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Autophagy Is Deficient and May be Negatively Regulated by SERPINB3 in Middle Ear Cholesteatoma
Kuen-Yao Ho1,2, Chih-Jen Huang3, Chih-Chang Hung4
1Department of Otolaryngology, Kaohsiung Medical University Hospital.
Hypothesis:
Whereas autophagy has been linked to various human diseases, whether it also plays a role in cholesteatoma is virtually unknown. This study aimed to investigate the activity and regulation of autophagy in cholesteatoma.
Background:
The treatment of middle ear cholesteatoma has been challenging due to an insufficient understanding of the underlying disease mechanism.
Methods:
Expression of microtubule-associated protein 1A/1B-light chain 3 (LC3), the autophagy protein marker, and phosphorylated Akt (p-Akt), and mammalian target of rapamycin (p-mTOR), the known autophagy regulators, in fresh retroauricular skin and cholesteatoma tissue samples was analyzed by immunoblotting. The results were further confirmed by immunohistochemistry and statistical analyses. Cell proliferation of primary retroauricular skin- and cholesteatoma-derived fibroblasts was evaluated by methyl thiazol tetrazolium (MTT) assay. Ectopic expression of serine proteinase inhibitor, clade B, member 3 (SERPINB3) in the fibroblasts was achieved by electroporation and the expression was detected by immunoblotting.
Results:
LC3 expression was significantly decreased in cholesteatoma in most of the 15 paired retroauricular skin/cholesteatoma tissue samples. However, p-Akt and p-mTOR expression in the cholesteatoma samples was not significantly different from that in the control subjects. Immunohistochemical studies further demonstrated an inverse correlation between LC3 expression and cholesteatoma. The cholesteatoma fibroblasts proliferated faster than the retroauricular skin fibroblasts, and had higher SERPINB3 but lower LC3 expression. Furthermore, overexpression of SERPINB3 in the retroauricular skin fibroblasts enhanced cell proliferation and downregulated LC3 expression.
Conclusion:
Autophagy is significantly suppressed in cholesteatoma tissues, which may not involve the Akt/mTOR signaling pathway. More importantly, SERPINB3 may promote cell proliferation and negatively regulate autophagy in cholesteatoma fibroblasts. Together, these findings warrant further investigation into the pathogenic mechanism of cholesteatoma.
Insights
Autophagy is suppressed in cholesteatoma, with decreased LC3 protein expression. The protein SERPINB3 promotes cholesteatoma fibroblast proliferation and inhibits autophagy, suggesting a new therapeutic target.
Area of Science:
- Cell biology
- Otolaryngology
- Molecular pathology
Background:
- Middle ear cholesteatoma treatment is challenging due to poorly understood disease mechanisms.
- Autophagy's role in cholesteatoma pathogenesis is largely unknown.
Purpose of the Study:
- To investigate autophagy activity and regulation in cholesteatoma.
- To explore the role of SERPINB3 in cholesteatoma fibroblast behavior.
Main Methods:
- Immunoblotting and immunohistochemistry to analyze autophagy markers (LC3) and regulators (p-Akt, p-mTOR) in cholesteatoma tissues.
- MTT assay to assess fibroblast proliferation.
- Gene expression analysis of SERPINB3 in fibroblasts.
Main Results:
- Cholesteatoma tissues showed significantly decreased LC3 expression compared to normal skin.
- Akt/mTOR signaling pathway did not appear to be involved in autophagy regulation in cholesteatoma.
- Cholesteatoma fibroblasts exhibited increased proliferation and SERPINB3 expression, with decreased LC3 expression.
- Overexpression of SERPINB3 enhanced fibroblast proliferation and reduced LC3 expression.
Conclusions:
- Autophagy is suppressed in cholesteatoma, potentially independent of the Akt/mTOR pathway.
- SERPINB3 may drive cholesteatoma progression by promoting cell proliferation and inhibiting autophagy.
- Further research into SERPINB3's role is warranted for understanding cholesteatoma pathogenesis.
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