TGF-β isoforms are important signalling molecules in wound repair in the skin.
Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In
both animal and human models the application of exogenous TGF-β3 causes a
reduction in the inflammatory response and improves the architecture of the
neodermis. Research into the influence of TGF-β on scarring has tended to focus on
fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a
result of their influence over the behaviour of fibroblasts and also by directly influencing
wound contraction. Thus, experiments were carried out to investigate the influence of
TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3
increased cell spreading and appeared to reduce cell-surface contacts indicated by
both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell
alignment response to microcontact printed protein patterns, in part due to the
deposition of laminin which is associated with the TGF-β induced cell migration.
There is evidence that TGF-β isoforms differentially influence the outcome of wound
healing. Similar to the results produce following addition of exogenous TGF-β3, the
neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult
wounds. During reepithelialisation keratinocytes experience a dynamic environment.
Both extracellular matrix proteins and growth factors influence the progression of
wound repair which includes both cell migration and proliferation. Few studies have
examined collective cell behaviour in response to TGF-β isoforms and ECM coated
substrates. Thus both wound closure and cell proliferation assays were conducted for
different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3.
Rates of wound closure were significantly reduced on laminin coated substrates while
cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in
wound closure rates. This appeared to correspond with an increase in the number of
cells independently migrating out from the wound margins. Only TGF-β3 caused a
significant decrease in cell proliferation over a 4 day period.
Laminin332 deposition is central to the reepithelialisation process and is known to be
induced in response to TGF-β. Thus experiments were carried out to investigate
HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an
immunofluorescence staining technique and an ELISA based semi-quantification
method was used. Following 4 day incubation all TGF-β isoforms significantly
increased laminin332 deposition; however TGF-β2 and 3 caused the most significant
increases.
Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known
to influence the expression of integrin subunits. Thus, experiments were carried out to
compare the influence of each TGF-β isoform on the expression of subunits α3, α2, α5,
β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3
caused the most significant increase in β4 and both TGF-β2 and 3 caused the most
significant increase in α2. While there were differences in cell responses to each
isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2
shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing
wound closure and LN332 deposition. These comparative studies have shown that
differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely
migration, laminin deposition and integrin expression. / EPSRC and DTA grant
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/5393 |
| Date | January 2011 |
| Creators | Berends, Rebecca F. |
| Contributors | Denyer, Morgan C.T., Youseffi, Mansour |
| Publisher | University of Bradford, School of Life Sciences |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Thesis, doctoral, PhD |
| Rights | <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-nd/3.0/88x31.png" /></a><br />The University of Bradford theses are licenced under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-nd/3.0/">Creative Commons Licence</a>. |
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