Abstract
Transforming growth factor β (TGFβ) potently activates hepatic stellate cells (HSCs), which promotes production and secretion of extracellular matrix (ECM) proteins and hepatic fibrogenesis. Increased ECM synthesis and secretion in response to TGFβ is associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). TGFβ and UPR signaling pathways are tightly intertwined during HSC activation, but the regulatory mechanism that connects these two pathways is poorly understood. Here, we found that TGFβ treatment of immortalized HSCs (i.e. LX-2 cells) induces phosphorylation of the UPR sensor inositol-requiring enzyme 1α (IRE1α) in a SMAD2/3-procollagen I-dependent manner. We further show that IRE1α mediates HSC activation downstream of TGFβ and that its role depends on activation of a signaling cascade involving apoptosis signaling kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK). ASK1-JNK signaling promoted phosphorylation of the UPR-associated transcription factor CCAAT/enhancer binding protein β (C/EBPβ), which is crucial for TGFβ- or IRE1α-mediated LX-2 activation. Pharmacological inhibition of C/EBPβ expression with the antiviral drug adefovir dipivoxil attenuated TGFβ-mediated activation of LX-2 or primary rat HSCs in vitro and hepatic fibrogenesis in vivo Finally, we identified a critical relationship between C/EBPβ and the transcriptional regulator p300 during HSC activation. p300 knockdown disrupted TGFβ- or UPR-induced HSC activation, and pharmacological inhibition of the C/EBPβ-p300 complex decreased TGFβ-induced HSC activation. These results indicate that TGFβ-induced IRE1α signaling is critical for HSC activation through a C/EBPβ-p300-dependent mechanism and suggest C/EBPβ as a druggable target for managing fibrosis.
Highlights
Transforming growth factor  (TGF) potently activates hepatic stellate cells (HSCs), which promotes production and secretion of extracellular matrix (ECM) proteins and hepatic fibrogenesis
We further show that IRE1␣ mediates HSC activation downstream of TGF and that its role depends on activation of a signaling cascade involving apoptosis signaling kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK)
TGF canonically signals through a pathway involving the transcription factors SMAD2/3, which up-regulates transcription of several genes involved in HSC activation and fibrogenesis, including procollagen 1␣1 and 1␣2 and fibronectin [20]
Summary
To better understand the relationship between UPR signaling and TGF, we first established a model to characterize and understand IRE1␣ signaling in the presence of TGF. Inhibition of ERK1/2 (U0126, 5 M), which acts downstream of TGF signaling, showed no effect on IRE1␣-mediated expression of fibronectin or collagen I (Fig. 2C, quantification adjacent) Together, these data suggest that TGF-induced IRE1␣ signaling drives HSC activation in an ASK1-JNK– dependent manner. In vivo analysis of p300 protein levels showed increased p300 in mice treated with CCl4, but this effect was blocked in mice receiving both CCl4 and adefovir dipivoxil (Fig. 8A, quantification below) This prompted us to study p300 in both TGF- and UPR-induced HSC activation. Helenalin acetate reduced TGF induction of collagen I and fibronectin in primary HSCs isolated from rats (Fig. 9D, quantification adjacent) Together, these data show that p300 is critical for mediating HSC activation downstream of TGF-IRE1␣ signaling and may act through a mechanism involving C/EBP
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