Downregulation of Heat Shock Protein 70 Impairs Osteogenic and Chondrogenic Differentiation in Human Mesenchymal Stem Cells

Chenghai Li,Sihong Wang,Kristifor Sunderic,Steven B Nicoll

doi:10.1038/s41598-017-18541-1

Abstract

Human mesenchymal stem cells (hMSCs) show promise for bone and cartilage regeneration. Our previous studies demonstrated that hMSCs with periodic mild heating had enhanced osteogenic and chondrogenic differentiation with significantly upregulated heat shock protein 70 (HSP70). However, the role of HSP70 in adult tissue regeneration is not well studied. Here, we revealed an essential regulatory mechanism of HSP70 in osteogenesis and chondrogenesis using adult hMSCs stably transfected with specific shRNAs to knockdown HSP70. Periodic heating at 39 °C was applied to hMSCs for up to 26 days. HSP70 knockdown resulted in significant reductions of alkaline phosphatase activity, calcium deposition, and gene expression of Runx2 and Osterix during osteogenesis. In addition, knockdown of HSP70 led to significant decreases of collagens II and X during chondrogenesis. Thus, downregulation of HSP70 impaired hMSC osteogenic and chondrogenic differentiation as well as the enhancement of these processes by thermal treatment. Taken together, these findings suggest a putative mechanism of thermal-enhanced bone and cartilage formation and underscore the importance of HSP70 in adult bone and cartilage differentiation.

Highlights

Considering non-specificity of chemical inhibitors, target-specific heat shock protein 70 (HSP70) shRNAs were used to knock down HSP70 expression in Human mesenchymal stem cells (hMSCs), and the role of HSP70 in osteogenic and chondrogenic differentiation of hMSCs was examined with and without HSP70 knockdown
It was found that HSP70 knockdown impaired osteogenic and chondrogenic differentiation and diminished heat-enhanced osteogenesis and chondrogenesis in hMSCs
Effects of periodic mild heating at 39 °C for one hour once every two days on hMSC chondrogenic differentiation at Day 17 are illustrated in Fig. 1B with upregulated type II collagen in pellet cultures using hMSCs transfected with scrambled shRNA

Summary

Introduction

Considering non-specificity of chemical inhibitors, target-specific HSP70 shRNAs were used to knock down HSP70 expression in hMSCs, and the role of HSP70 in osteogenic and chondrogenic differentiation of hMSCs was examined with and without HSP70 knockdown. An early study indicated that heating 1.5–3 °C above regular body temperature plays a role in bone growth stimulation in rats and dogs[15]. In this study hMSCs were exposed to mild heating at 39 °C for one hour once every other day during osteogenic and chondrogenic differentiation of 3 to 4 weeks. Osteogenic differentiation of hMSCs is typically assessed by monitoring alkaline phosphatase (ALP) activity, an early marker of osteogenic differentiation, and calcium deposition, a late indicator[22,23]. It was found that HSP70 knockdown impaired osteogenic and chondrogenic differentiation and diminished heat-enhanced osteogenesis and chondrogenesis in hMSCs. It was found that HSP70 knockdown impaired osteogenic and chondrogenic differentiation and diminished heat-enhanced osteogenesis and chondrogenesis in hMSCs Overall, these data demonstrate that HSP70 plays a direct and key role in heat-enhanced hMSC osteogenesis and chondrogenesis. This study may result in a potential new approach toward prevention and treatment of diseases related to bone and cartilage loss

Methods

Results

Conclusion