Tension enhances cell proliferation and collagen synthesis by upregulating expressions of integrin αvβ3 in human keloid-derived mesenchymal stem cells

Abstract

Aims

Keloids are a dermal fibrotic disease whose etiology remains totally unknown and for which there is no successful treatment. Mechanical tension, in addition, is closely associated with the germination and development of keloids. In this study, we investigated the influence of human keloid-derived mesenchymal stem cells (KD-MSCs) on cell proliferation, collagen synthesis, and expressions of integrin αvβ3 under tension.

Main methods

KD-MSCs and human normal skin-derived mesenchymal stem cells (NS-MSCs) were isolated and cultured in stem cell medium with a gradual increase in the serum concentration. Cell proliferation and collagen synthesis were detected by Cell Counting Kit-8 (CCK-8) assay and hydroxyproline content analysis under tension respectively. We investigated the messenger RNA expressions of nine integrin subunits, including integrin units α2, α3, α5, αv, α8, α10, α11, β1, and β3, in KD-MSCs stimulated with tension. Identification of differentially expressed genes was performed by Western blot analysis and immunocytochemistry staining.

Key findings

We obtained high-purity KD-MSCs and NS-MSCs using the culture method of decreasing serum concentration gradient gradually. Furthermore, we found that tension enhances cell proliferation and collagen synthesis and promotes expressions of integrin αvβ3 in KD-MSCs. In addition, blocking experiments showed that increased integrin αvβ3 expression affects cell proliferation and collagen synthesis of KD-MSCs under tension.

Significance

Our results suggest that integrin αvβ3 receptor may be sensitive molecules of mechanical tension and could contribute to the occurrence and development of keloids. It could lead to novel targets for therapeutic intervention, treatment, and prevention of recurrence for keloid disorders.

Introduction

Keloid disease, which is characterized by exaggerated response to injury and formation of excessive scar tissues, is a fibroproliferative cutaneous tumor of ill-defined pathogenesis characterized by clinical, behavioral, and histological heterogeneity [1,2]. Its main features include excessive fibroblast proliferation and the overproduction of extracellular matrix (ECM) components such as collagen [3,4]. Unlike hypertrophic scars, which may take years to form and tend to regress over time, keloid scars would appear many years later and extend beyond the site of injury [5]. Keloids can grow excessively and invade nearby normal skin, so the patient with keloid suffers from great physical and psychological pressure. However, most treatments for managing keloids, including surgery, drug therapy, radiotherapy, laser therapy, and cryotherapy, have very limited effectiveness, because keloids grow slowly but progressively and the recurrence rate is very high. Therefore, there is a pressing need to seek a new therapeutic regimen.

Mesenchymal stem cells (MSCs), which can be isolated from many different adult human tissues [6,7], are generally defined as plastic-adherent cells with a fibroblast-like morphology and the capacity for multipotent differentiation in vitro [8,9]. Lately, many efforts have focused on the MSCs that have been postulated as a therapeutic agent to prevent fibrosis [10]. Recent studies have demonstrated that keloid-derived mesenchymal stem cells (KD-MSCs) maintain a relatively higher proliferative rate than their normal skin counterparts [11], but it is still unfamiliar how keloid pathogenesis is affected. Therefore, it is important to research the KD-MSCs and its contribution to the keloid therapeutic strategies.

Through clinical and histopathologic observation, we found that keloid usually occurs at sites that are frequently subjected to mechanical tension, such as anterior chest and scapular regions. Furthermore, data from basic and clinical studies have shown that the pathogenesis of keloid is associated with local mechanical tension [[12], [13], [14]]. The conductive process of tension is a mechanical stimulus transformed into biochemical signals and participated in cellular responses. Coincidentally, integrins comprise one of the most important families of mechanical receptors, which connect the ECM with intracellular actin cytoskeleton and thereby mechanically integrate the extracellular and intracellular compartments. Integrins are heterodimeric transmembrane receptors composed of subunits α and β at a ratio of 1:1. To date, a total of 18 α subunits and 8 β subunits have been identified, and these can form 24 functional heterodimers, which recognize a specific set of ECM ligands [15,16]. They regulate many aspects of cell behavior, including providing positive or negative feedback on the synthesis of the extracellular proteins themselves. Therefore, faulty or absent integrins could be involved in the etiology of keloids. Previous studies found that mechanical tension and integrin α2β1 regulate fibroblast functions using a three-dimensional cultured dermal fibroblast [17]. However, the direct effects of pathologic mechanical tension on KD-MSCs are unclear.

In this study, we first examined cell proliferation and collagen synthesis in KD-MSCs under tension. On this basis we further detected the expressions of nine integrin subunits, including integrin units α2, α3, α5, αv, α8, α10, α11, β1, and β3 in KD-MSCs and investigated which subunits may be involved in cell proliferation and collagen synthesis. This research may improve our understanding of integrins working in complex biological systems on MSCs.