Neonatal human keratinocytes were cultured in CellnTec basal media (containing 0.07mM calcium) or basal media supplemented with 10μM Y-27632.

<p>Cells were assessed for cell viability at each passage, performed at 90% confluence. Trypan blue exclusion assay differentiated between viable and non-viable cells based upon membrane permeability and growth rate calculated as population doubling per day (A). Growth rate is represented graphically as population doubling over days in culture (B). Phase contrast images at 20x magnification demonstrate morphology of primary passage 3 and 10 HEKn cultured in CnT-07 alone and passage 3 and 10 HEKn-CaY supplemented with Y-27632 (C). n = 3 isolations from separate donors. Data is expressed as Mean +/- SEM. Images are representative of three experimental repeats.</p

Passage 10 HEK-CaY were seeded onto LabTek II Chamber slides for the 2D differentiation model.

<p>Cells were cultured for 72hrs in either 0.07 calcium and assessed for K14 (D), K10 (E) or Inv (F) expression by immunocytochemistry or 1.2mM calcium and K14 (G), K10 (H) or Inv (I) expression assessed. DAPI nuclear counterstain was also performed on cells grown in 0.07mM (A-C) and 1.2mM (J-L) calcium to confirm the presence of live cells. All images are taken at 20x magnification and are representative of three experimental repeats.</p

Diagram of human skin showing differentiated keratinocytes of the epidermis and expression of stratification markers Keratin 10 (K10), Keratin 14 (K14) and Involucrin (Inv).

<p>Diagram of human skin showing differentiated keratinocytes of the epidermis and expression of stratification markers Keratin 10 (K10), Keratin 14 (K14) and Involucrin (Inv).</p

mRNA was extracted from HaCaT, HEKn cells (passage 1 & 3) or HEK-CaY (passage 3 & 10) and expression of differentiation markers K10 (A) and Inv (B) or basal marker K14 (C) assessed by qRT-PCR normalised to multiple reference genes B2M and YWHAZ.

<p>Data is expressed as relative normalised expression, Mean +/- SEM, n = 3. *** = p<0.001, ns = not significant, p>0.05, ANOVA. Isolated protein was assessed by western blot to confirm expression with β-tubulin used as a loading control (D). Images are representative of three experimental repeats.</p

LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation

<div><p>Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor β (TGF-β), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-β, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9–14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.</p></div

FGF-2 has a single binding domain in the central region of LTBP-2.

<p><b>A</b>. Three recombinant fragments spanning the LTBP-2 molecule were tested for binding to FGF-2 in a solid phase assay. Full length LTBP-2(H), fragments LTBP-2 NT (H), LTBP-2C (H), LTBP-2 CT (H) or BSA control were coated onto wells at 100 ng/ml, followed by incubation with FGF-2 (100ng/ml) for 3h at 37°C. Strong specific binding to central fragment LTBP-2C(H) was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2A</a>. Mean values ± S.D. from triplicate wells are shown. <b>B</b>. A binding curve was produced for the FGF-2 interaction with fragment LTBP-2C(H) following the protocol described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>, with 400 ng/well (4.8 pmol) of LTBP-2C (H) or BSA control coated on the wells incubated with increasing concentrations FGF-2 (0–1.5 nM). The Kd for binding of FGF-2 to fragment LTBP-2C (H) was calculated as 1.02 ± 0.19 nM. Mean values ± S.D. from triplicate determinations are shown. <b>C</b>. Three sub-fragments F1, F2 and F3 spanning fragment LTBP-2 C(H) were produced and tested for FGF-2 binding as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>. LTBP-2C (H) (200 ng/well, 2.4 pmol) or sub-fragment (F1, F2 or F3) (66ng/well, 2.4 pmol) or BSA control was coated on the wells and incubated with FGF-2 (100 ng/ml). Strong specific binding of FGF-2 to sub-fragment LTBP-2C F2 was detected. Mean values ± S.D. from triplicate wells are shown. <b>D</b>. Subsequently binding curves were obtained for sub-fragments F1 (solid squares), F2 (open circles), F3 (solid circles) (35 ng/well, 1.2 pmol) coated on the wells and incubated with increasing concentrations of FGF-2 (0–30 ng / ml). Note specific FGF-2 binding to sub-fragment LTBP-2C F2 but no binding of fragments F1 and F3 above the BSA control (triangles). Mean values ± S.D. from triplicate determinations are shown. <b>E</b>. The Kd for the FGF-2 interaction with sub-fragment LTBP-2C F2 was calculated as 1.03 ± 0.10 nM which is similar to the Kds calculated for the interactions of FGF-2 with full-length LTBP-2 and fragment LTBP2C. Mean values ± S.D. from triplicate determinations are shown.</p

Recombinant LTBP-2 Fragments.

<p><b>A</b>. Schematic diagram of recombinant LTBP-2 fragments. Protein fragments generated specifically for this study (LTBP-2C(H) F1, F2 and F3) are highlighted within the blue box. FGF-2 binding was confined to a single central region of the LTBP-2 molecule consisting of 6 EGF-like repeats (fragment LTBP-2C(H) F2).<b>B</b>. SDS-PAGE of purified recombinant LTBP-2 fragments. Samples of purified fragments LTBP-2 C(H), LTBP-2 C(H) F1, LTBP-2 C(H) F2 and LTBP-2 C(H) F3 were analyzed on a 12% gel under non-reducing conditions and stained with Coomassie blue. The relative mobilities of protein standards are indicated by arrows.</p

FGF-2 has a single binding domain in the central region of LTBP-2.

