Subunit Gene Transcription Research Articles

EZH2 Gene Knockdown Inhibits Sheep Pituitary Cell Proliferation via Downregulating the AKT/ERK Signaling Pathway.

Pituitary gonadotropins perform essential functions in mammalian reproduction by stimulating gametogenesis and steroidogenesis in the ovaries and testicles. EZH2 is a histone methyltransferase that inhibits proliferation and aggravates apoptosis in stem cells subjected to pathological stimuli. However, the expression and molecular mechanisms of EZH2 in pituitary cells in vitro have not been extensively studied. In this study, the relative abundances of EZH2 mRNA (p < 0.01) and protein (p < 0.05) expression were larger in the pituitary cells of Hu sheep with relatively greater fecundity (GF) compared to those with lesser fecundity (LF). Loss-of-function examinations demonstrated that EZH2 gene knockdown led to an earlier induction of apoptosis in sheep pituitary cells (PCs). The relative abundance of CASP3, CASP9, and BAX was increased (p < 0.01), while BCL2's abundance was less decreased (p < 0.01) in PCs where there was EZH2 gene knockdown. Additionally, cell proliferation (p < 0.01) and viability (p < 0.01) were decreased in EZH2-knockdown sheep PCs, and the cell cycle was blocked compared to a negative control (NC). Notably, EZH2 gene knockdown led to reduced abundances of gonadotropin subunit gene transcripts (FSHβ, p < 0.05) and reduced FSH release (p < 0.01) from PCs. EZH2 gene knockdown led to reduced phosphorylation of AKT, ERK, and mTOR (p < 0.01). The results suggest that EZH2 regulates pituitary cell proliferation, apoptosis, and FSH secretion through modulation of the AKT/ERK signaling pathway, providing a foundation for further study of pituitary cell functions.

Activating Transcription Factor 3 Stimulates Follicle-Stimulating Hormone-β Expression In Vitro But Is Dispensable for Follicle-Stimulating Hormone Production in Murine Gonadotropes In Vivo.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHβ subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone β (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LβT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to increase endogenous Fshb expression in these cells. Nevertheless, FSH production was intact in gonadotrope-specific Atf3 knockout [conditional knockout (cKO)] mice. Ovarian follicle development, ovulation, and litter sizes were equivalent between cKOs and controls. Testis weights and sperm counts did not differ between genotypes. Following gonadectomy, increases in LH secretion were enhanced in cKO animals. Though FSH levels did not differ between genotypes, post-gonadectomy increases in pituitary Fshb and gonadotropin α subunit expression were more pronounced in cKO than control mice. These data indicate that ATF3 can selectively stimulate Fshb expression in vitro but is not required for FSH production in vivo.

P-092: Multiple Myeloma cells depend on the DDI2/NRF1-mediated proteasome stress response for survival

<h3>Background</h3> Multiple myeloma (MM) cells depend on the ubiquitin-proteasome system for survival. Proteasome inhibitors (PIs) are FDA-approved in MM and have radically improved patient survival. However, acquisition of resistance is inevitable. The proteasome stress response (PSR) contributes to proteostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency, it is deglycosylated by NGLY1, cleaved by the aspartic protease DDI2, and translocates to the nucleus, to induce proteasome subunit gene transcription. Pharmacological targeting of NGLY1 was recently shown to sensitize cells to PIs. <h3>Hypothesis</h3> We hypothesize that blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM and represent an innovative therapeutic strategy to overcome PI resistance. <h3>Methods</h3> We used MM cell lines with distinct baseline sensitivity to PIs, including AMO1-VR, an isogenic AMO1 cell line adapted to grow in continuous bortezomib. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2/NRF1. Cell viability or tumor growth of DDI2/NRF1 KO versus non-targeting gRNA-edited cells were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate. Proteasome subunit PSMA7, PSMB5, PSMB6 and PSMD11 transcription was evaluated via real time PCR. <h3>Results</h3> Full length and cleaved NRF1 is detectable in MM cell lines and positively correlates with polyubiquitinated proteins, suggesting constitutively active PSR in MM. DDI2/NRF1 KO is cytotoxic alone or in combination with PI carfilzomib in MM cells with distinct PI sensitivity, including de-novo PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NSG mice and results in prolongation of animal survival. DDI2 KO in AMO1-VR sensitizes to carfilzomib. Further, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome subunit transcription and CT-L proteasome activity in baseline conditions and following carfilzomib treatment, resulting in increased sensitivity to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. <h3>Conclusions</h3> MM cells exhibit baseline activation of NRF1 and are dependent upon DDI2 for survival. DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome subunit biogenesis and recovery of CT-L proteasome activity, thereby increasing sensitivity to PI. Add-back of wild-type, but not of catalytically-dead DDI2 fully rescues these phenotypes. Our study provides the preclinical rationale for development of novel therapeutics targeting DDI2/NRF1 in MM.

