bims-smemid 2023-11-26 papers

Free Radic Biol Med. 2023 Nov 16. pii: S0891-5849(23)01101-2. [Epub ahead of print]

Mitochondrial hyperfusion induces metabolic remodeling in lung endothelial cells by modifying the activities of electron transport chain complexes I and III.

Manivannan Yegambaram, Xutong Sun, Qing Lu, Yan Jin, Wojciech Ornatowski, Jamie Soto, Saurabh Aggarwal, Ting Wang, Kim Tieu, Haiwei Gu, Jeffrey R Fineman, Stephen M Black.

OBJECTIVE: Pulmonary hypertension (PH) is a progressive disease with vascular remodeling as a critical structural alteration. We have previously shown that metabolic reprogramming is an early initiating mechanism in animal models of PH. This metabolic dysregulation has been linked to remodeling the mitochondrial network to favor fission. However, whether the mitochondrial fission/fusion balance underlies the metabolic reprogramming found early in PH development is unknown.
METHODS: Utilizing a rat early model of PH, in conjunction with cultured pulmonary endothelial cells (PECs), we utilized metabolic flux assays, Seahorse Bioassays, measurements of electron transport chain (ETC) complex activity, fluorescent microscopy, and molecular approaches to investigate the link between the disruption of mitochondrial dynamics and the early metabolic changes that occur in PH.
RESULTS: We observed increased fusion mediators, including Mfn1, Mfn2, and Opa1, and unchanged fission mediators, including Drp1 and Fis1, in a two-week monocrotaline-induced PH animal model (early-stage PH). We were able to establish a connection between increases in fusion mediator Mfn1 and metabolic reprogramming. Using an adenoviral expression system to enhance Mfn1 levels in pulmonary endothelial cells and utilizing 13C-glucose labeled substrate, we found increased production of 13C lactate and decreased TCA cycle metabolites, revealing a Warburg phenotype. The use of a 13C5-glutamine substrate showed evidence that hyperfusion also induces oxidative carboxylation. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels secondary to the disruption of cellular bioenergetics and higher levels of mitochondrial reactive oxygen species (mt-ROS). The elevation in mt-ROS correlated with attenuated ETC complexes I and III activities. Utilizing a mitochondrial-targeted antioxidant to suppress mt-ROS, limited HIF-1α protein levels, which reduced cellular glycolysis and reestablished mitochondrial membrane potential.
CONCLUSIONS: Our data connects mitochondrial fusion-mediated mt-ROS to the Warburg phenotype in early-stage PH development.

Keywords: Glycolysis; Metabolomics; Mitochondrial function; Mitofusin; Pulmonary hypertension

DOI: https://doi.org/10.1016/j.freeradbiomed.2023.11.008

Curr Protoc. 2023 Nov;3(11): e921

Purification of Mouse Embryonic Fibroblasts (MEFs).

Rita Ferreira, Nadine Hein.

Mouse embryonic fibroblasts (MEFs) are primary fibroblasts purified from mouse embryos at a defined time post-fertilization. MEFs have versatile applications, including use as feeder cell layers or sources of untransformed primary cells for a variety of biological assays. MEFs are most commonly isolated between embryonic day (E)12.5 and E13.5 but can be isolated from embryos as early as E8.5 and as late as E15.5. The individual embryos are harvested by carefully removing uterine tissue, yolk sac, and placenta. The embryos are euthanized, and non-mesenchymal tissues, such as the fetal liver and heart, are removed before tissue homogenization. The remaining fetal tissue is homogenized by mechanical mincing using a sterile blade, followed by enzymatic digestion and resuspension. During tissue dissociation, the duration of trypsin-EDTA/DNase digestion and enzyme concentration are critical parameters to produce high-quality MEFs with the highest rates of cell viability and proliferation potential. MEFs can be cryopreserved at passage (P) 0 if >80% confluent, passaged for further expansion before freezing down, or directly utilized for downstream applications, i.e., preparation as feeder cell layers. Primary MEFs possess a limited proliferation capacity of ∼20 cell divisions, beyond which the percentage of senescent cells rapidly increases; thus, cultures should only be expanded/passaged to a maximum of P5. Critical for cell viability during cryopreservation and thawing of MEFs is the slow decrease in temperature when freezing, the rapid increase when thawing, the use of a cryoprotective agent, and an optimal cell density. While it is critical to generate high-quality MEFs to standardize and optimize preparation procedures and utilize fresh reagents, some variability in proliferation capacity and cell viability between MEF preparations remains. Thus, MEF preparation, culture, and cryopreservation procedures are continuously being optimized. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Purification, passaging, and expansion of MEFs Supporting Protocol: Cryopreservation and thawing of MEFs.

Keywords: cryopreservation; mouse embryonic fibroblasts (MEFs); passaging; purification; thawing; timed mating

DOI: https://doi.org/10.1002/cpz1.921

bioRxiv. 2023 Nov 07. pii: 2023.11.07.566074. [Epub ahead of print]

Mitochondrial One-Carbon Metabolism is Required for TGF-β-Induced Glycine Synthesis and Collagen Protein Production.

Angelo Y Meliton, Rengül Cetin-Atalay, Yufeng Tian, Jennifer C Houpy Szafran, Kun Woo D Shin, Takugo Cho, Kaitlyn A Sun, Parker S Woods, Obada R Shamaa, Bohao Chen, Alexander Muir, Gökhan M Mutlu, Robert B Hamanaka.

