Biology subjects

Dooley, S.

Publications and source records attributed to Dooley, S..

4 recordsLinked to original sources

bioRxiv2023

TGF-β1 inhibits cholesterol metabolism in hepatocytes to facilitate cell death, EMT and signals for HSC activation.

Background and Aims: Transforming growth factor-{beta}1 (TGF-{beta}1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the reciprocal regulation of TGF-{beta}1 signaling and cholesterol metabolism in MASLD is yet unknown. Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA-Seq of murine hepatocyte cell line (AML12) and mouse primary hepatocytes (MPH) treated with TGF-{beta}1. Functional assays were performed on AML12 cells (untreated, TGF-{beta}1 treated, or subjected to cholesterol enrichment (CE) or depletion (CD)), and on mice injected with adeno-associated virus 8 (AAV8)-Control/TGF-{beta}1. Results: TGF-{beta}1 inhibited mRNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, MPHs, and AAV8-TGF-{beta}1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue were also reduced upon TGF-{beta}1 treatment. Smad2/3 phosphorylation following 2 h TGF-{beta}1 treatment persisted after CE or CD and was mildly increased following CD, while TGF-{beta}1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 h incubation with TGF-{beta}1, including EMT, actin polymerization, and apoptosis. CD mimicked the outcome of long term TGF- {beta}1 administration, an effect that was blocked by an inhibitor of the type I TGF-{beta} receptor. Additionally, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. Conclusions: TGF-{beta}1 inhibits cholesterol metabolism while cholesterol attenuates TGF-{beta}1 downstream effects in hepatocytes.

cell biology↗

bioRxiv2023

Insulin determines the effects of TGF-beta on HNF4alpha transcription and epithelial-to-mesenchymal transition in hepatocytes

To date, epithelial-to-mesenchymal transition (EMT) has been observed in cultured hepatocytes, but not in vivo. TGF-{beta} is supposed to initiate EMT in hepatocytes by inhibiting HNF4 through the SMAD2/3 complex. We report that TGF-{beta} does not directly inhibit HNF4, but contributes to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CBP/p300 to the HNF4 promoter. The recruitment of CBP/p300 is indispensable for C/EBPa binding, another essential requirement for constitutive HNF4 expression in hepatocytes. In contrast to the observed induction of HNF4, SMAD2/3 inhibits C/EBP transcription. Therefore, long-term TGF-{beta} incubation results in C/EBP depletion, which abrogates HNF4 expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBP promoter is abolished by insulin. Thus, maintaining a high insulin concentration in culture medium ensures constitutive HNF4 and thereby prevents TGF-{beta}-induced hepatocyte EMT. Furthermore, insulin inhibits TGF-{beta}-induced SMAD2/3 binding to the promoters of core EMT transcription factors e.g., SNAI1. SNAI1 transcription requires both SMAD2/3 and FOXO1 in nuclei. Insulin inhibits SNAI1 transcription through impeding SMAD2/3 binding to its promoter and inducing FOXO1 phosphorylation. Hence, insulin is the key factor that prevents TGF-{beta}-induced EMT in hepatocytes.

cell biology↗

bioRxiv2022

Training induced improvements in knee extensor force accuracy are associated with reduced vastus lateralis motor unit firing variability

BackgroundMuscle force output during sustained submaximal isometric contractions fluctuates around an average value and is known to be influenced by variation in motor unit (MU) firing rates. MU firing rate variability seemingly reduces following exercise training interventions, however, much less is known with respect to peripheral MU properties. We therefore investigated whether targeted force accuracy training could lead to improved muscle functional capacity and control, in addition to determining any alterations of individual MU features. MethodsTen healthy participants (7 females, 3 males, 27{+/-}6 years, 170{+/-}8 cm, 69{+/-}16kg) underwent a 4-week supervised, unilateral, force accuracy training intervention. The coefficient of variation for force (FORCECoV) and sinusoidal wave force tracking accuracy (FORCESinu) were determined at 25% maximal voluntary contraction (MVC) pre- and post-training. Intramuscular electromyography was utilised to record individual MU potentials from the vastus lateralis (VL) muscles at 25% MVC during sustained contractions, pre- and post-training. ResultsKnee extensor muscle strength remained unchanged following training, with no improvements in unilateral leg-balance. FORCECoV and FORCESinu significantly improved in only the trained knee extensors by ~13% (p=0.01) and ~30% (p<0.0001) respectively. MU firing rate variability significantly reduced in the trained VL by ~16% (n=8; p=0.001), with no further alterations to MU firing rate or neuromuscular junction transmission instability. ConclusionOur results suggest muscle force control and tracking accuracy is a trainable characteristic in the knee extensors, which is likely explained by the reduction in MU firing rate variability apparent in the trained limb only.

physiology↗

bioRxiv2022

Repeated toxic injuries of murine liver are tolerated through microsteatosis and mild inflammation

The liver has a remarkable capacity to regenerate and thus compensates for repeated injuries through toxic chemicals, drugs, alcohol or malnutrition for decades. However, largely unknown is how and when alterations in the liver occur due to tolerable damaging insults. To that end, we induced repeated liver injuries over ten weeks in a mouse model injecting carbon tetrachloride (CCl4) twice a week. We lost 10% of the study animals within the first six weeks, which was accompanied by a steady deposition of extracellular matrix (ECM) regardless of metabolic activity of the liver. From week six onwards, all mice survived, and in these mice ECM deposition was rather reduced, suggesting ECM remodeling as a liver response contributing to better coping with repeated injuries. The data of time-resolved paired transcriptome and proteome profiling of 18 mice was subjected to multi-level network inference, using Knowledge guided Multi-Omics Network inference (KiMONo), identified multi-level key markers exclusively associated with the injury-tolerant liver response. Interestingly, pathways of cancer and inflammation were lighting up and were validated using independent data sets compiled of 1034 samples from publicly available human cohorts. A yet undescribed link to lipid metabolism in this damage-tolerant phase was identified. Immunostaining revealed an unexpected accumulation of small lipid droplets (microvesicular steatosis) in parallel to a recovery of catabolic processes of the liver to pre-injury levels. Further, mild inflammation was experimentally validated. Taken together, we identified week six as a critical time point to switch the liver response program from an acute response that fosters ECM accumulation to a tolerant "survival" phase with pronounced deposition of small lipid droplets in hepatocytes potentially protecting against the repetitive injury with toxic chemicals. Our data suggest that microsteatosis formation plus a mild inflammatory state represent biomarkers and probably functional liver requirements to resist chronic damage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/476054v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11ecc7aorg.highwire.dtl.DTLVardef@1027df5org.highwire.dtl.DTLVardef@9ba0f7org.highwire.dtl.DTLVardef@164f80a_HPS_FORMAT_FIGEXP M_FIG C_FIG The datasets generated via transcriptomics, proteomics as well as blood, histopathological and biochemical analysis were analyzed in an independent and integrative manner. The independent analysis was performed via state-of-the-art statistical approaches i.e. differential and consistently regulated genes and proteins. Combining the results identified three fibrosis phases. Using the KiMONo algorithm, a fibrosis specific multi-omic network was inferred. Within this network we identified several nodes connecting phase III specific features forming 13 distinct multi-omic modules suggesting a tolerance scheme. Some of these modules were experimentally validated and compared to 11 independent human studies of various liver diseases.

systems biology↗