The autophagy protein, FIP200 (RB1CC1) mediates progesterone responses governing uterine receptivity and decidualization†.
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ABSTRACT:
SUBMITTER: Oestreich AK
PROVIDER: S-EPMC7124967 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Progesterone is required for the establishment and maintenance of mammalian pregnancy and widely used for conservative treatment of luteal phase deficiency in clinics. However, there are limited solid evidences available for the optimal timing and dose of progesterone therapy, especially for the possible adverse effects on implantation and decidualization when progesterone is administrated empirically. In our study, mouse models were used to examine effects of excess progesterone on embryo implantation and decidualization. Our data indicate that excess progesterone is not only harmful for mouse implantation, but also impairs mouse decidualization. In excess progesterone-treated mice, the impaired LIF/STAT3 pathway and dysregulated endoplasmic reticulum stress may lead to the inhibition of embryo implantation and decidualization. It is possible that the decrease in birth weight of excess progesterone-treated mice is due to a compromised embryo implantation and decidualization. Furthermore, excess progesterone compromises in vitro decidualization of human endometrial stromal cells.
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Project description:Apoptosis is an important contributing factor in spinal cord injury (SCI). ZBTB38 is involved in the transcriptional regulation of multiple signaling pathways, is differentially expressed at different SCI stages, and may provide a therapeutic strategy for the treatment of patients with SCI. In this study, we found that autophagy is blocked in ZBTB38 knockdown SH-SY5Y cells and that the expression levels of LC3B II/I decreased and P62 increased. We used transcriptome high-throughput sequencing to identify the target in ZBTB38 knockdown cells. From the transcriptome profile, RB1CC1 (i.e., FIP200), a key component of the initiation machinery of autophagy (FIP200-ATG13-ULK1-ATG101), was found to decrease 4.2-fold following ZBTB38 knockdown. When RB1CC1-overexpressed plasmids were transfected into ZBTB38 knockdown cells, they rescued the phenotype of ZBTB38 knockdown cells. Cell proliferation and viability were significantly enhanced by RB1CC1 overexpression, and LC3B and P62 expression returned to their original levels. We also injected ZBTB38-overexpressed lentivirus into the injured center of the spinal cord and detected significant upregulation of RB1CC1 in the spinal cord. ZBTB38 overexpression can promote autophagy and partly rescue the secondary damage of SCI. Therefore, our findings provide a new strategy for the treatment of SCI.
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Project description:Previous in vitro studies have suggested that calreticulin (CALR), which is responsible for the folding and quality control of glycoproteins, may be associated with decidualization. However, its precise role in regulating decidualization has not been explored in vivo. Here, we used pregnant rat models to examine endometrial CALR expression during the peri-implantation period. We also examined whether polypectomy, a procedure that could ameliorate infertility, alters the endometrial expression levels of CALR and several implantation factors in women diagnosed as infertile. In rats, uterine CALR was expressed at a high level at the implantation site, and a marked increase in CALR expression was observed in decidual cells of normal pregnancy. In addition, endometrial CALR expression was enhanced by either administration of estradiol-17β in the delayed implantation rat model or the artificial induction of decidualization in the pseudopregnant rat. In cultured stromal cells, siRNA-mediated silencing of CALR inhibited the decidual stimulus-induced expression of prolactin, decidual/trophoblast prolactin-related protein, and connexin 43. In humans, the endometrial expression levels of the mRNAs encoding CALR and the implantation-related factor insulin-like growth factor binding protein (IGFBP)-7 tended to increase after polypectomy. The strongest positive correlation between expression levels before polypectomy was observed for IGFBP-7 and CALR, and the strength of this correlation increased after the surgery. Thus, endometrial CALR may play a role in the formation of decidua, and the polypectomy of infertile patients may result in the co-operative expression of endometrial factors, including CALR, that could enhance endometrial receptivity.
| S-EPMC8509037 | biostudies-literature
Project description:Retinoic acid (RA) signaling has long been speculated to regulate embryo implantation, because many enzymes and proteins responsible for maintaining RA homeostasis and transducing RA signals are tightly regulated in the endometrium during this critical period. However, due to a lack of genetic data, it was unclear whether RA signaling is truly required for implantation and which specific RA signaling cascades are at play. Herein we utilize a genetic murine model that expresses a dominant-negative form of RA receptor (RAR) specifically in female reproductive organs to show that functional RA signaling is fundamental to female fertility, particularly implantation and decidualization. Reduction in RA signaling activity severely affects the ability of the uterus to achieve receptive status and decidualize, partially through dampening follistatin expression and downstream activin B/bone morphogenetic protein 2 signaling. To confirm translational relevance of these findings to humans, human endometrial stromal cells (hESCs) were treated with a pan-RAR antagonist to show that in vitro decidualization is impaired. RNA interference perturbation of individual RAR transcripts in hESCs revealed that RARα in particular was essential for proper decidualization. These data provide direct functional evidence that uterine RAR-mediated RA signaling was crucial for mammalian embryo implantation, and its disruption led to failure of uterine receptivity and decidualization, resulting in severely compromised fertility.
