CK1α deficiency impairs mouse uterine adenogenesis by inducing epithelial cell apoptosis through GSK3ß pathway and inhibiting Foxa2 expression through p53 pathway†.

Uterine glands and their secretions are crucial for conceptus survival and implantation in rodents and humans. In mice, the development of uterine gland known as adenogenesis occurs after birth, whereas the adenogenesis in humans initiates from fetal life and completed at puberty. Uterine adenogenesis involves dynamic epithelial cell proliferation, differentiation, and apoptosis. However, it is largely unexplored about the mechanisms governing adenogenesis. CK1α plays important roles in regulating cell division, differentiation, and death, but it is unknown whether CK1α affects adenogenesis. In the current study, uterus-specific CK1α knockout female mice (Csnk1a1d/d) were infertile resulted from lack of uterine glands. Subsequent analysis revealed that CK1α deletion induced massive apoptosis in uterine epithelium by activating GSK3ß, which was confirmed by injections of GSK3ß inhibitor SB216763 to Csnk1a1d/d females, and the co-treatment of SB216763 and CK1 inhibitor d4476 on cultured epithelial cells. Another important finding was that our results revealed CK1α deficiency activated p53, which then blocked the expression of Foxa2, an important factor for glandular epithelium development and function. This was confirmed by that Foxa2 expression level was elevated in p53 inhibitor pifithrin-α injected Csnk1a1d/d mouse uterus and in vitro dual-luciferase reporter assay between p53 and Foxa2. Collectively, these studies reveal that CK1α is a novel factor regulating uterine adenogenesis by inhibiting epithelial cell apoptosis through GSK3ß pathway and regulating Foxa2 expression through p53 pathway. Uncovering the mechanisms of uterine adenogenesis is expected to improve pregnancy success in humans and other mammals.

Cysteine dioxygenase and taurine are essential for embryo implantation by involving in E2-ERα and P4-PR signaling in mouse.

BACKGROUND: Taurine performs multiple physiological functions, and the maintenance of taurine level for most mammals relies on active uptake from diet and endogenous taurine synthesis through its synthesis enzymes, including cysteine dioxygenase (CDO). In addition, uterus tissue and uterus fluid are rich in taurine, and taurine synthesis is regulated by estrogen (E2) and progesterone (P4), the key hormones priming embryo-uterine crosstalk during embryo implantation, but the functional interactions and mechanisms among which are largely unknown. The present study was thus proposed to identify the effects of CDO and taurine on embryo implantation and related mechanisms by using Cdo knockout (KO) and ovariectomy (OVX) mouse models. RESULTS: The uterine CDO expression was assayed from the first day of plugging (d 1) to d 8 and the results showed that CDO expression level increased from d 1 to d 4, followed by a significant decline on d 5 and persisted to d 8, which was highly correlated with serum and uterine taurine levels, and serum P4 concentration. Next, Cdo KO mouse was established by CRISPER/Cas9. It was showed that Cdo deletion sharply decreased the taurine levels both in serum and uterus tissue, causing implantation defects and severe subfertility. However, the implantation defects in Cdo KO mice were partly rescued by the taurine supplementation. In addition, Cdo deletion led to a sharp decrease in the expressions of P4 receptor (PR) and its responsive genes Ihh, Hoxa10 and Hand2. Although the expression of uterine estrogen receptor (ERα) had no significant change, the levels of ERα induced genes (Muc1, Ltf) during the implantation window were upregulated after Cdo deletion. These accompanied by the suppression of stroma cell proliferation. Meanwhile, E2 inhibited CDO expression through ERα and P4 upregulated CDO expression through PR. CONCLUSION: The present study firstly demonstrates that taurine and CDO play prominent roles in uterine receptivity and embryo implantation by involving in E2-ERα and P4-PR signaling. These are crucial for our understanding the mechanism of embryo implantation, and infer that taurine is a potential agent for improving reproductive efficiency of livestock industry and reproductive medicine.

RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells.

