Agborbesong, Ewud

Key Points Alanyl-transfer RNA synthetase 1 (AARS1) was upregulated and promoted cyst growth in autosomal dominant polycystic kidney disease. AARS1 promoted lysine lactylation of signal transducer and activator of transcription 3 (K140) and NF-κB p65 (K310), enhancing their phosphorylation, interaction, and transcriptional activity. AARS1 transcriptionally repressed its novel targets, Atg5 and Dusp4, to impair autophagy but activate polycystic kidney disease signaling pathways. Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder, characterized by metabolic reprogramming with enhanced glycolysis and lactate accumulation. However, the enzyme that senses lactate and uses it as a substrate for protein lactylation in ADPKD remains unknown. Alanyl-transfer RNA synthetase 1 (AARS1) is a lactate-responsive enzyme with lactyltransferase activity, while its role and mechanisms in ADPKD have not been defined. Methods To investigate the role of AARS1, we generated Pkd1 and Aars1 double conditional knockout Pkd1 fl/fl :Aars1 fl/fl :Ksp-Cre mice and evaluated the effect of AARS1 inhibitor β -alanine in two ADPKD mouse models. Coimmunoprecipitation and Cleavage Under Targets and Tagmentation analyses were performed to identify novel AARS1 substrates and downstream target genes involved in cystogenesis. Results AARS1 was elevated in Pkd1 mutant renal epithelial cells and kidneys, and genetic deletion of Aars1 significantly delayed cyst growth, preserved kidney function, and reduced renal lactylation in Pkd1 mutant mice. Mechanistically, AARS1 promoted lactylation-dependent activation of signal transducer and activator of transcription 3 (STAT3) and NF-κB (p65), specifically at STAT3 K140 and p65 K310, thereby amplifying pro-proliferative and proinflammatory transcriptional programs. AARS1 also associated with promoter regions and mediated histone H3 lysine 14 lactylation, thereby repressing the transcription of Atg5 to impair autophagy and suppressing the transcription of Dusp4 to sustain phosphorylation of cAMP response element–binding protein and retinoblastoma protein. Cytokine signaling through IL-6 and TNF- α further reinforced AARS1 expression through STAT3- and NF-κB–dependent feed-forward loops. Importantly, inhibition of AARS1 with β -alanine markedly slowed cyst progression in Pkd1 mutant mouse models. Conclusions Our study identified AARS1 as a central metabolic sensor linking lactate-driven lysine lactylation to transcriptional, epigenetic, and signaling pathways that drive ADPKD progression.

ABSTRACT Renal epithelial cell senescence and kidney aging have become the focus of scientific investigation. However, how epigenetic regulation in these processes remains elusive. Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, regulates trimethylation of histone H3 at lysine 27 (H3K27me3) and plays an important role in renal pathophysiology. In this study, we show that the expression of EZH2 is decreased in naturally aged and irradiation (IR)‐induced mouse kidneys, as well as in IR‐induced human renal cortical tubular epithelial (RCTE) cells through proteasome‐mediated degradation. Inhibition of EZH2 with its specific inhibitor 3‐DZNeP promotes tubular cell senescence and kidney aging characterized by an increase in the expression of senescence markers, including p16 and p21, in mouse kidneys and in IR‐induced RCTE cells. We show that EZH2 represses the transcription of p16 through trimethylation of H3K27me3, which directly binds to the promoter of p16. EZH2 represses the transcription of p21 through directly binding to the promoter of p21, and this process is involved in its interaction with p53 and its phosphorylation by ataxia‐telangiectasia mutated (ATM), a critical protein involved in the cellular response to DNA damage. Inhibition of ATM with its inhibitor decreased the phosphorylation of EZH2 and the binding of EZH2 to the promoter of p21 in IR‐treated RCTE cells in a p53‐dependent manner. This study suggests that EZH2 plays a critical role in preventing kidney aging and DNA‐damage‐induced renal tubular cellular senescence, in which senescence and kidney aging also result in the destabilization of EZH2, forming a negative feedback loop.

