Bile Acids Alter Male Fertility Through G-Protein-Coupled Bile Acid Receptor 1 Signaling Pathways in Mice

Bile acids (BAs) are signaling molecules that are involved in many physiological functions, such as glucose and energy metabolism. These effects are mediated through activation of the nuclear and membrane receptors, farnesoid X receptor (FXR-alpha) and TGR5 (G-protein-coupled bile acid receptor 1; GPBAR1). Although both receptors are expressed within the testes, the potential effect of BAs on testis physiology and male fertility has not been explored thus far. Here, we demonstrate that mice fed a diet supplemented with cholic acid have reduced fertility subsequent to testicular defects. Initially, germ cell sloughing and rupture of the blood-testis barrier occur and are correlated with decreased protein accumulation of connexin-43 (Cx43) and N-cadherin, whereas at later stages, apoptosis of spermatids is observed. These abnormalities are associated with increased intratesticular BA levels in general and deoxycholic acid, a TGR5 agonist, in particular. We demonstrate here that Tgr5 is expressed within the germ cell lineage, where it represses Cx43 expression through regulation of the transcriptional repressor, T-box transcription factor 2 gene. Consistent with this finding, mice deficient for Tgr5 are protected against the deleterious testicular effects of BA exposure. Conclusions: These data identify the testis as a new target of BAs and emphasize TGR5 as a critical element in testicular pathophysiology. This work may open new perspectives on the potential effect of BAs on testis physiology during liver dysfunction.

CO-REGULATION OF ENDOTHELIAL AND MYOGENIC CELL FATES THROUGH INTERCELLULAR CROSSTALK IN SKELETAL MUSCLE

Umji Lee

Skeletal muscle is one of the most dynamic organs in the human body, which has a vigorous potential to regenerate and adapt to environmental changes. Depending on environments, skeletal muscle enables essential life activities to survive by controlling movement and metabolism. The functional and structural adaptations of skeletal muscle crosstalk with other physiological systems enabling nutrition and oxygen delivery, and the elimination of metabolic waste products. The work in this dissertation focuses on the intercellular signals produced by myogenic cells to dynamically remodel skeletal muscle during exercise or muscle repair, and the reciprocal signals from the local niche and the systemic environment that regulate myogenic cell fate and metabolism according to physiological needs. This complex crosstalk is dissected in three specific chapters where we study the crosstalk of myogenic cells with endothelial cells and the vasculature to coordinate local niche interactions and systemic crosstalks. First, we demonstrate that an exercise-induced myokine called apelin is produced by muscle fiber and mediates intercellular signaling to endothelial cells through the apelin receptor. In addition, through a yeast one-hybrid screen of transcription factor binding to the apelin promoter, we identified the myogenic transcription factor TEA domain family member 1 (Tead1) as a regulator of Apln transcription. We observed via single-cell transcriptomic analysis of regenerating skeletal muscle that Aplnr (Apelin receptor) is enriched in muscle endothelial cells, whereas Tead1 is enriched in myogenic cells. Myofiber-specific over-expression of Tead1 suppresses apelin secretion at the whole-body level, and apelin secretion via Tead1 knock-down in muscle cells stimulates endothelial cell proliferation in co-cultures. By showing that apelin peptide supplementation in vivo enhances endothelial cell expansion following muscle injury, we conclude that paracrine crosstalk in which apelin secretion controlled by Tead1 in myogenic cells influences endothelial remodeling during skeletal muscle repair. Secondly, we show how tissue-resident support cells affect muscle progenitor activation in different metabolic environments. Skeletal muscle progenitors (SKMP) reside in close proximity to supportive cell types in the stem cell niche and dynamically interact with each other to adapt to environmental changes. Here, we studied how different niche cell types affect SKMPs in response to glycemic levels. Using co-cultures of SKMPs and niche cells, we discovered that endothelial cells synergistically enhance SKMP proliferation in a low glycemic environment, while fibroblasts and macrophages had no effect. We observed that the crosstalk between SKMPs and endothelial cells was mediated by direct cell-cell contacts independent of soluble paracrine signals. Transcriptomic analysis revealed that the endothelial alpha/beta hydrolase N-Myc Downstream Regulated1 (NDRG1) is induced by a low glycemic environment and is associated with biological adhesion. SKMPs co-cultured with Ndrg1 knock-down endothelial cells lose their synergistic functional relationship in response to glucose levels. Therefore, our findings suggest that Ndrg1 is a key mediator of endothelial cell-mediated glycemic control of SKMPs and provides a link between systemic energy levels and the skeletal muscle stem cell niche. Lastly, I developed intravital imaging to monitor muscle stem cells and vasculature.

