Elsevier

Cellular Signalling

Volume 71, July 2020, 109606
Cellular Signalling

Upregulation of HSP72 attenuates tendon adhesion by regulating fibroblast proliferation and collagen production via blockade of the STAT3 signaling pathway

https://doi.org/10.1016/j.cellsig.2020.109606Get rights and content

Highlights

  • HSP70 is downregulated in a rat model of tendon adhesion.

  • HSP70 elevation attenuates tendon adhesion.

  • HSP70 elevation inhibits phosphorylated STAT3.

  • The phosphorylated STAT3 facilitates tendon adhesion.

  • This study provides new therapeutic target for tendon adhesion.

Abstract

The proliferation of fibroblasts creates an environment favoring post-operative tendon adhesion, but targeted therapy of this pathology remains in its infancy. In this study, we explored the effect of heat shock protein 72 (HSP72), a major inducible member of the heat shock protein family that can protect cells against many cellular stresses including heat shock, on fibroblast proliferation in tendon adhesion, with its underlying mechanisms investigated. HSP72 expression was examined in an established rat model of tendon injury using RT-qPCR and immunoblot analysis. After conducting ectopic expression and depletion experiments in fibroblast NIH3T3 cells, we determined the effects of HSP72 on the expression of α-SMA and STAT3 signaling pathway-related genes, fibroblast proliferation, as well as collagen production. The mRNA (65.46%) and protein (63.65%) expression of HSP72 was downregulated in the rat model of tendon injury. The in vitro experiments revealed that overexpression of HSP72 inhibited fibroblast proliferation (42.57%) and collagen production (45.60%), as well as reducing α-SMA expression (42.49%) and the extent of STAT3 phosphorylation (55.46%). Moreover, we observed that HSP72 overexpression reduced inflammation as well as the number of inflammatory cell infiltration and fibroblasts in vivo. Furthermore, the inhibited extent of STAT3 phosphorylation contributed to the impaired fibroblast proliferation and collagen production evoked by upregulated HSP72. In summary, the present study unveils an inhibitory role of HSP72 in tendon adhesion via inactivation of the STAT3 signaling pathway. This finding may enable the development of new therapeutic strategies for the prevention against tendon adhesion.

Introduction

The tendons consist of a special collagen-rich connective tissue that links muscle to bone, thus transmitting mechanical force to the joint and enabling joint movement in the musculoskeletal system [1]. Tendon adhesion commonly occurs between the surgical tendon and the synovial sheath, and it contributes to tendon dysfunction following operation [2] and incurs common complications including peritendinous adhesion [3]. Various types of collagen in the extracellular matrix (ECM) of tendons exert important functions on the repair and regeneration of injured tissues, whereby fibroblasts, the main cells in the tendon are responsible for the maintenance and modification for the ECM in response to tendon injury [4]. However, functional problems may arise because of immoderate ECM production and tissue contraction following injury, and the persistent (myo) fibroblast proliferation and their excessive ECM production contribute to the pathology of scarring [5]. Elucidating cellular mechanisms that inhibit the proliferation of fibroblast is therefore critical to attenuating tendon adhesion.

The stress-inducible 70-kDa heat shock protein (HSP70) exerts multiple functions intracellularly and extracellularly, and elevation of its expression helps maintain muscle fiber integrity after injury, thereby accelerating the regeneration and recovery of muscle [6]. Heat shock protein 72 (HSP72), a member of the HSP70 family, is a stress-inducible ATPase molecular chaperone, which can stabilize and refold substrate proteins to maintain cellular homeostasis [7]. HSP72 can be synthesized as a cytoprotective response to cellular stress, and its upregulation in human fibroblasts by heat and mechanical stress is a response that involves its nuclear translocation [8]. Interestingly, HSP72 can induce antagonistic actions depending on its location; specifically, intracellular HSP72 exerts an anti-inflammatory function while extracellular HSP72 has the potency to activate pro-inflammatory-related pathways [9,10]. A previous study showed that repressed expression of HSP72 is related to the chronic inflammation in type 2 diabetes [11]. Interestingly, the overexpression of HSP72 induced by thermal preconditioning has the capacity to alleviate tendon adhesion [8]. The regulatory mechanism of HSP70 on signal transducer and activator of transcription (STAT3), a member of the STAT family, has been previously investigated in myeloid-derived suppressor cells [12]. STAT3 exerts effects on the transcription of genes mediating cell proliferation and conversion in phenotype [13]. The STAT3 signaling pathway modulated by aspirin is associated with attenuated inflammation and scar formation during tendon healing [14]. In addition, STAT3 phosphorylation is promoted mainly by proliferating blood vessels in ruptured rotator cuff tendon [15]. Inhibition of STAT3 signaling pathway decreases ECM production and fibroblast proliferation from hypertrophic scars [16]. The aforementioned findings present a possible mechanism by which HSP72 and the STAT3 signaling pathway interact in fibroblast proliferation and tendon adhesion. Thus, we established a mouse tendon injury model to explore the contribution of this pathway to tendon adhesion pathology.

Section snippets

Ethical approval

The study was approved by the Institutional Animal Care and Use Committee of Zhongnan Hospital of Wuhan University. Animal experiments were conducted in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All efforts were made to minimize the number and discomfort of the included animals.

Establishment of a rat model of tendon injury

A total of 120 male Sprague-Dawley rats (aged 9 weeks; weighing 250–300 g) were obtained from the Hubei Provincial

HSP72 elevation contributed to inhibited fibroblast proliferation and attenuated tendon adhesion in vivo

To verify the regulatory role of HSP72 in tendon adhesion, we initially established a rat model of tendon injury. As shown in Fig. 2A, markedly higher scores were observed in the rat model of tendon injury (4.54 + CI 2.03–2.803) than that in normal rats (2.32) (p = .001), while sham-operated rats (2.46 + CI - 0.06 - 0.723) exhibited no obvious disparity (p = .09). HE staining results (Fig. 2B) revealed that compared to normal rats, the rat tendon injury model exhibited disorder of collagen

Discussion

Tendon injuries commonly promote the formation of scar-like tissues with a poor biochemical structure and mechanical properties [23], and the resultant tendon adhesion is a leading cause of gliding dysfunction pain, or even the complex re-operative surgery [24]. Patients suffering from tendon rupture or tendinopathy would experience matrix degeneration and tendon healing [25]. The emerging field of epigenetics has the potential for revealing new molecular biomarkers to prevent or alleviate

Authors contributions

Zhengqi Pan, Qinfen Wu, Zhe Xie and Qiang Wu designed the study. Xinti Tan collated the data, designed and developed the database, carried out data analyses and produced the initial draft of the manuscript. Xin Wang, Zhengqi Pan and Qinfen Wu contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

Data availability statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This study was supported by Science and Technology Innovation and Cultivation Fund of Zhongnan Hospital of Wuhan University (No. znpy2018018).

Declaration of Competing Interest

The authors declare that they have no competing interests.

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