A hallmark of Idiopathic Pulmonary Fibrosis is the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by lung fibroblasts requires de novo synthesis of glycine, the most abundant amino acid in collagen protein. TGF-β upregulates the expression of the enzymes of the de novo serine/glycine synthesis pathway in lung fibroblasts through mTORC1 and ATF4- dependent transcriptional programs. SHMT2, the final enzyme of the de novo serine/glycine synthesis pathway, transfers a one-carbon unit from serine to tetrahydrofolate (THF), producing glycine and 5,10-methylene-THF (meTHF). meTHF is converted back to THF in the mitochondrial one-carbon (1C) pathway through the sequential actions of MTHFD2 (which converts meTHF to 10-formyl-THF), and either MTHFD1L, which produces formate, or ALDH1L2, which produces CO 2 . It is unknown how the mitochondrial 1C pathway contributes to glycine biosynthesis or collagen protein production in fibroblasts, or fibrosis in vivo . Here, we demonstrate that TGF-β induces the expression of MTHFD2 , MTHFD1L , and ALDH1L2 in human lung fibroblasts. MTHFD2 expression was required for TGF-β-induced cellular glycine accumulation and collagen protein production. Combined knockdown of both MTHFD1L and ALDH1L2 also inhibited glycine accumulation and collagen protein production downstream of TGF-β; however knockdown of either protein alone had no inhibitory effect, suggesting that lung fibroblasts can utilize either enzyme to regenerate THF. Pharmacologic inhibition of MTHFD2 recapitulated the effects of MTHFD2 knockdown in lung fibroblasts and ameliorated fibrotic responses after intratracheal bleomycin instillation in vivo . Our results provide insight into the metabolic requirements of lung fibroblasts and provide support for continued development of MTHFD2 inhibitors for the treatment of IPF and other fibrotic diseases.

DOI: https://doi.org/10.1101/2023.11.07.566074

Cells. 2023 Nov 16. pii: 2636. [Epub ahead of print]12(22):

ATF4 May Be Essential for Adaption of the Ocular Lens to Its Avascular Environment.

Jiawen Xiang, Anthony J Pompetti, Adam P Faranda, Yan Wang, Samuel G Novo, David Wan-Cheng Li, Melinda K Duncan.

The late embryonic mouse lens requires the transcription factor ATF4 for its survival although the underlying mechanisms were unknown. Here, RNAseq analysis revealed that E16.5 Atf4 null mouse lenses downregulate the mRNA levels of lens epithelial markers as well as known markers of late lens fiber cell differentiation. However, a comparison of this list of differentially expressed genes (DEGs) with other known transcriptional regulators of lens development indicated that ATF4 expression is not directly controlled by the previously described lens gene regulatory network. Pathway analysis revealed that the Atf4 DEG list was enriched in numerous genes involved in nutrient transport, amino acid biosynthesis, and tRNA charging. These changes in gene expression likely result in the observed reductions in lens free amino acid and glutathione levels, which would result in the observed low levels of extractable lens protein, finally leading to perinatal lens disintegration. These data demonstrate that ATF4, via its function in the integrated stress response, is likely to play a crucial role in mediating the adaption of the lens to the avascularity needed to maintain lens transparency.

Keywords: ATF4; CREB-2; amino acids; glutathione; lens development; transporter

DOI: https://doi.org/10.3390/cells12222636

Biochim Biophys Acta Mol Cell Res. 2023 Nov 21. pii: S0167-4889(23)00212-4. [Epub ahead of print] 119639

Proline and dihydroorotate dehydrogenase promote a hyper-proliferative state and dampen ferroptosis in cancer cells by rewiring mitochondrial redox metabolism.

Ryan J Mailloux.

Redox realignment is integral to the initiation, progression, and metastasis of cancer. This requires considerable metabolic rewiring to induce aberrant shifts in redox homeostasis that favor high hydrogen peroxide (H2O2) generation for the induction of a hyper-proliferative state. The ability of tumor cells to thrive under the oxidative burden imposed by this high H2O2 is achieved by increasing antioxidant defenses. This shift in the redox stress signaling threshold (RST) also dampens ferroptosis, an iron (Fe)-dependent form of cell death activated by oxidative distress and lipid peroxidation reactions. Mitochondria are central to the malignant transformation of normal cells to cancerous ones since these organelles supply building blocks for anabolism, govern ferroptosis, and serve as the major source of cell H2O2. This review summarizes advances in understanding the rewiring of redox reactions in mitochondria to promote carcinogenesis, focusing on how cancer cells hijack the electron transport chain (ETC) to promote proliferation and evasion of ferroptosis. I then apply emerging concepts in redox homeodynamics to discuss how the rewiring of the Krebs cycle and ETC promotes shifts in the RST to favor high rates of H2O2 generation for cell signaling. This discussion then focuses on proline dehydrogenase (PRODH) and dihydroorotate dehydrogenase (DHODH), two enzymes over expressed in cancers, and how their link to one another through the coenzyme Q10 (CoQ) pool generates a redox connection that forms a H2O2 signaling platform and pyrimidine synthesome that favors a hyper-proliferative state and disables ferroptosis.

Keywords: Dihydroorotate dehydrogenase;; Ferroptosis; Hydrogen peroxide; Proline dehydrogense; Redox stress signaling threshold

DOI: https://doi.org/10.1016/j.bbamcr.2023.119639