1. Inducible Lineage-Specific Deletion of TβRII in Fibroblasts Defines a Pivotal Regulatory Role during Adult Skin Wound Healing 
Christopher P. Denton, Korsa Khan, Rachel K. Hoyles, Xu Shiwen, Patricia Leoni, Yunliang Chen, Mark Eastwood, David J. Abraham 
Journal of Investigative Dermatology 
Volume 129, Issue 1, Pages (January 2009)
DOI: /jid
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2. Figure 1
Inducible fibroblast-specific deletion of TβRII. (a) A transgenic strain was generated in which tamoxifen-dependent Cre recombinase is expressed under the control of a fibroblast-specific expression cassette (Col1a2-Cre-ERT) and both alleles of TβRII were conditionally inactivatable due to LoxP sites flanking exon 4 (TβRIIflx/flx). When treated with tamoxifen, genetic recombination occurred in fibroblasts to generate the null allele (TβRII-null). (b) Neonatal compound mutant mice (Col1a2-Cre-ERT/TβRIIflx/flx) were treated with intraperitoneal tamoxifen or corn oil vehicle control. PCR analysis of tail DNA (derived from fibroblasts and non-fibroblastic cells) confirms that a 692bp PCR product from the null allele (P3, P5) is present only after tamoxifen administration. A 575bp product from the non-recombined allele is also present (P3, P4). (c) Lysates from fibroblasts cultured from skin biopsies after tamoxifen administration confirm the absence of TβRII protein. Control lanes show Coomassie staining of gel and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunolocalization. (d) Explanted skin fibroblasts from TβRII-null-fib show no significant immunostaining for TβRII compared with littermate wild-type fibroblasts. Nuclei are counterstained with DAPI. (e) Skin from mice aged 3 weeks shows scattered cells within the dermis expressing TβRII in wild-type control mice treated with corn oil (f, arrows) whereas no dermal staining is present in mice that had received tamoxifen in the neonatal period (bar=100μm).
Journal of Investigative Dermatology  , DOI: ( /jid )
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3. Figure 2
Fibroblasts are refractory to recombinant TGFβ1 after deletion of TβRII. (a) Expression of key TGFβ-regulated proteins was explored, including PAI-1, αSMA, type I collagen, fibronectin, and CTGF. Constitutive and TGFβ1 (2ngml−1, 16hours)-treated fibroblasts are compared. Basal CTGF level was elevated in the cell layer of fibroblasts lacking TβRII, but these cells were refractory to TGFβ1 stimulation. (b) Transient transfection of three TGFβ-regulated reporter plasmids confirms reduced responsiveness of TβRII-null-fib dermal fibroblasts to recombinant TGFβ1. Data are representative of three independent experiments for each construct. Units are in RLU (±SD) corrected for transfection efficiency. Basal values were compared with activity after TGFβ1 activation (***P<0.005; **P<0.01; *P<0.05, paired Student's t-test). See Materials and Methods for details of the reporter constructs.
Journal of Investigative Dermatology  , DOI: ( /jid )
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4. Figure 3
Perturbed signalling with increased basal ERK1/2 phosphorylation after TβRII deletion. (a) There was no increase in Smad2 phosphorylation at 30minutes after TGFβ1 simulation in fibroblasts lacking TβRII. Basal phosphorylation of ERK1/2 was increased in these fibroblasts and induction was delayed compared with wild-type control littermates. Data are representative of a series of three independent studies. (b) Immunostaining of dermal fibroblasts confirms increased phospho-ERK1/2 in TβRII-null cells compared with wild type. The inset shows staining of nuclei with DAPI. Data are representative of three independent studies (bar=50μm).
