LPS mediated Smad2 carboxyl phosphorylation is dependent on TGFBR1 activation.
LPS stimulates Smad2 carboxy-terminal phosphorylation (Ser465/467) and its nuclear translocation in cultured hepatic stellate cells (HSC‑T6) [42]. We have recently reported that LPS stimulates Smad2 linker region phosphorylation in HAVSMCs [6]; however, the role of LPS mediated Smad2 carboxy-terminal phosphorylation in HA‑VSMCs remains to be elucidated. HA-VSMCs were treated with LPS in a time-dependent manner (0-240 min), and TGF‑β was used as a control. Treatment with LPS for 30 mins caused a 1.4-fold increase in the phospho-Smad2C (Fig. 1a) with a peak stimulation of 1.8-fold observed at 60 mins. TGF-β, used as a control, resulted in an 11-fold increase in phospho-Smad2C.
Canonical Smad2 carboxyl-terminal phosphorylation occurs as a direct outcome of the activation of the TGFBR1 [43]. To assess whether LPS signalling via its respective TLR could transactivate the TGFBR1 we utilised a pharmacological approach using the TLR4 antagonist, LPS-RS [44–46], and TGFBR1 antagonist, SB431542 [47]. LPS treatment of HA-VSMCs resulted in a 2.2-fold increase in phospho-Smad2C (Fig. 1b) that was inhibited in the presence of SB431542. LPS-RS used as a control completely inhibited LPS mediated Smad2 phosphorylation. The cells were treated with TGF‑β in the presence and absence of receptor antagonists, as expected TGF-β mediated phospho-Smad2C was completely inhibited in the presence of SB431542 and unaffected in the presence of TLR4 antagonist (Fig. 2b). These findings demonstrate that LPS via its cognate receptor, TLR4 (trans)activates the TGFBR1 leading to phospho-Smad2C. These findings highlight the existence of TLR4 transactivation of the TGFBR1 in HA‑VSMCS.
TLR4 transactivation of TGFBR1 is independent of MyD88 and TRIF pathways
TLR signalling pathways are classified into two distinct types: the myeloid differentiation primary response protein-88 (MyD88) and the TIR domain-containing adaptor-inducing IFN (TRIF)-dependent pathways. We asked whether TLR4 transactivation of the TGFBR1 was occurring via MyD88 or TRIF dependent pathways (Fig. 2). Schaftoside was utilised as an inhibitor of MyD88 signalling, and amlexanox was exploited as a specific inhibitor of the TBK1 inhibiting TRIF dependent signalling pathways. LPS mediated phosphorylation of Smad2C was unaffected in schaftoside and amlexanox treated cells, however, it was abolished entirely in the presence of a TGFBR1 antagonist. This demonstrates that the LPS mediated transactivation of the TGFBR1 is independent of MyD88 and TRIF pathways. Therefore, we aimed to delineate the underlying mechanisms of TLR4 transactivation of the TGFBR1.
Tlr4 Activates Matrix Metalloproteinases Leading To Phosphorylation Of Smad2c
We have recently demonstrated that in HA-VSMCs, GPCR transactivation of the TGFBR1 relies on cytoskeletal rearrangement, which activates Rho/ROCK and integrin-dependent signalling leading to conformational changes in the TGF-β complex and the activation of the TGFBR1 [22, 23, 31, 48, 49]. To study if LPS transactivation of the TGFBR1 was dependent on ROCK signalling, we used the potent and selective ROCK inhibitor Y27632 (Fig. 3a). Treatment with LPS resulted in a 2.3-fold increase in phospho-Smad2C that was unaffected in the presence of the ROCK inhibitor. We used the TGFBR1 antagonist as a control, and as expected, LPS mediated Smad 2 phosphorylation was completely inhibited. We have shown that in VSMCs thrombin mediated Smad2 phosphorylation was dependent on Rho/ROCK pathways [31, 48], therefore we treated cells with thrombin in the presence and absence of Y27632 as a control. Treatment with thrombin stimulated Smad2 phosphorylation to 2.6-fold, which was completely inhibited in the presence of Y27632. These results demonstrate that LPS mediated transactivation of the TGFBR1 leading to Smad2 phosphorylation in these cells was not dependent on Rho/ROCK pathways.
