Changes in Porcine Corpus Luteum Proteome Associated with Development, Maintenance, Regression, and Rescue during Estrous Cycle and Early Pregnancy

Corpus luteum (CL), a transitory gland, undergoes rapid growth in a limited time to produce progesterone (P4) followed by its regression. A complex molecular signaling is involved in controlling luteal P4 production. In the present study, 2D gel electrophoresis-based proteomics and in silico functional analysis were used to identify changes in key proteins and pathways in CL along the different stages of the estrous cycle as its development progresses from early (Day 3) to mid-luteal phase (Day 9), effective functioning (Day 12) followed by regression (Day 15) or, in the case of pregnancy, rescue of function (Day 15). A total of 273 proteins were identified by MALDI-MS/MS analysis that showed significant changes in abundances at different stages of CL development or regression and rescue. Functional annotation of differentially abundant proteins suggested enrichment of several important pathways and functions during CL development and function maintenance including cell survival, endocytosis, oxidative stress response, estradiol metabolism, and angiogenesis. On the other hand, differentially abundant proteins during CL regression were associated with decreased steroid synthesis and metabolism and increased apoptosis, necrosis, and infiltration of immune cells. Establishment of pregnancy rescues CL from regression by maintaining the expression of proteins that support steroidogenesis as pathways such as the super-pathway of cholesterol biosynthesis, RhoA signaling, and functions such as fatty acid metabolism and sterol transport were enriched in CL of pregnancy. In this study, some novel proteins were identified along CL development that advances our understanding of CL survival and steroidogenesis.

The Role and Mechanism of AMIGO3 in the Formation of Aberrant Neural Circuits After Status Convulsion in Immature Mice

Leucine rich repeat and immunoglobulin-like domain-containing protein 1 (Lingo-1) has gained considerable interest as a potential therapy for demyelinating diseases since it inhibits axonal regeneration and myelin production. However, the results of clinical trials targeted at Lingo-1 have been unsatisfactory. Amphoterin-induced gene and open reading frame-3 (AMIGO3), which is an analog of Lingo-1, might be an alternative therapeutic target for brain damage. In the present study, we investigated the effects of AMIGO3 on neural circuits in immature mice after status convulsion (SC) induced by kainic acid. The expression of both AMIGO3 and Lingo-1 was significantly increased after SC, with levels maintained to 20 days after SC. Following SC, transmission electron microscopy revealed the impaired microstructure of myelin sheaths and Western blot analysis showed a decrease in myelin basic protein expression, and this damage was alleviated by downregulation of AMIGO3 expression. The ROCK/RhoA signaling pathway was inhibited at 20 days after SC by downregulating AMIGO3 expression. These results indicate that AMIGO3 plays important roles in seizure-induced damage of myelin sheaths as well as axon growth and synaptic plasticity via the ROCK/RhoA signaling pathway.

First person – Shafali Gupta

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Shafali Gupta is first author on ‘ Enhanced RhoA signaling stabilizes E-cadherin in migrating epithelial monolayers’, published in JCS. Shafali is a postdoc in the lab of Alpha S. Yap at Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia, investigating the critical mechanisms behind different cellular processes.

Salidroside Ameliorated Intermittent Hypoxia-Aggravated Endothelial Barrier Disruption and Atherosclerosis via the cAMP/PKA/RhoA Signaling Pathway

Background: Endothelial barrier dysfunction plays a key role in atherosclerosis progression. The primary pathology of obstructive sleep apnea-hypopnea syndrome is chronic intermittent hypoxia (IH), which induces reactive oxygen species (ROS) overproduction, endothelial barrier injury, and atherosclerosis. Salidroside, a typical pharmacological constituent of Rhodiola genus, has documented antioxidative, and cardiovascular protective effects. However, whether salidroside can improve IH-aggravated endothelial barrier dysfunction and atherosclerosis has not been elucidated.Methods and results: In normal chow diet-fed ApoE−/− mice, salidroside (100 mg/kg/d, p. o.) significantly ameliorated the formation of atherosclerotic lesions and barrier injury aggravated by 7-weeks IH (21%–5%–21%, 120 s/cycle). In human umbilical vein endothelial cells (HUVECs), exposure to IH (21%–5%–21%, 40 min/cycle, 72 cycles) decreased transendothelial electrical resistance and protein expression of vascular endothelial cadherin (VE-cadherin) and zonula occludens 1. In addition, IH promoted ROS production and activated ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) pathway. All of these effects of IH were reversed by salidroside. Similar to salidroside, ROCK-selective inhibitors Y26732, and Fasudil protected HUVECs from IH-induced ROS overproduction and endothelial barrier disruption. Furthermore, salidroside increased intracellular cAMP levels, while the PKA-selective inhibitor H-89 attenuated the effects of salidroside on IH-induced RhoA/ROCK suppression, ROS scavenging, and barrier protection.Conclusion: Our findings demonstrate that salidroside effectively ameliorated IH-aggravated endothelial barrier injury and atherosclerosis, largely through the cAMP/PKA/RhoA signaling pathway.

