UNASSIGNED: To examine the roles of microRNAs in the development of colitis, we conducted the RNA-sequencing studies using RNA derived from normal and colitogenic CD4+ T cells. Colitogenic CD4+ T cells demonstrated the increased expression of miR-150. We focused on the involvement of miR-150 in the colitis.
UNASSIGNED: We crossed miR-150 knockout mice and T-cell-specific Rap1KO mice, which is colitis model mice and spontaneously develop the colitis with tubular adenomas in microbiota-dependent manner.
UNASSIGNED: MiR-150 silencing completely inhibited the expansion of pathogenic Th17 cells and the development of colitis.
UNASSIGNED: MiR-150 is a potential therapeutic target of inflammatory bowel diseases.

Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but specifically causes extrusion and apoptosis of acinar progenitors at a time when they are shielded by myoepithelial cells from the basement membrane. Csk mutants can be phenocopied by constitutively active Yap and rescued by deleting Yap or Taz, indicating a significant functional overlap between Src and Yap signaling. Although Src-induced tyrosine phosphorylation has long been believed to regulate Yap activity, we find that mutating these tyrosine residues in both Yap and Taz fails to perturb mouse development or alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Further chemical genetics studies demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas removing Crk/CrkL or Rac1/Rap1 ameliorates the Csk mutant phenotype. These results show that Src controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to promote cell extrusion.

BACKGROUND: Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown.
RESULTS: Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome.
CONCLUSIONS: Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.

BACKGROUND: Osteoarthritis (OA) is a prevalent global health concern associated with the loss of articular cartilage and subchondral bone. The lack of disease-modifying drugs for OA necessitates the exploration of novel therapeutic options. Our previous study has demonstrated that traditional Chinese medical herb Trachelospermum jasminoides (Lindl.) Lem. extract suppressed osteoclastogenesis and identified trachelogenin (TCG) as a representative compound. Here, we delved into TCG\'s potential to alleviate OA.
METHODS: We initially validated the in vivo efficacy of TCG in alleviating OA using a rat OA model. Subsequently, we isolated primary bone marrow-derived macrophages in vitro to investigate TCG\'s impact on osteoclastogenesis. We further employed a small molecule pull-down assay to verify TCG\'s binding target within osteoclasts. Finally, we isolated primary mouse chondrocytes in vitro to study TCG\'s regulatory effects and mechanisms on chondrocyte survival.
RESULTS: TCG preserved subchondral bone integrity and protected articular cartilage in a rat OA model. Subsequently, in vitro experiments unveiled TCG\'s capability to inhibit osteoclastogenesis and function through binding to Ras association proximate 1 (Rap1) and inhibiting its activation. Further study demonstrated that TCG inhibited Rap1/integrin αvβ3/c-Src/Pyk2 signaling cascade, and consequently led to failed F-actin ring formation. Besides, TCG promoted the proliferation of mouse primary chondrocytes while suppressing apoptosis in vitro. This is attributed to TCG\'s ability to upregulate HIF1α, thereby promoting glycolysis.
CONCLUSIONS: TCG exerted inhibitory effects on osteoclastogenesis through binding to Rap1 and inhibiting Rap1 activation, consequently preventing subchondral bone loss. Moreover, TCG enhanced chondrocyte survival by upregulating HIF1α and promoting glycolysis. These dual mechanisms collectively provide a novel approach to prevented against cartilage degradation.

Although located at the chromosome end, telomeres are an essential chromosome component that helps maintain genome integrity and chromosome stability from protozoa to mammals. The role of telomere proteins in chromosome end protection is conserved, where they suppress various DNA damage response machineries and block nucleolytic degradation of the natural chromosome ends, although the detailed underlying mechanisms are not identical. In addition, the specialized telomere structure exerts a repressive epigenetic effect on expression of genes located at subtelomeres in a number of eukaryotic organisms. This so-called telomeric silencing also affects virulence of a number of microbial pathogens that undergo antigenic variation/phenotypic switching. Telomere proteins, particularly the RAP1 homologs, have been shown to be a key player for telomeric silencing. RAP1 homologs also suppress the expression of Telomere Repeat-containing RNA (TERRA), which is linked to their roles in telomere stability maintenance. The functions of RAP1s in suppressing telomere recombination are largely conserved from kinetoplastids to mammals. However, the underlying mechanisms of RAP1-mediated telomeric silencing have many species-specific features. In this review, I will focus on Trypanosoma brucei RAP1\'s functions in suppressing telomeric/subtelomeric DNA recombination and in the regulation of monoallelic expression of subtelomere-located major surface antigen genes. Common and unique mechanisms will be compared among RAP1 homologs, and their implications will be discussed.

