Proteomic Analysis of the Trabecular Meshwork of Rats in a Steroid-Induced Ocular Hypertension Model: Downregulation of Type I Collagen C-Propeptides

Background/Aims: In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress. In our previous studies, we found that periodic mechanical stress can promote proliferation and matrix synthesis through the integrin beta 1-mediated ERK1/2 pathway, and we used proteomic analysis to detect quantitative changes in chondrocytes under periodic mechanical stress. Despite these results, the effects and mechanisms of periodic mechanical stress are still not fully understood, so in this study we extended our study using phosphoproteomic techniques. Methods: We used phosphoproteomic techniques to detect phosphorylation changes in chondrocytes under periodic mechanical stress and combined the results with the quantitative proteomic data to further explore the underlying mechanisms. Data were obtained by phosphorylation inhibition, quantitative real-time PCR (qPCR) analysis, western blot analysis and immunofluorescence assay. Results: From phosphoproteomic analysis, a total of 1073 phosphorylated proteins and 2054 phosphopeptides were identified. The number of significant differentially expressed proteins and phosphopeptides was 97 and 108, respectively (ratio >1.20 or <0.83 at p<0.05). Periodic mechanical stress increased glycogen synthase kinase 3-beta (GSK3-beta) phosphorylation at Y216, promoted the phosphorylation of beta-catenin, decreased beta-catenin levels and suppressed the expression of type I collagen. In contrast, inhibition of GSK3-beta by TWS119, which specifically inhibits the phosphorylation of Y216, suppressed the phosphorylation of beta-catenin, which resulted in the accumulation of beta-catenin and an increase in the expression of type I collagen. Conclusions: We successfully constructed differentially expressed phosphoproteomic profiles of rat chondrocytes under periodic mechanical stress, and discovered a potential new therapeutic benefit in which periodic mechanical stress suppressed the formation of type I collagen in the matrix of chondrocytes via phosphorylation of GSK3-beta and beta-catenin.

Download Full-text

Proteomic Investigation of Dermal Fibroblasts Isolated from Affected and Unaffected Skin Samples from Patients with Limited Cutaneous Systemic Sclerosis: 2 Distinct Entities?

The Journal of Rheumatology ◽

10.3899/jrheum.160736 ◽

2016 ◽

Vol 44 (1) ◽

pp. 40-48 ◽

Cited By ~ 7

Author(s):

Claudio Corallo ◽

Annalisa Santucci ◽

Giulia Bernardini ◽

Natale Figura ◽

Roberto Leoncini ◽

...

Keyword(s):

Systemic Sclerosis ◽

Proteomic Analysis ◽

Type I Collagen ◽

Dermal Fibroblasts ◽

Type I ◽

Matrix Remodeling ◽

Mrna Levels ◽

Qrt Pcr ◽

Unaffected Skin ◽

Skin Samples

Objective.To identify using proteomic analysis the proteins of altered abundance in the affected and unaffected limited cutaneous systemic sclerosis (lcSSc) skin fibroblasts.Methods.Excision biopsies (3 mm) were obtained from the affected and unaffected skin of 5 patients with lcSSc. Dermal fibroblasts were isolated enzymatically. Two-dimensional gel electrophoresis was used to separate and define proteins in affected and unaffected fibroblast lysates. Proteins of altered abundance were identified by mass spectrometry. Differences among skin samples were confirmed also by immunohistochemistry (IHC) and by quantitative real-time PCR (qRT-PCR) for type I collagen (Col-1) and vimentin (VIM).Results.Proteomic analysis revealed different expressions of proteins involved in cytoskeleton organization (27%), extracellular matrix remodeling (11%), response to oxidative stress (22%), energy metabolism (19%), protein metabolism (5%), cellular homeostasis (5%), signal transduction (3%), and protein transcription, synthesis, and turnover (8%). IHC analysis showed that SSc-affected epidermis is thickened and the dermis is strongly reactive to Col-1 and VIM (typical markers of activated myofibroblasts) compared to SSc-unaffected skin, whose stainings are comparable to those of control healthy skin. Overexpression of Col-1 and VIM mRNA levels in affected lcSSc fibroblasts compared to unaffected lcSSc ones was confirmed by qRT-PCR.Conclusion.Consistent with previous studies, these findings are important for 2 reasons: first, because they reveal the opposite behavior of dermal fibroblasts in the unaffected and affected skin areas of the same patient with lcSSc; second, because they demonstrate the histological/histochemical similarities between unaffected skin from patients with lcSSc and healthy control skin.

Download Full-text

Up-regulation of type I collagen during tumorigenesis of colorectal cancer revealed by quantitative proteomic analysis

Journal of Proteomics ◽

10.1016/j.jprot.2013.10.020 ◽

2013 ◽

Vol 94 ◽

pp. 473-485 ◽

Cited By ~ 44

Author(s):

Xia Zou ◽

Bo Feng ◽

Taotao Dong ◽

Guoquan Yan ◽

Binbin Tan ◽

...

Keyword(s):

Colorectal Cancer ◽

Proteomic Analysis ◽

Type I Collagen ◽

Type I ◽

Quantitative Proteomic Analysis

Download Full-text

Ocular Hypertension in Mice with a Targeted Type I Collagen Mutation

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.02-0759 ◽

2003 ◽

Vol 44 (4) ◽

pp. 1581 ◽

Cited By ~ 50

Author(s):

Makoto Aihara ◽

James D. Lindsey ◽

Robert N. Weinreb

Keyword(s):

Ocular Hypertension ◽

Type I Collagen ◽

Type I

Download Full-text

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142670 ◽

1986 ◽

Vol 44 ◽

pp. 210-211

Author(s):

Arthur J. Wasserman ◽

Kathy C. Kloos ◽

David E. Birk

Keyword(s):

Type I Collagen ◽

Corneal Stroma ◽

Minor Component ◽

Homogeneous Distribution ◽

Type I ◽

Type V Collagen ◽

Fibril Morphology ◽

Type V ◽

In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

Download Full-text