How autophagy eats large mitochondria: Autophagosome formation coupled with mitochondrial fragmentation

Mitochondrial autophagy (mitophagy) is thought to be a multi-step pathway wherein mitochondria are first divided into small fragments, which are subsequently recognized by the phagophore. DNM1L (dynamin 1 like) plays a pivotal role in mitochondrial division; however, its role in mitophagy remains controversial. In our recent study, we examined the contribution of DNM1L to mitophagy and showed that mitophagy and mitochondrial division occur even in DNM1L-defective cells. Furthermore, time-lapse imaging of mitophagy showed that DNM1L-independent mitochondrial division occurs concomitantly with autophagosome formation. Upstream factors of autophagosome formation, i.e., RB1CC1/FIP200, ATG14, and WIPIs, are required for mitochondrial division, whereas ATG5 and ATG3 are dispensable. These results indicate that a portion of the tubular mitochondria is first recognized and then divided into small fragments by a phagophore-mediated event, independently of DNM1L. This autophagic process suggests that autophagy has the potential to degrade substrates larger than autophagosomes.     

Bone morphogenetic protein 2 functions via a conserved signaling pathway involving Wnt4 to regulate uterine decidualization in the mouse and the human

A critical role of progesterone (P) during early pregnancy is to induce differentiation of the endometrial stromal cells into specialized decidual cells that support the development of the implanting embryo. The P-induced signaling pathways that participate in the formation and function of the decidual cells remain poorly understood. We report here that the expression of the bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFbeta superfamily, is induced downstream of P action in the mouse uterine stroma during decidualization. To determine the function of BMP2 during this differentiation process, we employed a primary culture system in which undifferentiated stromal cells isolated from pregnant mouse uterus undergo decidualization. When recombinant BMP2 was added to these stromal cultures, it markedly advanced the differentiation program. We also found that siRNA-mediated silencing of BMP2 expression in these cells efficiently blocked the differentiation process. Gene expression profiling experiments identified Wnt4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization. Attenuation of Wnt4 expression by siRNAs greatly reduced stromal differentiation in vitro, indicating that it is a key mediator of BMP2-induced decidualization. We also observed a remarkable induction in the expression of BMP2 in human endometrial stromal cells during decidualization in vitro in response to steroids and cAMP. Addition of BMP2 to these cultures led to a robust enhancement of Wnt4 expression and stimulated the differentiation process. Collectively, our studies uncovered a unique conserved pathway involving BMP2 and Wnt4 that mediates P-induced stromal decidualization in the mouse and the human.     

Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.     

Epidermal growth factor inhibits 8-Br-cAMP-induced decidualization of human endometrial stromal cells

The effects of epidermal growth factor (EGF) on human endometrial stromal cells have not been characterized well, although production of EGF in endometrial epithelial and stromal cells and expression of EGF receptors in endometrial stromal cells have been reported. We investigated the effects of EGF on endometrial cell viability, 8-Br-cAMP-induced stromal decidualization, and prolactin secretion from decidualized endometrial stromal cells using an in vitro decidualization activity assay of human endometrial stromal cells. EGF did not show any significant effects on viable cell numbers of nondecidualized and 8-Br-cAMP-induced decidualized cells. Prolactin release from the 8-Br-cAMP-induced decidualized cells was not affected by EGF. However, EGF dose-dependently inhibited prolactin release from the stromal cells that were in the process of decidualization by co-stimulation with 8-Br-cAMP and EGF, though there was no significant change in viable cell numbers of the 8-Br-cAMP-stimulated decidualizing cells. Flow cytometric analysis revealed that 8-Br-cAMP enhanced EGF receptor expression on the endometrial stromal cells. These results indicate that endometrial EGF inhibits decidualization through autocrine/paracrine mechanisms.     

