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Regulation of fibroblast activity by keratinocytes, TGF-β and IL-1α: studies in two- and three dimensional in vitro models
Örebro University, School of Medical Sciences.ORCID iD: 0000-0001-8304-2772
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor-β (TGF-β) in dermal fibroblasts. Reepithelialisation and epidermal wound coverage counteract excessive scar formation. We have previously shown that interleukin-1α (IL-1α) derived from keratinocytes conteracts TGF-β-stimulated CTGF-expression. The aim of this thesis was to further explore the effects of keratinocytes and IL-1α on gene and protein expression, as well as pathways, in TGF-β stimulated fibroblasts. Fibroblasts were studied in vitro by conventional two dimensional cell culture models and in a three dimensional keratinocyte-fibroblast organotypic skin culture model.

The results showed that IL-1 suppresses basal and TGF-β-induced CTGF mRNA and protein, involving a possible TAK1 mechanism. Keratinocytes regulate the expression of fibroblast genes important for the turnover of the extracellular matrix. Most of the genes analysed (11/13) were regulated by TGF-β and counter regulated by keratinocytes. The overall results support a view that keratinocytes regulate fibroblasts to act catabolically (anti-fibrotic) on the extracellular matrix.

Transcriptional microarray and gene set enrichment analysis showed that antagonizing effects of IL-1α on TGF-β were much more prominent than the synergistic effects. The most confident of these pathways was the interferon signaling, which were inhibited by TGF-β and activated by IL-1α. A proteomics study confirmed that IL-1α preferentially conteracts TGF-β effects. Six new fibroblast proteins involved in synthesis/ regulation were identified, being regulated by TGF-β and antagonized by IL-1α. Pathway analysis confirmed counter-regulation of interferon signaling by the two cytokines. These findings have implications for understanding the role of fibroblasts for inflammatory responses and development of fibrosis in the skin.

Place, publisher, year, edition, pages
Örebro: Örebro university , 2016. , 83 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 133
Keyword [en]
Fibroblast, Keratinocyte, TGF-β, IL-1α, coculture, fibrosis CTGF/CNN 2, dermal, organotypic culture
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:oru:diva-48225ISBN: 978-91-7529-120-8 (print)OAI: oai:DiVA.org:oru-48225DiVA: diva2:902824
Public defence
2016-03-18, Universitetssjukhuset, Wilandersalen, Södra Grev Rosengatan, Örebro, 09:00 (English)
Opponent
Supervisors
Available from: 2016-02-12 Created: 2016-02-12 Last updated: 2017-10-17Bibliographically approved
List of papers
1. Inhibition of Connective Tissue Growth Factor/CCN2 Expression in Human Dermal Fibroblasts by Interleukin-1 alpha and beta
Open this publication in new window or tab >>Inhibition of Connective Tissue Growth Factor/CCN2 Expression in Human Dermal Fibroblasts by Interleukin-1 alpha and beta
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2010 (English)In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 110, no 5, 1226-1233 p.Article in journal (Refereed) Published
Abstract [en]

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)-beta and intimately involved with tissue repair and overexpressed in various fibrotic conditions We previously showed that keratmocytes in vitro downregulate TGF-beta-induced expression of CTGF in fibroblasts by an interleukin (IL)-1 alpha-dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL-1 alpha and beta Human dermal fibroblasts and NIH 3T3 cells were treated with IL-1 alpha or beta in presence or absence of TGF-beta 1. IL-1 suppressed basal and TGF-beta-induced CTGF mRNA and protein expression. IL-1 alpha and beta inhibited TGF-beta-stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3-binding CAGA elements Furthermore. IL-1 alpha and beta inhibited TGF-beta-stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition. RNA interference suggested that TGF-beta activated kinase1 (TAK1) is necessary for IL-1 inhibition of TGF-beta-stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell Biochem. 110: 1226-1233, 2010. (C) 2010 Wiley-Liss. Inc

Keyword
interleukin-1, connective tissue growth factor, transforming growth factor-beta, smad 3, tak1
National Category
Cell Biology Biochemistry and Molecular Biology
Research subject
Cell Research
Identifiers
urn:nbn:se:oru:diva-28379 (URN)10.1002/jcb.22637 (DOI)000280435900021 ()20544797 (PubMedID)2-s2.0-77954894569 (Scopus ID)
Available from: 2013-03-26 Created: 2013-03-14 Last updated: 2017-12-06Bibliographically approved
2. Regulation of fibroblast gene expression by keratinocytes in organotypic skin culture provides possible mechanisms for the antifibrotic effect of reepithelialization.
Open this publication in new window or tab >>Regulation of fibroblast gene expression by keratinocytes in organotypic skin culture provides possible mechanisms for the antifibrotic effect of reepithelialization.
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2010 (English)In: Wound Repair and Regeneration, ISSN 1067-1927, E-ISSN 1524-475X, Vol. 18, no 5, 452-459 p.Article in journal (Refereed) Published
Abstract [en]