Journal of Investigative Dermatology  , DOI: ( /jid )
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5. Figure 4
Defective wound healing in TβRII-null-fib mice. (a) Representative wound cross-sections stained by hematoxylin and eosin comparing TβRII-null-fib mice with wild-type littermates. Key abnormalities in TβRII-null-fib included the lack of any subdermal granulation tissue and scar at 7 days and absence of a fibrous dermal scar at 14 days, with replacement of the dermal defect by cellular granulation tissue at this time point. Data are representative of three independent studies each with at least two wild-type or mutant littermate mice at each time point (bar=100μm). (b) Differences between macroscopic healing of 4mm punch wounds on the lower back are confirmed by measuring maximum wound diameter, which was significantly larger at 7 and 14 days in TβRII-null-fib mice. Data summarize a series of wounds at three time points, each time point summarizing data from 3 to 6 wild-type or mutant littermate mice at each time point.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Fibroblast-specific deletion of TβRII is associated with increased epidermal proliferation. Postnatal deletion of TβRII in fibroblasts led to marked alteration in epidermal architecture at the site of full-thickness skin wounds. The panels show healing wounds at 7 days. (a) In wild-type skin, there was a well-organized multilayer epidermis with flattening of maturing keratinocytes at the skin surface and a well-organized 3- to 4-cell thick layer of mature cells. (b) Null mice had a much less organized multicellular epidermis without apparent maturation of keratinocytes and dense cellular aggregates in the basal layer. (c) The proliferation marker Ki-67 was expressed sporadically at this site in healing wild-type skin, but was much more uniformly present in null skin (d). Expression of TβRII is restricted to the central epidermis of wild-type wounds (e) compared with much more extensive expression (arrow) in null skin (f). Similarly, strong expression of TGFβ1 in wild-type skin is largely restricted to the basal epidermis in wild-type wounds (g) compared with staining throughout the epidermis (arrow) in null littermates (h). Data are representative of a series of three independent experiments. Original magnification for photomicrographs is indicated (bar=50μm (a–d, g, h), 100μm (e, f)).
Journal of Investigative Dermatology  , DOI: ( /jid )
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7. Figure 6
Impaired recruitment and differentiation of myofibroblasts in vivo after deletion of TβRII. There was high-level expression of αSMA in the dense subdermal granulation tissue of healing punch wounds at 7 days in wild-type mice compared with the TβRII-null-fib strain at low magnification (a, b). Higher magnification views show that expression within myofibroblastic cells within the granulation tissue (m, arrow(c, d)). Expression in the wounds of TβRII-null-fib mice was less and was mostly located in vascular structures (v). The lower panels are high-power views to show clusters of αSMA-expressing cells (pink) immediately beneath the epidermis of healing wounds at 7 days (e, f). These were more prominent in TβRII-null-fib mice. Some of these cells (magnified inset) also expressed CD34 (brown stain), suggesting that they are bone marrow-derived fibrocytes (c, arrow). Data are representative of three independent studies (bar=100μm (a–d), 50μm (e, f)).
Journal of Investigative Dermatology  , DOI: ( /jid )
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8. Figure 7
TβRII-null fibroblasts show impaired migration and collagen gel matrix contraction. (a) The scratch wound assay allowed direct evaluation of migration in vitro. Migration was reduced for fibroblasts lacking TβRII, as shown in the histogram summarizing three independent experiments with initial wound width taken as 100%. (b) TβRII-null fibroblasts showed reduced basal contraction of free-floating collagen lattices and were refractory to recombinant TGFβ1. Mean (±SD) data summarize three independent experiments. (c) Tethered FPCLs confirm attenuated contraction for TβRII-deleted fibroblasts and refractoriness to TGFβ1. Data summarize two independent experiments with littermate fibroblast cultures. The arrow in the inset shows addition of recombinant TGFβ1 as outlined in Materials and Methods.
Journal of Investigative Dermatology  , DOI: ( /jid )
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9. Figure 8
Attenuated cytoskeletal incorporation of αSMA after deletion of TβRII. Wild-type dermal fibroblasts showed more prominent αSMA-rich stress fibers after treatment with recombinant TGFβ1 (2ngml−1, 24hours) using an FITC conjugate (a), compared with fibroblasts cultured from TβRII-null-fib littermates (b). The upper panels show αSMA stain of fibroblasts at low magnification (× 10). Lower panels (c, d) are high-power (× 40) views. Dermal fibroblasts from TβRII-null-fib skin show a normal cytoskeleton (phalloidin-rhodamine conjugate) but few αSMA-rich stress fibers (FITC conjugate). There was no consistent αSMA staining in the absence of recombinant TGFβ1. Data are representative of three independent experiments (bar=100μm (a, b) 50μm (c, d)).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions