ATDC5 cells as a model of cartilage extracellular matrix neosynthesis, maturation and assembly
Graphical abstract
Introduction
The extracellular matrix (ECM) is primarily composed of proteoglycans (PGs) and fibrillar collagens, the most abundant proteins of the human body. It provides a critical scaffold for cells, is required for essential cell functions (motility, proliferation, survival and differentiation) [1] and supports most of the biomechanical properties of tissues and organs [2]. Although very distinct in their composition and elementary functions, both collagens and PGs consist in particularly large macromolecular complexes. Their building blocks carry multiple and tightly regulated post-translational modifications (PTMs) that perfectly cope with intrinsic needs and solicitations of each organ [3]. The crucial role of the ECM is highlighted by the wide spectrum of syndromes and diseases, from minor to lethal, that arise from genetic abnormalities in ECM proteins [[4], [5], [6]] and enzymes which modify them [[7], [8], [9]].
Over decades, remarkable improvements were achieved in describing ECM molecules [2,10], notably through mass spectrometry [[11], [12], [13]] and characterizing the molecular mechanisms of their processing [[14], [15], [16], [17]]. In vertebrates, cartilage and bone cells produce among the most abundant and dense ECM. In vivo high-throughput analysis of the chondrocyte transcriptome [18,19] and proteome [20,21] has brought a global picture of the major genes and proteins involved in cartilage ECM build-up. However, many of the processing mechanisms remain unsolved. In order to promote a more comprehensive characterization of the molecular mechanisms of cartilage ECM synthesis, the field would benefit from models of a lower complexity, such as established cell lines that recapitulate a substantial part of the sequential events occurring in vivo. Many established cell lines secrete procollagens and at least some of their processing intermediates into the medium. Nevertheless, very few are capable of assembling collagen into an insoluble network, and when they do, it is abnormally matured [22,23], demonstrating how unsatisfactorily they recapitulate in vivo situations.
The mouse embryonal carcinoma-derived cell line ATDC5 is a widely used cellular model of chondrogenesis [[24], [25], [26], [27], [28], [29], [30], [31]], referenced in more than 600 publications. It could potentially represent an instrumental model to investigate the mechanisms of ECM assembly in vitro. However, to date, the ATDC5 model has only been described at histological and transcriptomic levels [[32], [33], [34]]. Herein, for the first time, we inspected the secreted proteome of insulin-stimulated ATDC5 (iATDC5), compared to unstimulated ATDC5 (cATDC5) as controls, in order to determine to what extent these cells behave as a model of cartilage ECM synthesis.
Section snippets
Cell culture
The ATDC5 cell line was obtained from the Riken Cell Bank (Tsukubai, Japan) at passage 12 and cultured at all steps at 37 °C in a humidified atmosphere of 5% CO2–20% O2. ATDC5 cells were cultured in expansion medium consisting in DMEM/F-12 containing 5% FBS (11320–033 Gibco), 5.5 μg/ml human transferrin (T3309 Sigma), and 3.10−8 M sodium selenite (S5261 Sigma) as previously described [24]. Here, we used the most consensual differentiation method as follows: ATDC5 cells were plated at 3.105
The chondrogenic-like phenotype of iATDC5 confirmed at the proteome level by soluble secretome analysis
After confirming that the phenotype of our samples were in accordance with the literature [26,28] (Supplemental Data 2), we examined their soluble secretomes [43] as previously described [35,37]. 72 proteins being significantly up- or down-regulated were revealed. To obtain a comprehensive description of the related groups of regulated proteins, we performed Gene Ontology (GO) annotation enrichment analyses, focusing on biological processes. Fig. 1. A shows the significant top four enriched GO
Discussion
Since its first description in 1990 [24], the ATDC5 model has become truly popular in the field of chondrogenesis and spread through worldwide laboratories, allowing pertinent functional characterization of multiple actors of chondrogenesis without the complexity of in vivo experiments [25,27,28,[51], [52], [53]]. Noticeably, iATDC5 cells accumulate fibrillar collagen-resembling structures into their ECM, as shown by electron microscopy [31].
However, the ATDC5 proteome remained largely unknown
Formatting of funding sources
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
None.
Acknowledgements
We are grateful to Frédéric Cailotto and David Moulin, from our laboratory, for their critical reading of the manuscript. The proteomics core facility of UMS 2008 UL-CNRS-INSERM IBSLor acknowledges the funding from CPER IT2MP from Région Grand Est.
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