Modulation of Type II TGF-β Receptor Degradation by Integrin-Linked Kinase  Linda Vi, Stellar Boo, Samar Sayedyahossein, Randeep K. Singh, Sarah McLean, Gianni M. Di Guglielmo, Lina Dagnino  Journal of Investigative Dermatology  Volume 135, Issue 3, Pages 885-894 (March 2015) DOI: 10.1038/jid.2014.427 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 ILK association with type II TGF-β receptors. (a) Ilkf/f fibroblasts were cultured in serum-free medium for 4 hours, followed by a 1-hour incubation period with or without TGF-β1 (10 ng ml−1). Cell lysates were prepared and subjected to immunoprecipitation with antibodies against TβRII or ILK. Immunecomplexes were resolved by denaturing gel electrophoresis and transferred to membranes, which were probed with the indicated antibodies. (b) Forty hours after transfection with vectors encoding V5-ILK and/or HA-TβRII, IMDF cells were cultured as described in panel a. Lysates were then prepared and immunoprecipitated with anti-HA or anti-V5 antibodies. The immunoblot results are representative of six experiments, using IMDF or four different dermal fibroblast isolates. IMDF (c) or Rat-2 fibroblasts (d) cells treated as in b were processed for confocal microscopy. ILK and TβRII were visualized using, respectively, anti-V5 and anti-HA antibodies. Arrows indicate areas shown at higher magnification in the insets. The confocal microscopy results are representative of three experiments conducted in triplicate. Bar = 10 μm. HA, hyaluronic acid; ILK, integrin-linked kinase; TGF-β, transforming growth factor-β. Journal of Investigative Dermatology 2015 135, 885-894DOI: (10.1038/jid.2014.427) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Protein regions involved in the interactions between ILK and TβRII. (a, b) IMDF cells were transfected with vectors encoding the indicated V5-ILK and/or HA-TβRII proteins and cultured for 40 hours. Cell lysates were immunoprecipitated with anti-HA antibodies and analyzed by immunoblot with anti-V5 antibodies. (c) Primary dermal fibroblasts were cultured in serum-free HyQ DMEM-RS medium for 3.5 hours. At this time, SB431542 (10 nM) or dimethylsulfoxide (vehicle) was added, and incubation proceeded for 30 minutes, followed by addition of TGF-β1 (10 ng ml−1, final) to the culture medium, and incubation for one additional hour. Cell lysates were immunoprecipitated with anti-ILK antibodies or unrelated IgG, followed by immunoblot analysis with anti-TβRII antibodies. P-SMAD 2, phospho-SMAD 2. The results are representative of five experiments, using IMDF or four different dermal fibroblast isolates. HA, hyaluronic acid; ILK, integrin-linked kinase; TGF-β1, transforming growth factor-β1; TβRII, type II TGF-β receptor; Wt, wild type. Journal of Investigative Dermatology 2015 135, 885-894DOI: (10.1038/jid.2014.427) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of Ilk gene inactivation on TβRII abundance and signaling. (a) Ilkf/f fibroblasts were infected with Ad-βgal or Ad-Cre, and 96 hours later cell lysates were prepared and analyzed by immunoblot with antibodies against TβRII or TβRI. (b) Cells were transduced as in a and 96 hours after infection were incubated in serum-free medium for 4 hours and then in medium with or without TGF-β1 for 1 hour. Lysates were prepared and immunoprecipitated with anti-TβRI antibodies. The immunecomplexes were analyzed by immunoblot, using anti-TβRII antibodies. The asterisk indicates a nonspecific band. The results are representative of five experiments, using three different dermal fibroblast isolates. ILK, integrin-linked kinase; TGF-β1, transforming growth factor-β1; TβRII, type II TGF-β receptor. Journal of Investigative Dermatology 2015 135, 885-894DOI: (10.1038/jid.2014.427) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 TβRII turnover in ILK-deficient cells. (a) Ilkf/f fibroblasts were infected with Ad-βgal or Ad-Cre and 96 hours later were incubated with cycloheximide. TβRII levels in ILK-expressing (dashed line) or ILK-deficient (solid line) cells were determined by densitometry and are expressed as the mean+SD (n=4), relative to those in each cell type at t=0 (set to 100%). The asterisks represent P<0.05, relative to the corresponding ILK-expressing samples (analysis of variance). (b) Fibroblasts infected with Ad-βgal or Ad-Cre were cultured for 96 hours. Lysates were prepared and immunoprecipitated with anti-TβRII antibodies. The immunecomplexes were analyzed by immunoblot with anti-TβRII and anti-ubiquitin antibodies. (c) Immunoblot analysis of lysates from fibroblasts infected as in b and treated 90 hours later with or without MG132 for 8 hours. The results are representative of four experiments, using three different dermal fibroblast isolates. ILK, integrin-linked kinase; TβRII, type II TGF-β receptor. Journal of Investigative Dermatology 2015 135, 885-894DOI: (10.1038/jid.2014.427) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inhibition of TβRII degradation and distribution to lipid rafts in ILK-deficient fibroblasts. Primary fibroblasts were infected with Ad-βgal or Ad-Cre. Ninety hours after infection, cells were incubated in serum-free medium for 4 hours, followed by a 4-hour incubation with MG132 (a) or nystatin (b). TGF-β1 was then added, and culture proceeded for 1 hour. Lysates were then prepared and analyzed by immunoblot with the indicated antibodies. The histograms show the levels of P-SMAD 2 normalized to total SMAD 2/3 (mean+SEM; n=4). The asterisks indicate P<0.05 relative to the corresponding sample incubated without TGF-β1; #P<0.05 (analysis of variance). ILK, integrin-linked kinase; TGF-β1, transforming growth factor-β1; TβRII, type II TGF-β receptor. Journal of Investigative Dermatology 2015 135, 885-894DOI: (10.1038/jid.2014.427) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of SCC conditioned medium on α-SMA expression. (a) Fibroblasts were transduced with Ad-βgal or Ad-Cre or were left untreated. Ninety-six hours after transduction, the cells were rinsed and incubated for 24 hours in serum-free medium containing BSA, TGF-β1 (10 ng ml−1), TGF-β1 (10 ng ml−1) with SB431542 (10 nM), or in serum-free conditioned medium prepared from SCC-25 cells (CMSCC25), with or without SB431542. The cells were fixed and processed for indirect immunofluorescence microscopy, using anti-GFP or anti-α-SMA antibodies. Bar = 20 μm. (b) The percentage of fibroblasts treated as described in a that exhibited α-SMA-positive actin fibers was scored. The results are expressed as the mean+SEM (n=4). The asterisks indicate P<0.05 relative to the corresponding cell sample cultured in BSA-containing medium (analysis of variance). GFP, green fluorescent protein; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1. Journal of Investigative Dermatology 2015 135, 885-894DOI: (10.1038/jid.2014.427) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions