TGF-β signaling affects the growth of human MM cells. (A) Immunohistochemical staining for p-Smad2 of MM tissues derived from patients. Three representative sections, namely sarcomatoid, biphasic, and epithelioid subtype tumors, are shown. Azan staining was performed to visualize collagen fibers. H&E, hematoxylin and eosin. (B) Immunohistochemical staining for p-Smad2 in normal mesothelial cells (closed arrowheads) from normal lungs and reactivated normal mesothelial cells (open arrowheads) adjacent to MM tumors. Asterisks show cells with positive nuclear staining in normal mesothelial cells. (C) Cell numbers were counted 3 d after treatment with TGF-β type I receptor kinase inhibitor (SD-208). Results are expressed as mean ± SEM and are representative of three independent assays. (D) Soft agar colony formation assay was performed using MeT-5A cells treated with 4 ng/ml TGF-β for 7 d. The panel is representative of three independent assays. (E) NCI-H290 cells were infected with the indicated lentiviral expression vectors and stained with Giemsa after 14 d. HA-tagged luciferase (luc) was used as a control. The panel is representative of three independent assays. TβRIIDNR, dominant-negative form of the TGF-β type II receptor; ALK5KR, dominant-negative form of the TGF-β type I receptor; ALK5TD, constitutively activated TGF-β type I receptor.

The CTGF expression level was modulated by the TGF-β and Hippo pathways in MM cells. (A) NCI-H290 cells, which show homologous deletion of NF2 with concomitant YAP translocation to the nucleus, were transfected with plasmids containing shYAP. After 48 h of puromycin selection, TGF-β was added, followed by mRNA extraction after 2 and 24 h to perform real-time RT-PCR to evaluate gene expression. A plasmid with a nontarget sequence (NT) was used as the control. (B) Lentiviral vectors containing full-length and truncated NF2 that lack the ability to phosphorylate YAP on Ser 127 were used to infect NCI-H290 cells. Real-time RT-PCR was performed using mRNA extracted 2 h after TGF-β treatment. −, untreated or uninfected; NF2tr, truncated NF2. (A and B) Results are expressed as mean ± SEM and are representative of three independent assays.

Smad3 interacts with YAP and enhances the transactivation activity of the CTGF promoter. (A) The human CTGF promoter region contains a Smad-binding site (−227 to −220) and a TEAD-binding site (−219 to −214) adjacent to each other. A luciferase reporter plasmid was linked to the CTGF promoter with (CAGA, −245 to 16) or without (w/o CAGA, −206 to 16) the Smad/TEAD-binding sites indicated. (B–D) NCI-H290 cells were transfected with CTGF-luciferase reporter plasmids with or without the Smad/TEAD-binding sites, together with the indicated plasmids. After 24 h, either the TGF-β or TβRI inhibitor was added to the medium and incubated for an additional 24 h. Fold induction of transcriptional response relative to untreated cells is shown. Results are expressed as mean ± SEM and are representative of three independent assays. (E) Effects of TGF-β treatment on the activation of the CTGF promoter by YAP and Smads in NCI-H290 cells. Fold induction of transcriptional response relative to untreated cells is shown. Results are expressed as mean ± SEM and are representative of three independent assays. (F) Y-MESO-27 cells were lentivirally infected with either nontarget (NT) or YAP shRNA (shYAP) vector, followed by treatment with TGF-β type I receptor kinase inhibitor. Protein levels were determined by Western blotting. (G) Y-MESO-27 cells were lentivirally infected to express YAP protein and were treated with TGF-β. A lentivirus vector containing HA-luciferase was used as a control. Protein levels were determined by Western blotting. (H) HEK293 cells were transiently transfected with flag-tagged Smad vectors, and lysates were subjected to immunoprecipitation (IP), followed by Western blotting (WB) with anti-YAP antibody. Expression levels of exogenous Smads were confirmed in the bottom panel. (I) Schematic representation of the deletion constructs of YAP. CC, CC domains; FL, full length; TB, TEAD-binding domain; WW, WW domains. (J) HEK293 cells were transiently transfected with the indicated plasmids and subjected to immunoprecipitation. Western blotting was performed to confirm the expression level of YAP and Smad3 (top). NCI-H290 cells were transfected with the CTGF-luciferase reporter plasmid and combinations of Smad3 and YAP deletion constructs (bottom). Results are mean ± SEM and are representative of three independent assays.

