Na+, K+-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms

Ali Mobasheri,Mari-Francis Arteaga,Pablo Martín-Vasallo,Elisa Trujillo

doi:10.3390/ijms13045019

Abstract

Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons). Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4) using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes.

Highlights

Chondrocytes are specialized resident cells of articular cartilage responsible for the maintenance and turnover of highly charged extracellular matrix (ECM) macromolecules that endow cartilage with its unique load bearing properties [1,2]
Selected antibodies against the α1, α2 and α3 isoforms (α6F, McB2 and XVIF9G10 monoclonal antibodies) were characterized by western blotting to confirm their cross-reactivity with their respective protein targets in human brain and skeletal muscle (Figure 1)
Maintaining an optimal Na+:K+ ratio is essential for intracellular homeostasis and for the synthesis of a mechanically resilient extracellular matrix

Summary

Introduction

Chondrocytes are specialized resident cells of articular cartilage responsible for the maintenance and turnover of highly charged extracellular matrix (ECM) macromolecules that endow cartilage with its unique load bearing properties [1,2]. Chondrocytes must survive in an unusual ionic and osmotic environment that makes the maintenance of intracellular [Na+], [K+] and pH a high priority if the physiological turnover of cartilage matrix is to be accomplished [3]. The mechanical performance of cartilage relies on the biochemical properties of matrix macromolecules and any alterations to the ionic and osmotic extracellular environment of chondrocytes in turn influence the volume, intracellular pH and ionic content of the cells [12]. These changes in turn modify the synthesis and degradation of extracellular matrix macromolecules [13]. Physiological ion homeostasis is fundamental to the routine functioning of cartilage and the factors that control the integrity of this highly evolved and specialized tissue

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