Abstract
Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3β (GSK3β) as a novel key mediator of FGF inhibitory response in chondrocytes.
Highlights
The family of fibroblast growth factors (FGFs) is represented by 23 members
To understand the determinants of chondrocyte FGF inhibitory response we sought to identify the set of proteins whose phosphorylation status is changed upon FGF1 treatment
Rat ChondroSarcoma (RCS) cells were used for our experiments as a well-accepted model of proliferative chondrocytes which respond to FGF signaling with growth arrest [5, 19]
Summary
The family of fibroblast growth factors (FGFs) is represented by 23 members. Most of them (excluding FGF11-FGF14) activate FGF receptors (FGFRs) that trigger multiple signaling cascades [1, 2]. Unlike most cell types, where FGF signaling induces proliferation, chondrocytes undergo growth arrest when exposed to FGFs [4,5,6,7]. We utilized tandem mass tag (TMT) labeling to catalogue FGF1-induced changes in the chondrocyte phosphoproteome. This data set provides an array of proteins whose phosphorylation status is changed upon FGF1 treatment. Immediate FGF1 response was previously characterized by immunoblotting and some differences between signaling pathways induced by FGF1 in chondrocytes compared to cells with proliferative response were reported [4, 17]. We chose to investigate FGF1 response at intermediate time points to identify proteins which are targeted by FGF signaling in chondrocytes and might play a role in FGF inhibitory response
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