Autosomal dominant polycystic kidney disease (ADPKD) is a predominant genetic disease, which is caused by mutations in PKD genes and is associated with DNA damage in cystic cells. The intrinsic stimulator of interferon genes (STING) pathway is crucial for recognizing damaged DNA in the cytosol, triggering the expression of inflammatory cytokines to activate defense mechanisms. However, the precise roles and mechanisms of STING in ADPKD remain elusive. In this study, we show that Pkd1 mutant mouse kidneys show upregulation of STING, which is stimulated by the DNAs of nuclear and mitochondrial origin. The activation of STING promotes cyst growth through increasing (1) the activation of NF-κB in Pkd1 mutant cells and (2) the recruitment of macrophages in the interstitial and peri-cystic regions in Pkd1 mutant mouse kidneys via NF-κB mediating the upregulation of TNF-α and MCP-1. Targeting STING with its specific inhibitor C-176 delays cyst growth in an early-stage aggressive Pkd1 conditional knockout mouse model and a milder long-lasting Pkd1 mutant mouse model. Targeting STING normalizes mitochondrial structure and function, decreases the formation of micronuclei, induces Pkd1 mutant renal epithelial cell death via p53 signaling, and decreases renal fibrosis in Pkd1 mutant mouse kidneys. These results support that STING is a novel therapeutic target for ADPKD treatment.

The primary cilium, an antenna-like sensory organelle that protrudes from the surface of most eukaryotic cell types, has become a signaling hub of growing interest given that defects in its structure and/or function are associated with human diseases and syndromes, known as ciliopathies. With the continuously expanding role of primary cilia in health and diseases, identifying new players in ciliogenesis will lead to a better understanding of the function of this organelle. It has been shown that the primary cilium shares similarities with the immune synapse, a highly organized structure at the interface between an antigen-presenting or target cell and a lymphocyte. Studies have demonstrated a role for known cilia regulators in immune synapse formation. However, whether immune synapse regulators modulate ciliogenesis remains elusive. Here, we find that programmed death ligand 1 (PD-L1), an immune checkpoint protein and regulator of immune synapse formation, plays a role in the regulation of ciliogenesis. We found that PD-L1 is enriched at the centrosome/basal body and Golgi apparatus of ciliated cells and depleting PD-L1 enhanced ciliogenesis and increased the accumulation of ciliary membrane trafficking proteins Rab8a, BBS5, and sensory receptor protein PC-2. Moreover, PD-L1 formed a complex with BBS5 and PC-2. In addition, we found that depletion of PD-L1 resulted in the ciliary accumulation of Gli3 and the downregulation of Gli1. Our results suggest that PD-L1 is a new player in ciliogenesis, contributing to PC-2-mediated sensory signaling and the Hh signaling cascade.

The primary cilium is a microtubule-based sensory organelle that plays a critical role in signaling pathways and cell cycle progression. Defects in the structure and/or function of the primary cilium result in developmental diseases collectively known as ciliopathies. However, the constituents and regulatory mechanisms of the primary cilium are not fully understood. In recent years, the activity of the epigenetic modifier SMYD3 has been shown to play a key role in the regulation of cell cycle progression. However, whether SMYD3, a histone/lysine methyltransferase, contributes to the regulation of ciliogenesis remains unknown. Here, we report that SMYD3 drives ciliogenesis via the direct and indirect regulation of cilia-associated components. We show that SMYD3 is a novel component of the distal appendage and is required for centriolar appendage assembly. The loss of SMYD3 decreased the percentage of ciliated cells and resulted in the formation of stumpy cilia. We demonstrated that SMYD3 modulated the recruitment of centrosome proteins (Cep164, Fbf1, Ninein, Ttbk2 and Cp110) and the trafficking of intraflagellar transport proteins (Ift54 and Ift140) important for cilia formation and maintenance, respectively. In addition, we showed that SMYD3 regulated the transcription of cilia genes and bound to the promoter regions of C2cd3, Cep164, Ttbk2, Dync2h1 and Cp110. This study provides insights into the role of SMYD3 in cilia biology and suggests that SMYD3-mediated cilia formation/function may be relevant for cilia-dependent signaling in ciliopathies.