KLF6 negatively regulates HIF-1α in extravillous trophoblasts under hypoxia.

INTRODUCTION: HIF-1α, the master regulator of hypoxia cellular response, is stabilized under low oxygen levels and degraded in the presence of oxygen but its transcription, translation, and degradation are tightly regulated by numerous pathways. KLF6 is a transcription factor involved in proliferation, differentiation, and apoptosis in several cell systems. Under hypoxia it is upregulated in a HIF-1α-dependent manner in extravillous trophoblasts. Considering the importance of hypoxia modulation of EVT behavior through HIF1-α we aimed to study whether KLF6 modulates HIF-1α expression in HTR8/SVneo cells. METHODS: HTR8/SVneo cells were cultured in a 1 % oxygen chamber or in 3D format where a spontaneous oxygen gradient is generated. qRT-PCR and Western blot were performed to analyze mRNA and protein expression, respectively. SiRNA, shRNA, or plasmids were used to down- or up-regulate gene expression. Wound healing assay was performed under hypoxia to evaluate migration. The NFκB pathway was modulated with dominant negative mutants and a chemical inhibitor. Cobalt chloride was used to block HIF-1α degradation. RESULTS: KLF6 up- and down-regulation in HTR8/SVneo cells exposed to acute hypoxia revealed a negative regulation on HIF-1α. KLF6 silencing led to a partially HIF-1α-dependent increase in MMP9 and VEGF. The NF-κB pathway and HIF-1α degradation were involved in KLF6-dependent HIF-1α regulation. HTR8/SVneo-3D culture showed that KLF6 negatively regulates HIF-1α in a microenvironment with naturally generated hypoxia. DISCUSSION: Present results reveal that KLF6 contributes to a fine tune modulation of HIF-1α level under hypoxia. Thus, sustaining a HIF-1α homeostatic level, KLF6 might contribute to control EVT adaptation to hypoxia.

Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling.

BACKGROUND: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems-the ubiquitin proteasome system (UPS) and macroautophagy-mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless, how Ate1 operates through this intricate mechanism remains elusive. METHODS: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofluorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform were used as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofluorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy. RESULTS: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specific inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic flux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical significance was assessed using a two-sided unpaired t test with a significance threshold set at P<0.05. CONCLUSIONS: Our findings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.

StarD7 deficiency switches on glycolysis and promotes mitophagy flux in C2C12 myoblasts.

StarD7 is a member of the START protein family required for phosphatidylcholine delivery to the mitochondria, thus key to maintain mitochondrial structure. Its deficiency has been associated with an impairment of cellular processes, such as proliferation and migration, and it has also been reported that it is needed in myogenic differentiation. Here, we show that StarD7 deficiency in C2C12 muscle cells results in the accumulation of abnormal mitochondria, a reduced number of mitochondria per cell area and increased glycolysis. In addition, StarD7-deficient cells undergo an increase in mitochondria-ER contact sites, reduced connexin 43 expression, and disturbances in lipid handling, evidenced by lipid droplet accumulation and decreased levels in phosphatidylserine synthase 1 and 2 expression. Interestingly, StarD7-deficient cells showed alterations in mitophagy markers. We observed accumulation of LC3B-II and BNIP3 proteins in mitochondria-enriched fractions and accumulation of autophagolysosomal and lysosomal vesicles in StarD7-deficient cells. Furthermore, live-cell imaging experiments of StarD7 knockdown cells expressing mitochondria-targeted mKeima indicated an enhanced mitochondria delivery into lysosomes. Importantly, StarD7 reconstitution in StarD7-deficient cells restores LC3B-II expression in mitochondria-enriched fractions at similar levels to those observed in control cells. Collectively, these findings suggest that StarD7-deficient C2C12 myoblasts are associated with altered cristae structure, disturbances in neutral lipid accumulation, glucose metabolism, and increased mitophagy flux. The alterations mentioned above allow for the maintenance of mitochondrial function.

StarD7 deficiency hinders cell motility through p-ERK1/2/Cx43 reduction.

StarD7 belongs to START protein family involved in lipid traffic, metabolism, and signaling events. Its precursor, StarD7.I which is important for mitochondrial homeostasis, is processed to the StarD7.II isoform that lacks the mitochondrial targeting sequence and is mainly released to the cytosol. StarD7 knockdown interferes with cell migration by an unknown mechanism. Here, we demonstrate that StarD7 silencing decreased connexin 43 (Cx43), integrin ß1, and p-ERK1/2 expression in the non-tumoral migratory HTR-8/SVneo cells. StarD7-deficient cells exhibited Golgi disruption and reduced competence to reorient the microtubule-organizing center. The migratory capacity of StarD7-silenced cells was reestablished when Cx43 level was resettled, while p-ERK1/2 expression remained low. Importantly, ectopic expression of the StarD7.II isoform not only restored cell migration but also ERK1/2, Cx43, and integrin ß1 expression. Thus, StarD7 is implicated in cell migration through an ERK1/2/Cx43 dependent mechanism but independent of the StarD7.I function in the mitochondria.

