Li, Linda Xiaoyan

Aging occurs heterogeneously across organs, leading to progressive tissue dysfunction. Cellular senescence is a stress response triggered by age-associated insults, yet the mechanisms regulating senescence and organ aging remain incompletely understood. Here, we defined a role for lysine-specific demethylase 1 (LSD1) in DNA damage–mediated senescence and organ aging. LSD1 was upregulated in aged organs and senescent cells. In response to natural aging or ionizing radiation–induced DNA damage, LSD1 interacted with and demethylated ATM at lysine 3,016, as confirmed using a newly generated ATM-K3016me antibody. This modification sustained ATM phosphorylation, amplified DNA damage signaling, and delayed checkpoint recovery, promoting senescence and organ aging. Inhibition of LSD1 accelerated ATM dephosphorylation via WIP1, enhanced DNA repair, reduced senescence and DNA damage, and prevented irradiation-induced hair graying. Elimination of senescent cells with senolytics reduced LSD1 protein in aged organs, indicating a feedback loop between LSD1 and senescence. Mechanistically, LSD1 underwent autophagosome-lysosome degradation through interaction with LC3 and Beclin1, and autophagy impairment during DNA damage contributed to LSD1 accumulation in senescent cells. This study revealed LSD1 as a key regulator of DNA damage–induced senescence and organ aging and suggested that targeting LSD1 may attenuate senescence, delay organ aging, and prevent hair graying.

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 Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst growth, inflammation, fibrosis, and renal failure. Klotho is a renoprotective protein with antiproliferative, anti-inflammatory, and antioxidant properties, but its mechanistic role in ADPKD remains poorly defined. Methods Klotho expression was analyzed in human ADPKD kidneys, public bulk and single-cell RNA sequencing datasets, Pkd1 mutant renal epithelial cells, and Pkd1 mouse models. The therapeutic effects of recombinant Klotho were evaluated in late-onset Pkd1fl/fl:tamoxifen-Cre mice. Molecular mechanisms were investigated using cell viability assays, western blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry, ferroptosis assays, DNA methylation analysis, and chromatin immunoprecipitation assay. Results Klotho expression was markedly reduced in human ADPKD kidneys and Pkd1-deficient cells and mice, particularly in distal nephron segments. Recombinant Klotho treatment significantly reduced cyst burden, kidney enlargement, and blood urea nitrogen levels, while decreasing epithelial proliferation and increasing apoptosis in cyst-lining cells. Mechanistically, Klotho suppressed PKD-associated signaling pathways, including protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3), and retinoblastoma protein (Rb), reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated inflammation via downregulation of Smyd2, and attenuated transforming growth factor-beta (TGF-β)-Smad-dependent renal fibrosis. Klotho also inhibited ferroptosis by upregulating GPX-4 and reducing lipid peroxidation. Epigenetic analyses revealed DNA methyltransferase 1 (DNMT1)-mediated hypermethylation of the Klotho promoter and gene body in ADPKD kidneys, contributing to Klotho silencing. Inhibition of DNMT1 restored Klotho expression, and recombinant Klotho reduced DNMT1 levels, suggesting a negative feedback loop. Conclusions Klotho is epigenetically repressed in ADPKD and functions as a central regulator of cyst growth, inflammation, fibrosis, and ferroptosis. Restoration of Klotho signaling markedly attenuates disease progression in Pkd1 mutant mice, identifying the Klotho–DNMT1 axis as a promising therapeutic target for ADPKD.

Key Points α Klotho as an antiaging protein can extend the lifespan of healthy animals; serum α Klotho was lower in patients with autosomal dominant polycystic kidney disease. Transgenic α Klotho and recombinant α Klotho delayed cyst growth in Pkd1 mutant mouse kidneys. α Klotho exerted its proapoptotic and anti-inflammatory function through interacting with and destabilizing cellular inhibitor of apoptosis protein 1. Background α -Klotho ( α Klotho) is expressed mainly in the kidney, which is encoded by the KL gene, existing in a membrane-bound form and a soluble circulating form. Soluble serum α Klotho was lower in patients with autosomal dominant polycystic kidney disease (ADPKD). However, the role of α Klotho in ADPKD remains unknown. Methods To investigate the roles of α Klotho in ADPKD, we generated transgenic α Klotho and Pkd1 mutant Pkd1-HOMO-KL Tg mice to evaluate cyst growth and investigated underlying mechanisms with single-cell RNA sequencing analysis. Results We showed that α Klotho was lower in diverse kidney cell types in Pkd1 mutant kidneys, which was restored by transgenic α Klotho. We identified differential expression genes and pathways mediated by transgenic α Klotho at single-cell resolution and determined the cytokines and their receptors mediated cell-to-cell communication networks in Pkd1 mutant and Pkd1-HOMO-KL Tg kidneys by CellChat analysis. We further showed that α Klotho exerted a proapoptotic and anti-inflammatory function through interacting with and destabilizing cellular inhibitor of apoptosis protein 1, which disturbed TNF and its receptor mediated prosurvival complex, leading to ( 1 ) the formation of predeath complex to increase cystic cell death and ( 2 ) the decrease of the activation of NF-κB, the expression of cytokines, and the recruitment of macrophages, resulting in a delay of cyst growth. Treatment with recombinant α Klotho also delayed cyst growth in Pkd1 mutant kidneys. Conclusions This study defined an unknown role and mechanism of α Klotho in the regulation of a TNF-dependent cystic cell apoptosis and inflammation in Pkd1 mutant kidneys.

