Elucidating the Role and Regulation of Proteostasis in Hepatic Fibrogenesis

Liver cirrhosis is a major health crisis caused by chronic liver injury. Liver injury leads to fibrosis which impairs liver function and can progress to cirrhosis and end-stage liver disease if unchecked. Fibrosis can reverse with removal of the injury; however, no therapeutics effectively targeted fibrosis. The major fibrogenic cells in the liver are hepatic stellate cells (HSCs), which produce and secrete matrix proteins into the extracellular space to drive fibrogenesis. Production of matrix proteins in fibrogenic HSCs exceeds the folding capacity of the endoplasmic reticulum (ER), leading to ER stress and induction of the Unfolded Protein Response (UPR). The UPR propa- gates signaling cascades to increase protein folding demands, restore proteostasis, and promote cell survival (adaptive UPR); however prolonged ER stress leads to apoptosis. We propose that targeting the adaptive UPR in HSCs is an effective anti-fibrotic strategy, through disrupting proteostasis, promting apoptosis, and limiting fibrogenesis. A major effector of the adaptive UPR is ATF6α. We found that ATF6α is necessary and sufficient to promote HSC activation in vitro, and HSC-specific loss of ATF6α limited fibrogenesis in vivo; however, the pro-fibrotic mechanisms downstream of ATF6α are unclear. To gain insight into these mechanisms, we performed RNAseq on HSCs isolated from fibrotic Atf6aHSCΔ/Δ mice, revealing disruption of several pathways compared to Atf6afl/fl HSCs, including pathways crucial for maintaining proteostasis through autophagy, protein secretion, and cell survival. We hypothesize that ATF6α drives fibrogenesis through regulating proteosta- sis in fibrogenic HSCs. Based on RNAseq as well as proteomics data from patient derived HSCs, this proposal will focus on how ATF6α regulates proteostasis to drive fibrogenesis through three distinct mechanisms: protein degradation, regulation of pro-survival signals, and protein trafficking. Degrading misfolded proteins is crucial for relieving ER stress. Misfolded proteins in the ER can undergo ER-to-lysosomal associated degradation (ER- LAD), with ERLAD receptors targeting misfolded proteins for lysosomal degradation. ERLAD activity and ERLAD receptors increased in fibrogenic HSCs in an ATF6α-dependent manner. Aim 1 will study how ATF6α regulates ERLAD to reduce ER stress and promote HSC survival and fibrogenesis. Second, ATF6α is crucial for survival of secretory cells, and we show that ATF6α induces expression of GADD45A, a protein which binds to and regulates several kinases. We show that GADD45A loss limits HSC activation in vitro, thus Aim 2 will study how ATF6α regulates GADD45A expression and association with pro-survival kinases to promote HSC survival and fibrogenesis in vitro and in vivo. Finally, we identified a potential role for ATF6α in procollagen I secretion through SORT1. Aim 3 will utilize a combination of in vitro mechanistic studies, proteomics, and in vivo studies to reveal how ATF6α regulation of SORT1 promotes procollagen I trafficking and secretion to drive fibrogenesis. Together these studies will elucidate how ATF6α coordinates signaling mechanisms to restore proteostasis in activated HSCs, and identify novel therapeutic targets to limit liver fibrosis in vivo.