Ionic complexation improves wound healing in deep second-degree burns and reduces in-vitro ciprofloxacin cytotoxicity in fibroblasts.

The development of new treatments capable of controlling infections and pain related to burns continues to be a challenge. Antimicrobials are necessary tools, but these can be cytotoxic for regenerating cells. In this study, antibiotic-anesthetic (AA) smart systems obtained by ionic complexation of polyelectrolytes with ciprofloxacin and lidocaine were obtained as films and hydrogels. Ionic complexation with sodium alginate and hyaluronate decreased cytotoxicity of ciprofloxacin above 70% in a primary culture of isolated fibroblasts (p < 0.05). In addition, the relative levels of the proteins involved in cell migration, integrin ß1 and p-FAK, increased above 1.5 times (p < 0.05) with no significant differences in cell mobility. Evaluation of the systems in a deep second-degree burn model revealed that reepithelization rate was AA-films = AA-hydrogels > control films > no treated > reference cream (silver sulfadiazine cream). In addition, appendage conservation and complete dermis organization were achieved in AA-films and AA-hydrogels. Encouragingly, both the films and the hydrogels showed a significantly superior performance compared to the reference treatment. This work highlights the great potential of this smart system as an attractive dressing for burns, which surpasses currently available treatments.

The 19S proteasome subunit Rpt5 reversibly associates with cold-stable microtubules in glial cells at low temperatures.

The ubiquitin-proteasome system (UPS) degrades intracellular proteins through the 26S proteasome. We analysed how cold stress affects the UPS in glial cells. Together with a reduction in the 20S proteolytic activity and increased levels of polyubiquitinated proteins, exposure of glial cell cultures to cold induces a partial disassembly of the 26S proteasome. In particular, we found that Rpt5, a subunit of the 19S proteasome, relocates to cold-stable microtubules, although no apparent cytoskeletal redistribution was detected for other analysed subunits of the 19S or 20S complexes. Furthermore, we demonstrate that both the expression of the microtubule-associated protein MAP6 and the post-translational acetylation of α-tubulin modulate the association of Rpt5 with microtubules. This reversible association could be related to functional preservation of the proteolytic complex during cold stress.

Role of the lipid transport protein StarD7 in mitochondrial dynamics.

Mitochondria are dynamic organelles crucial for cell function and survival implicated in oxidative energy production whose central functions are tightly controlled by lipids. StarD7 is a lipid transport protein involved in the phosphatidylcholine (PC) delivery to mitochondria. Previous studies have shown that StarD7 knockdown induces alterations in mitochondria and endoplasmic reticulum (ER) with a reduction in PC content, however whether StarD7 modulates mitochondrial dynamics remains unexplored. Here, we generated HTR-8/SVneo stable cells expressing the precursor StarD7.I and the mature processed StarD7.II isoforms. We demonstrated that StarD7.I overexpression altered mitochondrial morphology increasing its fragmentation, whereas no changes were observed in StarD7.II-overexpressing cells compared to the control (Ct) stable cells. StarD7.I (D7.I) stable cells were able to transport higher fluorescent PC analog to mitochondria than Ct cells, yield mitochondrial fusions, maintained the membrane potential, and produced lower levels of reactive oxygen species (ROS). Additionally, the expression of Dynamin Related Protein 1 (Drp1) and Mitofusin (Mfn2) proteins were increased, whereas the amount of Mitofusin 1 (Mfn1) decreased. Moreover, transfections with plasmids encoding Drp1-K38A, Drp1-S637D or Drp1-S637A mutants indicated that mitochondrial fragmentation in D7.I cells occurs in a fission-dependent manner via Drp1. In contrast, StarD7 silencing decreased Mfn1 and Mfn2 fusion proteins without modification of Drp1 protein level. These cells increased ROS levels and presented donut-shape mitochondria, indicative of metabolic stress. Altogether our findings provide novel evidence indicating that alterations in StarD7.I expression produce significant changes in mitochondrial morphology and dynamics.

Effect of Chlorpyrifos on human extravillous-like trophoblast cells.

