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

Morphology of DD cells under isometric tension in FPCLs. Depicted are attached (A) FPCL cultures of one representative patient-matched disease and control primary cell line (DUP36). FPCLs were harvested at the indicated attached time points (A0 – A48 hours). Lattices were fixed and stained for F-actin and DNA, using phalloidin-Alexa 488 (green) and DAPI (blue), respectively (Molecular Probes Eugene ON, USA). Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera. The scale bar denotes 100 μ.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC183833&req=5

Figure 8: Morphology of DD cells under isometric tension in FPCLs. Depicted are attached (A) FPCL cultures of one representative patient-matched disease and control primary cell line (DUP36). FPCLs were harvested at the indicated attached time points (A0 – A48 hours). Lattices were fixed and stained for F-actin and DNA, using phalloidin-Alexa 488 (green) and DAPI (blue), respectively (Molecular Probes Eugene ON, USA). Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera. The scale bar denotes 100 μ.

Mentions: Given that Fn levels increase much earlier in the disease FPCL cultures, and that Fn matrix can regulate β-catenin levels, it is possible that disease cell cultures develop a peri-cellular Fn matrix (fibronexus) much earlier, and perhaps to a much greater extent, than control cells. Since previous studies have shown that the formation of filamentous actin (F-actin) networks parallels Fn matrix assembly [55], we probed FPCLs for F-actin expression using phalloidin-Alexa 488. Immunocytochemistry (ICC) analysis shows that the disease cells form extensive stress-fibres Fig. 5 and attach and spread within the collagen matrix much earlier than control cells Fig. 8, suggesting that the disease cells form a Fn matrix much earlier than control cells within attached-matrix cultures. While it is not clear whether β-catenin up regulates cellular Fn production, and/or cellular Fn stimulates increased β-catenin stability, it does appear that this altered metabolic state of the disease cells promotes cell-collagen attachment and cell-mediated collagen contraction. The build-up of isometric tension within the FPCL cultures likely accelerated this process since isometric tension can directly modulate cell signalling events important to fibroblast contractility [59], as well as stimulate the expression of contractile markers such as Fn and α-SM actin in vivo [60]. Moreover, the well recognized role that integrins (cell-ECM receptors) play in helping to generate isometric tension and Fn matrix assembly [51,61], suggests that integrin-mediated signalling factors, like ILK, may provide a possible mechano-chemical pathway linking isometric tension with changes in the β-catenin and Fn levels. Nevertheless, the possible relationship between β-catenin, Fn, ILK and other signalling factors remains to be examined.


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)

Morphology of DD cells under isometric tension in FPCLs. Depicted are attached (A) FPCL cultures of one representative patient-matched disease and control primary cell line (DUP36). FPCLs were harvested at the indicated attached time points (A0 – A48 hours). Lattices were fixed and stained for F-actin and DNA, using phalloidin-Alexa 488 (green) and DAPI (blue), respectively (Molecular Probes Eugene ON, USA). Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera. The scale bar denotes 100 μ.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Morphology of DD cells under isometric tension in FPCLs. Depicted are attached (A) FPCL cultures of one representative patient-matched disease and control primary cell line (DUP36). FPCLs were harvested at the indicated attached time points (A0 – A48 hours). Lattices were fixed and stained for F-actin and DNA, using phalloidin-Alexa 488 (green) and DAPI (blue), respectively (Molecular Probes Eugene ON, USA). Digital images were acquired on a Nikon eclipse TE-200 inverted fluorescent microscope (10x objective) using a Photometrics series 300 cooled CCD camera. The scale bar denotes 100 μ.
Mentions: Given that Fn levels increase much earlier in the disease FPCL cultures, and that Fn matrix can regulate β-catenin levels, it is possible that disease cell cultures develop a peri-cellular Fn matrix (fibronexus) much earlier, and perhaps to a much greater extent, than control cells. Since previous studies have shown that the formation of filamentous actin (F-actin) networks parallels Fn matrix assembly [55], we probed FPCLs for F-actin expression using phalloidin-Alexa 488. Immunocytochemistry (ICC) analysis shows that the disease cells form extensive stress-fibres Fig. 5 and attach and spread within the collagen matrix much earlier than control cells Fig. 8, suggesting that the disease cells form a Fn matrix much earlier than control cells within attached-matrix cultures. While it is not clear whether β-catenin up regulates cellular Fn production, and/or cellular Fn stimulates increased β-catenin stability, it does appear that this altered metabolic state of the disease cells promotes cell-collagen attachment and cell-mediated collagen contraction. The build-up of isometric tension within the FPCL cultures likely accelerated this process since isometric tension can directly modulate cell signalling events important to fibroblast contractility [59], as well as stimulate the expression of contractile markers such as Fn and α-SM actin in vivo [60]. Moreover, the well recognized role that integrins (cell-ECM receptors) play in helping to generate isometric tension and Fn matrix assembly [51,61], suggests that integrin-mediated signalling factors, like ILK, may provide a possible mechano-chemical pathway linking isometric tension with changes in the β-catenin and Fn levels. Nevertheless, the possible relationship between β-catenin, Fn, ILK and other signalling factors remains to be examined.

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