A critical role of superoxide anion in selenite-induced mitophagic cell death

Mitochondria, which are a major source of intracellular reactive oxygen species (ROS), are extremely vulnerable to oxidative stress. We recently reported that selenite treatment of various glioma cells induced a non-apoptotic cell death accompanied by excessive mitophagy (selective autophagy of damaged mitochondria). Examination of various ROS revealed that the superoxide anion played a key role in selenite-induced mitochondrial damage, mitophagy and cell death. Treatment with superoxide generators (diquat and paraquat) was sufficient to trigger mitophagy in glioma cells. Small interfering RNA-mediated knockdown of ATG6 or ATG7 attenuated selenite-induced mitophagy and cell death, demonstrating that the mitophagic pathway contributes to selenite-induced cell death. The effect of selenite in glioma cells may thus provide an example of superoxide-mediated mitophagic cell death, i.e., cell death caused by excessive mitophagy.     

A critical role of superoxide anion in selenite-induced mitophagic cell death

Mitochondria, which are a major source of intracellular reactive oxygen species (ROS), are extremely vulnerable to oxidative stress. We recently reported that selenite treatment of various glioma cells induced a non-apoptotic cell death accompanied by excessive mitophagy (selective autophagy of damaged mitochondria). Examination of various ROS revealed that the superoxide anion played a key role in selenite-induced mitochondrial damage, mitophagy and cell death. Treatment with superoxide generators (diquat and paraquat) was sufficient to trigger mitophagy in glioma cells. Small interfering RNA-mediated knockdown of ATG6 or ATG7 attenuated selenite-induced mitophagy and cell death, demonstrating that the mitophagic pathway contributes to selenite-induced cell death. The effect of selenite in glioma cells may thus provide an example of superoxide-mediated mitophagic cell death, i.e., cell death caused by excessive mitophagy.

Addendum to: Kim EH, Sohn S, Kwon HJ, Kim SU, Kim MJ, Lee SJ, Choi KS. Sodium selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death in malignant glioma cells. Cancer Res 2007; 67:6314-24     

Autophagy proteins LC3B, ATG5 and ATG12 participate in quality control after mitochondrial damage and influence lifespan

Mitochondrial health is maintained by the quality control mechanisms of mitochondrial dynamics (fission and fusion) and mitophagy. Decline of these processes is thought to contribute to aging and neurodegenerative diseases. To investigate the role of mitochondrial quality control in aging on the cellular level, human umbilical vein endothelial cells (HUVEC) were subjected to mitochondria-targeted damage by combining staining of mitochondria and irradiation. This treatment induced a short boost of reactive oxygen species, which resulted in transient fragmentation of mitochondria followed by mitophagy, while mitochondrial dynamics were impaired. Furthermore, targeted mitochondrial damage upregulated autophagy factors LC3B, ATG5 and ATG12. Consequently these proteins were overexpressed in HUVEC as an in vitro aging model, which significantly enhanced the replicative life span up to 150% and the number of population doublings up to 200%, whereas overexpression of LAMP-1 did not alter the life span. Overexpression of LC3B, ATG5 and ATG12 resulted in an improved mitochondrial membrane potential, enhanced ATP production and generated anti-apoptotic effects, while ROS levels remained unchanged and the amount of oxidized proteins increased. Taken together, these data relate LC3B, ATG5 and ATG12 to mitochondrial quality control after oxidative damage, and to cellular longevity.     

PTEN-induced kinase 1 (PINK1) and Parkin: Unlocking a mitochondrial quality control pathway linked to Parkinson's disease

Dissection of the function of two Parkinson's disease-linked genes encoding the protein kinase, PTEN-induced kinase 1 (PINK1) and ubiquitin E3 ligase, Parkin, has illuminated a highly conserved mitochondrial quality control pathway found in nearly every cell type including neurons. Mitochondrial damage-induced activation of PINK1 stimulates phosphorylation-dependent activation of Parkin and ubiquitin-dependent elimination of mitochondria by autophagy (mitophagy). Structural, cell biological and neuronal studies are unravelling the key steps of PINK1/Parkin-dependent mitophagy and uncovering new insights into how the pathway is regulated. The emerging role for aberrant immune activation as a driver of dopaminergic neuron degeneration after loss of PINK1 and Parkin poses new exciting questions on cell-autonomous and noncell-autonomous mechanisms of PINK1/Parkin signalling in vivo.     

