The roles of Gas6 and Axl in endochondral bone growth

Philip Newton

Research output: ThesisDoctoral Thesis

Abstract

During endochondral ossification, mesenchymal cells undergo differentiation into chondrocytes followed by further differentiation to a state of hypertrophy. The chondrocytes secrete specific proteins and proteoglycans to form an extracellular matrix which becomes mineralised within the hypertrophic zone. The control of endochondral ossification is critical to normal linear bone growth. The tyrosine kinase, Axl, and its ligand, growth arrest-specific 6 (Gas6), can fulfil a broad range of functions in various cell types. A study using the mesenchymal cell-line C3H10T1/2 has shown that Gas6 transcription decreased upon chondrogenic differentiation. In addition, Gas6/Axl signalling has been shown to promote the growth and survival of articular chondrocytes, in vitro. During vascular calcification, Axl signalling can prevent mineralisation by inhibiting the apoptosis of osteogenic vascular smooth muscle cells. Gas6/Axl signalling is also able to inhibit vascular calcification by osteogenic pericytes. Hence, the aim of this project was to test the hypothesis that Gas6 and Axl could regulate the process of endochondral ossification.
This thesis reports the detection of Gas6 and Axl mRNA expression by murine growth plate chondrocytes. Furthermore, Gas6 mRNA was spatially localised to the proliferating chondrocytes of 28-day old mice. Gas6 protein was immuno-detected in all zones of the murine growth plate, but was most concentrated in the proliferating zone chondrocytes. An in vitro model of chondrocyte differentiation and extracellular matrix mineralisation during endochondral ossification was developed and characterised using the ATDC5 cell-line. In this model, Gas6 (mRNA) and Axl (mRNA and protein) expression patterns were comparable to the current in vivo data, when compared with the expression of chondrocytes markers. In addition, a temporal pattern of Axl phosphorylation was detected in this model, indicating that signal transduction via Axl occurred. By inhibiting Axl signalling using an Axl-specific inhibitor, the rate of chondrocyte marker expression significantly increased. This indicated that Axl signalling reduced the rate of chondrocyte differentiation in vitro. Growth plate organisation, histomorphometry and mineralisation was similar in wild-type and Axl-/- mice. The tibial lengths of litter-mate pups resulting from Axl+/- crossed mice were not significantly different at 28-days of age between the resultant Axl-genotypes. However, Axl-deficient mice had significantly reduced body mass at 28-days of age compared with wild-type controls.
These results demonstrate that Axl and Gas6 are expressed by growth plate chondrocytes in a differentiation-dependent manner. Furthermore, Axl signalling appears to regulate chondrocyte function and ultimately, the process of endochondral ossification.
Original languageEnglish
Awarding Institution
  • The University of Manchester
Supervisors/Advisors
  • Farquharson, Colin, Supervisor
  • Canfield, Anne, Supervisor, External person
Thesis sponsors
Publication statusPublished - 2012
Externally publishedYes

Keywords

  • Gas6
  • Axl
  • bone
  • growth plate
  • cartilage

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