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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.

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Western analysis of β-catenin and Fn in attached FPCLs. Attached FPCLs (3 lattices per cell line) were harvested at the indicated time points, homogenized, and protein extract prepared for western analysis. Membranes were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), 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 (ratios), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point. Two patient-matched disease and normal/control primary cultures were examined.
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Figure 7: Western analysis of β-catenin and Fn in attached FPCLs. Attached FPCLs (3 lattices per cell line) were harvested at the indicated time points, homogenized, and protein extract prepared for western analysis. Membranes were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), 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 (ratios), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point. Two patient-matched disease and normal/control primary cultures were examined.

Mentions: To further explore β-catenin and its relation to mechanical stress, we also analyzed β-catenin levels within attached-matrix (see Fig. 3) FPCL cultures. As shown in Fig. 7, western analysis of attached-matrices showed an accumulation of β-catenin in the disease cells over time, relative to control Hsp47 levels, while β-catenin levels remain relatively unchanged in the control cells. Although Fn levels in both control and disease cultures increased over the incubation period, Fn levels accumulated much faster within the disease FPCLs compared to control FPCL cultures (Fig. 7). Although the exact mechanism responsible for the tension stimulated accumulation of β-catenin is not clear it may be related to proliferative nature of attached-matrix cultures [58].


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 attached FPCLs. Attached FPCLs (3 lattices per cell line) were harvested at the indicated time points, homogenized, and protein extract prepared for western analysis. Membranes were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), 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 (ratios), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point. Two patient-matched disease and normal/control primary cultures were examined.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Western analysis of β-catenin and Fn in attached FPCLs. Attached FPCLs (3 lattices per cell line) were harvested at the indicated time points, homogenized, and protein extract prepared for western analysis. Membranes were sequentially probed with β-catenin (1:750; clone 14, Transduction Laboratories), 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 (ratios), and plotted (bar graphs, lower panel) as the mean ratio intensity ± SEM per time point. Two patient-matched disease and normal/control primary cultures were examined.
Mentions: To further explore β-catenin and its relation to mechanical stress, we also analyzed β-catenin levels within attached-matrix (see Fig. 3) FPCL cultures. As shown in Fig. 7, western analysis of attached-matrices showed an accumulation of β-catenin in the disease cells over time, relative to control Hsp47 levels, while β-catenin levels remain relatively unchanged in the control cells. Although Fn levels in both control and disease cultures increased over the incubation period, Fn levels accumulated much faster within the disease FPCLs compared to control FPCL cultures (Fig. 7). Although the exact mechanism responsible for the tension stimulated accumulation of β-catenin is not clear it may be related to proliferative nature of attached-matrix cultures [58].

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