HSP90α higher level of expression in TβRII
+/αSMA
+ cells prompted us to investigate its putative role in the development of the ERMs in vitro using the spontaneously immortalized Müller cell line MIO-M1. We chose this cell line as there is evidence that at least part of ERM cells may derive from Müller cells as previously reported.
9 Our results show that HSP90 overexpression changes substantially TGF-β1-induced signal transduction pathway with an increased expression of TβRII
+, SMAD2, and SMAD3 and with enhanced levels of SMAD2 phosphorylation. TGF-β1, previously reported in iERMs,
7 can therefore activate SMAD2/3 intracellular transduction pathway more efficiently when cells express HSP90. It is well known that TGF-β1 is a profibrotic cytokine. It increases αSMA protein expression and type-I collagen synthesis by binding to TβRII and activating the SMAD pathway, which is crucial for tissue fibrosis.
24 This pathway is partially quenched by Smurf-dependent TβRII ubiquitination, which directs the receptor to the proteasome system.
42 As HSP90 is capable of protecting TβRII from proteasome degradation, it is also able to prolong TβRII phosphorylating activity, to maintain active the SMAD pathway and to increase TGF-β1 target gene expression.
43 Thus HSP90 enhances the profibrotic weight of TGF-β1. Our finding that SMAD2 is translocated to the nucleus of HSP90
+ even in vivo suggests that the well-established mechanisms underlying TGF-β1-induced matrix deposition in several fibrotic diseases
44–47 likely operate in iERMs as well. The use of Epon-embedded samples in this research has certainly reached the goal to allow us to make several different reactions on the same samples and gather more information from the same iERM. In addition, glutaraldehyde fixation and osmium post-fixation achieved a high level of tissue preservation unveiling many structural details that more conventional approaches could have missed. However, this technique has its drawbacks that also represent the limit of the present study. In particular, we could not use regular DNA dyes that do not work with such a strong fixation and on the harsh procedures required to deplasticize sections and to retrieve antigenicity (121°C). Moreover, although many antibodies have been successfully employed in this study, many others fail to bind their antigens in our setting despite that they are known to work with more conventional immunohistochemical approaches (i.e., formalin-fixed paraffin embedded samples). For instance, a convincing labeling of the ILM could not be achieved, although we tried several antibodies against the components of the membrane. Nevertheless, we could achieve a deeper characterization of αSMA
+ myofibroblast-like cells that resulted in GFAP
−/αSMA
+/vimentin
+/HSP90α
+ and in almost half of the cases contained type-I collagen. The positive effects of HSP90 on the activation of the TGF-β1-induced SMAD-dependent transduction pathway has also been confirmed in vitro with HSP90-overexpressing MIO-M1 cells.