scholarly journals Proteomic Analysis Reveals a New Benefit of Periodic Mechanical Stress on Chondrocytes

2017 ◽  
Vol 44 (4) ◽  
pp. 1578-1590 ◽  
Author(s):  
Zeng Li ◽  
Zhen Wang ◽  
Shun Xu ◽  
Wenwei Liang ◽  
Weimin Fan
Keyword(s):  
Mechanical Stress ◽  
Proteomic Analysis ◽  
Type I Collagen ◽  
Glycogen Synthase ◽  
Therapeutic Benefit ◽  
Immunofluorescence Assay ◽  
Differentially Expressed ◽  
Type I ◽  
Beta Catenin ◽  
Underlying Mechanisms

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.

scholarly journals Nutrient Intake Prior to Exercise Is Necessary for Increased Osteogenic Marker Response in Diabetic Postmenopausal Women

Nutrients ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1494 ◽  
Author(s):  
Katarina T. Borer ◽  
Qingyun Zheng ◽  
Akram Jafari ◽  
Saba Javadi ◽  
Thomas Kernozek
Keyword(s):  
Mechanical Stress ◽  
Postmenopausal Women ◽  
Type I Collagen ◽  
Serum Insulin ◽  
Insulin Response ◽  
Osteogenic Potential ◽  
Type I ◽  
Downhill Exercise ◽  
Osteogenic Response

Type 2 diabetes increases bone fracture risk in postmenopausal women. Usual treatment with anti-resorptive bisphosphonate drugs has some undesirable side effects, which justified our interest in the osteogenic potential of nutrition and exercise. Since meal eating reduces bone resorption, downhill locomotion increases mechanical stress, and brief osteogenic responsiveness to mechanical stress is followed by several hours of refractoriness, we designed a study where 40-min of mechanical stress was manipulated by treadmill walking uphill or downhill. Exercise preceded or followed two daily meals by one hour, and the meals and exercise bouts were 7 hours apart. Fifteen subjects each performed two of five trials: No exercise (SED), uphill exercise before (UBM) or after meals (UAM), and downhill exercise before (DBM) or after meals (DAM). Relative to SED trial, osteogenic response, defined as the ratio of osteogenic C-terminal propeptide of type I collagen (CICP) over bone-resorptive C-terminal telopeptide of type-I collagen (CTX) markers, increased in exercise-after-meal trials, but not in exercise-before-meal trials. CICP/CTX response rose significantly after the first exercise-after-meal bout in DAM, and after the second one in UAM, due to a greater CICP rise, and not a decline in CTX. Post-meal exercise, but not the pre-meal exercise, also significantly lowered serum insulin response and homeostatic model (HOMA-IR) assessment of insulin resistance.

scholarly journals NANOG improves type I collagen expression in human fetal scleral fibroblasts

2019 ◽  
Vol 71 (1) ◽  
pp. 63-70
Author(s):  
Xuyan Li ◽  
Tianfei Yu ◽  
Ming Li ◽  
Youqi Wang ◽  
Bo Meng ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Embryonic Stem ◽  
Coiled Coil ◽  
Structure And Function ◽  
Type I ◽  
Transfected Cells ◽  
Collagen Expression ◽  
Nanog Expression ◽  
Underlying Mechanisms ◽  
And Function

Human fetal scleral fibroblasts (HFSFs) are components of the sclera and play important roles in its structure and function. In myopia, scleral remodeling reduces collagen fibers and the sclera begins to thin. NANOG is a key transcription factor essential for pluripotent and self-renewing phenotypes of undifferentiated embryonic stem cells. To determine whether NANOG improves human fetal scleral fibroblast quality and the underlying mechanisms in these cells, we established stable NANOG-overexpressing HFSFs. We studied type I collagen (COL1A 1) and Rho-associated coiled-coil protein kinase 1 (ROCK1) expression in transfected cells. We also investigated POU5F1, SOX2, KLF4, MYC and SALL4 expression in NANOG stably-overexpressed fibroblasts. Our data show that NANOG expression increased proliferation rates in fibroblasts. When compared to controls, expression of COL1A 1 in transfected fibroblasts was increased and the expression of ROCK1 was decreased. Similarly, the expression of POU5F1, SOX2 and KLF4 was downregulated, the expression of MYC was upregulated and there was no significant change in the expression of SALL4 in transfected fibroblasts. Our results suggest that in fibroblasts, NANOG regulates ROCK1 expression and improves COL1A 1 expression to delay scleral remodeling.