| S-EPMC8492326 | biostudies-literature
Project description:RB1CC1/FIP200 is an essential macroautophagy/autophagy protein that plays an important role in a variety of biological and disease processes through its canonical autophagy-dependent and -independent functions. However, it remains largely unknown whether post-translational modifications could regulate RB1CC1 and its associated autophagy functions. Here, we report acetylation of several lysine residues of RB1CC1 by acetyltransferase CREBBP (CREB binding protein), with K276 as the major CREBBP acetylation site. K276 is also identified as a ubiquitination site by mass spectrometry, and acetylation at this site reduces ubiquitination of RB1CC1 to inhibit its ubiquitin-dependent degradation. We also find that RB1CC1 contains an N-terminal intrinsically disordered region (IDR) capable of forming liquid-liquid phase separation (LLPS) in vitro, which may drive formation of RB1CC1 puncta with LLPS properties in cells independent of SQSTM1/p62 and other autophagy receptors CALCOCO2/NDP52, NBR1, TAX1BP1 and OPTN. Mutational analysis shows that both K276 acetylation and the N-terminal IDR containing it are important for maintaining canonical autophagy function of RB1CC1 in breast cancer cells. Our findings demonstrate regulation of RB1CC1 by a new post-translational mechanism and suggest potential therapeutic application of inducing RB1CC1 degradation through blocking K276 acetylation in the treatment of cancer and other diseases.Abbreviations: Baf-A1: bafilomycin A1; CREBBP/CBP: CREB binding protein; CHX: cycloheximide; EP300/p300: E1A binding protein p300; FRAP: fluorescence recovery after photobleaching; HADCs: histone deacetylases; IDR: intrinsically disordered region; LLPS: liquid-liquid phase separation; KAT2A/GCN5: lysine acetyltransferase 2A; KAT2B/PCAF: lysine acetyltransferase 2B; KAT5/TIP60: lysine acetyltransferase 5; KAT8/MOF: lysine acetyltransferase 8; NAM: nicotinamide; PAS: phagophore assembly site; PEG-8000: polyethylene glycol 8000; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TSA: trichostatin A.
| S-EPMC10262773 | biostudies-literature
Project description:Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types and their hormonal regulation, cell type, and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR and in situ hybridization studies demonstrated that among the known Akp2, Akp3, Akp5, and Akp6 murine AP isozyme genes, hamster uteri express only Akp2 and Akp6; both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone (P₄) is the major uterine Akp2 inducer, both P₄ and estrogen are strong Akp6 regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation, Akp2 showed reduced expression in luminal epithelial cells surrounding the implanted embryo. By contrast, expression of Akp6 and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of only Akp2 and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection.
| S-EPMC3805369 | biostudies-literature
Project description:In this report, we investigated the consequences of neonatal progesterone exposure on adult rat uterine function. Female pups were subcutaneously injected with vehicle or progesterone from postnatal days 3 to 9. Early progesterone exposure affected endometrial gland biogenesis, puberty, decidualization, and fertility. Because decidualization and pregnancy success are directly linked to progesterone action on the uterus, we investigated the responsiveness of the adult uterus to progesterone. We first identified progesterone-dependent uterine gene expression using RNA sequencing and quantitative RT-PCR in Holtzman Sprague-Dawley rats and progesterone-resistant Brown Norway rats. The impact of neonatal progesterone treatment on adult uterine progesterone responsiveness was next investigated using quantitative RT-PCR. Progesterone resistance affected the spectrum and total number of progesterone-responsive genes and the magnitude of uterine responses for a subset of progesterone targets. Several progesterone-responsive genes in adult uterus exhibited significantly dampened responses in neonatally progesterone-treated females compared with those of vehicle-treated controls, whereas other progesterone-responsive transcripts did not differ between female rats exposed to vehicle or progesterone as neonates. The organizational actions of progesterone on the uterus were dependent on signaling through the progesterone receptor but not estrogen receptor 1. To summarize, neonatal progesterone exposure leads to disturbances in endometrial gland biogenesis, progesterone resistance, and uterine dysfunction. Neonatal progesterone effectively programs adult uterine responsiveness to progesterone.