Smad3, YAP, TEAD, and p300 are components of a functional complex on the CTGF promoter. (A and B) HEK293 cells were transiently cotransfected with indicated plasmids, and lysates were subjected to immunoprecipitation (IP) followed by Western blotting (WB). (C) HEK293 cells were transfected with expression vectors as indicated. Cell lysates were divided and subjected to immunoprecipitation using p300, YAP, and TEAD4 antibodies. Samples subjected to Western blot are shown in the lowest panels. (D) Interaction between endogenous TEAD4 and p-Smad3 was examined using TGF-β–treated Y-MESO-27 cells. (E) Y-MESO-27 cells were infected with a TEAD4 lentiviral vector. After 4 d, the cells were treated with TGF-β, and lysates were immunoprecipitated with Smad2/3 antibodies and detected by YAP antibodies. (F) NCI-H290 cells were transfected with the CTGF-luciferase reporter plasmid and combinations of TGF-β, YAP, and p300. Results are expressed as mean ± SEM and are representative of three independent assays. (G) NCI-H290 cells were transfected with the CTGF-luciferase reporter together with the indicated plasmids. Luciferase activity (top) and protein levels (bottom) are shown. Results are expressed as mean ± SEM and are representative of three independent assays. (H) ChIP analysis was performed using MSTO-211H cells by pulling down endogenous Smad2/3, p300, YAP, and TEAD4. CTGF promoter with Smad- and TEAD-binding adjacent regions was amplified by PCR. The value was normalized by input. The results shown are representative of three independent assays. (I) ChIP-reChIP assay was performed using TGF-β–treated MSTO-211H cell lysates, followed by quantitative PCR (qPCR). The first and second primary antibodies used for immunoprecipitation are indicated. (H and I) Results are expressed as mean ± SEM and are representative of three independent assays.

CTGF expression affects the growth and malignancy of MM cells. (A) NCI-H290 cells were lentivirally infected with shRNA against CTGF (shCTGF). Cell proliferation analysis was performed 4 d after lentiviral shRNA transduction. The endogenous protein level of CTGF was confirmed by Western blotting (left). Cell number was counted and normalized to the nontarget (NT) control (right). (B) NCI-H290 cells were infected with the indicated lentivirus expression vectors and stained with Giemsa after 14 d. HA-tagged luciferase (luc) was used as a control for CTGF and nontarget control for shCTGF. The results shown are representative of three independent assays. (C) Soft agar colony formation assay was performed using shRNA lentivirus–transduced NCI-H290 cells and stained with 0.5 mg/ml p-iodonitrotetrazolium after 10 d. The lower panel shows the mean size of colonies in each well. Colony number was counted in a range with a >100-µm diameter. (D) Real-time RT-PCR was performed using NCI-H290 cells infected by shRNA lentivirus 24 h after the treatment of TGF-β. (A, C, and D) Results are expressed as mean ± SEM and are representative of three independent assays. (E) NCI-H290 cells infected with the shCTGF lentivirus were kept under puromycin selection. Cells were then infected with the ALK5 lentivirus and stained using Giemsa after 14 d. The results shown are representative of three independent assays.

CTGF expression associates with the deposition of ECM protein in MM tumors. (A) Immunohistochemical staining of MM cells implanted in the thoracic cavities of athymic nude mice using p-Smad2, YAP, and CTGF antibodies. Collagen fibers were visualized in blue by azan staining (left). The right panel shows the scoring of the immunohistochemical staining. Staining intensity was scored as follows: 1, normal; 2, mild; 3, moderate; and 4, severe. (B) The Kaplan-Meier method was used to monitor the survival of athymic nude mice after the thoracic implantation of the NCI-H290 cells, followed by initiation of treatment with the TβRI inhibitor SD-208 (60 mg/kg) 2 d later (left). SD-208 was administered daily by oral gavage for 10 d (n = 9), and then tumor tissues were excised and subjected to Western blotting (right). The results shown are representative of two independent experiments. (C) NCI-H290 cells lentivirally transduced with shRNA constructs were implanted into the thoracic cavities of athymic nude mice, and survival was monitored (n = 8). The results shown are representative of three independent experiments. (D and E) Immunohistochemical staining of CTGF and YAP in MM tissues derived from patients with sarcomatoid as well as biphasic and epithelioid subtypes. Normal mesothelial cells (closed arrowheads) and reactivated normal mesothelial cells (open arrowheads) are shown in the right panels.