Selective deletion of the soluble Colony-Stimulating Factor 1 isoform in vivo prevents estrogen-deficiency bone loss in mice

Gang-Qing Yao,Karl L Insogna,Nancy Troiano,Christine A Simpson

doi:10.1038/boneres.2017.22

Abstract

Neutralizing CSF1 in vivo completely prevents ovariectomy (OVX)-induced bone loss in mice. There are two isoforms of CSF1, soluble (sCSF1), and membrane-bound (mCSF1), but their individual biological functions are unclear. It had been previously reported that mCSF1 knockout (K/O) and wild type (Wt) female mice experience the same degree of bone loss following OVX. In Wt mice the expression of sCSF1 was elevated fourfold in skeletal tissue following OVX while expression of mCSF1 was unchanged. To examine the role of sCSF1 in OVX-induced bone loss, mice were engineered in which sCSF1 was not expressed but expression of mCSF1 was unaffected (sCSF1 K/O). Isoform-specific reverse transcription PCR confirmed the absence of transcripts for sCSF1 in bone tissue isolated from these animals and no circulating CSF1 was detected by ELISA. Surprisingly, there were no significant differences in bone mineral density (BMD) between sCSF1 K/O mice and Wt controls as assessed by dual-energy X-ray absorptiometry and micro-CT. However, one month after OVX, femoral, spinal and total BMD had declined by 11.2%, 8.9%, and 8.7% respectively in OVX-Wt animals as compared to Sham-OVX. In contrast OVX sCSF1 K/O mice showed changes of +0.1%, −2.4%, and +2.3% at the same 3 sites compared to Sham-OVX sCSF1 K/O mice. These data indicate important non-redundant functions for the two isoforms of CSF1 and suggest that sCSF1, but not mCSF1, plays a key role in estrogen-deficiency bone loss.

Highlights

Alternative splicing is the major source of proteome diversity.[1,2] Mutations in splicing sites and changes in the relative expression levels of isoforms have been implicated in the pathogenesis of human diseases.[3,4]Colony-stimulating factor 1 (CSF1) is one of two cytokines absolutely required for osteoclastogenesis
Confirmation of isoform-selective deletion of sCSF1 To determine whether our molecular strategy resulted in isoform-selective deletion of sCSF1, RNA was isolated from tibiae of eight-week-old wild type (Wt) and sCSF1 K/O mice
Isoform-specific quantitative reverse transcription PCR confirmed the absence of sCSF1 transcripts in sCSF1 K/O mice

Summary

Introduction

Alternative splicing is the major source of proteome diversity.[1,2] Mutations in splicing sites and changes in the relative expression levels of isoforms have been implicated in the pathogenesis of human diseases.[3,4]Colony-stimulating factor 1 (CSF1) is one of two cytokines absolutely required for osteoclastogenesis. Alternative splicing is the major source of proteome diversity.[1,2] Mutations in splicing sites and changes in the relative expression levels of isoforms have been implicated in the pathogenesis of human diseases.[3,4]. Multiple human CSF1 mRNA species (4.0, 3.0, 2.3, 1.9, and 1.6 kb) are transcribed from the CSF1 gene,[5,6,7,8,9] and molecular cloning of cDNAs derived from these transcripts has demonstrated that the size differences are due to alternative splicing in exon 6 and the alternative use of the 3′-end of exons 9 and 10.6–8 The 1.6 and 3.0 kb CSF1 cDNAs give rise, by alternative splicing, to a short exon 6 in which the site for proteolytic cleavage of the CSF1 precursor has been spliced out. The products of the 1.6- and 3.0 kb cDNAs are cell-surface (membrane-bound) glycoproteins, which are slowly and inefficiently released from the cell surface by extracellular proteolysis.[9,10] By contrast, the products of the 1.9, 2.3, and 4.0 kb cDNAs have intermediate or long versions of exon 6 in which the proteolytic cleavage site is intact, giving rise to a soluble, rapidly secreted growth factor.[11,12] Similar findings have been reported for the murine CSF1 gene.[13,14]

Methods

Results

Conclusion