scholarly journals A Mouse Model of Brittle Cornea Syndrome caused by mutation in Zfp469

2021 ◽  
Author(s):  
Chloe M. Stanton ◽  
Amy S. Findlay ◽  
Camilla Drake ◽  
Mohammad Z. Mustafa ◽  
Philippe Gautier ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Therapeutic Benefit ◽  
Type I ◽  
Loss Of Function ◽  
Brittle Cornea Syndrome ◽  
Vitro Model ◽  
Biomechanical Strength ◽  
Corneal Thinning

Brittle Cornea Syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene, Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the type I:type V collagen ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of type I collagen offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus where the cornea thins progressively.

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

2016 ◽  
Vol 44 (1) ◽  
pp. 40-48 ◽  
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.

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

2013 ◽  
Vol 94 ◽  
pp. 473-485 ◽  
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

Examination of the Dynamic Mechanical Properties of Tissue Engineering Scaffolds

2004 ◽  
Vol 844 ◽  
Author(s):  
Heather Kauth ◽  
Catherine Klapperich
Keyword(s):  
Gene Expression ◽  
Tissue Engineering ◽  
Mechanical Stress ◽  
Gas Flow ◽  
Type I Collagen ◽  
Loss Modulus ◽  
Mechanical Tests ◽  
Type I ◽  
Preparation Technique ◽  
Dynamic Mechanical

AbstractScaffolds of both natural and synthetic materials have been widely utilized to provide a three dimensional environment for cell growth. The characteristics of these scaffolds play a vital, but sometimes ambiguous, role in tissue engineering constructs. Collagen-glycosaminoglycan matrices have been used clinically as artificial skin implants. Previous research in our laboratory on this model has shown that gene expression is regulated by mechanical stress applied to the matrix. In this work we begin the process of quantifying the effects of mechanical stress and preparation technique on a matrix comprised of type I collagen and chondroitin-6-sulfate. Compressive loads were applied to the matrices using a dynamic mechanical analyzer at 37 degrees Celsius and retrofitted with 5% carbon dioxide gas flow. All samples were tested hydrated in either simulated body fluid or PBS. Storage and loss modulus data are presented. Mechanical tests were performed on collagen-GAG meshes both seeded with humandermal fibroblasts and not seeded with cells. Continuing work includes gene expression analysis of the cells on the seeded matrices in order to identify differential gene expression induced by mechanicalloading.

scholarly journals A Mouse Model of Brittle Cornea Syndrome caused by mutation in Zfp469.

2021 ◽  
Author(s):  
Chloe M. Stanton ◽  
Amy S. Findlay ◽  
Camilla Drake ◽  
Mohammad Z. Mustafa ◽  
Philippe Gautier ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Therapeutic Benefit ◽  
Type I ◽  
Loss Of Function ◽  
Brittle Cornea Syndrome ◽  
Vitro Model ◽  
Biomechanical Strength ◽  
Corneal Thinning

Brittle Cornea Syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene, Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the type I:type V collagen ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of type I collagen offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus where the cornea thins progressively.

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

2007 ◽  
Vol 39 (6) ◽  
pp. 330-337 ◽  
Author(s):  
Manabu Shinzato ◽  
Yoshito Yamashiro ◽  
Nariko Miyara ◽  
Akihiro Iwamatsu ◽  
Kouji Takeuchi ◽  
...  
Keyword(s):  
Ocular Hypertension ◽  
Trabecular Meshwork ◽  
Proteomic Analysis ◽  
Type I Collagen ◽  
Type I

scholarly journals Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins.

1995 ◽  
Vol 131 (6) ◽  
pp. 1903-1915 ◽  
Author(s):  
O Langholz ◽  
D Röckel ◽  
C Mauch ◽  
E Kozlowska ◽  
I Bank ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Specific Protein ◽  
Type I ◽  
Collagen Gels ◽  
Lattice Contraction ◽  
Ecm Remodeling ◽  
Signal Transduction Inhibitors ◽  
Underlying Mechanisms

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down-regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine-specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.

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

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.

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