Journal of Clinical Investigation最新文献

How β-Catenin (βCat) mediates tissue hyperplasia is poorly understood. To explore this, we employed the adrenal cortex as a model system given its stereotypical spatial organization and the important role βCat plays in homeostasis and disease. For example, excessive production of aldosterone by the adrenal cortex (primary aldosteronism, PA) constitutes a major cause of cardiovascular morbidity and is associated with βCat gain-of-function (βCat-GOF). Adherens junctions (AJs) connect the actin cytoskeletons of adjacent zona Glomerulosa (zG) cells via a cadherin-βCat-α-Catenin complex and mediate aldosterone production. Whether βCat-GOF drives zG hyperplasia, a key feature of PA, via AJs is unknown. Here, we showed that aldosterone secretagogues (K+, AngII) and βCat-GOF mediated AJ formation via Rho/ROCK/actomyosin signaling. In addition, Rho/ROCK inhibition led to altered zG rosette morphology and decreased aldosterone production. Mice with zG-specific βCat-GOF demonstrated increased AJ formation and zG hyperplasia, which was blunted by Rho/ROCK inhibition and deletion of α-Catenin. βCat also impacted AJ formation independently of its role as a transcription factor. Furthermore, analysis of human aldosterone-producing adenomas revealed high levels of βCat expression were associated with increased membranous expression of K-Cadherin. Together, our findings identified Rho/ROCK signaling and αCat as key mediators of AJ formation and βCat-driven hyperplasia.

Protectin DX (PDX) is a member of the superfamily of specialized proresolving mediators and exerts anti-inflammatory actions in animal models; however, its signaling mechanism remains unclear. Here, we demonstrate the analgesic actions of PDX in a mouse model of tibial fracture-induced postoperative pain (fPOP). Intravenous early- and late-phase treatment of PDX (100 ng/mouse) effectively alleviated fPOP. Compared with protectin D1 (PD1)/neuroprotectin D1, DHA, steroids, and meloxicam, PDX provided superior pain relief. While dexamethasone and meloxicam prolonged fPOP, PDX shortened the pain duration. The analgesic effects of PDX were abrogated in Gpr37-/- mice, which displayed deficits in fPOP resolution. PDX was shown to bind GPR37 and induce calcium responses in peritoneal macrophages. LC-MS/MS-based lipidomic analysis revealed that endogenous PDX levels were approximately 10-fold higher than those of PD1 in muscle at the fracture site. PDX promoted macrophage polarization via GPR37-dependent phagocytosis and efferocytosis through calcium signaling in vitro, and it further enhanced macrophage viability and efferocytosis in vivo via GPR37. Finally, PDX rapidly modulated nociceptor neuron responses by suppressing C-fiber-induced muscle reflex in vivo and calcium responses in DRG neurons ex vivo and by reducing TRPA1/TRPV1-induced acute pain and neurogenic inflammation in vivo. Our findings highlight multiple benefits of PDX to manage postoperative pain and promote perioperative recovery.

MRE11, a breast tumor suppressor and component of the MRE11-RAD50-NBS1 (MRN) complex, plays a critical role in DNA end resection and initiation of ATM-dependent DNA damage signaling. However, the precise mechanisms governing MRE11 function in the DNA damage response (DDR) remain incompletely understood. Here, we found that MRE11 is deacetylated by the SIRT2 sirtuin deacetylase and breast tumor suppressor, which promotes DNA binding to facilitate DNA end resection and ATM-dependent signaling. SIRT2 deacetylase activity promoted DNA end resection. SIRT2 further complexed with and deacetylated MRE11 at conserved lysine (K) 393 in response to DNA double-strand breaks (DSBs), which promoted MRE11 localization and DNA binding at DSBs but not interaction with RAD50, NBS1, or CtIP. Moreover, MRE11 K393 deacetylation by SIRT2 promoted ATM-dependent signaling. Our findings define a mechanism regulating MRE11 binding to DNA through SIRT2 deacetylation, elucidating a critical upstream signaling event directing MRE11 function in the DDR and providing insight into how SIRT2 dysregulation leads to genomic instability and tumorigenesis.