<p><b>A</b>. Three recombinant fragments spanning the LTBP-2 molecule were tested for binding to FGF-2 in a solid phase assay. Full length LTBP-2(H), fragments LTBP-2 NT (H), LTBP-2C (H), LTBP-2 CT (H) or BSA control were coated onto wells at 100 ng/ml, followed by incubation with FGF-2 (100ng/ml) for 3h at 37°C. Strong specific binding to central fragment LTBP-2C(H) was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2A</a>. Mean values ± S.D. from triplicate wells are shown. <b>B</b>. A binding curve was produced for the FGF-2 interaction with fragment LTBP-2C(H) following the protocol described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>, with 400 ng/well (4.8 pmol) of LTBP-2C (H) or BSA control coated on the wells incubated with increasing concentrations FGF-2 (0–1.5 nM). The Kd for binding of FGF-2 to fragment LTBP-2C (H) was calculated as 1.02 ± 0.19 nM. Mean values ± S.D. from triplicate determinations are shown. <b>C</b>. Three sub-fragments F1, F2 and F3 spanning fragment LTBP-2 C(H) were produced and tested for FGF-2 binding as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.g002" target="_blank">Fig 2</a>. LTBP-2C (H) (200 ng/well, 2.4 pmol) or sub-fragment (F1, F2 or F3) (66ng/well, 2.4 pmol) or BSA control was coated on the wells and incubated with FGF-2 (100 ng/ml). Strong specific binding of FGF-2 to sub-fragment LTBP-2C F2 was detected. Mean values ± S.D. from triplicate wells are shown. <b>D</b>. Subsequently binding curves were obtained for sub-fragments F1 (solid squares), F2 (open circles), F3 (solid circles) (35 ng/well, 1.2 pmol) coated on the wells and incubated with increasing concentrations of FGF-2 (0–30 ng / ml). Note specific FGF-2 binding to sub-fragment LTBP-2C F2 but no binding of fragments F1 and F3 above the BSA control (triangles). Mean values ± S.D. from triplicate determinations are shown. <b>E</b>. The Kd for the FGF-2 interaction with sub-fragment LTBP-2C F2 was calculated as 1.03 ± 0.10 nM which is similar to the Kds calculated for the interactions of FGF-2 with full-length LTBP-2 and fragment LTBP2C. Mean values ± S.D. from triplicate determinations are shown.</p

LTBP-2 blocks FGF-2-induced cell proliferation.

<p><b>A</b>. The effect of LTBP-2 on the bio-activity of FGF-2 was tested in a cell proliferation assay (see experimental). Human foreskin fibroblasts were treated with FGF-2 with and without follistatin (white columns), or FGF-2 and follistatin pre-incubated with 5 or 10 fold molar excess of full length LTBP-2 or fragment LTBP-2C F2 (cross-hatched). Negative controls (black columns), included cells only and cells incubated with follistatin, LTBP-2 or fragment LTBP-2C F2. Mean values ± S.D. from triplicate determinations. Note 5 fold molar excess of full-length LTBP-2 completely blocked FGF-2 induced cell proliferation (p = 0.0001) and 5-fold molar excess of fragment LTBP-2C F2 partially blocked the activity (p = 0.0001). <b>B</b>. Immunoblot analysis FGF receptor (FGFR1) phosphorylation. Human foreskin fibroblasts were treated for 2 hours with FGF-2 (10 ng / ml) only or with FGF-2 plus 10-fold molar excess of full length LTBP-2 (LTBP-2 FL) or fragment F2 (LTBP-2C F2). Control cells had no FGF-2 or LTBP-2 added. Cellular proteins were extracted and duplicate samples were analysed by SDS-PAGE and immunoblotting with anti-phospho-FGFR1 antibody, and anti-total FGFR1 antibody. Bands were visualised using the LI-COR Odyssey Infrared Imaging System. <b>C</b>. The band intensity was measured using ImageJ 1.48 software [National Institutes of Health (NIH), Bethesda, MD] and normalised to the internal β actin signal. The ratio of the phospho-FGFR1 to total FGFR1 value for each sample is expressed relative to the average FGF-2 only control value (= 100%). Note the strong FGFR1 activation by FGF-2 was substantially blocked by both LTBP-2 C and LTBP-2C F2 fragments. Mean values ± S.D. of duplicate lanes.</p

The FGF-2 binding site is close to the central heparin binding site on LTBP-2.

<p>In a previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135577#pone.0135577.ref032" target="_blank">32</a>] we identified LTBP-2 C(H) as a heparin-binding fragment of LTBP-2. To further define the location of this heparin binding activity, the three sub-fragments F1, F2, F3 spanning LTBP-2 C(H), were assayed for heparin binding using a heparin-albumin conjugate (HAC). HAC or BSA control (400 ng) was coated on wells followed by incubation with equimolar concentrations (23.5 nM) of LTBP-2C(H) or sub-fragment F1, F2 or F3. Specific binding was detected using anti-His₄ antibody targeting the poly-His tag on each recombinant fragment. Fragment F2 showed strong specific binding to the heparin conjugate in contrast to F1 and F3 which showed no binding above background. Mean values ± S.D. from triplicate wells are shown.</p