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.

The progression of autosomal dominant polycystic kidney disease (ADPKD), an inherited kidney disease, is associated with renal interstitial inflammation and fibrosis. CD74 has been known not only as a receptor of macrophage migration inhibitory factor (MIF) it can also have MIF independent functions. In this study, we report unknown roles and function of CD74 in ADPKD. We show that knockout of CD74 delays cyst growth in Pkd1 mutant kidneys. Knockout and knockdown of CD74 (1) normalize PKD associated signaling pathways, including ERK, mTOR and Rb to decrease Pkd1 mutant renal epithelial cell proliferation, (2) decrease the activation of NF-κB and the expression of MCP-1 and TNF-alpha (TNF-α) which decreases the recruitment of macrophages in Pkd1 mutant kidneys, and (3) decrease renal fibrosis in Pkd1 mutant kidneys. We show for the first time that CD74 functions as a transcriptional factor to regulate the expression of fibrotic markers, including collagen I (Col I), fibronectin, and α-smooth muscle actin (α-SMA), through binding on their promoters. Interestingly, CD74 also regulates the transcription of MIF to form a positive feedback loop in that MIF binds with its receptor CD74 to regulate the activity of intracellular signaling pathways and CD74 increases the expression of MIF in ADPKD kidneys during cyst progression. We further show that knockout of MIF and targeting MIF with its inhibitor ISO-1 not only delay cyst growth but also ameliorate renal fibrosis through blocking the activation of renal fibroblasts and CD74 mediated the activation of TGF-β-Smad3 signaling, supporting the idea that CD74 is a key and novel upstream regulator of cyst growth and interstitial fibrosis. Thus, targeting MIF-CD74 axis is a novel therapeutic strategy for ADPKD treatment.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder worldwide and progresses to end-stage renal disease (ESRD). However, its precise mechanism is not fully understood. In recent years, epigenetic reprogramming has drawn increasing attention regarding its effect on cyst growth. However, considering the complexity of epigenetic mechanisms and the broad range of alterations of epigenetic components in ADPKD, identifying more specific epigenetic factors and understanding how they are mechanistically linked to promote cyst growth is relevant for the development of treatment for ADPKD. Here, we find that the histone methyltransferase SMYD3, which activates gene transcription via histone H3 lysine 4 trimethylation (H3K4me3), is upregulated in PKD1 mutant mouse and human ADPKD kidneys. Genetic knockout of SMYD3 in a PKD1 knockout mouse model delayed cyst growth and improved kidney function compared with PKD1 single knockout mouse kidneys. Immunostaining and Western blot assays indicated that SMYD3 regulated PKD1-associated signaling pathways associated with proliferation, apoptosis, and cell cycle effectors in PKD1 mutant renal epithelial cells and tissues. In addition, we found that SMYD3 localized to the centrosome and regulated mitosis and cytokinesis via methylation of α-tubulin at lysine 40. In addition, SMYD3 regulated primary cilia assembly in PKD1 mutant mouse kidneys. In summary, our results demonstrate that overexpression of SMYD3 contributes to cyst progression and suggests targeting SMYD3 as a potential therapeutic strategy for ADPKD.

ADPKD is a genetic disorder with a molecular complexity that remains poorly understood. In this study, we sampled renal cells to construct a comprehensive and spatiotemporally resolved gene expression atlas in whole Pkd1 mutant polycystic mouse kidneys at single-cell resolution. We characterized cell diversity and identified novel collecting duct (CD) cell subtypes in cystic kidneys. We further found that CD cells appear to take different cell fate trajectories, and the first and the most important step might take place around day 14 in Pkd1 homozygous kidneys. After that day, increased numbers of CD cells showed highly proliferative and fibrotic characteristics, as detected in later-stage Pkd1 homozygous kidneys, both of which should contribute to cyst growth and renal fibrosis. With a newly developed modeling algorithm, called CellChat Explorer, we identify cell-to-cell communication networks mediated by the ligand receptor, such as MIF-CD44/CD74, in cystic kidneys, and confirm them via the expression patterns of ligands and receptors in four major cell types, which addresses the key question as to whether and how Pkd1 mutant renal epithelial cells affect their neighboring cells. The allele-specific gene expression profiles show that the secretion of cytokines by Pkd1 mutant epithelial cells may affect the gene expression profiles in recipient cells via epigenetic mechanisms, and vice versa. This study can be used to drive precision therapeutic targeting of ADPKD.

TP53 is the most common mutated gene in human cancer. Mutant p53 protein loses its tumor-suppressor properties and gains oncogenic activity. Mutant p53 is a therapeutic target in a broad range of cancer types. However, how mutant p53 is epigenetically regulated during tumor progression remains elusive. In this study, we found that the upregulation of mutant p53 is mediated by bromodomain protein BRD4 in triple-negative breast cancer (TNBC) cells. Inhibition of BRD4 with its inhibitor JQ1 or knockdown of BRD4 suppressed the transcription of mutant p53, which led to the re-expression of p21, the inhibition of S-phase entry, and colony formation in TNBC cells. BRD4 also positively regulated the transcription of wild-type p53, whereas JQ1 treatment and knockdown of BRD4 decreased the expression of p21 in MCF-7 cells. Knockdown of BRD4 resulted in attenuation of TNBC tumor growth in vivo. Taken together, our results uncover a novel regulatory mechanism of mutant p53 via BRD4, and suggest that the bromodomain inhibitor suppresses tumorigenesis through targeting mutant p53 in TNBC.