Ac106/107 affects production of infectious progeny BV by regulating transcription of late viral genes and host cell energy metabolism.

AcMNPV is a model organism of baculovirus, and Spodoptera frugiperda is one of its hosts. Disclosing the role of ac106/107 in AcMNPV infecting Spodoptera frugiperda 9 (Sf9) cells is of great significance for modifying AcMNPV as a microbial insecticide. This work constructed recombinant baculovirus that knocking out, repairment and overexpression of ac106/107 and explored the effects of Ac106/107 on the proliferation of progeny viruses. Moreover, the potential mechanism and targets of ac106/107 were further revealed. First, compared with the Bacmid-EGFP transfection group, the progeny virus does not proliferate after knocking out of ac106/107, and the proliferation ability increases by 14.5% at 72 h post transfection (h p.t.) when overexpression of ac106/107. However, knockout, repairment and overexpression of ac106/107 have no effect on viral DNA replication. Secondly, Ac106/107-EGFP was located in the cytoplasm and nucleus. Transcription level of late viral genes and viral RNA polymerase subunit genes in the Bacmidac106/107KO -EGFP transfection group and Bacmid-Ac106/107-EGFP transfection group was reduced and increased, respectively. Thirdly, AcMNPV would increase the glucose utilization and lactate consumption of the host Sf9 cells, and Bacmidac106/107KO -EGFP transfection group had lower glucose consumption and lactic acid accumulation than Bacmid-EGFP, Bacmidac106/107KO -Ac106/107(rep)-EGFP and Bacmid-Ac106/107-EGFP transfection groups. Ac106/107 can enter the nucleus and affect transcription of viral RNA polymerase subunit genes, which in turn affects the transcription of late genes, and ultimately affects virus proliferation and energy metabolism in host cells. © 2021 Society of Chemical Industry.