An increased risk of pregnancy disorders has been reported in women and animal models exposed to organophosphate pesticides. However, less information is available on impacts to human placental function. Here, we addressed the impact of chlorpyrifos (CPF) on extravillous cytotrophoblasts (evCTB) employing HTR8/SVneo cells as an in vitro model. Cell proliferation, migration and invasion were not affected by CPF under conditions where cell viability was not compromised; however, we observed reduced expression of genes for vascular endothelial growth factor receptor 1, hypoxia-inducible factor 1-alpha, peroxisome proliferator activated receptor gamma, and the ß-subunit of human chorionic gonadotropin. These results are the first effects reported by organophosphate pesticide in evCTB cells and show altered expression of several genes important for placental development that could serve as potential biomarkers for future research.

Hexosamine pathway regulates StarD7 expression in JEG-3 cells.

StarD7 is a lipid binding protein involved in the delivery of phosphatidylcholine to the mitochondria whose promoter is activated by Wnt/ß-catenin signaling. Although the majority of glucose enters glycolysis, ~ 2-5% of it can be metabolized via the hexosamine biosynthetic pathway (HBP). Considering that HBP has been implicated in the regulation of ß-catenin we explored if changes in glucose levels modulate StarD7 expression by the HBP in trophoblast cells. We found an increase in StarD7 as well as in ß-catenin expression following high-glucose (25 mM) treatment in JEG-3 cells; these effects were abolished in the presence of HBP inhibitors. Moreover, since HBP is able to promote unfolded protein response (UPR) the protein levels of GRP78, Ire1α, calnexin, p-eIF2α and total eIF2α as well as XBP1 mRNA was measured. Our results indicate that a diminution in glucose concentration leads to a decrease in StarD7 expression and an increase in the UPR markers: GRP78 and Ire1α. Conversely, an increase in glucose is associated to high StarD7 levels and low GRP78 expression, phospho-eIF2α and XBP1 splicing, although Ire1α remains high when cells are restored to high glucose. Taken together these findings indicate that glucose modulates StarD7 and ß-catenin expression through the HBP associated to UPR, suggesting the existence of a link between UPR and HBP in trophoblast cells. This is the first study reporting the effects of glucose on StarD7 in trophoblast cells. These data highlight the importance to explore the role of StarD7 in placenta disorders related to nutrient availability.

Chlorpyrifos induces endoplasmic reticulum stress in JEG-3 cells.

Chlorpyrifos (CPF) is an organophosphorous pesticide widely used in agricultural, industrial, and household applications. We have previously shown that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway. Considering that there is a relationship between oxidative stress and endoplasmic reticulum stress (ER), herein we investigated whether CPF also induces ER stress in JEG-3 cells. Cells were exposed to 50µM or 100µM CPF during 24h in conditions where cell viability was not altered. Western blot and PCR assays were used to explore the protein and mRNA levels of ER stress biomarkers, respectively. CPF induced an increase of the typical ER stress-related proteins, such as GRP78/BiP and IRE1α, a sensor for the unfolded protein response, as well as in phospho-eIF2α and XBP1 mRNA splicing. Additionally, CPF led to a decrease in p53 protein expression. The downregulation of p53 levels induced by CPF was partially blocked when cells were exposed to CPF in the presence of the proteasome inhibitor MG132. Altogether, these findings point out that CPF induces ER stress in JEG-3 cells; however these cells are able to attenuate it downregulating the levels of the pro-apoptotic protein p53.

Suppression of StarD7 promotes endoplasmic reticulum stress and induces ROS production.

StarD7 is an intracellular lipid transport protein identified as up-regulated in the choriocarcinoma JEG-3 cell line. StarD7 facilitates the delivery of phosphatidylcholine (PC) to the mitochondria, and StarD7 knockdown causes a reduction in phospholipid synthesis. Since inhibition of PC synthesis may lead to endoplasmic reticulum (ER) stress we hypothesized that StarD7 may be involved in maintaining cell homeostasis. Here, we examined the effect of StarD7 silencing on ER stress response and on the levels of reactive oxygen species (ROS) in the human hepatoma cell line HepG2. StarD7 knockdown induced alterations in mitochondria and ER morphology. These changes were accompanied with an ER stress response as determined by increased expression of inositol-requiring enzyme 1α (IRE1α), calnexin, glucose regulated protein 78/immunoglobulin heavy chain-binding protein (Grp78/BiP), protein kinase-like ER kinase (PERK) as well as the phosphorylated eukaryotic translation initiation factor 2, subunit 1α (p-eIF2α). Additionally, a downregulation of the tumor suppressor p53 by a degradation mechanism was observed in StarD7 siRNA cells. Furthermore, StarD7 silencing induced ROS generation and reduced cell viability after H2O2 exposure. Decreased expression of StarD7 was associated to increased levels of the heme oxygenase-1 (HO-1) and catalase enzymes as well as in catalase enzymatic activity. Finally, no changes in levels of autophagy and apoptosis markers were observed in StarD7 siRNA treated cells respect to control cells. Taken together, these results indicate that StarD7 contributes to modulate cellular redox homeostasis.

