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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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Related in: MedlinePlus

ICC of FPCL cultures. Depicted are representative images from disease (d) and normal/control (c) matched primary culture (patient DUP48). The cells were grown as attached FPCLs for 2 days (105 cells/ml of matrix). Cells were then fixed and stained with phalloidin-Alexa 488 (green) and DAPI (blue) to label both F-actin and the nucleus, respectively. Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera, and deconvolved using softWoRx (v 2.5) software (Applied Precision Inc., Issaquah WA, USA). Arrows identify prominent F-actin networks (stress fibres) within the diseased cells. The scale bar denotes 100 μ.
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Figure 5: ICC of FPCL cultures. Depicted are representative images from disease (d) and normal/control (c) matched primary culture (patient DUP48). The cells were grown as attached FPCLs for 2 days (105 cells/ml of matrix). Cells were then fixed and stained with phalloidin-Alexa 488 (green) and DAPI (blue) to label both F-actin and the nucleus, respectively. Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera, and deconvolved using softWoRx (v 2.5) software (Applied Precision Inc., Issaquah WA, USA). Arrows identify prominent F-actin networks (stress fibres) within the diseased cells. The scale bar denotes 100 μ.

Mentions: Stressed-collagen lattices stained with phalloidin-Alexa 488, a fluorescent probe that binds filamentous actin, revealed more prominent stress fiber networks in the disease FPCLs compared to control FPCLs (Fig. 5), suggesting that the disease cells may be more inherently responsive to factors that promote collagen contraction. Although the exact nature of the cell signalling networks responsible for the enhanced contractile activity of the disease cells is unclear (e.g. cell-ECM interactions, growth factor pathways), the three-dimensional collagen matrix environment appears to differentially regulate the activity of the control and disease cells.


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)

ICC of FPCL cultures. Depicted are representative images from disease (d) and normal/control (c) matched primary culture (patient DUP48). The cells were grown as attached FPCLs for 2 days (105 cells/ml of matrix). Cells were then fixed and stained with phalloidin-Alexa 488 (green) and DAPI (blue) to label both F-actin and the nucleus, respectively. Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera, and deconvolved using softWoRx (v 2.5) software (Applied Precision Inc., Issaquah WA, USA). Arrows identify prominent F-actin networks (stress fibres) within the diseased cells. The scale bar denotes 100 μ.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: ICC of FPCL cultures. Depicted are representative images from disease (d) and normal/control (c) matched primary culture (patient DUP48). The cells were grown as attached FPCLs for 2 days (105 cells/ml of matrix). Cells were then fixed and stained with phalloidin-Alexa 488 (green) and DAPI (blue) to label both F-actin and the nucleus, respectively. Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera, and deconvolved using softWoRx (v 2.5) software (Applied Precision Inc., Issaquah WA, USA). Arrows identify prominent F-actin networks (stress fibres) within the diseased cells. The scale bar denotes 100 μ.
Mentions: Stressed-collagen lattices stained with phalloidin-Alexa 488, a fluorescent probe that binds filamentous actin, revealed more prominent stress fiber networks in the disease FPCLs compared to control FPCLs (Fig. 5), suggesting that the disease cells may be more inherently responsive to factors that promote collagen contraction. Although the exact nature of the cell signalling networks responsible for the enhanced contractile activity of the disease cells is unclear (e.g. cell-ECM interactions, growth factor pathways), the three-dimensional collagen matrix environment appears to differentially regulate the activity of the control and disease cells.

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