Journal: Cell Communication and Signaling : CCS

Article Title: Divergent signaling pathways cooperatively regulate TGFβ induction of cysteine-rich protein 2 in vascular smooth muscle cells

doi: 10.1186/1478-811X-12-22

Figure Lengend Snippet: Type II TGFβ receptor is crucial in mediating TGFβ-induced ATF2 signaling. (A-C) VSMCs were transfected with 20 nM control siRNA or siRNA to different types of TGFβ 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 TGFβ receptor expression levels and phosphorylation of Smad2 and ATF2. (A) TβRI knockdown inhibits Smad2 phosphorylation but not that of ATF2. # P < 0.05 vs. TGFβ-stimulated siControl group. (B) TβRIII knockdown does not affect TGFβ activation of ATF2 or Smad2. (C) TβRII knockdown attenuates TGFβ activation of ATF2 and Smad2. # P < 0.05 vs. TGFβ-stimulated siControl group. (D) Dominant-negative TβRII (DN-TβRII) impairs Smad2 but not ATF2 activation. VSMCs were electroporated with control vector or HA-tagged DN-TβRII (HA-TβRII(∆Cyt)) and then stimulated with or without TGFβ for 10 min. Western blot analysis was performed to assess phosphorylation of Smad2 and ATF2. Overexpression of DN-TβRII was evaluated by probing Western blots with HA antibody. # P < 0.05 vs. TGFβ-stimulated vector control group. (E) DN-TβRII impairs TGFβ-induced CRP2 induction. VSMCs were electroporated with control vector or HA-TβRII(∆Cyt) and stimulated with TGFβ 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-TβRII(∆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.

Article Snippet: Dominant-negative mutant of type II TGFβ receptor pCMV5 HA-TβRII(∆Cyt) that lacks C-terminal kinase domain and is unable to activate TβRI [ ] was obtained from Addgene (plasmid 14051).

Techniques: Transfection, Control, Western Blot, Expressing, Phospho-proteomics, Knockdown, Activation Assay, Dominant Negative Mutation, Plasmid Preparation, Over Expression, Isolation

Journal: Cell Communication and Signaling : CCS

Article Title: Divergent signaling pathways cooperatively regulate TGFβ induction of cysteine-rich protein 2 in vascular smooth muscle cells

doi: 10.1186/1478-811X-12-22

Figure Lengend Snippet: Schematic model of TGFβ signaling pathways for CRP2 induction in VSMCs. Upon TGFβ stimulation, two signaling axes are activated: the canonical TβRI-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 TβRII binding of TGFβ, TβRII (without the requirement of TβRII’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 TGFβ.

Article Snippet: Dominant-negative mutant of type II TGFβ receptor pCMV5 HA-TβRII(∆Cyt) that lacks C-terminal kinase domain and is unable to activate TβRI [ ] was obtained from Addgene (plasmid 14051).

Techniques: Protein-Protein interactions, Binding Assay, Activation Assay, Phospho-proteomics, Control