The Lipid Transfer Protein StarD7: Structure, Function, and Regulation.

The steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain proteins constitute a family of evolutionarily conserved and widely expressed proteins that have been implicated in lipid transport, metabolism, and signaling. The 15 well-characterized mammalian START domain-containing proteins are grouped into six subfamilies. The START domain containing 7 mRNA encodes StarD7, a member of the StarD2/phosphatidylcholine transfer protein (PCTP) subfamily, which was first identified as a gene overexpressed in a choriocarcinoma cell line. Recent studies show that the StarD7 protein facilitates the delivery of phosphatidylcholine to the mitochondria. This review summarizes the latest advances in StarD7 research, focusing on the structural and biochemical features, protein-lipid interactions, and mechanisms that regulate StarD7 expression. The implications of the role of StarD7 in cell proliferation, migration, and differentiation are also discussed.

StarD7 knockdown modulates ABCG2 expression, cell migration, proliferation, and differentiation of human choriocarcinoma JEG-3 cells.

BACKGROUND: StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (-26.4% and -41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and (3)H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin ß-subunit (ßhCG) protein synthesis and secretion as well as ßhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7. CONCLUSIONS/SIGNIFICANCE: Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.

Chlorpyrifos modifies the expression of genes involved in human placental function.

The effects of organophosphate pesticides on human placenta remain poorly investigated although an increased risk of pregnancy alterations has been reported in women chronically exposed to these pesticides. Here, we have addressed whether chlorpyrifos (CPF) modifies the expression of genes relevant for placental function. Human placental JEG-3 cells were exposed to increasing CPF concentrations up to 100 µM for 24 and 48 h and cell viability, mRNA, protein and hormone levels were analyzed. Quantitative RT-PCR assays revealed that CPF increased the expression of ABCG2, GCM1 and, even more significantly, ßhCG mRNAs in conditions where cell viability and morphology were not compromised. In addition, ßhCG protein synthesis and secretion were time-dependently augmented. Present results may reflect a CPF nocive effect on placenta cells or a placental-defense mechanism to preserve its function. These novel CPF trophoblast target genes should be considered in future studies of pregnancy outcomes associated with in vivo exposures.

StarD7 gene expression in trophoblast cells: contribution of SF-1 and Wnt-beta-catenin signaling.

Steroidogenic acute regulatory protein-related lipid transfer domain containing 7 (StarD7) is a poorly characterized member of the steroidogenic acute regulatory protein-related lipid transfer proteins, up-regulated in JEG-3 cells, involved in intracellular transport and metabolism of lipids. Previous studies dealing with the mechanisms underlying the human StarD7 gene expression led us to define the cis-acting regulatory sequences in the StarD7 promoter using as a model JEG-3 cells. These include a functional T cell-specific transcription factor 4 (TCF4) site involved in Wnt-ß-catenin signaling. To understand these mechanisms in more depth, we examined the steroidogenic factor 1 (SF-1) contribution to StarD7 expression. Cotransfection experiments in JEG-3 cells point out that the StarD7 promoter is activated by SF-1, and this effect is increased by forskolin. EMSA using JEG-3 nuclear proteins demonstrated that SF-1 binds to the StarD7 promoter. Additionally, chromatin immunoprecipitation analysis indicated that SF-1 and ß-catenin are bound in vivo to the StarD7 promoter. Reporter gene assays in combination with mutations in the SF-1 and TCF4 binding sites revealed that the StarD7 promoter is synergistically activated by SF-1 and ß-catenin and that the TCF4 binding site (-614/-608) plays an important role in this activation. SF-1 amino acid mutations involved in the physical interaction with ß-catenin abolished this activation; thus demonstrating that the contact between the two proteins is necessary for an efficient StarD7 transcriptional induction. Finally, these data suggest that ß-catenin could function as a bridge between SF-1 and TCF4 forming a ternary complex, which would stimulate StarD7 expression. The SF-1 and ß-catenin pathway convergence on StarD7 expression may have important implications in the phospholipid uptake and transport, contributing to the normal trophoblast development.