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Project description:Macroautophagy/autophagy degrades proteins and organelles to generate macromolecular building blocks. As such, some cancer cells are particularly dependent on autophagy. In a previous paper, we found that even highly autophagy-dependent cancer cells can adapt to circumvent autophagy inhibition. However, it remains unclear if autophagy-dependent cancer cells could survive the complete elimination of autophagosome formation. We extended our previous findings to show that knockout (KO) of both the upstream autophagy regulator RB1CC1/FIP200 and the downstream regulator and mediator of LC3 conjugation, ATG7, strongly inhibits growth in highly autophagy-dependent cells within one week of editing. However, rare clones survived the loss of ATG7 or RB1CC1 and maintained growth even under autophagy-inducing conditions. Autophagy-dependent cells circumvent the complete loss of autophagy that is mediated by RB1CC1 KO, similar to the loss of ATG7, by upregulating NFE2L2/NRF2 signaling. These results indicate that cancer cell lines could adapt to the complete loss of autophagy by changing their biology to adopt alternative ways of dealing with autophagy-mediated cellular functions.AbbreviationsCGS: CRISPR growth score; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: Earl's balanced salt solution; EEF2: eukaryotic translation elongation factor 2; FOXO3/FOXO3a: forkhead box O3; GFP: green fluorescent protein; KEAP1: kelch Like ECH associated protein 1; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NLS: nuclear localization signal; PCNA: proliferating cell nuclear antigen; PE: phosphatidylethanolamine; POLR2A: RNA polymerase II subunit A; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SNARE: soluble NSF attachment protein receptor; SQSTM1: sequestosome 1; STX17: syntaxin 17; TBHP: tert-butyl hydroperoxide; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; WT: wild type.
| S-EPMC7469620 | biostudies-literature
Project description:The present investigation examined the spatiotemporal expression of estrogen receptors (ER-alpha and ER-beta) and progesterone receptor (PR) in the periimplantation mouse uterus (days 1-8). ER-alpha messenger RNA (mRNA) was detected at much higher levels in the periimplantation uterus compared with that of ER-beta mRNA, the levels of which were very low in all uterine cells during this period. Results of in situ hybridization demonstrated expression of ER-alpha mRNA primarily in the luminal and glandular epithelia on days 1 and 2 of pregnancy. On days 3 and 4, the accumulation was localized primarily in stromal cells in addition to its presence in the epithelium. Following implantation on day 5, the accumulation of this mRNA was more condensed in the luminal and glandular epithelia, but declined in the subluminal epithelial stroma at the sites of implanting embryos. On days 6-8, the accumulation of ER-alpha mRNA was primarily localized in the secondary decidual zone (SDZ) with more intense localization in the subepithelial cells at the mesometrial pole. In contrast, signals were very low to undetectable in the primary decidual zone (PDZ), and no signals were detected in implanting embryos. The undifferentiated stroma underneath the myometrium also showed positive signals. The immunolocalization of ER-alpha protein correlated with the mRNA localization. Western blot analysis showed down-regulation of ER-alpha in day 8 decidual cell extracts consistent with the down-regulation of ER-alpha mRNA in decidual cells immediately surrounding the embryo on this day. The expression pattern of PR was also dynamic in the periimplantation uterus. On day 1, the accumulation of PR mRNA was very low to undetectable, whereas only a modest level of accumulation in the epithelium was noted on day 2. On days 3 and 4, the accumulation of this mRNA was detected in both the epithelium and stroma. In contrast, the expression was restricted only to the stroma with increased signals at the sites of implantation on day 5. On days 6-8, PR mRNA accumulation increased dramatically throughout the deciduum. The localization of immunoreactive PR correlated with the mRNA distribution in the periimplantation uterus. Taken together, the results demonstrate that the expression of ER-alpha, ER-beta, and PR is differentially regulated in the periimplantation mouse uterus. This compartmentalized expression of ER and PR provides information regarding the sites of coordinated effects ofestrogen and progesterone in the preparation of the uterus for implantation and decidualization during early pregnancy.
| S-EPMC4280800 | biostudies-literature
Project description:Decidualization, the process by which fibroblastic human endometrial stromal cells (HESC) differentiate into secretory decidual cells, is a critical event during the establishment of pregnancy. It is dependent on the steroid hormone progesterone acting through the nuclear progesterone receptor (PR). Previously, we identified insulin receptor substrate 2 (IRS2) as a factor that is directly regulated by PR during decidualization. IRS2 is an adaptor protein that functionally links receptor tyrosine kinases, such as insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R), and their downstream effectors. IRS2 expression was induced in HESC during in vitro decidualization and siRNA-mediated downregulation of IRS2 transcripts resulted in attenuation of this process. Further use of siRNAs targeted to IR or IGF1R transcripts showed that downregulation of IR, but not IGF1R, led to impaired decidualization. Loss of IRS2 transcripts in HESC suppressed phosphorylation of both ERK1/2 and AKT, downstream effectors of insulin signaling, which mediate gene expression associated with decidualization and regulate glucose uptake. Indeed, downregulation of IRS2 resulted in reduced expression and membrane localization of the glucose transporters GLUT1 and GLUT4, resulting in lowered glucose uptake during stromal decidualization. Collectively, these data suggest that the PR-regulated expression of IRS2 is necessary for proper insulin signaling for controlling gene expression and glucose utilization, which critically support the decidualization process to facilitate pregnancy. This study provides new insight into the mechanisms by which steroid hormone signaling intersects with insulin signaling in the uterus during decidualization, which has important implications for pregnancy complications associated with insulin resistance and infertility.
| S-EPMC6986554 | biostudies-literature