Yap is essential for uterine decidualization through Rrm2/GSH/ROS pathway in response to Bmp2

Yap is required for ovarian follicle and early embryo development, but little information is available regarding its physiological significance in decidualization. Here we determine the effects of YAP on decidualization, mitochondrial function, cell apoptosis and DNA damage, and explore its interplay with Bmp2, Rrm2, GSH and ROS. The results exhibited that Yap was abundant in decidual cells and its inactivation impaired the proliferation and differentiation of stromal cells along with the deferral of G1/S phase transition, indicating Yap importance in decidualization. Bmp2 via Alk2 receptor promoted nuclear translocation of Yap where it might interact with Tead and then bind to the promoter of Rrm2 whose activation rescued the faultiness of differentiation program and attenuated oxidative DNA damage caused by Yap impediment. Meanwhile, Yap had an important part in the crosstalk between Bmp2 and Rrm2. Furthermore, inactivation of Yap resulted in an obvious accumulation of intracellular ROS followed by the abnormal GR activity and GSH content dependent on Rrm2. Replenishment of GSH counteracted the regulation of Yap inactivation on stromal differentiation and DNA damage with distinct reduction for intracellular ROS. Additionally, blockage of Yap caused the enhancement of stromal cell apoptosis and brought about mitochondrial dysfunction as indicated by the aberration for ATP level, mtDNA copy number and mitochondrial membrane potential concomitant with the opening of mitochondrial permeability transition pore, but these abnormalities were neutralized by GSH. Administration of mitochondrial antioxidant Mito-TEMPO rescued the fault of stromal differentiation conferred by Yap inactivation. Collectively, Yap was essential for uterine decidualization through Rrm2/GSH/ROS pathway in response to Bmp2.     

Anandamide targets aromatase: A breakthrough on human decidualization

In each menstrual cycle endometrial stromal cells (hESC) proliferate and differentiate into specialized decidual cells, a process termed decidualization, which regulates endometrial receptivity. Decidualization is mainly controlled by sex ovarian hormones, estradiol (E₂) and progesterone. E₂ plays an important role in the expression of the progesterone receptor and promotes the endometrial stromal cells differentiation. Our group previously reported that anandamide (AEA) impairs decidualization through cannabinoid receptor 1 (CB1). In this study, we hypothesized whether AEA inhibitory effect on cell decidualization could be mediated through interaction with aromatase and consequent interference in estradiol production/signaling. We used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) derived from human term placenta. In cells exposed to a differentiation stimulus, AEA-treatment prevents the increase of the expression of CYP19A1 gene encoding aromatase, E₂ levels and of estradiol receptor expression, that are observed in differentiating cells. Regarding CYP19A1 mRNA levels, the effect was partially reverted by a CB1 receptor antagonist and by a COX2 inhibitor. In addition, we report that AEA presents anti-aromatase activity in placental microsomes, the nature of the inhibition being the uncommon mixed type as revealed by the kinetic studies. Structural analysis of the AEA-Aromatase complexes determined that AEA may bind to the active site pocket of the enzyme. In overall we report that AEA inhibits aromatase activity and may affect E₂ signaling crucial for the decidualization process, indicating that a deregulation of the endocannabinoid system may be implicated in endometrial dysfunction and in fertility/infertility disorders.     