Background: The v-raf-leukemia viral oncogene 1 (RAF1) plays an essential physiological role in reproduction and development through the mediation of steroid hormone synthesis. Follicle-stimulating hormone (FSH) signaling pathway was not involved in the majority of RAF1 studies, whether RAF1 takes part in the signaling events of gonadotropic hormones such as FSH in ovarian tissue is unknown. Methods: The process is blocked by treating granulosa cells (GCs) with the RAF1 inhibitor, RAF709. Inhibition of RAF1 activity by RAF709 decreased extracellular regulated protein kinases (ERK) phosphorylation and suppressed the expression of the cytochrome P450 subfamily 19 member 1 (CYP19A1), which is a major rate-limiting enzyme that participates in the last step of E2 biosynthesis. Results: We found that RAF1, acting as a downstream molecule, mediates FSH signalling to stimulate estradiol (E2) synthesis and secretion in mouse ovarian GCs. Gene expression of RAF1 was induced by FSH and the secretion of E2 increased into the bloodstream of mice and into the supernatant of primary GCs. Our in vitro and in vivo studies clearly illustrate RAF1 plays an important medium adjusting role in the FSH signaling pathway, and RAF1 acting as a downstream molecule to trigger ERK phosphorylation to stimulate GC E2 synthesis and secretion. Conclusions: RAF1 plays a pivotal mediating role in the FSH signaling pathway by inducing the phosphorylation of ERK and promoting E2 synthesis.

GALNT4 primes monocytes adhesion and transmigration by regulating O-Glycosylation of PSGL-1 in atherosclerosis.

Atherosclerosis is a major underlying cause of cardiovascular disease. Genome wide association studies have predicted that GalNAc-T4 (GALNT4), which responsible for initiating step of mucin-type O-glycosylation, plays a causal role in the susceptibility to cardiovascular diseases, whereas the precise mechanism remains obscure. Thus, we sought to determine the role and mechanism of GALNT4 in atherosclerosis. Firstly, we found the expression of GALNT4 and protein O-glycosylation were both increased in plaque as atherosclerosis progressed in ApoE-/- mice by immunohistochemistry. And the expression of GALNT4 was also increased in human monocytes treated with ACS (acute coronary syndrome) sera and subjected to LPS and ox-LDL in vitro. Moreover, silencing expression of GALNT4 by shRNA lentivirus alleviated atherosclerotic plaque formation and monocyte/macrophage infiltration in ApoE-/- mice. Functional investigations demonstrate that GALNT4 knockdown inhibited P-selectin-induced activation of ß2 integrin on the surface of monocytes, decreased monocytes adhesion under flow condition with P-selectin stimulation, as well as suppressed monocytes transmigration triggered by monocyte chemotactic protein- 1(MCP-1). In contrast, GALNT4 overexpression enhanced monocytes adhesion and transmigration. Furthermore, Vicia Villosa Lectin (VVL) pull down and PSGL-1 immunoprecipitation assays showed that GALNT4 overexpression increased O-Glycosylation of PSGL-1 and P-selectin induce phosphorylation of Akt/mTOR and IκBα/NFκB on monocytes. Conversely, knockdown of GALNT4 decreased VVL binding and attenuated the activation of Akt/mTOR and IκBα/NFκB. Additionally, mTOR inhibitor rapamycin blocked these effects of GALNT4 overexpression on monocytes. Collectively, GALNT4 catalyzed PSGL-1 O-glycosylation that involved in P-selectin induced monocytes adhesion and transmigration via Akt/mTOR and NFκB pathway. Thus, GALNT4 may be a potential therapeutic target for atherosclerosis.

Cyclin-dependent kinase inhibitor 1B acts as a novel molecule to mediate testosterone synthesis and secretion in mouse Leydig cells by luteinizing hormone (LH) signaling pathway.

Cyclin-dependent kinase inhibitor 1B (Cdkn1b, p27) plays important regulatory roles in many cellular processes. p27 is highly expressed in the mouse testis, but its roles and underlying mechanisms for testosterone synthesis and secretion remain not well understood. In the current study, we found that p27 located in Leydig cells and Sertoli cells of adult mouse testis. To explore the function of p27 in Leydig cells, p27 inhibitor and activator were injected into the adult mice, primary Leydig cells and TM3 cells. Our in vivo and in vitro results showed that change in the expression of p27 significantly alters the testosterone in both globe serum and culture medium. Meanwhile, the steroidogenesis-related gene expression was significantly regulated too. Moreover, our in vitro study showed that luteinizing hormone (LH) significantly increased p27 mRNA levels. Furthermore, our results proved that altering the mRNA expression of p27 leads to the synchronized changes of Lhcgr, Star, Cyp11a1, Hsd3b6, Cyp11a1, and Hsd17b3. Alterations of p27 also result in synchronously changes of RAF1 and ERK1/2 phosphorylation. These findings indicate that p27 plays vital roles in LH-induced testosterone production, providing a novel mechanism that p27 acts as an upstream molecule to elevate ERK1/2 phosphorylation to promote the expression of StAR and other cholesterol-metabolizing enzymes.