To investigate the mechanisms behind the antifibrotic effect associated with epidermal regeneration, the expression of 12 fibroblast genes important for the modulation of the extracellular matrix (ECM), as well as α-smooth muscle actin, was studied in a keratinocyte-fibroblast organotypic skin culture model. The study was performed over time during epidermal generation and in the presence or absence of the profibrotic factor transforming growth factor-β. the Presence of epidermal differentiation markers in the model was essentially coherent with that of native skin. Fibroblast gene expression was analyzed with real-time polymerase chain reaction after removal of the epidermal layer. After 2 days of air-exposed culture, 11 out of the 13 genes studied were significantly regulated by keratinocytes in the absence or presence of transforming growth factor-β. The regulation of connective tissue growth factor, collagen I and III, fibronectin, plasmin system regulators, matrix metalloproteinases and their inhibitors as well as α-smooth muscle actin was consistent with a suppression of ECM formation or contraction. Overall, the results support a view that keratinocytes regulate fibroblasts to act catabolically on the ECM in epithelialization processes. This provides possible mechanisms for the clinical observations that reepithelialization and epidermal wound coverage counteract excessive scar formation.

Place, publisher, year, edition, pages
The Wound Healing Society, 2010
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-48462 (URN)10.1111/j.1524-475X.2010.00605.x (DOI)000282263500004 ()20731800 (PubMedID)2-s2.0-77956625499 (Scopus ID)
Available from: 2016-02-22 Created: 2016-02-22 Last updated: 2017-11-30Bibliographically approved
3. IL-1α Counteract TGF-β Regulated Genes and Pathways in Human Fibroblasts
Open this publication in new window or tab >>IL-1α Counteract TGF-β Regulated Genes and Pathways in Human Fibroblasts
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2016 (English)In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 117, no 7, 1622-1632 p.Article in journal (Refereed) Published
Abstract [en]

Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor-β (TGF-β) in dermal fibroblasts. We previously showed that interleukin-1 (IL-1α) counteracts TGF-β-stimulated CTGF mRNA and protein expression in these cells. The aim of this study was to explore the effects of IL-1α on further genes and pathways in TGF-β regulated fibroblasts. Transcriptional microarray and multiple comparison analysis showed that the antagonizing effects of IL-1α was much more prominent than the synergistic effects, both with respect to number of genes and extent of changes in gene expression. Moreover, comparing canonical pathways by gene set enrichment analysis and the Ingenuity Pathway Analysis tool revealed that IL-1α counteracted TGF-β in the top six most confident pathways regulated by both cytokines. Interferon and IL-1 signaling, as well as two pathways involved in apoptosis signaling were suppressed by TGF-β and activated by IL-1α. Pathways involving actin remodeling and focal adhesion dynamics were activated by TGF-β and suppressed by IL-1α. Analyzing upstream regulators in part corroborate the comparison of canonical pathways and added cell cycle regulators as another functional group regulated by IL-1α. Finally, gene set enrichment analysis of fibrosis-related genes indicated that IL-1 moderately counteracts the collective effect of TGF-β on these genes. Microarray results were validated by qPCR. Taken together, the results indicate prominent antagonistic effects of IL-1α on TGF-β regulated interferon signaling, as well as on a wide variety of other genes and pathways in fibroblasts. This article is protected by copyright. All rights reserved.

Place, publisher, year, edition, pages
Hoboken, USA: Wiley-Blackwell, 2016
Keyword
Connective tissue growth factor, transforming growth factor-beta, interleukin-1, interferon, fibroblast, fibrosis and ingenuity pathway analysis
National Category
Other Basic Medicine Cell Biology Biochemistry and Molecular Biology
Research subject
Cell Research
Identifiers
urn:nbn:se:oru:diva-48463 (URN)10.1002/jcb.25455 (DOI)000375916800014 ()26629874 (PubMedID)2-s2.0-84964773875 (Scopus ID)
Note

Funding Agencies:

Örebro County Council Research Committee of the Örebro University Hospital OLL-550071

Nyckelfonden AE56340

Available from: 2016-02-22 Created: 2016-02-22 Last updated: 2017-11-30Bibliographically approved
4. IL-1α counteracts TGF-β regulated protein expression in human dermal fibroblasts
Open this publication in new window or tab >>IL-1α counteracts TGF-β regulated protein expression in human dermal fibroblasts
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-48464 (URN)
Available from: 2016-02-22 Created: 2016-02-22 Last updated: 2017-10-17Bibliographically approved

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