Gunther S, Alexander RW, Atkinson WJ, Gimbrone MA, Jr.: Functional

angiotensin II receptors in cultured vascular smooth muscle cells. J Cell Biol 1982, 92:289-298.

Figures

Figure 1 TGF induces CRP2 expression through Smad2/3 and ATF2. (A) TβRI kinase activity contributes to CRP2 induction. VSMCs were pretreated with vehicle or TβRI kinase inhibitor SB431542 (10 µM) for 30 min before stimulation with TGFβ (10 ng/ml) for 24 h. Total proteins were then harvested for Western blot analysis to detect CRP2 expression. SB431542 significantly decreased TGFβ-induced CRP2 expression.

Values are mean ± S.E. of at least three experiments. *P<0.05 vs. control (– TGFβ);

#P<0.05 vs. TGFβ-stimulated vehicle group. (B) TβRI kinase activity is required for

activation of Smad2/3 but not ATF2. VSMCs were pretreated with vehicle, SB431542, PI3K inhibitor Wortmannin (1 µM), or LY294002 (10 µM) for 30 min prior to stimulation with or without TGFβ for 15 min. Activation of Smad2/3 and ATF2 was then determined with Western blot analysis. (C-D) VSMCs were transfected with 20 nM control siRNA, Smad2/3 siRNA (C) or ATF2 siRNA (D) using Lipofectamine RNAiMAX transfection reagent and then stimulated with or without TGFβ for 24 h. Western blot analysis was

performed to detect CRP2, Smad2/3, or ATF2 levels. The membranes were

subsequently probed with actin for loading control. Values are mean ± S.E. of at least three experiments. ^#P<0.05 vs. TGFβ-stimulated siControl group.

Figure 2 Neither TAK1 nor TRAF6 mediates TGFβ-induced ATF2 activation.

VSMCs were transfected with 20 nM TAK1 siRNA (A) or TRAF6 siRNA (B) using

Lipofectamine RNAiMAX transfection reagent and then stimulated with or without TGFβ for 10 min. Control siRNA was also transfected as a control. Western blot analysis was performed to detect TAK1, TRAF6, and phosphorylation of ATF2 and Smad2. The membranes were subsequently probed with actin for loading control. (C) VSMCs were transfected with control or TRAF6 siRNA and then treated with or without TGFβ for 24 h. Total proteins were then isolated for Western blot analysis to detect TRAF6 and CRP2 expression. The membranes were subsequently probed with actin for loading control. Representative blots of at least three independent experiments are shown.

Figure 3 Type II TGF receptor is crucial in mediating TGF-induced ATF2 signaling. (A-C) VSMCs were transfected with 20 nM control siRNA or siRNA to different types of TGF receptors. Following 24 h recovery in growth media and 24 h serum-starvation, cells were stimulated with or without TGFβ for 10 min. Western blot analysis was performed to detect different TGF receptor expression levels and phosphorylation of Smad2 and ATF2. (A) TRI knockdown inhibits Smad2

phosphorylation but not that of ATF2. ^#P<0.05 vs. TGFβ-stimulated siControl group. (B) TRIII knockdown does not affect TGF activation of ATF2 or Smad2. (C) TRII

knockdown attenuates TGF activation of ATF2 and Smad2. ^#P<0.05 vs.

TGFβ-stimulated siControl group. (D) Dominant-negative TRII (DN-TRII) impairs Smad2 but not ATF2 activation. VSMCs were electroporated with control vector or HA-tagged DN-TRII (HA-DN-TRII(∆Cyt)) and then stimulated with or without TGF for 10 min. Western blot analysis was performed to assess phosphorylation of Smad2 and ATF2.

Overexpression of DN-TRII was evaluated by probing Western blots with HA antibody.

#P<0.05 vs. TGFβ-stimulated vector control group. (E) DN-TRII impairs TGF-induced

CRP2 induction. VSMCs were electroporated with control vector or HA-TRII(∆Cyt) and stimulated with TGF for 24 h. Total proteins were then isolated for Western blot

analysis to detect CRP2 protein levels. The blots were then probed with HA antibody to detect HA-TRII(∆Cyt) expression. ^#P<0.05 vs. TGFβ-stimulated vector control group.

