Limits...
Elevated levels of beta-catenin and fibronectin in three-dimensional collagen cultures of Dupuytren's disease cells are regulated by tension in vitro.

Howard JC, Varallo VM, Ross DC, Roth JH, Faber KJ, Alman B, Gan BS - BMC Musculoskelet Disord (2003)

Bottom Line: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands.Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University of Western Ontario, London, Ontario, Canada. jhoward@lri.sjhc.london.on.ca

ABSTRACT

Background: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands. Although the molecular pathology of DD is unknown, recent evidence suggests that beta-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in beta-catenin and fibronectin levels.

Methods: Western blots of beta-catenin and fibronectin levels were determined for control and disease primary cell cultures grown within stressed- and attached-collagen matrices. Collagen contraction was quantified, and immunocytochemistry analysis of filamentous actin performed.

Results: Disease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in beta-catenin and fibronectin levels, including a transient increase in beta-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and beta-catenin levels increased in attached collagen-matrix cultures of disease cells, while control cultures showed only increases in fibronectin levels. Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.

Conclusion: Three-dimensional collagen matrix cultures of primary disease cell lines are more contractile and express a more extensive filamentous actin network than patient-matched control cultures. The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

Show MeSH

Related in: MedlinePlus

Western analysis of β-catenin and Fn in contracting FPCLs. The upper panel shows the western analysis results of contracting 'stressed' FPCLs of three patient-matched disease (D) and normal/control (C) primary cell lines (n = 3). FPCLs (3 lattices per cell line) were harvested at the indicated time points following mechanical release and homogenized for protein extraction. The resulting western blots were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), and Fn (clone IST-4, 1:500, Sigma) and Hsp47 (1:500, StressGen) antibodies. Antibody-specific bands for β-catenin and fibronectin and Hsp47 were quantified using NIH Imaging software, normalized to Hsp47 levels (ratio), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC183833&req=5

Figure 6: Western analysis of β-catenin and Fn in contracting FPCLs. The upper panel shows the western analysis results of contracting 'stressed' FPCLs of three patient-matched disease (D) and normal/control (C) primary cell lines (n = 3). FPCLs (3 lattices per cell line) were harvested at the indicated time points following mechanical release and homogenized for protein extraction. The resulting western blots were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), and Fn (clone IST-4, 1:500, Sigma) and Hsp47 (1:500, StressGen) antibodies. Antibody-specific bands for β-catenin and fibronectin and Hsp47 were quantified using NIH Imaging software, normalized to Hsp47 levels (ratio), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point.

Mentions: Since β-catenin is aberrantly expressed in vivo and in vitro (FPCL cultures) in DD [13], we examined its levels in relation to changes in mechanical stress (stressed- or attached-matrices). Fibronectin (Fn) levels were also examined since it plays an important role in cell-collagen interactions [43,50,51], and the regulation and function of β-catenin [52,53]. During the contraction of stressed-FPCLs β-catenin levels transiently increased in disease cells, peaking at 1 hr post-mechanical release (R1, Dis/ctrl ratio = 3.6) and decreasing thereafter to levels equivalent to, or below those seen in control FPCLs, while in control matrix cultures β-catenin levels did not significantly change throughout the incubation period (Fig. 6). By contrast, Fn levels in disease FPCLs (R0, Dis/ctrl ratio = 2.7) steadily decreased upon mechanical release of the matrices, while Fn levels in control lattices showed a slight increase during contraction (Fig. 6).


Elevated levels of beta-catenin and fibronectin in three-dimensional collagen cultures of Dupuytren's disease cells are regulated by tension in vitro.

Howard JC, Varallo VM, Ross DC, Roth JH, Faber KJ, Alman B, Gan BS - BMC Musculoskelet Disord (2003)

Western analysis of β-catenin and Fn in contracting FPCLs. The upper panel shows the western analysis results of contracting 'stressed' FPCLs of three patient-matched disease (D) and normal/control (C) primary cell lines (n = 3). FPCLs (3 lattices per cell line) were harvested at the indicated time points following mechanical release and homogenized for protein extraction. The resulting western blots were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), and Fn (clone IST-4, 1:500, Sigma) and Hsp47 (1:500, StressGen) antibodies. Antibody-specific bands for β-catenin and fibronectin and Hsp47 were quantified using NIH Imaging software, normalized to Hsp47 levels (ratio), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183833&req=5

Figure 6: Western analysis of β-catenin and Fn in contracting FPCLs. The upper panel shows the western analysis results of contracting 'stressed' FPCLs of three patient-matched disease (D) and normal/control (C) primary cell lines (n = 3). FPCLs (3 lattices per cell line) were harvested at the indicated time points following mechanical release and homogenized for protein extraction. The resulting western blots were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), and Fn (clone IST-4, 1:500, Sigma) and Hsp47 (1:500, StressGen) antibodies. Antibody-specific bands for β-catenin and fibronectin and Hsp47 were quantified using NIH Imaging software, normalized to Hsp47 levels (ratio), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point.
Mentions: Since β-catenin is aberrantly expressed in vivo and in vitro (FPCL cultures) in DD [13], we examined its levels in relation to changes in mechanical stress (stressed- or attached-matrices). Fibronectin (Fn) levels were also examined since it plays an important role in cell-collagen interactions [43,50,51], and the regulation and function of β-catenin [52,53]. During the contraction of stressed-FPCLs β-catenin levels transiently increased in disease cells, peaking at 1 hr post-mechanical release (R1, Dis/ctrl ratio = 3.6) and decreasing thereafter to levels equivalent to, or below those seen in control FPCLs, while in control matrix cultures β-catenin levels did not significantly change throughout the incubation period (Fig. 6). By contrast, Fn levels in disease FPCLs (R0, Dis/ctrl ratio = 2.7) steadily decreased upon mechanical release of the matrices, while Fn levels in control lattices showed a slight increase during contraction (Fig. 6).

Bottom Line: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands.Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University of Western Ontario, London, Ontario, Canada. jhoward@lri.sjhc.london.on.ca

ABSTRACT

Background: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands. Although the molecular pathology of DD is unknown, recent evidence suggests that beta-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in beta-catenin and fibronectin levels.

Methods: Western blots of beta-catenin and fibronectin levels were determined for control and disease primary cell cultures grown within stressed- and attached-collagen matrices. Collagen contraction was quantified, and immunocytochemistry analysis of filamentous actin performed.

Results: Disease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in beta-catenin and fibronectin levels, including a transient increase in beta-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and beta-catenin levels increased in attached collagen-matrix cultures of disease cells, while control cultures showed only increases in fibronectin levels. Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.

Conclusion: Three-dimensional collagen matrix cultures of primary disease cell lines are more contractile and express a more extensive filamentous actin network than patient-matched control cultures. The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

Show MeSH
Related in: MedlinePlus