Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice

Abstract

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths—called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton. Here, we carried out a retrospective analysis of cervical spine MRI and CT scans from 50 consecutive HME patients that included cranial skeletal images. Interestingly, nearly half of the patients displayed moderate defects or osteochondroma-like outgrowths in the cranial base and specifically in the clivus. In good correlation, osteochondromas developed in the cranial base of mutant Ext1f/f;Col2-CreER or Ext1f/f;Aggrecan-CreER mouse models of HME along the synchondrosis growth plates. Osteochondroma formation was preceded by phenotypic alteration of cells at the chondro-perichondrial boundary and was accompanied by ectopic expression of major cartilage matrix genes -collagen 2 and collagen X- within the growing ectopic masses. Because chondrogenesis requires bone morphogenetic protein (BMP) signaling, we asked whether osteochondroma formation could be blocked by a BMP signaling antagonist. Systemic administration with LDN-193189 effectively inhibited osteochondroma growth in conditional Ext1-mutant mice. In vitro studies with mouse embryo chondrogenic cells clarified the mechanisms of LDN-193189 action that turned out to include decreases in canonical BMP signaling pSMAD1/5/8 effectors but interestingly, concurrent increases in such anti-chondrogenic mechanisms as pERK1/2 and Chordin, Fgf9 and Fgf18 expression. Our study is the first to reveal that the cranial base can be affected in patients with HME and that osteochondroma formation is amenable to therapeutic drug intervention.

Aberrant perichondrial BMP signaling mediates multiple osteochondromagenesis in mice

“…In this study, we demonstrate the suppressive effect of LDN-193189 in two MHE models based on conditional KO of Ext1. While this paper was being revised, Sinha et al (39) reported a similar effect of LDN-193189 on osteochondroma-like bone outgrowth that develops in another Ext1-conditional KO model (Acan-Cre ER ;Ext1 flox/flox ). Thus, the molecular mechanism of osteochondromagenesis shown in this paper appears to be applicable to at least three MHE mouse models.…”

Dwarfism in homozygous Agc1CreERT mice is associated with decreased expression of aggrecan

“…One article reported the use of Agc Cre/Cre mice for their analysis (Maihiot et al, 2015). However, most of the articles do not state if Agc +/Cre or Agc Cre/Cre mice were used for phenotypic analysis (Shen et al, 2017; Liao et al, 2017; Hu et al, 2017; Sinha et al, 2017; Jing et al, 2015, 2016; Liu et al, 2015; Zhou et al 2014; Ono et al, 2014; Jing et al 2014; Henry et al, 2012). Therefore, caution must be exercised when interpreting data using this model as Agc Cre/Cre mice themselves have a skeletal phenotype.…”

“…Tamoxifen treatment results in nuclear translocation of the Cre-ERT fusion protein and subsequent excision of a floxed gene. Several studies have utilized the Agc Cre model to investigate the role of target genes in developing cartilage tissue (Shen et al, 2017; Liao et al, 2017; He et al, 2017; Hu et al, 2017; Sinha et al, 2017; Jing et al 2016; Liu et al, 2015; Maihiot et al, 2015; Zhou et al 2014; Ono et al, 2014; Jing et al 2014; 2013; Henry et al, 2012). Prior to breeding with our floxed mice, we noted variable weight and size among the Agc Cre littermates.…”

Domains with highest heparan sulfate–binding affinity reside at opposite ends in BMP2/4 versus BMP5/6/7: Implications for function

“…HME is characterized by cartilaginous tumors (called exostoses or osteochondromas) that develop within perichondrium flanking the growth plates of long bones, ribs and other elements, but the underlying mechanisms had long remained unclear (62,63). Using mouse models, we found that conditional Ext1 ablation in perichondrial cells (and concurrent drop in HS levels) caused a sharp decrease in local ERK/FGF signaling and a reciprocal increase in canonical BMP signaling (64,65). These opposing signaling changes were followed by the development of cartilaginous tumors in very good correlation with the fact that normally, BMP signaling is prochondrogenic while ERK/FGF signaling is anti-chondrogenic (66,67).…”