Abstract LB110: Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival

Abstract Introduction: We previously showed that an imbalance between polyubiquitinated proteins awaiting proteasomal degradation and proteasome activity is a main determinant of proteasome inhibitor (PI) sensitivity in MM. While PIs have radically improved survival of MM patients, resistance is inevitable over time and understanding the molecular mechanisms underlying it is critically important to develop novel therapies to improve patient outcomes. The proteasome stress response (PSR) is an evolutionarily conserved homeostatic mechanism triggered by insufficient proteasome activity and aimed at restoring homeostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency is cleaved by the aspartic protease DDI2, translocates to the nucleus, and induces proteasome subunit gene transcription. It is unknown whether blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM. Materials and methods: We used human MM cell lines with distinct baseline sensitivity to PIs. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2 and NRF1. Cell viability or tumor growth of DDI2/NRF1 KO cells versus cells edited with a non-targeting gRNA were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate upon incubation with protein lysates from DDI2 WT, KO, or addback cells. Results: We detected full-length and cleaved NRF1 in human MM cell lines, but not other hematologic cancer cell lines, suggesting constitutively active PSR in MM. Query of the dependency map (depmap.org) shows that MM is the cell line type most highly dependent on DDI2. Consistently, DDI2 or NRF1 KO is cytotoxic alone or in combination with PI across a panel of MM cells with distinct PI sensitivity, including de novo, PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NOD SCID mice and results in prolongation of animal survival. While biallelic DDI2 or NRF1 KO monoclones could not be generated across 7 distinct MM cell lines, consistent with these genes being essential, we successfully established DDI2 KO monoclones in AMO1-VR, an isogenic AMO1 cell line variant adapted to grow in continuous bortezomib. Even in this cell line model of acquired PI resistance, DDI2 KO sensitized cells to PI carfilzomib. In this model, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome recovery after irreversible PI treatment and sensitizing to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. Conclusions: Our data show that MM cells are intrinsically dependent on an intact DDI2/NRF1 axis to survive in baseline conditions and upon proteasomal inhibition. We propose that DDI2 is an unexplored, promising molecular target for MM treatment by disrupting the PSR and exacerbating proteotoxicity. This study provides the preclinical rationale for the development of novel therapeutics targeting the DDI2/NRF1 pathway to overcome acquired and de novo PI resistance and improve patient outcomes in MM. Citation Format: Tianzeng Chen, Matthew Ho, Jenna Briere, Maria Moscvin, Peter G. Czarnecki, Kenneth C. Anderson, T. Keith Blackwell, Giada Bianchi. Multiple myeloma cells depend on the NRF1-DDI2 proteasome stress response pathway for survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB110.

Insulin‑receptor substrate 1 protects against injury inendothelial cell models of ox‑LDL‑induced atherosclerosis by inhibiting ER stress/oxidative stress‑mediated apoptosis and activating the Akt/FoxO1 signaling pathway.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell (EC) injury is a risk factor for atherosclerosis. Therefore, the present study aimed to investigate the effects of insulin-receptor substrate 1 (IRS-1) on injury to ox-LDL-exposed ECs. For this purpose, thoracic aorta tissues were isolated from rats and cultured to obtain ECs, which were then identified using immunohistochemical staining. IRS-1 overexpression plasmid (pcDNA3.1-IRS-1) and IRS-1-small interfering RNA were synthesized and transfected into ECs pre-exposed to ox-LDL. MTT and TUNEL assays were performed to evaluate the cell proliferative activity and apoptosis. Intracellular reactive oxygen species (ROS) production was determined by a flow cytometry assay. Reverse transcription-quantitative PCR was conducted to measure the peroxisome proliferator-activated receptor gamma co-activator 1 alpha (Ppargcla), phosphoenol-pyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) gene transcription levels. Western blot analysis was then performed to determine the IRS-1, forkhead box O1 (FoxO1), phosphorylated (p-)FoxO1, 78-kDa glucose-regulated protein (GRP78), p-eukaryotic translation initiation factor 2A (eIF2α), CHOP, Akt and p-Akt expression levels. Immunofluorescence staining was used to evaluate p-FoxO1 nuclear localization. The results indicated that IRS-1 significantly enhanced the proliferative activity, whereas it inhibited the apoptosis of ECs in a model of ox-LDL-induced atherosclerosis compared with ECs without IRS-1 treatment (P<0.05). IRS-1 significantly decreased the p-FoxO1/FoxO1 ratio compared with ECs without ox-LDL treatment (P<0.05). IRS-1 significantly downregulated the expression of ER stress biomarkers, including GRP78, CHOP and the p-eIF2α/eIF2α ratio in ox-LDL-exposed ECs compared with ECs without ISR-1 treatment (P<0.05). IRS-1 significantly reduced the intracellular ROS levels in the EC models of ox-LDL-induced atherosclerosis compared with ECs without IRS-1 treatment (P<0.05). Moreover, IRS-1 promoted the phosphorylation of Akt in the EC models of ox-LDL-induced atherosclerosis. IRS-1 also significantly suppressed the transcription of atherosclerosis-associated genes in ox-LDL-exposed ECs compared with ECs without IRS-1 treatment (P<0.05). Furthermore, IRS-1 significantly increased the cytoplasmic localization of p-FoxO1 in EC models of ox-LDL-induced atherosclerosis. On the whole, the findings of the present study demonstrate that IRS-1 exerts protective effects in an EC model of ox-LDL-induced atherosclerosis by inhibiting ER stress/oxidative stress-mediated apoptosis and activating the Akt/FoxO1 signaling pathway.

