Upregulation of phosphorylated HSP27, PRDX2, GRP75, GRP78 and GRP94 in acquired middle ear cholesteatoma growth

Abstract

Cholesteatoma is a destructive and expanding growth of keratinizing squamous epithelium in the middle ear or petrous apex. The molecular and cellular processes of the pathogenesis of acquired middle ear cholesteatoma have not been fully understood. In this study, comparative proteomic analysis was conducted to investigate the roles of specific proteins in the pathways regarding keratinocyte proliferation in cholesteatoma. The differential proteins were detected by comparing the two-dimension electrophoresis (2-DE) maps of the epithelial tissues of 12 attic cholesteatomas with those of retroauricular skins. There were 14 upregulated proteins in the epithelial tissues of cholesteatoma in comparison with retroauricular skin. The modulation of five crucial proteins, HSP27, PRDX2, GRP75, GRP78 and GRP94, was further determined by RT-PCR, Western blot and immunohistochemistry. Phosphorylation of HSP27 at Ser-82 was identified by mass spectroscopy. The results of this study suggested that phosphorylated HSP27 is the end expression of two potential signal-transduction pathways, and together with PRDX2, they are very likely involved in the proliferation of keratinocytes in cholesteatoma. Upregulations of GRP75, GRP78 and GRP94 in keratinocytes may be able to counter endoplasmic reticulum stress, to inhibit cell apoptosis, to prevent protein unfolding and to promote cholesteatoma growth.

Figure 1

2-DE maps of (A) cholesteatoma tissues (pI 4–7) and (B) retroauricular skin (pI 4–7). A total of 23 differential proteins were identified by LC-MS/MS analysis (Table 2).

Figure 2

2D side-by-side comparison of the 2-DE Western blot images of HSP27, PRDX2, GRP78 and GRP75 in cholesteatoma tissues and retroauricular skin run with pI 4–7 and pI 3–10, respectively. Images A, B, C and D are for HSP27. Images E, F, G and H are for PRDX2. Images I, J, K and L are for GRP78. Images M, N, O and P are for GRP75.

Figure 3

MS/MS profile and immunoblot data of phosphorylated HSP27. (A) MS/MS spectrum of phosphorylated HSP27 indicated the phosphorylation site at QLpSSGVSEIR; (B) Verification of phosphorylation-HSP27 (Ser-82) by Western blotting analysis. Cholesteatoma is presented by C, and retroauricular skin is presented by S. β-actin was used for normalization.

Figure 4

Validation of HSP27, GRP75, GRP78, GRP94 and PRDX2 by Western blotting analysis and RT-PCR. The tissues of cholesteatoma and retroauricular skin were collected from six individual patients. Cholesteatoma is presented by C, and retroauricular skin is presented by S. β-actin was used for normalization.

Figure 5

Validation of Ras, Raf, ERK1/2, MEK1/2, p38MAPK and MAPKAPK2 by Western blotting analysis. The tissues of cholesteatoma and retroauricular skin were from six individual patients. Cholesteatoma is presented by C, and retroauricular skin is presented by S. β-actin was used for normalization.

Figure 6

Immunoreactive staining of HSP27 for patient 5. (A,C) A strong and homogenous positive brownish staining in all layers of cholesteatoma epithelium (arrows). (B,D) Heterogeneous positive brownish staining in retroauricular skin (arrows) (C), (D). Immunoreactivity was confined to the cytoplasm of keratinocytes (arrows). The negative control was represented as (E). Magnification: (A and B) ×100; (A inset, C) ×200; (B inset, D) ×200; (E) ×200.