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CCN2 promotes keratinocyte adhesion and migration via integrin alpha 5 beta 1
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Plastic Surgery.
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2013 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 319, no 19, 2938-2946 p.Article in journal (Refereed) Published
Abstract [en]

Background: CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration. Materials and methods: Adhesion assays were performed by coating wells with 10 mu g/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 mu l arginine-glycine-aspartic acid (RGD), 500 mu M arginine-glycine-glutamate-serine (RGES), and 10 mu g/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 mu M PD98059 (MEN-1 inhibitor) or 20 mu M PF573228 (FAN inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2. Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin alpha 5 beta 1. The addition of anti-integrin alpha 5 beta 1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits alpha 5 and beta 1 CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration. Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin alpha 5 beta 1 and activation of the FAK-MAPK signaling cascade.

Place, publisher, year, edition, pages
2013. Vol. 319, no 19, 2938-2946 p.
Keyword [en]
CCN2, Cell adhesion, Cell migration, Fibronectin, Integrin, Keratinocyte
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-212856DOI: 10.1016/j.yexcr.2013.08.021ISI: 000326853900004OAI: oai:DiVA.org:uu-212856DiVA: diva2:680320
Available from: 2013-12-17 Created: 2013-12-16 Last updated: 2017-12-06Bibliographically approved
In thesis
1. CCN2 – Keratinocyte Interactions In Vitro and In Vivo
Open this publication in new window or tab >>CCN2 – Keratinocyte Interactions In Vitro and In Vivo
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cutaneous wound healing is a complex process involving the migration of inflammatory cells to the wound site, deposition of extracellular matrix, and the reestablishment of an intact epithelial barrier. Re-epithelialization depends on the proliferation and directional migration of keratinocytes from the wound edges. Initially, keratinocytes migrate over a provisional wound matrix that is rich in fibronectin, and as the wound heals the provisional matrix becomes replaced by one consisting of collagen and proteoglycans. Re-epithelialization is tightly regulated by a variety of peptides such as growth factors, cytokines and proteases, and abnormalities may result in chronic non-healing wounds or hypertrophic scars. CCN2 (Connective Tissue Growth Factor) is a multifunctional protein with effects on cells and their interactions with the connective tissue. CCN2 is expressed in a variety of cell types and regulates numerous cell functions including proliferation, differentiation, adhesion, migration and stimulation of collagen production. While the importance of CCN2 for the fibrotic response has been well studied, its involvement in keratinocyte function has not yet been fully explored. Using an in vivo wound model, the expression of CCN2 was captured at the leading keratinocyte edge during re-epithelialization. In vitro, exogenous addition of CCN2 to human keratinocyte cultures promoted keratinocyte migration. Subsequently, integrin a5b1 was identified as an important mediator of CCN2 enhancement of keratinocyte adhesion to fibronectin. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1 specific inhibitor PD98059 markedly reduced CCN2-promoted keratinocyte migration. In vitro, CCN2 expression was induced by TGF-β1. Compared with inhibiting the SMAD pathway, blocking MAPK was more effective in reducing TGF-β1-induced CCN2 mRNA and protein expression. In addition, CCN2-induced keratinocyte spreading required FAK. Treatment with CCN2 led to actin disassembly and altered the activity of the Rho proteins and p190RhoGAP in keratinocytes. Furthermore, Cdc42 mediated CCN2-induced cell polarity. In conclusion, using in vivo and in vitro models, CCN2 was shown to regulate keratinocyte function by promoting keratinocyte adhesion, spreading and migration. A complete understanding of CCN2 expression in keratinocytes is crucial in order to develop novel therapies for wound healing.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 968
Keyword
CCN2, connective tissue growth factor, keratinocytes, re-epithelialization, cell migration, cell signaling
National Category
Medical and Health Sciences
Research subject
Plastic Surgery
Identifiers
urn:nbn:se:uu:diva-213566 (URN)978-91-554-8853-6 (ISBN)
Public defence
2014-02-28, Skoogsalen, Akademiska sjukhuset; Ingång 78-79, Uppsala, 13:15 (English)
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Available from: 2014-02-06 Created: 2013-12-28 Last updated: 2014-02-10

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Kiwanuka, ElizabethGerdin, Bengt

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