THE SENESCENCE-ASSOCIATED SECRETOME OF HEDGEHOG-DEFICIENT HEPATOCYTES DRIVES MASLD PROGRESSION

MASLD and other inflammatory liver diseases induce hepatocyte DNA damage and senescence. MASLD severity correlates with the burden of senescent hepatocytes but the mechanisms driving hepatocyte senescence, and how this exacerbates MASLD, are unknown. We recently discovered hepatocytes become senescent when Smoothened (Smo, an obligatory Hedgehog pathway component) is disrupted and showed that liver accumulation of Smo-depleted hepatocytes increases in parallel with MASLD severity in people. Smo-depleted hepatocytes remain viable but their state change has a robust effect on hepatocyte-secreted proteins. Therefore, we hypothesized that the secretome of Hedgehog-deficient hepatocytes promotes senescence to drive MASLD progression. Adult Smo^flox/flox mice were treated with control- or AAV8-TBG-Cre vectors to delete Smo specifically in hepatocytes. Chow-fed mice were analyzed one week after vector injection. Other mice were analyzed after being fed with CDA-HFD for 6 weeks to induce MASLD. Blood and livers were harvested for serological, histological, molecular, and proteome-profiler analysis. Secreted proteins that were differentially abundant in sera of chow-fed Smo (-) and Smo (+) mice, and that were also differentially expressed in conditioned medium of hepatocytes from these two groups, were identified. AML-12, Huh-7, and primary Smo(-) hepatocytes were cultured with oleic and palmitic acid to model lipotoxic stress and treated with vehicle or the protein that was most suppressed by Smo deletion; effects on lipotoxicity and senescence were compared. Wild type mice were also fed with CDA-HFD for 6 weeks and treated with this protein or vehicle during the last week of diet exposure. Compared to Smo(+) mice fed CDA-HFD, Smo(-) mice fed the same diet demonstrated lower anti-oxidant defense markers (Nrf2 and HO1) and developed significantly worse DNA damage (8OHDG and γH2AX), senescence (p16, p21, and β-galactosidase), steatosis (4-HNE, MDA), inflammation (F4/80) and fibrosis (α-SMA, Vimentin, Desmin, and Col1). Sera of Smo(-) mice and conditioned media of Smo(-) hepatocytes were similarly depleted of a protein that is known to maintain mitochondrial fitness. Adding this protein to cultures of Smo(-) hepatocytes reduced their senescence and rescued them from lipotoxicity. Inhibiting this protein in cultures of Smo(+) hepatocytes induced senescence and lipotoxicity. CDA-HFD wild type mice accumulated more senescent hepatocytes and developed worse steatohepatitis and liver fibrosis when this protein was inhibited. Therefore, inhibiting Hedgehog signaling in hepatocytes exacerbates MASLD by suppressing hepatocyte production of proteins that normally protect cells from lipotoxicity and senescence during metabolic stress.