WHIM syndrome is an immunodeficiency caused by autosomal dominant hyperfunctional mutations in chemokine receptor CXCR4 that promote panleukopenia due to BM retention. We previously reported a preclinical gene therapy protocol involving allele-nonspecific Cxcr4 CRISPR/Cas9 inactivation, leveraging the known in vivo dominance of Cxcr4+/o (+, WT; o, inactivated) hematopoietic stem cells (HSCs) for autologous BM engraftment and leukocyte reconstitution over HSCs with other Cxcr4 genotypes. Here, we show that without BM conditioning, this approach is not able to correct leukopenia in WHIM mice. We therefore modified the protocol by adding conditioning with a non-genotoxic CD117-targeted immunotoxin, CD117-antibody-saporin-conjugate (CD117-ASC). With this change, donor-derived blood cells rapidly reached ~95% chimerism after transplantation, which was stable without adverse events for more than 400 days. Mice receiving edited HSCs showed rapid normalization of absolute myeloid cell counts, the key blood subset responsible for WHIM syndrome. In competitive transplants using equal numbers of edited and unedited donor HSCs, over 80% of blood cells originated from the edited population, predominantly with the Cxcr4+/o genotype. These results provide proof of principle that CRISPR/Cas9-mediated inactivation of the Cxcr4 disease allele, combined with non-genotoxic HSC-targeted conditioning, may offer a safe and effective gene therapy strategy generalizable to all WHIM mutations.

Large-cohort genome-wide association studies (GWAS) for alcohol use disorder (AUD) drug treatment outcomes and AUD risk have repeatedly identified genetic loci which are splicing quantitative trait loci for the fibronectin III domain containing 4 (FNDC4) gene in the brain. However, FNDC4 function in the brain and how it might contribute to AUD pathophysiology remain unclear. In the present study, we characterized GWAS loci-associated FNDC4 splice isoforms and demonstrated that FNDC4 alternative splicing results in loss-of-function for FNDC4. We also investigated FNDC4 function using CRISPR/cas9 editing, and the creation of human induced pluripotent stem cell (iPSC)-derived neural organoids joined with single-nucleus RNA sequencing, a series of studies which showed that FNDC4 knock-out (KO) resulted in a striking shift in the relative proportions of glutamatergic and GABAergic neurons in iPSC-derived forebrain organoids as well as changes in their electrical activity. We further explored potential mechanism(s) of FNDC4-dependent neurogenesis with results that suggested a role for FNDC4 in mediating neural cell surface interactions. In summary, this series of experiments indicates that FNDC4 plays a role in regulating cerebral cortical neurogenesis in the brain. This regulation may contribute to the response to AUD pharmacotherapy as well as the effects of alcohol on the brain.

The urokinase plasminogen activator receptor (uPAR) is a membrane-bound protein found on the surface of immune cells. Through the action of proteases, uPAR is cleaved to produce several circulating proteins in the bloodstream, including the soluble form suPAR and the fragments D1 and D2D3. Initially studied in the context of infectious diseases and cancer, recent research has revealed roles for suPAR and its related proteins as mediators linking innate immunity to the pathogenesis of kidney and cardiovascular diseases, as well as insulin-dependent diabetes. While these proteins have long been recognized as prognostic biomarkers, growing clinical, experimental, and genetic evidence highlights their active involvement in the onset and progression of these diverse conditions. This Review examines suPAR's evolution from its discovery as a modulator of innate immunity to its current status as a key driver in chronic kidney and cardiovascular diseases. Furthermore, we explore the molecular mechanisms through which suPAR and D2D3 contribute to multiorgan damage, emphasizing emerging opportunities for therapeutic interventions across interconnected organ systems.