Role of heat shock protein 47 in transdifferentiation of human tenon's fibroblasts to myofibroblasts

Abstract

Background: Heat shock protein 47 (Hsp47) is a well-known molecular chaperone in collagen synthesis and maturation. The aim of this study is to investigate its putative role in the transdifferentiation of Tenon's fibroblasts to myofibroblasts.

Methods: Primary cultured human Tenon's fibroblasts were exposed to transforming growth factor-β1 (TGF-β1) for up to 48 hours. The mRNA levels of Hsp47 and α smooth muscle actin (αSMA) were determined by quantitative real time RT-PCR. After delivery of small interfering RNA (siRNA) molecules targeting Hsp47 into the cells, the expression of Hsp47 and αSMA proteins was determined by western immunoblotting.

Results: TGF-β1 increased the mRNA expressions of both Hsp47 and αSMA in human Tenon's fibroblasts, as determined by quantitative real time RT-PCR. However, it induced the protein expression of only αSMA but not Hsp47, as determined by western immunoblots. When siRNAs specific for Hsp47 were introduced into those cells, the TGF-β1-induced expression of αSMA was significantly attenuated on western immunoblots; after 48 hours of exposure to TGF-β1, the relative densities of immunobands were 11.58 for the TGF-β1 only group and 2.75 for the siRNA treatment group, compared with the no treatment control group (p < 0.001).

Conclusions: Our data suggest that Hsp47 may be related to the TGF-β1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts.

Figure 1

Quantitative real time RT-PCR for heat shock protein 47 (Hsp47) and α smooth muscle actin (αSMA) after exposure to transforming growth factor-β1 (TGF-β1) for up to 48 hours. The level of target mRNA was calculated using a relative ratio to β-actin of no treatment control and expressed as the mean ± S.E.M. (n = 16 for each group).

Figure 2

Representative sequential bands of western immunoblots (A) and densitometric data (B) for heat shock protein 47 (Hsp47), α smooth muscle actin (αSMA), and β-actin. Human Tenon’s fibroblasts were exposed to transforming growth factor-β1 (TGF-β1) for up to 48 hours with or without siRNAs targeting Hsp47. The densitometric data are expressed as the mean ± S.E.M. (n = 16 for each group) *p < 0.001.