BACKGROUND: Rasal1 is a Ras GTPase-activating protein which contains C2 domains necessary for dynamic membrane association following intracellular calcium elevation. Membrane-bound Rasal1 inactivates Ras signaling through its RasGAP activity, and through such mechanisms has been implicated in regulating various cellular functions in the context of tumors. Although highly expressed in the brain, the contribution of Rasal1 to neuronal development and function has yet to be explored.
RESULTS: We examined the contributions of Rasal1 to neuronal development in primary culture of hippocampal neurons through modulation of Rasal1 expression using molecular tools. Fixed and live cell imaging demonstrate diffuse expression of Rasal1 throughout the cell soma, dendrites and axon which localizes to the neuronal plasma membrane in response to intracellular calcium fluctuation. Pull-down and co-immunoprecipitation demonstrate direct interaction of Rasal1 with PKC, tubulin, and CaMKII. Consequently, Rasal1 is found to stabilize microtubules, through post-translational modification of tubulin, and accordingly inhibit dendritic outgrowth and branching. Through imaging, molecular, and electrophysiological techniques Rasal1 is shown to promote NMDA-mediated synaptic activity and CaMKII phosphorylation.
CONCLUSIONS: Rasal1 functions in two separate roles in neuronal development; calcium regulated neurite outgrowth and the promotion of NMDA receptor-mediated postsynaptic events which may be mediated both by interaction with direct binding partners or calcium-dependent regulation of down-stream pathways. Importantly, the outlined molecular mechanisms of Rasal1 may contribute notably to normal neuronal development and synapse formation.

Following stimulation, the T cell receptor (TCR) and its coreceptors integrate multiple intracellular signals to initiate T cell proliferation, migration, gene expression, and metabolism. Among these signaling molecules are the small GTPases RAS and RAP1, which induce MAPK pathways and cellular adhesion to activate downstream effector functions. Although many studies have helped to elucidate the signaling intermediates that mediate T cell activation, the molecules and pathways that keep naive T cells in check are less understood. Several recent studies provide evidence that RASA2 and RASA3, which are GAP1-family GTPase-activating proteins (GAPs) that inactivate RAS and RAP1, respectively, are crucial molecules that limit T cell activation and adhesion. In this review we describe recent data on the roles of RASA2 and RASA3 as gatekeepers of T cell activation and migration.

After endocytosis, many plasma membrane components are recycled via membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that Syndapin/PACSIN-family protein SDPN-1 is required in vivo for basolateral endocytic recycling in the C. elegans intestine. Here, we document an interaction between the SDPN-1 SH3 domain and a target sequence in PXF-1/PDZ-GEF1/RAPGEF2, a known exchange factor for Rap-GTPases. We found that endogenous mutations engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling in vivo, as does the loss of the PXF-1 target RAP-1. In some contexts, Rap-GTPases negatively regulate RhoA activity, suggesting a potential for Syndapin to regulate RhoA. Our results indicate that in the C. elegans intestine, RHO-1/RhoA is enriched on SDPN-1- and RAP-1-positive endosomes, and the loss of SDPN-1 or RAP-1 elevates RHO-1(GTP) levels on intestinal endosomes. Furthermore, we found that depletion of RHO-1 suppressed sdpn-1 mutant recycling defects, indicating that control of RHO-1 activity is a key mechanism by which SDPN-1 acts to promote endocytic recycling. RHO-1/RhoA is well known for controlling actomyosin contraction cycles, although little is known about the effects of non-muscle myosin II on endosomes. Our analysis found that non-muscle myosin II is enriched on SDPN-1-positive endosomes, with two non-muscle myosin II heavy-chain isoforms acting in apparent opposition. Depletion of nmy-2 inhibited recycling like sdpn-1 mutants, whereas depletion of nmy-1 suppressed sdpn-1 mutant recycling defects, indicating that actomyosin contractility controls recycling endosome function.

Zyxin (ZYX) is an actin-interacting protein with unknown biological functions in patients with osteosarcoma. This research sought to understand how ZYX affects the biological behavior of osteosarcoma cells and to identify the associated mechanism. Firstly, ZYX expression was decreased in osteosarcoma, and its higher expression indicated better outcomes in patients with osteosarcoma. ZYX overexpression significantly inhibited the proliferation, migration, and invasion of osteosarcoma cells, whereas ZYX silencing resulted in the opposite trend. Subsequently, we found that the Rap1 signaling pathway was significantly correlated with ZYX expression as reported in The Cancer Genome Atlas\'s database using bioinformatic analysis. Moreover, we found that ZYX overexpression regulated the Rap1/MEK/ERK axis, and osteosarcoma cell growth, migration, and invasion were consequently restrained. Additionally, by administering tumor cells subcutaneously to nude mice, a mouse model of transplanted tumors was created. Compared to the control group, the ZYX overexpression group\'s tumors were lighter and smaller, and the ZYX/Rap1 axis was activated in the ZYX overexpression group. Taken together, our results suggest that ZYX inhibits osteosarcoma cell proliferation, migration, and invasion by regulating the Rap1/MEK/ERK signaling pathway. ZYX might be crucial in the clinical management of osteosarcoma and is a promising novel therapeutic target in patients with this disease.

UNASSIGNED: Extravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP-binding protein Rap1 in EGF- and HB-EGF-stimulated EVT invasion.
UNASSIGNED: Expression of Rap1 in the first-trimester placenta was examined by immunohistochemistry. Effect of EGF or HB-EGF on Rap1 activation (GTP-Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR-8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined.
UNASSIGNED: Rap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB-EGF activated Rap1 and promoted invasion of HTR-8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF- and HB-EGF-induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF- and HB-EGF-induced Rap1 activation and reduced EGFR expression.
UNASSIGNED: Rap1 may function as a mediator of EGF and HB-EGF signaling pathways and can modulate EGFR expression in EVTs during placental development.