Sox9 Determines Translational Capacity During Early Chondrogenic Differentiation of ATDC5 Cells by Regulating Expression of Ribosome Biogenesis Factors and Ribosomal Proteins.

Marjolein M J Caron,Andy Cremers,Mandy J Peffers,Berta Cillero-Pastor,Guus Van Den Akker,Bas Housmans,Maxime Eveque,Tim J M Welting,Kasper Derks,Ron M A Heeren,Lodewijk W Van Rhijn

doi:10.3389/fcell.2021.686096

Abstract

IntroductionIn addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study, we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation.MaterialsSox9 expression in ATDC5 cells was knocked down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 h and 7 days of differentiation. The transcriptomes (RNA-seq approach) and proteomes (Label-free proteomics approach) were compared using pathway and network analyses. Total protein translational capacity was evaluated with the SuNSET assay, active ribosomes were evaluated with polysome profiling, and ribosome modus was evaluated with bicistronic reporter assays.ResultsEarly Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown.ConclusionHere, we identified an essential new function for Sox9 during early chondrogenic differentiation. A role for Sox9 in regulation of ribosome amount, activity, and/or composition may be crucial in preparation for the demanding proliferative phase and subsequent cartilage extracellular matrix production of chondroprogenitors in the growth plate in vivo.

Highlights

In addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late phase expression
Induction of Sox9 expression is biphasic during chondrogenic differentiation of progenitor cells in vitro (Caron et al, 2012; Spaapen et al, 2013)
Since we identified the differential expression of ribosomal protein subunits and ribosome biogenesis factors, combined with unaltered ribosomal RNAs (rRNAs) expression levels, we hypothesized that ribosomes of early Sox9 knockdown ATDC5 cells are functionally distinct

Summary

Introduction

In addition to the well-known cartilage extracellular matrix-related expression of Sox, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox: an immediate early and a late (extracellular matrix associated) phase expression. Chondrogenesis, or chondrogenic differentiation, is the differentiation path of progenitor cells via early mesenchymal condensation into chondrocytes that synthesize a cartilaginous extracellular matrix (ECM) (Kronenberg, 2003; Lefebvre and Smits, 2005; Mackie et al, 2008). Development of the long bones of the mammalian skeleton depends on the activity of growth plates, cartilaginous entities at the ends of developing bones in which chondrocytes differentiate from progenitor cells (Kronenberg, 2003; Lefebvre and Smits, 2005; Mackie et al, 2008). In addition to high amounts of oligosaccharides (mostly hyaluronic acid, heparan sulfate, and chondroitin sulfate), important cartilage ECM proteins are type II collagen (Col2a1) and aggrecan (Acan) (Mankin et al, 2000; de Crombrugghe et al, 2001; Lefebvre and Smits, 2005)

Objectives

Methods

Results

Conclusion