Biology subjects

Lagna, G.

Publications and source records attributed to Lagna, G..

6 recordsLinked to original sources

bioRxiv2025

Essential Role of Protein Kinase R in the Pathogenesis of Pulmonary Veno-occlusive Disease

Pulmonary veno-occlusive disease (PVOD) is a rare and severe subtype of pulmonary arterial hypertension, marked by progressive remodeling of small pulmonary arteries and veins with no therapies. Using a mitomycin C (MMC)-induced rat model, we previously demonstrated that protein kinase R (PKR)-mediated integrated stress response (ISR) drives endothelial dysfunction and vascular remodeling. To determine if PKR is the sole mediator of ISR and the pathogenesis, we treated control (Ctrl) and PKR knockout (KO) mice with the same dose of MMC. Consistent with rat data, Ctrl mice displayed ISR activation, vascular remodeling, and pulmonary hypertension after MMC treatment, while KO mice showed none of these phenotypes. Proteomic analysis revealed that MMC-mediated ISR activation attenuates protein synthesis in Ctrl but not in KO mice. These findings underscore the significance of PKR-dependent ISR activation and subsequent perturbation of proteostasis as central mechanisms driving PVOD pathogenesis and identifying PKR as a promising therapeutic target.

cell biology↗

bioRxiv2024

Depletion of Protein Phosphatase 1 Results in Persistent Activation of the Integrated Stress Response and Age-Associated Exacerbation of Pulmonary Veno-Occlusive Disease

Pulmonary veno-occlusive disease (PVOD) is a form of pulmonary hypertension that affects individuals across the age spectrum. PVOD is characterized by the obstruction of small pulmonary vessels, causing increased pulmonary artery (PA) pressure and leading to right ventricular heart (RV) failure. Previous research showed that the administration of Mitomycin-C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase PKR and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. In this study, we reveal that older rats experience more severe pulmonary vascular remodeling and RV hypertrophy than younger rats after MMC treatment due to lower levels of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and ISR activation. We demonstrate that pharmacological blocking of the PKR-ISR pathway mitigates PVOD symptoms in both age groups, suggesting targeting the PKR-ISR axis as a potential PVOD therapeutic strategy.

cell biology↗

bioRxiv2023

Reversal of pulmonary veno-occlusive disease phenotypes by inhibition of the integrated stress response

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension arising from EIF2AK4 gene mutations or mitomycin C (MMC) administration. The lack of effective PVOD therapies is compounded by a limited understanding of the mechanisms driving the vascular remodeling in PVOD. We show that the administration of MMC in rats mediates the activation of protein kinase R (PKR) and the integrated stress response (ISR), which lead to the release of the endothelial adhesion molecule VE-Cadherin in the complex with Rad51 to the circulation, disruption of endothelial barrier, and vascular remodeling. Pharmacological inhibition of PKR or ISR attenuates the depletion of VE-Cadherin, elevation of vascular permeability, and vascular remodeling instigated by MMC, suggesting potential clinical intervention for PVOD. Finally, the severity of PVOD phenotypes was increased by a heterozygous BMPR2 mutation that truncates the carboxyl tail of BMPR2, underscoring the role of deregulated BMP signal in the development of PVOD.

cell biology↗

bioRxiv2022

Essential role of the amino-terminal region of Drosha for the Microprocessor function

The ribonuclease (RNase) III enzyme Drosha enables processing of microRNA (miRNA) primary transcripts (pri-miRNAs) and control of ribosomal protein (RP) biogenesis by the Microprocessor. The extensively studied carboxyl-terminal half of Drosha is sufficient to crop pri-miRNAs in vitro, but the function of the evolutionarily conserved Drosha amino-terminal region (NTR) is unknown, despite it harboring mutations linked to a hereditary vascular disorder. Here, we provide evidence that, when replacing the endogenous Drosha, a mutant missing the NTR ({Delta}N-Drosha) fails to associate with endogenous pri-miRNAs and to support the expression of all miRNAs, except the miR-183 cluster. Surprisingly, Argonaute2 (Ago2) associates with and cleaves pri-miR-183 in {Delta}N-Drosha cells and in Drosha-depleted cells. {Delta}N-Drosha is also unable to inhibit RP biogenesis upon serum starvation. Thus, Drosha NTR is essential for pri-miRNA processing and RP biogenesis in vivo, and Ago2 can process the miR-183 cluster in the absence of Drosha.

molecular biology↗

bioRxiv2020

Control of ribosomal protein synthesis by Microprocessor complex

Ribosome biogenesis in eukaryotes requires stoichiometric production and assembly of 80 ribosomal proteins (RPs) and 4 ribosomal RNAs, and its rate must be coordinated with cellular growth. The indispensable regulator of RP biosynthesis is the 5-terminal oligopyrimidine (TOP) motif, spanning the transcription start site of all RP genes. Here we show that the Microprocessor complex, previously linked to the first step of processing microRNAs (miRNAs), coregulates RP expression by binding the TOP motif of nascent RP mRNAs and stimulating transcription elongation via resolution of DNA/RNA hybrids. Cell growth arrest triggers nuclear export and degradation of the Microprocessor protein Drosha by the E3 ubiquitin ligase Nedd4, accumulation of DNA/RNA hybrids at RP gene loci, decreased RP synthesis, and ribosome deficiency, hence synchronizing ribosome production with cell growth. Conditional deletion of Drosha in erythroid progenitors phenocopies human ribosomopathies, in which ribosomal insufficiency leads to anemia. Outlining a miRNA-independent role of the Microprocessor complex at the interphase between cell growth and ribosome biogenesis offers a new paradigm by which cells alter their protein biosynthetic capacity and cellular metabolism.

molecular biology↗

bioRxiv2019

Tissue-specific 3 prime end-adenylation of miR-125b mediates cell survival

Next-generation sequencing has uncovered microRNAs (miRNAs) that undergo sequence modifications, known as isomiRs. Their physiological significance, however, remains uncertain, partly because they generally comprise a small fraction of total miRNAs. Here we report that more than 60% of miR-125b, one of the most abundant miRNAs in vascular smooth muscle cells (vSMC), exists as an edited isoform containing a non-templated adenosine residue at the 3 prime-end (miR-125b+A). The properties of miR-125b+A, such as stability and subcellular localization, are similar to those of canonical miR-125b, but miR-125b+A more potently inhibits the expression of a subgroup of targets, including the apoptosis effector Caspase-6 (CASP6). In the CASP6 transcript, adenylated miR125b preferentially targets a conserved, atypical site, with an unusual 36 nucleotides loop between seed sequence and 3 prime-end supplementary site. PAP associated domain containing 2 (PAPD2) is responsible for monoadenylation of miR-125b. Downregulation of PAPD2 results in the conversion of miR-125b+A to miR-125b, derepression of CASP6, and sensitization of vSMC to apoptotic stimuli. Thus, atypical site recognition by a tissue-specific isomiR fulfills a pro-survival role.

molecular biology↗