The ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation

Hum Mol Genet. 2013 Aug 1;22(15):3048-62. doi: 10.1093/hmg/ddt162. Epub 2013 Apr 9.

Abstract

The severe defects in growth plate development caused by chondrocyte extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) gain or loss-of-function suggest that tight spatial and temporal regulation of mitogen-activated protein kinase signaling is necessary to achieve harmonious growth plate elongation and structure. We provide here evidence that neurofibromin, via its Ras guanosine triphosphatase -activating activity, controls ERK1/2-dependent fibroblast growth factor receptor (FGFR) signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking neurofibromin 1 (Nf1) in type II collagen-expressing cells, (Nf1col2(-/-) mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-of-function mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, matrix metalloproteinase 9 (Mmp9) and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1(-/-) chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that return to the basal level is delayed in Nf1(-/-) chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide to post-natal pups for 18 days was able to correct the short stature of Nf1col2(-/-) mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / genetics
  • Cell Proliferation
  • Chondrocytes / metabolism
  • Chondrogenesis / physiology*
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • Gene Expression
  • Growth Plate / metabolism
  • Mice, Knockout
  • Neurofibromin 1 / genetics
  • Neurofibromin 1 / metabolism*
  • Osteogenesis / physiology*
  • Receptors, Fibroblast Growth Factor / metabolism*
  • Signal Transduction*

Substances

  • Neurofibromin 1
  • Receptors, Fibroblast Growth Factor
  • Extracellular Signal-Regulated MAP Kinases