Abstract
Previous expression study using quantitative proteomics has shown that immune-mediated pathway may not be the main mechanism inducing alopecia areata (AA). Nevertheless, functional impact of such expression data set remained unknown and unexplored. This study thus aimed to define potentially novel mechanisms of the AA pathogenesis by functional investigations of the differentially expressed proteins previously identified from lesional biopsies. From 122 altered proteins, protein–protein interactions network analysis revealed that downregulated heat shock protein 90 (HSP90) and lamin A/C served as the central nodes of protein–protein interactions involving in several crucial biological functions, including cytoskeleton organization, extracellular matrix organization, and tissue development. Interaction between HSP90 and lamin A/C in dermal papilla cells (DPCs) was confirmed by reciprocal immunoprecipitation and immunofluorescence co-staining. Small-interfering RNA (siRNA) targeting to HSP90 (siHSP90) and lamin A/C (siLamin A/C) effectively reduced levels of HSP90 and lamin A/C, respectively and vice versa, comparing to non-transfected and siControl-transfected cells, strengthening their interactive roles in DPCs. Functional investigations revealed that DPCs transfected with siHSP90 and siLamin A/C had defective cell proliferation and growth, prolonged doubling time, cell cycle arrest at G0/G1 phase, and defective self-aggregation formation. Moreover, siHSP90-transfected cells had less spindle index, reduced levels of vimentin (mesenchymal marker) and fibronectin (extracellular matrix), and defective migratory activity. Our data have demonstrated for the first time that HSP90 and lamin A/C physically interact with each other. Moreover, both of them are essential for growth, migration, and self-aggregation of DPCs and can be linked to the disease mechanisms of AA.
Highlights
Based on histopathology that frequently shows inflammatory lymphocytes around hair follicles and on clinical response after immunosuppressive treatment, autoimmune mechanism has been hypothesized to play a major role in Official journal of the Cell Death Differentiation AssociationThanomkitti et al Cell Death Discovery (2018)4:53our previous unbiased proteomics study of lesional vs. non-lesional biopsies taken from AA patients has shown that immune-mediated pathway may not be the main mechanism inducing AA pathogenesis[6]
The central nodes of such protein–protein interactions network were validated by reciprocal immunoprecipitation and immunofluorescence co-staining, and subjected to protein knockdown using small-interfering RNA in dermal papilla cells (DPCs) followed by functional investigations of various cellular functions that might be linked to the pathogenic mechanisms of AA
Global protein–protein interactions network analysis Global protein–protein interactions network analysis of all differentially expressed proteins in lesional vs. nonlesional biopsies of AA patients identified from our previous proteomics study[6] revealed that heat shock protein (HSP90) and lamin A/C, both of which were decreased in lesional biopsies[6], served as the central nodes of protein–protein interactions involving in several crucial biological functions, including cytoskeleton organization, extracellular matrix organization, and tissue development (Fig. 1a)
Summary
Based on histopathology that frequently shows inflammatory lymphocytes around hair follicles and on clinical response after immunosuppressive treatment, autoimmune mechanism has been hypothesized to play a major role in Official journal of the Cell Death Differentiation AssociationThanomkitti et al Cell Death Discovery (2018)4:53our previous unbiased proteomics study of lesional vs. non-lesional biopsies taken from AA patients has shown that immune-mediated pathway may not be the main mechanism inducing AA pathogenesis[6]. Most of the differentially expressed proteins identified from this study seem to get involved in potentially novel mechanisms of AA6. Our present study aimed to define potentially novel mechanisms of the AA pathogenesis by functional investigations of such large-scale expression proteomics data set. All the differentially expressed proteins identified from lesional vs non-lesional biopsies were subjected to global protein–protein interactions network analysis. The central nodes of such protein–protein interactions network were validated by reciprocal immunoprecipitation and immunofluorescence co-staining, and subjected to protein knockdown using small-interfering RNA (siRNA) in dermal papilla cells (DPCs) (which are the major compositions of hair follicles and are crucial for hair growth) followed by functional investigations of various cellular functions that might be linked to the pathogenic mechanisms of AA
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