Identification of Chondrocyte Genes and Signaling Pathways in Response to Acute Joint Inflammation

Mengxi Lv,X Lucas Lu,Liyun Wang,Yilu Zhou,Lin Han,Meiqing Wang,Leo Q Wan,Shawn W Polson

doi:10.1038/s41598-018-36500-2

Abstract

Traumatic joint injuries often result in elevated proinflammatory cytokine (such as IL-1β) levels in the joint cavity, which can increase the catabolic activities of chondrocytes and damage cartilage. This study investigated the early genetic responses of healthy in situ chondrocytes under IL-1β attack with a focus on cell cycle and calcium signaling pathways. RNA sequencing analysis identified 2,232 significantly changed genes by IL-1β, with 1,259 upregulated and 973 downregulated genes. Catabolic genes related to ECM degeneration were promoted by IL-1β, consistent with our observations of matrix protein loss and mechanical property decrease during 24-day in vitro culture of cartilage explants. IL-1β altered the cell cycle (108 genes) and Rho GTPases signaling (72 genes) in chondrocytes, while chondrocyte phenotypic shift was observed with histology, cell volume measurement, and MTT assay. IL-1β inhibited the spontaneous calcium signaling in chondrocytes, a fundamental signaling event in chondrocyte metabolic activities. The expression of 24 genes from 6 calcium-signaling related pathways were changed by IL-1β exposure. This study provided a comprehensive list of differentially expressed genes of healthy in situ chondrocytes in response to IL-1β attack, which represents a useful reference to verify and guide future cartilage studies related to the acute inflammation after joint trauma.

Highlights

Due to its important role in OA pathology, IL-1β-treated articular cells or tissues have been widely adopted as in vitro models to study OA initiation or PTOA6,11
Loss was defined as the total sGAG released into the culture medium divided by total sGAG content in explant and culture medium. (c) Accumulative collagen loss from cartilage explant in 24-day treatment of 1 ng/mL IL1β. (d) The total sGAG content of cartilage explants, which includes the sGAG released into medium and sGAG left in explants at the end of 8-day culture. (e) The total collagen content and distribution of collagen in medium and explant after 24-day culture. (f) Safranin O staining of the superficial zone of cartilage explant after 2- and 8-day culture
Chondrocytes demonstrate drastic genetic changes before cartilage shows visible degradation, whilst they adapt to the degenerated extracellular environment with little chondrogenic features at the late stage of OA5,24

Summary

Introduction

Due to its important role in OA pathology, IL-1β-treated articular cells or tissues have been widely adopted as in vitro models to study OA initiation or PTOA6,11 For both isolated and in situ chondrocytes, IL-1β has been observed to induce transient concentration changes of intracellular calcium ([Ca2+]i) and small GTPases. The coordination of Rho GTPases signaling and [Ca2+]i signaling plays a fundamental role in cytoskeleton organization, regulating the chondrocyte phenotypic shift and cartilage ECM homeostasis[8,9] Many of these studies focused on specific genes/pathways in chondrocytes. To maintain the natural environment of chondrocytes and perform longitudinal evaluation of cells and ECM, fresh cartilage explants were cultured in vitro and treated with IL-1β. To verify and understand the changes in genetic profiles, we tracked the loss and synthesis of ECM components longitudinally, the poroelastic properties of ECM, the proliferation of cells, the change of cell volume and calcium signaling of in situ chondrocytes

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