Figure 1.
(A–C) On day 0, A549 cells were seeded at 4×105 cells per 60-mm dish. On day 1, cells were treated with 1 ng/ml TGF-β for the indicated time. For treatment longer than 24 h, cells were changed to fresh medium containing TGF-β once every 24 h. (A and B) Cells were harvested and separated into nuclear and membrane fractions, and analyzed by immunoblot analysis with indicated antibodies. Immunoblot analysis with antibodies against calnexin and lysine-specific demethylase 1 (LSD1) served as loading controls for membrane and nuclear fractions, respectively. (C) Cell lysate was subjected to immunoblot analysis with indicated antibodies.
Figure 2.
Sustained induction of collagen synthesis by TGF-β requires CREB3L1.
(A–F) On day 0, A549 cells were seeded at 1×105 cells per 60 mm dish. On day 1, the cells were transfected with indicted siRNAs. (A) On day 3, cells were harvested for quantification of CREB3L1 mRNA by real time-quantitative PCR (RT-QPCR). The amount of the mRNA in cells transfected with the control siRNA is set to 1. (B–F) On day 3, cells were treated with 0.5 ng/ml TGF-β for the indicated time as described in Fig. 1. Cells were then harvested for quantification of indicated mRNA through RT-QPCR. The amount of the indicated mRNA in cells transfected with the control siRNA immediately before the TGF-β treatment is set to 1. (A–F) Results are reported as mean ± S.E.M. of three independent experiments.
Figure 3.
Smad4 is a co-factor for CREB3L1 to induce transcription of COL1A1 and SPARC.
(A) Quantification of Smad4 mRNA through RT-QPCR following transfection of indicated siRNA was performed as described in Fig. 2A. (B) On day 0, A549 cells were seeded at 1×105 cells per 60 mm dish. On day 1, the cells were transfected with indicted siRNAs. On day 3, cells were treated with or without 1 ng/ml TGF-β. On day 4, 24 h after the treatment, cells were harvested and analyzed as in Fig. 1A. (C–D) Quantification of the indicated mRNA following transfection with the indicated siRNA and treatment with TGF-β for the indicated time was performed as described in Fig. 2C. (E) On day 0, A549 cells were seeded at 4×105 cells per 60 mm dish. On day 1, cells were treated with or without 1 ng/ml TGF-β. On day 2, 24 h after the treatment, cells were harvested. Cell lysates were subjected to immunoprecipitation with the indicated antibodies. The immunoprecipitates (pellet) from 2 dishes of the cells and supernatant (sup) from 0.7 dishes of the cells were analyzed by immunoblot analysis with the indicated antibodies. (F–H) On day 0, Huh7 cells were seeded at 5×104 cells per 60 mm dish. On day 1, cells were transfected with indicted siRNAs. On day 3, cells were treated with or without 500 nM doxorubicin. (F and H) On day 4, 24 h after the treatment, cells were harvested for quantification of indicated mRNA by RT-QPCR. The amount of the mRNA in cells that were not treated with doxorubicin and transfected with the control siRNA is set to 1. (G) On day 4, 24 h after the treatment, cells were harvested and RIP of CREB3L1 was analyzed as described in Fig. 1A. (A, C, D, F and H) Results are reported as mean ± S.E.M. of three independent experiments.
Figure 4.
TGF-β induces CREB3L1 cleavage by inhibiting expression of TM4SF20.
(A) On day 0, A549 cells were seeded at 1×105 cells per 60 mm dish. On day 1, the cells were transfected with indicted siRNAs. On day 3, cells were treated with or without 1 ng/ml TGF-β for 12 h. cells were then harvested for quantification of TM4SF20 mRNA by RT-QPCR. The amount of the mRNA in cells that were not treated with TGF-β and transfected with the control siRNA is set to 1. (B) On day 0, A549 cells were seeded at 4×105 cells per 60 mm dish. On day 1, the cells were treated with 1 ng/ml TGF-β for the indicated time. Cells were then harvested for quantification of TM4SF20 mRNA by RT-QPCR. The amount of the mRNA in cells immediately before the TGF-β treatment is set to 1. (C and D) On day 0, A549 cells were seeded at 1×105 cells per 60 mm dish. On day 1, the cells were transfected with indicted siRNAs. On day 3, cells were treated with or without 1 ng/ml TGF-β. On day 4, 24 h after the treatment, cells were harvested for quantification of TM4SF20 mRNA as described in A (C), and analysis of RIP of CREB3L1 as described in Fig. 1A (D). (E and F) On day 0, A549 and A549/pTM4SF20 cells were seeded at 4×105 cells per 60 mm dish. On day 1, cells were treated with or without 1 ng/ml TGF-β. On day 2, 24 h after the treatment, cells were harvested for quantification of TM4SF20 mRNA by RT-QPCR, with the amount of the mRNA in A549 cells that were not treated with TGF-β set to 1 (E), and analysis of RIP of CREB3L1 as described in Fig. 1A (F). (A–G) Bar graphs are reported as mean ± S.E.M. of three independent experiments.
Figure 5.
TGF-β induces cleavage of CREB3L1 through activation of ERKs.
(A) A549 cells were set up, treated, and analyzed with immunoblot with indicated antibodies as described in Fig. 1C. (B–D) On day 0, A549 cells were seeded at 4×105 cells per 60 mm dish. On day 1, cells were treated with 0.5 µM RDEA119 or PD0325901 for 3 h followed by treatment with 1 ng/ml TGF-β as indicated. On day 2, 24 h after the TGF-β treatment, cells were harvested for immunoblot analysis with indicated antibodies (B and D) and quantification of TM4SF20 mRNA with RT-QPCR, with the amount of the mRNA in untreated cells set to 1 (Results are reported as mean ± S.E.M. of three independent experiments) (C).
Figure 6.
A model illustrating the role of CREB3L1 in TGF-β-induced collagen synthesis.
In the absence of TGF-β, Smad2 and Smad3 (Smad2/3) are not phosphorylated. RIP of CREB3L1 is blocked by TM4SF20. In the absence of activation of these transcription factors, expression of collagen is not induced. Acute exposure of cells to TGF-β results in heterodimerization of two TGF-β receptors, namely TGFβRI and TGFβRII. Consequently, Smad2/3 are phosphorylated by the activated receptor, allowing them to form a complex with Smad4 to drive transcription of collagen. TGF-β treatment also leads to phosphorylation of ERKs, which in turn inhibit transcription of TM4SF20. In cells chronically exposed to TGF-β, the amount of phosphorylated Smad2/3 is drastically reduced. Owing to depletion of TM4SF20, CREB3L1 is cleaved by S1P and S2P. This cleavage releases the NH2-terminal domain of CREB3L1 from membranes, allowing it to form a complex with Smad4 to continue activating transcription of collagen.