Direct effect of gonadotropins on decidualization of human endometrial stromal cells

Decidualization of stromal cells isolated from proliferative human endometrium was achieved by adding to the culture medium human gonadotropins (FSH, FSH + LH, hCG). In addition to changes in the morphology of the stromal cells to the decidual phenotype, decidualization was evident from the expression of prolactin (PRL), demonstrated immunocytochemically, by Western blotting analysis, and by measuring its output into the medium through solid phase enzyme immunoassay. Gonadotropins also induced cAMP formation in the endometrial stromal cells under the same experimental conditions. This finding suggests that the mechanism by which gonadotropins promote decidualization of human endometrial stromal cells in vitro involves the introduction of cAMP, a compound that we have found to elicit the expression of PRL in this system. PRL is likely to be a key intermediate in the process of decidualization since it is by itself capable of inducing differentiation of the endometrial stromal cells to the decidual phenotype. Awareness of direct actions of gonadotropins on the endometrial cells and, in particular, of the decidualizing effects of FSH (Metrodin), FSH + LH (Pergonal) and hCG may contribute to the understanding of physiologic as well as pathophysiologic conditions relevant to endometrial functions and fertility.     

Interaction between insulin and androgen signalling in decidualization,cell migration and trophoblast invasion in vitro

Finely tuned decidualization of endometrial stromal fibroblasts into decidual cells is crucial for successful implantation and a healthy pregnancy. Both insulin and androgens are known to modulate decidualization, however, their complex effect on this process has not been fully elucidated. As hyperinsulinemia and hyperandrogenism are associated in clinical conditions, we aimed to investigate the interaction between insulin and androgens on decidualization. Primary human endometrial stromal cells were decidualized in vitro in the presence of insulin and/or androgens (dihydrotestosterone (DHT), testosterone). Gene or protein expressions of decidualization markers were measured, and cells size characteristics were determined. Migration of decidualizing endometrial stromal cells and invasion of HTR-8/SVneo trophoblast spheroids were assessed. We found that insulin and androgens in combination enhanced the upregulation of several decidualization markers including prolactin, tissue factor, tissue inhibitor of matrix metalloproteinase 3 and connexin-43, and also interacted in modulating cell size characteristics resulting in enlarged decidualizing cells. However, insulin and DHT together restricted the migration of decidualizing cells and invasion of trophoblast spheroids. Our findings suggest that insulin and androgens interact to potentiate the process of decidualization. On the other hand, inhibited cell migration and trophoblast invasion might negatively impact the function of decidualizing endometrial stromal cells.     

Ubiquitin-specific protease 7 (USP7) is essential for endometrial stromal cell decidualization in mice

Ubiquitin-specific protease 7 (USP7), a member of the deubiquitinating (DUB) enzyme family, regulates protein stability and has a well-characterized function in tumorigenesis. Given its critical role in growth and development, it was speculated to be involved in modulating processes in the female reproductive system but its exact role has not been elucidated. Decidualization is one of the key processes in pregnancy and aberrant decidualization is a cause of pregnancy failure. The uterine endometrium layer undergoes significant structural and functional changes during decidualization in preparation for and after embryo implantation. Here, we hypothesized that USP7 could be involved in mediating endometrial stromal cell (ESC) decidualization and set out to determine its function with a primary stromal cell culture. Using in situ hybridization and immunohistochemical techniques, we observed increased USP7 expression during uterine decidualization and found that it was predominantly localized to the decidual zone in the post-implantation uterus. Since the ovarian hormones, progesterone (P4) and estrogen (E2), function in promoting stroma decidualization, we investigated their relationship with USP7 expression and found that they exert minimal influence. Moreover, increased USP7 expression observed during deciduoma development was found to be independent of blastocyst attachment. Using a specific USP7 inhibitor, HBX19818, we demonstrated an additional novel role for USP7 in endometrial stroma decidualization in mice during early pregnancy. Our findings could potentially be applied towards future research and development in female infertility.     

