Up-regulation of HSP70 by the fibronectin-derived peptide PHSRN in human corneal epithelial cells

https://doi.org/10.1016/j.bbrc.2008.03.093Get rights and content

Abstract

HSP70 is a member of the heat shock protein family and is induced by various types of cellular stress. We examined whether HSP70 might play a role in the healing of corneal epithelial wounds. Given that the PHSRN peptide, which corresponds to the second cell-binding domain of fibronectin, promotes corneal epithelial migration, we investigated the effect of this peptide on HSP70 expression in cultured human corneal epithelial cells. Reverse transcription–polymerase chain reaction and immunoblot analyses revealed that PHSRN increased the amounts of HSP70 mRNA and protein in these cells in a concentration- and time-dependent manner, whereas a control peptide (NRSHP) had no such effects. Furthermore, the PHSRN-induced up-regulation of HSP70 was blocked by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), but it was not affected by PD98059 or SP600125, inhibitors of signaling by the MAPKs ERK and JNK, respectively. Our results suggest that induction of HSP70 expression may contribute to the promotion of corneal epithelial migration by PHSRN and hence to corneal epithelial wound healing.

Section snippets

Materials and methods

Antibodies and reagents. Mouse monoclonal antibodies to HSP70 and to α-tubulin were obtained from Abcam (Cambridge, MA) and Sigma (St. Louis, MO), respectively. Horseradish peroxidase-conjugated secondary antibodies were obtained from Promega (Madison, WI). SB203580 and PD98059 were obtained from Calbiochem (San Diego, CA), and SP600125 was from Merck (White House Station, NJ). The peptides PHSRN and NRSHP were obtained from Peptide Institute (Osaka, Japan).

Cells. A simian virus 40-transformed

Effects of PHSRN on HSP expression in HCE cells

We first investigated the effects of the PHSRN peptide on the expression of HSP27, HSP47, HSP70, and HSP90 in HCE cells. RT–PCR analysis revealed that the amount of HSP70 mRNA in HCE cells exposed to PHSRN (1 μg/ml) for 12 h was markedly increased compared with that in cells exposed to the control peptide NRSHP or incubated alone (Fig. 1). In contrast, PHSRN had no effect on the amounts of HSP27, HSP47, or HSP90 mRNAs.

The effect of PHSRN on the abundance of HSP70 mRNA was concentration dependent,

Discussion

We have shown that the PHSRN peptide up-regulated expression of HSP70, but not that of other HSPs (HSP27, HSP47, HSP90), in HCE cells. This effect of PHSRN was apparent at both the protein and mRNA levels and appeared to be mediated by the p38 MAPK signaling pathway, but not by the ERK or JNK signaling pathways. Our results thus suggest that up-regulation of HSP70 expression by PHSRN, corresponding to the second cell-binding domain of fibronectin, may contribute to the stimulation of corneal

Acknowledgments

We thank Shizuka Murata, Yukari Mizuno, and Yasumiko Akamatsu for technical assistance.

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