Enhanced RhoA signaling stabilizes E-cadherin in migrating epithelial monolayers

Epithelia migrate as physically coherent populations of cells. Earlier studies revealed that mechanical stress accumulates in these cellular layers as they move. These stresses are characteristically tensile in nature and have often been inferred to arise when moving cells pull upon the cell-cell adhesions that hold them together. We now report that epithelial tension at adherens junctions between migrating cells also increases due to an increase in RhoA-mediated junctional contractility. We find that active RhoA levels were stimulated by p114 RhoGEF at the junctions between migrating MCF-7 monolayers, and this was accompanied by increased levels of actomyosin and mechanical tension. Applying a strategy to restore active RhoA specifically at adherens junctions by manipulating its scaffold, anillin, we found that this junctional RhoA signal was necessary to stabilize junctional E-cadherin during epithelial migration and promoted orderly collective movement. We suggest that stabilization of E-cadherin by RhoA serves to increase cell-cell adhesion against the mechanical stresses of migration.

Ginsenoside Rg1 Ameliorated Colitis by Regulating the Homeostasis of M1/M2 Macrophage Polarization and Intestinal Flora

Background: Aberrant M1/M2 macrophage polarization and intestinal flora disruption are involved in the pathological processes associated with ulcerative colitis (UC). Ginsenoside Rg1 has good immunomodulatory and anti-inflammatory effects and is effective in treating UC of humans and animals. However, it is unclear how ginsenoside Rg1 regulate the homeostasis of M1/M2 macrophage polarization and intestinal flora.Methods: BALB/c mice were randomly divided into 4 groups: Control, DSS, DSS+Rg1, DSS+Y27632 groups. In this study, experiment colitis was induced in BALB/c mice using sodium dextran sulfate (DSS). Mice of DSS+Rg1, DSS+Y27632 groups were treated respectively with ginsenoside Rg1 and Rock inhibitor Y27632 for 14 consecutive days. On day 21, all mice were sacrificed. Histopathological analysis of the colon tissues was performed by Hematoxylin Eosin sinning. Cytokines (IL-6, IL-33, CCL-2, TNF-α, IL-4 and IL-10) were detected by Elisa. Flow cytometry was used to analyse macrophage activation and M1/M2 macrophage polarisation. Western blotting were applied to detect the levels of Macrophage polarization-associated protein (Arg-1, MIF-1, PIM-1, TLR2) and Nogo-B/RhoA signaling molecules (Rock1, RhoA and Nogo-B). The fecal microbial populations were analyzed using 16S gene sequencing. Results: After ginsenoside Rg1 and Y27632 treatment, the changes of body weight, colon length, colonic weight index and colonic mucosal injury of colitis mice were effectively improved, accompanied by less ulcer formation and inflammatory cell infiltration, lower levels of pro-inflammatory cytokines (IL-6, IL-33, CCL-2, TNF-α) and higher anti-inflammatory cytokines (IL-4 and IL-10). Importantly, the percentage of CD11b+F4/80+, CD11b+F4/80+Tim-1+, CD11b+F4/80+TLR4+, and CD11b+F4/80+iNOS+ cells and the expression levels of MIF-1 and PIM-1 proteins were down-regulated significantly after ginsenoside Rg1 and Y27632 treatment, and CD11b+F4/80+CD206+ and CD11b+F4/80+CD163+ cells and Arg-1 up-regulated significantly. Intestinal flora composition were effectively improved after administration of ginsenoside Rg1. The Nogo-B/RchoA signaling pathway were obviously inhibited after ginsenoside Rg1 and Y27632 treatment, and the levels of Rock1, RhoA and Nogo-B proteins were significantly reduced. Conclusions: Ginsenoside Rg1 has the protective effect on UC by inhibiting macrophage activation, restoring the balance of M1/M2 macrophage polarization, and improving intestinal flora composition, associated with inhibition of the Nogo-B/RhoA signaling pathway.

Resveratrol ameliorates neuronal apoptosis and cognitive impairment by activating the SIRT1/RhoA pathway in rats after anesthesia with sevoflurane

Studies have shown that long-term exposure to sevoflurane (SEV) may cause postoperative cognitive dysfunction. This study aimed to investigate the effects of resveratrol (RES) treatment on the changes in the cognitive function of rats after prolonged anesthesia with SEV. Seventy-six adult male rats were used in this study. The SEV model was established under continuous anesthesia for 6 h. Rats were randomly classified into four groups as follows: control, SEV+vehicle, SEV+pre-RES (RES was administered 24 h before establishing the SEV model), and SEV+post-RES (RES was administered 1 h after establishing the SEV model) groups. Neurobehavioral outcomes and the potential mechanism underlying RES-mediated neuroprotection through the SIRT1/RhoA signaling pathway were evaluated. The water maze test showed that long-term exposure to SEV may lead to loss of learning and memory ability in rats (p<0.05). Compared with the SEV+vehicle group, the RES treatment groups showed significantly improved neurobehavioral scores (p<0.05). Additionally, the SEV+pre-RES group had a better outcome than the SEV+vehicle group on days 1 or 2 (p<0.05), unlike the SEV+post-RES group (p>0.05). Western blotting showed that SIRT1, RhoA, and cleaved Caspase-3 (CC3) expression significantly increased in the SEV+vehicle group (p<0.05), while Bcl2 expression decreased (p < 0.05). RES treatment further upregulated SIRT1 and Bcl2 expression and downregulated the expression of RhoA and CC3 (p<0.05). In conclusion, RES treatment improved cognitive dysfunction by reducing neuronal apoptosis in adult rats exposed to SEV. RES partly exerted a neuroprotective effect through the activation of the SIRT1/RhoA signaling pathway.