Matrine promotes liver cancer cell apoptosis by inhibiting mitophagy and PINK1/Parkin pathways

Matrine is a natural alkaloid isolated from the root and stem of the legume plant Sophora. Its anti-proliferative and pro-apoptotic effects on several types of cancer have been well-documented. However, the role of matrine in regulating mitochondrial homeostasis, particularly mitophagy in liver cancer apoptosis, remains uncertain. The aim of our study was to explore whether matrine promotes liver cancer cell apoptosis by modifying mitophagy. HepG2 cells were used in the study and treated with different doses of matrine. Cell viability and apoptosis were determined by MTT assay, TUNEL staining, western blotting, and LDH release assay. Mitophagy was monitored by immunofluorescence assay and western blotting. Mitochondrial function was assessed by immunofluorescence assay, ELISA, and western blotting. The results of our study indicated that matrine treatment dose-dependently reduced cell viability and increased the apoptotic rate of HepG2 cells. Functional studies demonstrated that matrine treatment induced mitochondrial dysfunction and activated mitochondrial apoptosis by inhibiting protective mitophagy. Re-activation of mitophagy abolished the pro-apoptotic effects of matrine on HepG2 cells. Molecular investigations further confirmed that matrine regulated mitophagy via the PINK1/Parkin pathways. Matrine blocked the PINK1/Parkin pathways and repressed mitophagy, whereas activation of the PINK1/Parkin pathways increased mitophagy activity and promoted HepG2 cell survival in the presence of matrine. Together, our data indicated that matrine promoted HepG2 cell apoptosis through a novel mechanism that acted via inhibiting mitophagy and the PINK1/Parkin pathways. This finding provides new insight into the molecular mechanism of matrine for treating liver cancer and offers a potential target to repress liver cancer progression by modulating mitophagy and the PINK1/Parkin pathways.     

Human endometrial stromal interleukin-1 beta: autocrine secretion and inhibition by interleukin-1 receptor antagonist

Decidualization of human endometrial stromal cells is suppressed by endometrial IL-1 in an autocrine or paracrine manner, indicating that constant suppression of stromal decidualization by IL-1 requires a neutralizing mechanism for IL-1 action to accept embryo implantation. Since production of IL-1ra in human endometrium is reported to be 10- to 30-fold higher than that of IL-1 alpha/beta, we investigated whether endogenous IL-1 beta secreted from human endometrial stromal cells can be inhibited by IL-1ra by using an in vitro decidualization culture. Human stromal cells were cultured with 8-Br-cAMP to induce decidualization, and concentrations of IL-1 beta, IL-8, and prolactin in the culture supernatants were assayed before and after decidualization. There was no significant difference in mean IL-1 beta concentrations measured before and after decidualization. Addition of IL-1ra to endometrial stromal cell cultures strongly inhibited endogenous IL-8 secretion from the cells. Although IL-1 beta showed a biphasic effect on cell proliferation and a suppressive effect on decidualization of stromal cells, these effects were completely inhibited by IL-1ra. The results imply that a high in vivo concentration of IL-1ra in human endometrial tissues may regulate IL-1 effects on decidualization and cell proliferation of human endometrial stromal cells.     

Selective degradation of mitochondria by mitophagy

Mitochondria are the essential site of aerobic energy production in eukaryotic cells. Reactive oxygen species (ROS) are an inevitable by-product of mitochondrial metabolism and can cause mitochondrial DNA mutations and dysfunction. Mitochondrial damage can also be the consequence of disease processes. Therefore, maintaining a healthy population of mitochondria is essential to the well-being of cells. Autophagic delivery to lysosomes is the major degradative pathway in mitochondrial turnover, and we use the term mitophagy to refer to mitochondrial degradation by autophagy. Although long assumed to be a random process, increasing evidence indicates that mitophagy is a selective process. This review provides an overview of the process of mitophagy, the possible role of the mitochondrial permeability transition in mitophagy and the importance of mitophagy in turnover of dysfunctional mitochondria.     

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE₂ stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley-Liss, Inc.