Cholesterol modulates type I/II TGF-ß receptor complexes and alters the balance between Smad and Akt signaling in hepatocytes.

Cholesterol mediates membrane compartmentalization, affecting signaling via differential distribution of receptors and signaling mediators. While excessive cholesterol and aberrant transforming growth factor-ß (TGF-ß) signaling characterize multiple liver diseases, their linkage to canonical vs. non-canonical TGF-ß signaling remained unclear. Here, we subjected murine hepatocytes to cholesterol depletion (CD) or enrichment (CE), followed by biophysical studies on TGF-ß receptor heterocomplex formation, and output to Smad2/3 vs. Akt pathways. Prior to ligand addition, raft-dependent preformed heteromeric receptor complexes were observed. Smad2/3 phosphorylation persisted following CD or CE. CD enhanced phospho-Akt (pAkt) formation by TGF-ß or epidermal growth factor (EGF) at 5 min, while reducing it at later time points. Conversely, pAkt formation by TGF-ß or EGF was inhibited by CE, suggesting a direct effect on the Akt pathway. The modulation of the balance between TGF-ß signaling to Smad2/3 vs. pAkt (by TGF-ß or EGF) has potential implications for hepatic diseases and malignancies.

The Interplay of TGF-ß1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation.

BACKGROUND & AIMS: Transforming growth factor-ß1 (TGF-ß1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis and hepatocellular carcinoma. However, the reciprocal regulation of TGF-ß1 signaling and cholesterol metabolism in MASLD is yet unknown. METHODS: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA sequencing of murine hepatocyte cell line (alpha mouse liver 12/AML12) and mouse primary hepatocytes treated with TGF-ß1. Functional assays were performed on AML12 cells (untreated, TGF-ß1 treated, or subjected to cholesterol enrichment [CE] or cholesterol depletion [CD]), and on mice injected with adenovirus-associated virus 8-control/TGF-ß1. RESULTS: TGF-ß1 inhibited messenger RNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, mouse primary hepatocytes, and adenovirus-associated virus-TGF-ß1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue also were reduced upon TGF-ß1 treatment. Smad2/3 phosphorylation after 2 hours of TGF-ß1 treatment persisted after CE or CD and was mildly increased after CD, whereas TGF-ß1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 hours of incubation with TGF-ß1, including epithelial-mesenchymal transition, actin polymerization, and apoptosis. CD mimicked the outcome of long-term TGF-ß1 administration, an effect that was blocked by an inhibitor of the type I TGF-ß receptor. In addition, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. CONCLUSIONS: TGF-ß1 inhibits cholesterol metabolism whereas cholesterol attenuates TGF-ß1 downstream effects in hepatocytes.

Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers.

Hypoxia, a driver of tumor growth and metastasis, regulates angiogenic pathways that are targets for vessel normalization and ovarian cancer management. However, toxicities and resistance to anti-angiogenics can limit their use making identification of new targets vital. Inhibin, a heteromeric TGFß ligand, is a contextual regulator of tumor progression acting as an early tumor suppressor, yet also an established biomarker for ovarian cancers. Here, we find that hypoxia increases inhibin levels in ovarian cancer cell lines, xenograft tumors, and patients. Inhibin is regulated primarily through HIF-1, shifting the balance under hypoxia from activins to inhibins. Hypoxia regulated inhibin promotes tumor growth, endothelial cell invasion and permeability. Targeting inhibin in vivo through knockdown and anti-inhibin strategies robustly reduces permeability in vivo and alters the balance of pro and anti-angiogenic mechanisms resulting in vascular normalization. Mechanistically, inhibin regulates permeability by increasing VE-cadherin internalization via ACVRL1 and CD105, a receptor complex that we find to be stabilized directly by inhibin. Our findings demonstrate direct roles for inhibins in vascular normalization via TGF-ß receptors providing new insights into the therapeutic significance of inhibins as a strategy to normalize the tumor vasculature in ovarian cancer.