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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

Canonical Wnt/β-catenin pathway. β-catenin is a component of cell-cell adhesion structures (adherens junctions) [20-22] and a key signaling factor in the Wnt pathway [10]. As shown here canonical Wnt signalling (Wnt/β-catenin) is defined by its inhibition of glycogen synthase kinase-3β (GSK-3β) catalyzed phosphorylation of β-catenin. Several factors, including Cer (cerebrus), WIF-1 (wnt-interacting protein), and sFRPs (secreted frizzled related proteins) and dickkopf-1 (Dkk) are known to antagonize Wnt signalling [63-69]. However, upon Wnt stimulation several Fz-LRP downstream signalling components, including the phosphoprotein dishevelled (Dvl) [70-72], GBP/Frat1 (GSK-3β Binding Protein) [73] and casein kinase I (CKIε) [74,75], somehow co-operate to inhibit GSK-3β. This ultimately this leads to an increase in the cytosolic 'free' levels of β-catenin (uncomplexed to cadherin), and its accumulation within the nucleus where it binds to members of the Tcf/Lef (T-cell factor-lymphoid enhancer factor) transcription factor family to activate gene transcription in a cell-context dependent manner [28-34]. In the absence of Wnt signalling, Axin/conductin [76,77] in co-operation with the product of the tumour suppressor gene adenomatous polyposis coli (APC) [78,79] facilitate GSK-3β mediated phosphorylation of β-catenin on N-terminal serine and threonine residues [80]. This hyper-phosphorylated form of β-catenin then binds to the F-box protein β TrCP, which targets β-catenin for degradation via the ubiquitin-proteosome pathway [81-84].
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Figure 2: Canonical Wnt/β-catenin pathway. β-catenin is a component of cell-cell adhesion structures (adherens junctions) [20-22] and a key signaling factor in the Wnt pathway [10]. As shown here canonical Wnt signalling (Wnt/β-catenin) is defined by its inhibition of glycogen synthase kinase-3β (GSK-3β) catalyzed phosphorylation of β-catenin. Several factors, including Cer (cerebrus), WIF-1 (wnt-interacting protein), and sFRPs (secreted frizzled related proteins) and dickkopf-1 (Dkk) are known to antagonize Wnt signalling [63-69]. However, upon Wnt stimulation several Fz-LRP downstream signalling components, including the phosphoprotein dishevelled (Dvl) [70-72], GBP/Frat1 (GSK-3β Binding Protein) [73] and casein kinase I (CKIε) [74,75], somehow co-operate to inhibit GSK-3β. This ultimately this leads to an increase in the cytosolic 'free' levels of β-catenin (uncomplexed to cadherin), and its accumulation within the nucleus where it binds to members of the Tcf/Lef (T-cell factor-lymphoid enhancer factor) transcription factor family to activate gene transcription in a cell-context dependent manner [28-34]. In the absence of Wnt signalling, Axin/conductin [76,77] in co-operation with the product of the tumour suppressor gene adenomatous polyposis coli (APC) [78,79] facilitate GSK-3β mediated phosphorylation of β-catenin on N-terminal serine and threonine residues [80]. This hyper-phosphorylated form of β-catenin then binds to the F-box protein β TrCP, which targets β-catenin for degradation via the ubiquitin-proteosome pathway [81-84].

Mentions: β-catenin was first identified as a component of cell-cell adhesion structures (adherens junctions) that physically couples cadherins to the cytoskeleton via α-catenin (Fig. 2) [20-22]. It is also a key signalling factor within the canonical Wnt pathway [10], which is involved in growth and development of numerous cell types [23]. In the canonical Wnt pathway (Wnt/β-catenin), these secreted ligands bind to receptor complexes, consisting of a Frizzled (Fz) receptor and a low-density lipoprotein receptor-related protein (LRP) [24-27]. Upon Wnt stimulation glycogen synthase kinase-3β (GSK-3β) catalyzed phosphorylation of β-catenin is inhibited resulting in an increase in the 'free' (uncomplexed to cadherin) cytosolic levels of β-catenin. This in turn leads to its subsequent accumulation within the nucleus, where it binds to members of the Tcf/Lef (T-cell factor-lymphoid enhancer factor) transcription factor family [28,29] to regulate gene expression [30-34].


