The Role of Protein-Coupled Receptor GPR56 and Its Protective Mechanism in Ferroptosis-Induced Liver Injury

In recent years, ferroptosis has emerged as a new form of programmed cell death, becoming a hot topic in scientific research. It is characterized by cell death due to phospholipid peroxidation and is significantly different from traditional apoptosis and necrosis. Ferroptosis plays an important role under various pathological conditions, including ischemia-reperfusion injury (IRI) and drug-induced tissue damage. Despite the gradual elucidation of the mechanisms underlying ferroptosis, there are relatively few inhibitory pathways targeting it, especially within the G protein-coupled receptors (GPCRs) family, where the specific role of such receptors in ferroptosis remains unclear.

This study, conducted by Lin Hui, Ma Chuanshun, and others, from institutions including the School of Basic Medical Sciences at Shandong University, was published in the journal “Cell Metabolism” on November 5, 2024. The research team discovered that the adhesion-type G protein-coupled receptor GPR56 can resist ferroptosis by regulating CD36-mediated lipid metabolism. Moreover, the study identified the steroid hormone 17α-hydroxypregnenolone (17-OH Preg) as an endogenous agonist of GPR56, effectively inhibiting ferroptosis-induced liver injury, providing new targets and ideas for liver injury treatment.

Research Background and Significance

Ferroptosis relies on the peroxidation of intracellular phospholipids, particularly phospholipids containing polyunsaturated fatty acids (PUFA), which are major markers of ferroptosis. In the regulatory mechanisms of ferroptosis, factors like peroxidase (GPX4) and ferroptosis suppressor protein 1 (FSP1) play crucial roles. Ferroptosis is thought to be associated with various diseases, including cardiovascular and neurodegenerative diseases. Previous research has indicated that inhibiting ferroptosis can effectively alleviate symptoms in ischemia-reperfusion injuries and drug-induced tissue damage. The GPCRs family is one of the largest membrane protein families in the human body, involved in transmitting various physiological signals. This study is the first to propose that the adhesion-type GPCR family member GPR56 can maintain liver homeostasis by inhibiting ferroptosis.

Research Methods and Process

The study initially used mouse models to verify the role of GPR56 in various ferroptosis-induced liver injury models, followed by genetic knockout and pharmacological intervention to further reveal the specific mechanisms of GPR56. The research methodology involved several key steps:

1. Construction of Ferroptosis Induction Models

In the experiments, two ferroptosis induction models were used: one was a doxorubicin (Dox)-induced liver injury model, and the other was an ischemia-reperfusion injury (IRI) model. In the Dox-induced ferroptosis model, PTGS2 mRNA levels, a marker of ferroptosis, significantly increased in the liver tissue of mice after Dox injection, and the application of the ferroptosis inhibitor Ferrostatin-1 (Fer-1) effectively alleviated liver injury. Furthermore, by detecting the expression levels of markers such as PTGS2 and 4-HNE in mouse liver, the study further verified the importance of GPR56 in resisting ferroptosis.

2. Construction and Functional Verification of GPR56 Deficient Models

The research team successfully knocked out the GPR56 gene in the liver by injecting Cre recombinase into mice with specific GPR56 knockout, and observed a significant increase in PTGS2 levels in the liver of GPR56-deficient mice after Dox treatment, with significantly aggravated hepatocyte damage and lipid peroxidation. Another knockout model, Alb-Cre+/− GPR56fl/fl mice, showed more severe ferroptosis and lipid oxidative damage after Dox treatment compared to the control group, with higher serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels.

3. Inhibitory Effect of GPR56 on Cellular Ferroptosis

In vitro experiments were conducted by the research team using human fibrosarcoma (HT1080) and liver cancer cells (HCCLM3) to verify the inhibitory effect of GPR56 on ferroptosis. Experiments showed that overexpression of GPR56 effectively resisted ferroptosis induced by Erastin and RSL3, while knocking down GPR56 significantly increased the sensitivity of cells to ferroptosis. Moreover, the absence of GPR56 led to a significant increase in lipid peroxidation levels.

4. GPR56 Regulation of CD36-Mediated Lipid Metabolism Pathway

Further analysis showed that GPR56 reduces intracellular PUFA content by promoting CD36 endocytosis and lysosomal degradation, thereby decreasing susceptibility to ferroptosis. The knockout of GPR56 increased CD36 protein levels, exacerbating lipid peroxidation reactions. The study also found that GPR56 downregulates the expression of CD36 through the G12/G13 signaling pathway, a regulation process independent of proteasome and autophagy pathways, but dependent on the endocytosis-lysosomal degradation pathway.

5. 17-OH Preg as an Endogenous Agonist of GPR56

To find the endogenous agonist of GPR56, the research team screened various steroid hormones and found that 17-OH Preg can activate the G12/G13 signaling pathway of GPR56, significantly inhibiting ferroptosis. In vitro experiments showed that 17-OH Preg effectively reduced lipid peroxidation damage induced by Dox or IRI. In GPR56 knockout mice, this protective effect was significantly weakened, indicating that GPR56 is crucial in the anti-ferroptosis effect mediated by 17-OH Preg. Furthermore, molecular dynamics simulations showed that 17-OH Preg binds to specific amino acid sites on GPR56, triggering conformational changes in GPR56 and activating downstream signaling pathways.

Research Results and Analysis

The research results indicate that GPR56 reduces cellular sensitivity to ferroptosis by regulating CD36-mediated lipid metabolism. 17-OH Preg, as an endogenous agonist of GPR56, exerts anti-ferroptosis effects via the GPR56-G12 signaling axis. The experiment also found that the absence of GPR56 significantly increased the content of PUFA-containing phospholipids, making hepatocytes more sensitive to ferroptosis. Simultaneously, 17-OH Preg reduced PUFA absorption by inhibiting CD36 expression, thereby mitigating liver damage from ferroptosis.

The research team also validated the therapeutic potential of the GPR56-17-OH Preg-CD36 axis in clinical models. The experiment demonstrated that in a Dox-induced acute liver injury model, the application of 17-OH Preg significantly alleviated lipid oxidative damage in hepatocytes and reduced levels of liver injury markers. Additionally, 17-OH Preg effectively inhibited acute renal injury induced by ischemia-reperfusion, indicating its potential application in various tissue damage scenarios.

Research Significance and Outlook

This study first revealed the important role of GPR56 in ferroptosis-induced liver injury and identified 17-OH Preg as an endogenous agonist of GPR56, providing new targets for liver injury treatment.