The membranes were subsequently probed with actin for loading control.

Representative blots of at least three independent experiments are shown.

Figure 4 Src family kinase mediates TRII-dependent TGF activation of RhoA and ATF2 in VSMCs. (A) Src family kinase inhibitor SU6656 dose-dependently reduces TGF activation of ATF2 but not that of Smad2. VSMCs were pretreated with increasing concentrations of SU6656 for 30 min and then stimulated with TGF for 10 min.

Phosphorylation of ATF2 and Smad2 was determined by Western blot analysis. The membranes were subsequently probed with actin for loading control. ^#P<0.05 vs. TGFβ-stimulated but without SU6656 treatment group. (B) SU6656 abolishes TGF-induced RhoA activation. VSMCs were pretreated with 5 µM SU6656 for 30 min prior to

stimulation with TGF for 10 min. RhoA activation was then determined by

GST-Rhoteckin-RBD assays. GTP-RhoA was subsequently eluted and subjected to Western blot analysis with an anti-RhoA antibody. To verify equal loading, 15 µg of cell lysates were run on separate gels and blots probed with an anti-RhoA antibody for total RhoA.

*P<0.05 vs. control (– TGFβ and – SU6656); ^#P<0.05 vs. TGFβ-stimulated vehicle group. (C) RhoA knockdown reduces TGFβ activation of ATF2 but not Smad2. VSMCs were transfected with 20 nM control siRNA or RhoA siRNA and then stimulated with or without TGFβ for 10 min. Western blot analysis was performed to detect RhoA and phosphorylation of ATF2 and Smad2. ^#P<0.05 vs. TGFβ-stimulated siControl group. (D) RhoA knockdown reduces TGFβ-induced CRP2 expression. VSMCs were transfected with 20 nM control siRNA or RhoA siRNA and then stimulated with or without TGFβ for 24 h. Western analysis was performed to detect RhoA and CRP2 protein levels. The membranes were subsequently probed with actin for loading control. ^#P<0.05 vs. TGFβ-stimulated siControl group. Representative blots of at least three independent

experiments are shown.

Figure 5 ROCK mediates Smad-independent ATF2 activation by TGFβ. (A) VSMCs were pretreated with vehicle or ROCK inhibitor Y-27632 (10 µM) for 30 min before stimulation with TGFβ for 10 min. Western blot analyses were then performed to examine phosphorylation of ATF2 and Smad2. ^#P<0.05 vs. TGFβ-stimulated vehicle group. (B) VSMCs were pretreated with vehicle or Y-27632 for 30 min before

stimulation with or without TGFβ for 24 h. CRP2 expression was detected by Western blot analysis. ^#P<0.05 vs. TGFβ-stimulated vehicle group. Representative blots of at least three independent experiments are shown.

Figure 6 JNK activation is required for TGF-induced ATF2 phosphorylation and CRP2 induction. (A) JNK activation is required for TGFβ-induced phosphorylation of ATF2 but not Smad2/3. VSMCs were pretreated with vehicle, JNK inhibitor SP600125 (10 µM), p38 inhibitor SB203580 (10 µM), or ERK1/2 inhibitor U0126 (10 µM) for 30 min before stimulation with TGFβ (10 ng/ml). Phosphorylation of ATF2, JNK, p38, and ERK1/2 was determined 15 min after TGFβ stimulation. The membranes were subsequently probed with antibodies against total proteins for ATF2, JNK, p38, and ERK1/2 for loading control. (B) VSMCs were pretreated with vehicle or SP600125 for 30 min before stimulation with TGFβ. Cell lysates were harvested at the indicated time points and the phosphorylation of ATF2, JNK, and Smad2 examined. The membranes were subsequently probed with antibodies against total proteins for ATF2, JNK, and Smad2 for loading control. (C) JNK activity contributes to CRP2 induction. VSMCs were pretreated with vehicle or SP600125 for 30 min before stimulation with TGFβ for 24 h.