TGF-β can be cleaved and liberated from the large latent complex by proteolytic cleavage of matrix metalloproteinases (MMP) [50, 51]. To investigate whether LPS mediated phosphorylation of Smad2 is occurring via proteolytic activation of TGFBR1, we utilised the broad-spectrum MMP inhibitor, GM6001 (Fig. 3B). LPS treatment of VSMCs increased phospho-Smad2C to 2.7‑fold compared to the non‑treated control. This response was inhibited in the presence of GM6001 (1–10µM) with a complete inhibition observed with 3µM GM6001. HA‑VSMCs pre‑treated with SB431542 also showed complete inhibition of LPS mediated phosphorylation of Smad2 carboxy-terminal. These results demonstrate that TLR4 signals via MMPs to activate TGFBR1, which leads to Smad2 carboxy-terminal phosphorylation.
MMP2 but not MMP-9 is involved in LPS stimulation of phospho-Smad2C
We have identified above that MMPs are involved in LPS transactivation of the TGF-β receptor. Specifically, MMP2, 3, 9, 13 and 14 are involved in the release of TGFβ from the TGF-β complex. LPS increases the activity of MMP2 and MMP9 in rat aortic VSMCs [52]. We sought to investigate the role of MMP2 and MMP9 in TLR4 mediated transactivation of the TGFBR1 using a pharmacological and biochemical approach (Fig. 4a). HA-VSMCs were treated with LPS with MMP‑2 specific inhibitor, pyridoxatin or MMP‑9 specific inhibitor, Ab1421180. Treatment with LPS increased phosphorylation of Smad2 by 3.3-fold that was completely inhibited in the presence of pyridoxatin and unaffected by the presence of AB1421180. We thus demonstrate that LPS is signalling via MMP2 dependent pathways to stimulate phospho-Smad2 in HA-VSMCs.
We next investigated whether LPS mediated MMP-2 secretion from HA-VSMCs (Fig. 4b). LPS treatment of HA-VSMCs resulted in a 1.7-fold increase in the secretion of pro-MMP2, and a 2.4-fold increase in the secretion of active MMP-2 was observed over a 60 min treatment. These findings demonstrate that LPS stimulated MMP‑2 activity in HA-VSMCs, which endorses the involvement of MMPs in TLR4 transactivation of TGFBR1.
p38 via MMP2 is involved in TLR4 transactivation of TGFBR1 in VSMCs
TGFβ mediated Smad2 carboxy terminal phosphorylation does not involve Erk, p38 and/or Jnk MAPKs [53]; rather, Smad2 carboxy-terminal phosphorylation is a direct readout of TGFBR1 activation [54, 55]. In HSCT6 cell lines LPS mediated Smad2 carboxy-terminal phosphorylation involves Erk, p38 and Jnk driven pathways [42]. Here we investigated the role of Erk, p38 and Jnk in LPS mediated Smad2 carboxy-terminal phosphorylation utilising kinase-specific pharmacological inhibitors (Fig. 5a). LPS mediated Smad2 phosphorylation was unaffected in the presence of Mek1/2 inhibitor, U0126, and Jnk inhibitor SP600125. In the presence of p38 inhibitor SB202190, LPS mediated Smad2 phosphorylation was completely inhibited. MMP inhibitor GM6001 was used as a control, and completely inhibited LPS stimulated phospho-Smad2.
We next investigated the role of MAPKs on MMP2 secretion in HA-VSMCs (Fig. 5b). LPS treatment increased the secretion of pro-MMP2 and active-MMP2 by 2-fold. MMP2 secretion was unaffected in the presence of U0126, and SP600125, however it was completely inhibited in the presence of p38 inhibitor SB202190. GM6001 was used as a control which showed complete inhibition of LPS mediated MMP2 secretion. These data demonstrate that LPS via TLR4 transactivates the TGFBR1 to phosphorylate Smad2 via p38 and MMP2 dependent pathways.
LPS stimulates PAI1 mRNA expression in VSMCs via transactivation dependent pathway
PAI‑1 plays a role in regulating the fibrinolytic system and is present in human atherosclerotic lesions. PAI-1 is markedly increased in patients with sepsis, leading to an increase in thrombus formation and severe organ dysfunction [56]. We investigated whether LPS mediated PAI-1 expression is occurring via transactivation dependent pathways. HA-VSMCs were treated with LPS in the presence and absence of TGFBR inhibitor SB431542. We observed a 2.2-fold stimulation in PAI-1 mRNA expression (Fig. 5) that was completely inhibited in the presence of SB431542 and LPS-RS. These data demonstrate that LPS via TLR4 transactivates the TGFBR1 to stimulate PAI-1 mRNA expression in HA-VSMCs.