EPFL2021

In Vivo Studies on the Control of Gene Expression and Protein Degradation

Karolina Bojkowska

Cellular homeostasis is maintained by tightly regulated gene networks, controlled notably at the levels of transcription, mRNA processing, and protein post-translational modification and turnover. This thesis focuses on two of these regulatory steps, namely gene transcription and protein stability. I first explored the impact of the KRAB-ZFP/KAP1 gene regulation system on liver function. KRAB-ZFPs constitute the largest family of transcription factors encoded by mouse and humans, yet their physiological roles and gene targets remain mostly elusive. Conversely, their molecular mechanism of action has been rather well characterized, as they can recognize DNA in a sequence-specific manner and recruit the universal co-factor KAP1 (also known as TRIM28), which in turn serves as a scaffold for various heterochromatin-inducing effectors. Several studies have shed some light on the roles played by this system in embryonic tissues, but its functions in the adult remain almost completely unknown. As chromatin regulation has been shown to be a major factor in the control of metabolism, we decided to investigate the role of KRAB-ZFP/KAP1 in the liver, an organ central to this process. For this, we generated conditional liver-specific Kap1 KO mice, the study of which revealed a strikingly sex-specific phenotype, with early-onset steatosis and age-related tumorigenesis restricted to males, contrasting with the mild metabolic disturbances recorded in Kap1-deleted females. Underlying this phenotype, our combined transcriptome and chromatin analyses revealed that KAP1 controls sexually dimorphic genes notably involved in hormone, drug and xenobiotic metabolism. Finally, by using a recently developed technique for direct RNA quantification, we established the range of KRAB-ZFPs expressed in the liver and likely responsible for at least part of the observed effects. This study thus demonstrates the central role of the KRAB/KAP1 system in control of sexual dimorphism, responses to xenobiotic stress and metabolic control in the liver, and further links disturbances in these processes with hepatic carcinogenesis. In parallel, in an effort to pursue a multidisciplinary training combining skills in biology and chemistry, I worked towards the development of a method to study the half-life of proteins in vivo. Protein turnover critically influences many biological functions, yet methods have been lacking to assess this parameter in vivo. Capitalizing on an in vitro technology previously developed in Kai Johnsson's laboratory, I demonstrated how chemical labeling can be used to measure the half-life of resident intracellular and extracellular proteins in living mice. Our approach is based on labeling of SNAP-tag-fusion proteins with fluorescent probes. First, we verified that SNAP-tag substrates have wide bio-availability in mice and can be used for the specific in vivo labeling of SNAP-tag fusion proteins. We then applied near-infrared probes to perform non-invasive imaging of in vivo labeled tumors. Finally, we used SNAP-mediated chemical pulse-chase labeling to measuring in vivo the half-life of different extra- and intracellular proteins, including KAP1. Importantly, this tagging technology can be applied to any protein amenable to expression as a fusion partner, whether cell-associated or secreted. Furthermore, because the SNAP-tag chemical labeling allows for a large array of probes also suited for experiments aimed at manipulating and monitoring protein function in vivo, such as cross-linking, pull-down or FRET, this methodology opens broad perspectives for studying protein function in living animals.

EPFL2011

Macrophage NCOR1 Deficiency Ameliorates Myocardial Infarction and Neointimal Hyperplasia in Mice

Johan Auwerx, Yan Liu, Hongwei Zhu

Background NCOR1 (nuclear receptor corepressor 1) is an essential coregulator of gene transcription. It has been shown that NCOR1 in macrophages plays important roles in metabolic regulation. However, the function of macrophage NCOR1 in response to myocardial infarction (MI) or vascular wire injury has not been elucidated. Methods and Results Here, using macrophage Ncor1 knockout mouse in combination with a mouse model of MI, we demonstrated that macrophage NCOR1 deficiency significantly reduced infarct size and improved cardiac function after MI. In addition, macrophage NCOR1 deficiency markedly inhibited neointimal hyperplasia and vascular remodeling in a mouse model of arterial wire injury. Inflammation and macrophage proliferation were substantially attenuated in hearts and arteries of macrophage Ncor1 knockout mice after MI and arterial wire injury, respectively. Cultured primary macrophages from macrophage Ncor1 knockout mice manifested lower expression of inflammatory genes upon stimulation by interleukin-1 beta, interleukin-6, or lipopolysaccharide, together with much less activation of inflammatory signaling cascades including signal transducer and activator of transcription 1 and nuclear factor-kappa B. Furthermore, macrophage Ncor1 knockout macrophages were much less proliferative in culture, with inhibited cell cycle progression compared with control cells. Conclusions Collectively, our data have demonstrated that NCOR1 is a critical regulator of macrophage inflammation and proliferation and that deficiency of NCOR1 in macrophages attenuates MI and neointimal hyperplasia. Therefore, macrophage NCOR1 may serve as a potential therapeutic target for MI and restenosis.