Total proteins were then harvested for Western blot analysis to detect CRP2

expression. Values are mean ± S.E. of at least three experiments. *P<0.05 vs. control (–

TGFβ); ^#P<0.05 vs. TGFβ-stimulated vehicle group. (D) Constitutively active FLAG-C2/ATF2 increases CRP2 expression. VSMCs were electroporated with control vector or FLAG-C2/ATF2 and total proteins prepared 24 h later. Western blot analysis was performed to assess CRP2 levels. Overexpression of FLAG-C2/ATF2 was evaluated by probing Western blots with FLAG antibody. (E) Overexpression of FLAG-C2/ATF2 rescues SP600125-inhibited TGFβ induction of CRP2. VSMCs were electroporated with control vector or FLAG-C2/ATF2, serum-starved, pretreated with vehicle or JNK

inhibitor SP600125 for 30 min before stimulation with TGFβ for 24 h. Western blot analysis was performed to assess CRP2 expression. FLAG-C2/ATF2 expression was evaluated with FLAG antibody. (F) VSMCs were pretreated with vehicle or ROCK inhibitor Y-27632 (10 µM) for 30 min before stimulation with or without TGFβ for 24 h.

CRP2 expression was detected by Western blot analysis. The membranes were subsequently probed with actin for loading control. *P<0.05 vs. control (– TGFβ);

#P<0.05 vs. TGFβ-stimulated vehicle group. Representative blots of at least three

independent experiments are shown.

Figure 7 SBE445 and CRE461 are functionally important for basal and TGF-induced Csrp2 promoter activity. (A) Two putative SBE sites at bp –681 and –445 of the mouse Csrp2 promoter (bold and underlined). The CRE site at –461 is also

indicated (italic and underlined). (B) The putative SBE site at –445 and CRE at –461 are important for Csrp2 promoter activity. The –795 Csrp2 wild type and SBE and CRE mutant promoter constructs are schematically depicted in the left panel. VSMCs were transiently transfected with Csrp2 luciferase reporter constructs containing –795,

SBE681mut, SBE445mut, CREmut, or CREmut/SBE445mut in triplicate using FuGENE 6 transfection reagent. Two hours after transfection, cells were treated with or without TGF (10 ng/ml) for 24 h. Cells were then harvested for luciferase activity and protein assays. Luciferase activity is expressed relative to –795 without TGF treatment.

Values are mean ± S.E. of at least three experiments. (C) Conservation of the CRE and SBE sites among species. Sequence alignment of the corresponding regions of human and rat promoter sequences to the mouse promoter. The CRE site is in italic and SBE

site in bold type. (D) VSMCs were transiently cotransfected with –795Csrp2-luc reporter with empty vector or expression plasmids Smad2, C2/ATF2, or both. Cells were then harvested 24 h later for luciferase activity and protein assays. Luciferase activity is expressed relative to –795 with empty expression vector. Values are mean ± S.E. of at least three experiments. *P<0.05 vs. empty expression vector. (E) VSMCs were

electroporated with control vector or expression plasmids Smad2, C2/ATF2, or both.

Total RNA was prepared 12 h later for real-time PCR analysis to assess CRP2 mRNA expression and β-actin was used as an internal control for normalization. Quantification was performed by the comparative CT method. CRP2 mRNA is expressed relative to empty expression vector. Values are mean ± S.E. of at least three experiments. *P<0.05 vs. empty expression vector.

Figure 8 Schematic model of TGF signaling pathways for CRP2 induction in VSMCs. Upon TGF stimulation, two signaling axes are activated: the canonical TRI-dependent Smad2/3 pathway and the non-canonical ATF2 pathway. In the canonical pathway, activated Smad2/3 form a complex with Smad4, translocates into the nucleus, and binds to SBE site at –445 of the Csrp2 promoter. In the non-canonical pathway, following TRII binding of TGF, TRII (without the requirement of TRII’s cytoplasmic kinase domain) activates Src family kinase (SFK), which in turn activates RhoA.

Enhanced RhoA activation leads to ROCK activation and increased JNK

phosphorylation, resulting in enhanced ATF2 phosphorylation. Activated ATF2 dimer binds to CRE site at –461. The two signaling pathways (ATF2 and Smad) converge on the CRE and SBE sites of the Csrp2 promoter to cooperatively control CRP2 induction

in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGF.