Beclin 1, LC3 and P62 Expression in Equine Sarcoids.

Manuela Martano,Pierluigi Liguori,Paola Maiolino,Giuseppe Borzacchiello,Karen Power,Brunella Restucci,Gennaro Altamura

doi:10.3390/ani12010020

Manuela Martano, Pierluigi Liguori + Show 5 more

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https://doi.org/10.3390/ani12010020

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Abstract

Simple SummaryEquine sarcoids, caused by bovine papillomaviruses, are equine skin tumors of fibroblastic origin. It is well known that bovine papillomaviruses are able to interfere with the survival and proliferation of cells by regulating autophagy, a mechanism implicated in the breakdown and reuse of old and damaged cellular material. The present study focused on the evaluation in equine sarcoids and normal skins of the expression level of some of the main proteins involved in the autophagic pathway, such as Beclin 1, LC3 and P62, by immunohistochemical and biochemical techniques. Results obtained in equine sarcoids suggested an alteration of the autophagic process which could lead to a predominance of a particular population of fibroblast. Those fibroblasts could survive longer in a hypoxic microenvironment and produce more and/or altered collagen, giving an origin to the equine sarcoid. Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material. Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples. Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples. Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

Highlights

Normal cell growth requires a balance between the rates of protein synthesis and its degradative processes [1]
The present study focused on the evaluation, by immunohistochemical and biochemical analysis, of the expression level of some of the main autophagy-related proteins, such as the Beclin 1, light chain 3 (LC3) and P62 in BPV1/2 positive equine sarcoids and BPV negative normal skins
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Summary

Introduction

Normal cell growth requires a balance between the rates of protein synthesis and its degradative processes [1]. Autophagy is a complex process that consists of several sequential steps: (1) initiation; (2) elongation; (3) maturation; (4) degradation [2,5,6] All these steps are controlled by several proteins, encoded by more than 30 autophagy-related genes, which lead to newly synthesized doublemembrane vesicles encapsulating cytosolic material, which is delivered to the lysosomes for proteolytic digestion [4,7]. Beclin 1 binds protein light chain 3 (LC3), which is associated with autophagosomal membranes that engulf cytoplasmic content for subsequent degradation During this process, the cytosolic isoform LC3I is converted to the membrane-bound LC3II form [10] and binds to the adaptor protein P62 sequestrome (SQSTM1), which is selectively degraded by autophagy [11]. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen

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