Progesterone Receptor Modulates Extraembryonic Mesoderm and Cardiac Progenitor Specification during Mouse Gastrulation.

Progesterone treatment is commonly employed to promote and support pregnancy. While maternal tissues are the main progesterone targets in humans and mice, its receptor (PGR) is expressed in the murine embryo, questioning its function during embryonic development. Progesterone has been previously associated with murine blastocyst development. Whether it contributes to lineage specification is largely unknown. Gastrulation initiates lineage specification and generation of the progenitors contributing to all organs. Cells passing through the primitive streak (PS) will give rise to the mesoderm and endoderm. Cells emerging posteriorly will form the extraembryonic mesodermal tissues supporting embryonic growth. Cells arising anteriorly will contribute to the embryonic heart in two sets of distinct progenitors, first (FHF) and second heart field (SHF). We found that PGR is expressed in a posterior-anterior gradient in the PS of gastrulating embryos. We established in vitro differentiation systems inducing posterior (extraembryonic) and anterior (cardiac) mesoderm to unravel PGR function. We discovered that PGR specifically modulates extraembryonic and cardiac mesoderm. Overexpression experiments revealed that PGR safeguards cardiac differentiation, blocking premature SHF progenitor specification and sustaining the FHF progenitor pool. This role of PGR in heart development indicates that progesterone administration should be closely monitored in potential early-pregnancy patients undergoing infertility treatment.

The chromatin-remodeling subunit Baf200 promotes homology-directed DNA repair and regulates distinct chromatin-remodeling complexes.

The efficiency and type of pathway chosen to repair DNA double-strand breaks (DSBs) are critically influenced by the nucleosome packaging and the chromatin architecture surrounding the DSBs. The Swi/Snf (PBAF and BAF) chromatin-remodeling complexes contribute to DNA damage-induced nucleosome remodeling, but the mechanism by which it contributes to this function is poorly understood. Herein, we report how the Baf200 (Arid2) PBAF-defining subunit regulates DSB repair. We used cytological and biochemical approaches to show that Baf200 plays an important function by facilitating homologous recombination-dependent processes, such as recruitment of Rad51 (a key component of homologous recombination) to DSBs, homology-directed repair, and cell survival after DNA damage. Furthermore, we observed that Baf200 and Rad51 are present in the same complex and that this interaction is mediated by C-terminal sequences in both proteins. It has been recognized previously that the interplay between distinct forms of Swi/Snf has profound functional consequences, but we understand little about the composition of complexes formed by PBAF protein subunits. Our biochemical analyses reveal that Baf200 forms at least two distinct complexes. One is a canonical form of PBAF including the Swi/Snf-associated Brg1 catalytic subunit, and the other contains Baf180 but not Brg1. This distinction of PBAF complexes based on their unique composition provides the foundation for future studies on the specific contributions of the PBAF forms to the regulation of DNA repair.

Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation.

Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm.

Post-translational protein arginylation in the normal nervous system and in neurodegeneration.

Post-translational arginylation of proteins is an important regulator of many physiological pathways in cells. This modification was originally noted in protein degradation during neurodegenerative processes, with an apparently different physiological relevance between central and peripheral nervous system. Subsequent studies have identified a steadily increasing number of proteins and proteolysis-derived polypeptides as arginyltransferase (ATE1) substrates, including ß-amyloid, α-synuclein, and TDP43 proteolytic fragments. Arginylation is involved in signaling processes of proteins and polypeptides that are further ubiquitinated and degraded by the proteasome. In addition, it is also implicated in autophagy/lysosomal degradation pathway. Recent studies using mutant mouse strains deficient in ATE1 indicate additional roles of this modification in neuronal physiology. As ATE1 is capable of modifying proteins either at the N-terminus or middle-chain acidic residues, determining which proteins function are modulated by arginylation represents a big challenge. Here, we review studies addressing various roles of ATE1 activity in nervous system function, and suggest future research directions that will clarify the role of post-translational protein arginylation in brain development and various neurological disorders. Arginyltransferase (ATE1), the enzyme responsible for post-translational arginylation, modulates the functions of a wide variety of proteins and polypeptides, and is also involved in the main degradation pathways of intracellular proteins. Regulatory roles of ATE1 have been well defined for certain organs. However, its roles in nervous system development and neurodegenerative processes remain largely unknown, and present exciting opportunities for future research, as discussed in this review.

Mouse vascularized adipose spheroids: an organotypic model for thermogenic adipocytes.

Adipose tissues, particularly beige and brown adipose tissue, play crucial roles in energy metabolism. Brown adipose tissues' thermogenic capacity and the appearance of beige cells within white adipose tissue have spurred interest in their metabolic impact and therapeutic potential. Brown and beige fat cells, activated by environmental factors like cold exposure or by pharmacology, share metabolic mechanisms that drive non-shivering thermogenesis. Understanding these two cell types requires advanced, yet broadly applicable in vitro models that reflect the complex microenvironment and vasculature of adipose tissues. Here we present mouse vascularized adipose spheroids of the stromal vascular microenvironment from inguinal white adipose tissue, a tissue with 'beiging' capacity in mice and humans. We show that adding a scaffold improves vascular sprouting, enhances spheroid growth, and upregulates adipogenic markers, thus reflecting increased adipocyte maturity. Transcriptional profiling via RNA sequencing revealed distinct metabolic pathways upregulated in our vascularized adipose spheroids, with increased expression of genes involved in glucose metabolism, lipid metabolism, and thermogenesis. Functional assessment demonstrated increased oxygen consumption in vascularized adipose spheroids compared to classical 2D cultures, which was enhanced by ß-adrenergic receptor stimulation correlating with elevated ß-adrenergic receptor expression. Moreover, stimulation with the naturally occurring adipokine, FGF21, induced Ucp1 mRNA expression in the vascularized adipose spheroids. In conclusion, vascularized inguinal white adipose tissue spheroids provide a physiologically relevant platform to study how the stromal vascular microenvironment shapes adipocyte responses and influence activated thermogenesis in beige adipocytes.

