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CCN2 – Keratinocyte Interactions In Vitro and In Vivo
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Plastic Surgery.
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 [en]
CCN2, connective tissue growth factor, keratinocytes, re-epithelialization, cell migration, cell signaling
National Category
Medical and Health Sciences
Research subject
Plastic Surgery
Identifiers
URN: urn:nbn:se:uu:diva-213566ISBN: 978-91-554-8853-6 (print)OAI: oai:DiVA.org:uu-213566DiVA: diva2:685086
Public defence
2014-02-28, Skoogsalen, Akademiska sjukhuset; Ingång 78-79, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-02-06 Created: 2013-12-28 Last updated: 2014-02-10
List of papers
1. CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway
Open this publication in new window or tab >>CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway
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2013 (English)In: Journal of Surgical Research, ISSN 0022-4804, E-ISSN 1095-8673, Vol. 185, no 2, E109-E119 p.Article in journal (Refereed) Published
Abstract [en]

Background: CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing. Materials and methods: Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/ mitogen-activated protein kinase pathway to human keratinocytes were studied. Results: The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration. Conclusions: CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway.

Keyword
Wound healing, Re-epithelialization, CCN2, Keratinocyte, Cell migration
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-212843 (URN)10.1016/j.jss.2013.05.065 (DOI)000326938500006 ()
Available from: 2013-12-18 Created: 2013-12-16 Last updated: 2017-12-06Bibliographically approved
2. CCN2 promotes keratinocyte adhesion and migration via integrin alpha 5 beta 1
Open this publication in new window or tab >>CCN2 promotes keratinocyte adhesion and migration via integrin alpha 5 beta 1
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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.

Keyword
CCN2, Cell adhesion, Cell migration, Fibronectin, Integrin, Keratinocyte
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-212856 (URN)10.1016/j.yexcr.2013.08.021 (DOI)000326853900004 ()
Available from: 2013-12-17 Created: 2013-12-16 Last updated: 2017-12-06Bibliographically approved
3. TGF-β1 regulates the expression of CCN2 in human keratinocytes via Smad-ERK signaling interplay
Open this publication in new window or tab >>TGF-β1 regulates the expression of CCN2 in human keratinocytes via Smad-ERK signaling interplay
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-214169 (URN)
Available from: 2014-01-07 Created: 2014-01-07 Last updated: 2015-03-17
4. Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes
Open this publication in new window or tab >>Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes
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2016 (English)In: International Wound Journal, ISSN 1742-4801, E-ISSN 1742-481X, Vol. 13, no 3, 372-381 p.Article in journal (Refereed) Published
Abstract [en]

Cell migration requires spatiotemporal integration of signals that regulate cytoskeletal dynamics. In response to a migration-promoting agent, cells begin to polarise and extend protrusions in the direction of migration. These cytoskeletal rearrangements are orchestrated by a variety of proteins, including focal adhesion kinase (FAK) and the Rho family of GTPases. CCN2, also known as connective tissue growth factor, has emerged as a regulator of cell migration but the mechanism by which CCN2 regulates keratinocyte function is not well understood. In this article, we sought to elucidate the basicmechanism of CCN2-induced cellmigration in human keratinocytes. Immunohistochemical staining was used to demonstrate that treatment with CCN2 induces a migratory phenotype through actin disassembly, spreading of lamellipodia and re-orientation of the Golgi. In vitro assays were used to show that CCN2-induced cell migration is dependent on FAK, RhoA and Cdc42, but independent of Rac1. CCN2-treated keratinocytes displayed increased Cdc42 activity and decreased RhoA activity up to 12 hours post-treatment, with upregulation of p190RhoGAP. An improved understanding of how CCN2 regulates cell migration may establish the foundation for future therapeutics in fibrotic and neoplastic diseases.

Keyword
RhoGTPase, Actin, CCN2, CTGF, Keratinocyte migration
National Category
Medical and Health Sciences
Research subject
Plastic Surgery
Identifiers
urn:nbn:se:uu:diva-213565 (URN)10.1111/iwj.12315 (DOI)000379940300011 ()25185742 (PubMedID)
External cooperation:
Available from: 2013-12-28 Created: 2013-12-28 Last updated: 2017-12-06Bibliographically approved

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