conditioned for 24 h by MC3T3E1 cells (left lane), primary osteoblasts prepared from neonatal k/o mice (middle lane) or from wt littermates (right lane) was concentrated, desalted, and analyzed by Western blot using a polyclonal antibody to CSF1

Fig. 4

Flow cytometric analysis demonstrating the absence of mCSF1 protein expression in macrophages isolated from the k/o mice. Macrophages prepared from k/o mice (tinted) or from wt littermates (black) were analyzed by flow cytometry using a polyclonal antibody to CSF1. There is no staining of the k/o cells

Fig. 5

Femur spine and total body BMD determined by DXA (PIXImus) in 22 wt (12 male and 10 female) and 22 k/o (11 male and 11 female) mice. Mean values at the three sites were 734 ± 27 versus 784 ± 20; 552 ± 15 versus 641 ± 12 and 489 ± 12 versus 523 ± 6, g/cm² × 10³; wt versus ko for femur, spine and total body respectively; *P < 0.05, **P < 0.01 compared to wt. Results are expressed as M ± SEM

Fig. 6

a TRAP stain of co-cultures prepared using wt marrow and osteoblasts isolated from k/o (plates on the left) or wt (plates on the right) mice. b Numbers of TRAP-positive multinucleated OCLs formed in the co-cultures. The mean values were 95 ± 20 versus 32 ± 4 per well; wt vs. k/o, respectively. The results shown are the M ± SEM of three independent experiments with each determination performed in duplicate. **P < 0.01. c Numbers of TRAP-positive multinucleated OCLs generated for each genotype from osteoclast precursors cultured under osteoclastogenic conditions. d Mean values for the experiment shown panel C were 12.6 ± 0.8 versus 4.9 ± 0.4 cells per 130 mm²; n = 25 for both genotypes; ***P < 0.001. e Number of nuclei per cell; 8.3 ± 1.1 versus 5.8 ± 0.6, wt versus k/o; n = 18 for both genotypes; ^#P = 0.06

Fig. 7

Percent change in BMD in k/o and wt mice 1 month following OVX. BMD was reduced to similar extent in k/o and wt mice after OVX. The changes in femur, spine and total body BMD in wt mice compared so sham OVX wt mice were: −7.6, −12.6 and −5, respectively. Similar changes were observed in the OVX k/o mice compared to their sham OVX controls with declines of −7.5, −12.3 and −4.4% at the femur, spine and total body (P = NS for the difference by genotype at each site.)

Fig. 8

Effect of OVX on expression of the two CSF-1 isoforms in bone. Tibiae and femurs were isolated 1 month after surgery, the marrow was flushed, the bone snap frozen and pulverized while frozen, and RNA extracted. RNA was analyzed by isoform-specific real-time PCR. Mean fold induction in the sCSF1 isoform and the mCSF1 isoform were 4.0 ± 1.2 (*P < 0.05), and 1.4 ± 0.3 (P = NS)