Mouse Chd4-NURD is required for neonatal spermatogonia survival and normal gonad development.

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of Chd4 and Chd3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of chd4 early in gametogenesis, but not chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that Chd4 controls expression of dmrt1 and its downstream target plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of Chd4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

Blockade of beta-adrenergic receptors reduces cancer growth and enhances the response to anti-CTLA4 therapy by modulating the tumor microenvironment.

The development of immune checkpoint inhibitors (ICI) marks an important breakthrough of cancer therapies in the past years. However, only a limited fraction of patients benefit from such treatments, prompting the search for immune modulating agents that can improve the therapeutic efficacy. The nonselective beta blocker, propranolol, which for decades has been prescribed for the treatment of cardiovascular conditions, has recently been used successfully to treat metastatic angiosarcoma. These results have led to an orphan drug designation by the European Medicines Agency for the treatment of soft tissue sarcomas. The anti-tumor effects of propranolol are suggested to involve the reduction of cancer cell proliferation as well as angiogenesis. Here, we show that oral administration of propranolol delays tumor progression of MCA205 fibrosarcoma model and MC38 colon cancer model and increases the survival rate of tumor bearing mice. Propranolol works by reducing tumor angiogenesis and facilitating an anti-tumoral microenvironment with increased T cell infiltration and reduced infiltration of myeloid-derived suppressor cells (MDSCs). Using T cell deficient mice, we demonstrate that the full anti-tumor effect of propranolol requires the presence of T cells. Flow cytometry-based analysis and RNA sequencing of FACS-sorted cells show that propranolol treatment leads to an upregulation of PD-L1 on tumor associated macrophages (TAMs) and changes in their chemokine expression profile. Lastly, we observe that the co-administration of propranolol significantly enhances the efficacy of anti-CTLA4 therapy. Our results identify propranolol as an immune modulating agent, which can improve immune checkpoint inhibitor therapies in soft tissue sarcoma patients and potentially in other cancers.

Impaired glucocorticoid receptor expression in liver disrupts feeding-induced gene expression, glucose uptake, and glycogen storage.

The transition from a fasted to a fed state is associated with extensive transcriptional remodeling in hepatocytes facilitated by hormonal- and nutritional-regulated transcription factors. Here, we use a liver-specific glucocorticoid receptor (GR) knockout (L-GRKO) model to investigate the temporal hepatic expression of GR target genes in response to feeding. Interestingly, in addition to the well-described fasting-regulated genes, we identify a subset of hepatic feeding-induced genes that requires GR for full expression. This includes Gck, which is important for hepatic glucose uptake, utilization, and storage. We show that insulin and glucocorticoids cooperatively regulate hepatic Gck expression in a direct GR-dependent manner by a 4.6 kb upstream GR binding site operating as a Gck enhancer. L-GRKO blunts preprandial and early postprandial Gck expression, which ultimately affects early postprandial hepatic glucose uptake, phosphorylation, and glycogen storage. Thus, GR is positively involved in feeding-induced gene expression and important for postprandial glucose metabolism in the liver.

Arginylated Calreticulin Increases Apoptotic Response Induced by Bortezomib in Glioma Cells.

After retrotranslocation from the endoplasmic reticulum to the cytoplasm, calreticulin is modified by the enzyme arginyltransferase-1 (ATE1). Cellular levels of arginylated calreticulin (R-CRT) are regulated in part by the proteasomal system. Under various stress conditions, R-CRT becomes associated with stress granules (SGs) or reaches the plasma membrane (PM), where it participates in pro-apoptotic signaling. The mechanisms underlying the resistance of tumor cells to apoptosis induced by specific drugs remain unclear. We evaluated the regulatory role of R-CRT in apoptosis of human glioma cell lines treated with the proteasome inhibitor bortezomib (BT). Two cell lines (HOG, MO59K) displaying distinctive susceptibility to apoptosis induction were studied further. BT efficiency was found to be correlated with a subcellular distribution of R-CRT. In MO59K (apoptosis-resistant), R-CRT was confined to SGs formed following BT treatment. In contrast, HOG (apoptosis-susceptible) treated with BT showed lower SG formation and higher levels of cytosolic and PM R-CRT. Increased R-CRT level was associated with enhanced mobilization of intracellular Ca2+ and with sustained apoptosis activation via upregulation of cell death receptor DR5. R-CRT overexpression in the cytoplasm of MO59K rendered the cells susceptible to BT-induced, DR5-mediated cell death. Our findings suggest that R-CRT plays an essential role in the effect of BT treatment on tumor cells and that ATE1 is a strong candidate target for future studies of cancer diagnosis and therapy.