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)

Canonical Wnt/β-catenin pathway. β-catenin is a component of cell-cell adhesion structures (adherens junctions) [20-22] and a key signaling factor in the Wnt pathway [10]. As shown here canonical Wnt signalling (Wnt/β-catenin) is defined by its inhibition of glycogen synthase kinase-3β (GSK-3β) catalyzed phosphorylation of β-catenin. Several factors, including Cer (cerebrus), WIF-1 (wnt-interacting protein), and sFRPs (secreted frizzled related proteins) and dickkopf-1 (Dkk) are known to antagonize Wnt signalling [63-69]. However, upon Wnt stimulation several Fz-LRP downstream signalling components, including the phosphoprotein dishevelled (Dvl) [70-72], GBP/Frat1 (GSK-3β Binding Protein) [73] and casein kinase I (CKIε) [74,75], somehow co-operate to inhibit GSK-3β. This ultimately this leads to an increase in the cytosolic 'free' levels of β-catenin (uncomplexed to cadherin), and its accumulation within the nucleus where it binds to members of the Tcf/Lef (T-cell factor-lymphoid enhancer factor) transcription factor family to activate gene transcription in a cell-context dependent manner [28-34]. In the absence of Wnt signalling, Axin/conductin [76,77] in co-operation with the product of the tumour suppressor gene adenomatous polyposis coli (APC) [78,79] facilitate GSK-3β mediated phosphorylation of β-catenin on N-terminal serine and threonine residues [80]. This hyper-phosphorylated form of β-catenin then binds to the F-box protein β TrCP, which targets β-catenin for degradation via the ubiquitin-proteosome pathway [81-84].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC183833&req=5

Figure 2: Canonical Wnt/β-catenin pathway. β-catenin is a component of cell-cell adhesion structures (adherens junctions) [20-22] and a key signaling factor in the Wnt pathway [10]. As shown here canonical Wnt signalling (Wnt/β-catenin) is defined by its inhibition of glycogen synthase kinase-3β (GSK-3β) catalyzed phosphorylation of β-catenin. Several factors, including Cer (cerebrus), WIF-1 (wnt-interacting protein), and sFRPs (secreted frizzled related proteins) and dickkopf-1 (Dkk) are known to antagonize Wnt signalling [63-69]. However, upon Wnt stimulation several Fz-LRP downstream signalling components, including the phosphoprotein dishevelled (Dvl) [70-72], GBP/Frat1 (GSK-3β Binding Protein) [73] and casein kinase I (CKIε) [74,75], somehow co-operate to inhibit GSK-3β. This ultimately this leads to an increase in the cytosolic 'free' levels of β-catenin (uncomplexed to cadherin), and its accumulation within the nucleus where it binds to members of the Tcf/Lef (T-cell factor-lymphoid enhancer factor) transcription factor family to activate gene transcription in a cell-context dependent manner [28-34]. In the absence of Wnt signalling, Axin/conductin [76,77] in co-operation with the product of the tumour suppressor gene adenomatous polyposis coli (APC) [78,79] facilitate GSK-3β mediated phosphorylation of β-catenin on N-terminal serine and threonine residues [80]. This hyper-phosphorylated form of β-catenin then binds to the F-box protein β TrCP, which targets β-catenin for degradation via the ubiquitin-proteosome pathway [81-84].
Mentions: β-catenin was first identified as a component of cell-cell adhesion structures (adherens junctions) that physically couples cadherins to the cytoskeleton via α-catenin (Fig. 2) [20-22]. It is also a key signalling factor within the canonical Wnt pathway [10], which is involved in growth and development of numerous cell types [23]. In the canonical Wnt pathway (Wnt/β-catenin), these secreted ligands bind to receptor complexes, consisting of a Frizzled (Fz) receptor and a low-density lipoprotein receptor-related protein (LRP) [24-27]. Upon Wnt stimulation glycogen synthase kinase-3β (GSK-3β) catalyzed phosphorylation of β-catenin is inhibited resulting in an increase in the 'free' (uncomplexed to cadherin) cytosolic levels of β-catenin. This in turn leads to its subsequent accumulation within the nucleus, where it binds to members of the Tcf/Lef (T-cell factor-lymphoid enhancer factor) transcription factor family [28,29] to regulate gene expression [30-34].

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