Differential Expression of Ormdl Genes in the Islets of Mice and Humans with Obesity

SummaryThe orosomucoid-like (Ormdl) proteins play a critical role in sphingolipid homeostasis, inflammation, and ER stress, all of which are associated with obesity and βcell dysfunction. However, their roles in β cells and obesity remain unknown. Here, we show that islets from overweight/obese human donors displayed marginally reduced ORMDL1-2 expression, whereas ORMDL3 expression was significantly downregulated compared with islets from lean donors. In contrast, Ormdl3 was substantially upregulated in the islets of leptin-deficient obese (ob/ob) mice compared with lean mice. Treatment of ob/ob mice and their islets with leptin markedly reduced islet Ormld3 expression. Ormdl3 knockdown in a β cell line induced expression of pro-apoptotic markers, which was rescued by ceramide synthase inhibitor fumonisin B1. Our results reveal differential expression of Ormdl3 in the islets of a mouse model and humans with obesity, highlight the potential effect of leptin in this differential regulation, and suggest a role for Ormdl3 in β cell apoptosis.

Gonadotropin-releasing hormone regulated transcription of gonadotropin subunit genes

Gonadotropin-releasing hormone regulated transcription of gonadotropin subunit genes

Differential effects of synthetic media on long-term growth, starch accumulation and transcription of ADP-glucosepyrophosphorylase subunit genes in Landoltia punctata

Murashige & Skoog (MS) and Hoagland’s media were previously used for in vitro culture of Landoltia punctata. During subsequent ex vitro culture, the use of MS medium resulted in a higher growth rate, compared to Hoagland’s medium. Thus, a higher starch content of L. punctata in MS medium was previously hypothesized. Here, L. punctata strain 5632 was isolated and characterized using morphological characteristics and the atpF-atpH intergenic region. During early cultivation stage, fresh weight and relative growth rate in MS medium were lower than Hoagland’s medium. Conversely, starch content in MS medium was considerably higher than in Hoagland’s medium. Medium effects on expression of genes coding for starch-biosynthesis ADP-glucosepyrophosphorylase (AGPase) were determined. Genomic fragments of small (LeAPS) and large (LeAPL1) AGPase subunits were characterized. Differential expression between each AGPase subunit genes was observed in both media. Additionally, in MS medium, the highest correlation coefficients between starch content and gene expression was found with LeAPS (0.81) and followed by LeAPL3 (0.67), LeAPL2 (0.65) and LeAPL1 (0.28). In Hoagland’s medium, the coefficients of LeAPL3 (0.83) and LeAPL2 (0.62) were higher than LeAPS (0.18) and LeAPL1 (−0.62). This suggested different levels of contributions of these genes in starch biosynthesis in both media.

Interferon-γ potentiates GABAA receptor-mediated inhibitory currents in rat hippocampal CA1 pyramidal neurons

The neural transmission and plasticity can be differentially modulated by various elements of the immune system. Interferon-γ (IFN-γ) is a “pro-inflammatory” cytokine mainly produced by T lymphocytes, activates its corresponding receptor and plays important roles under both homeostatic and inflammatory conditions. However, the impact of IFN-γ on the γ-aminobutyric acid (GABA)-mediated currents in the hippocampus, a major brain region involved in the cognitive function, has not been investigated. Here we detected abundant expression of both IFN-γ receptor subunit gene transcripts (Ifngr1 and Ifngr2) in the rat hippocampus by quantitative PCR. In addition, we pre-incubated rat hippocampal slices with IFN-γ (100 ng/ml) and recorded GABA-activated spontaneous and miniature postsynaptic inhibitory currents (sIPSCs and mIPSCs) and tonic currents in hippocampal CA1 pyramidal neurons by the whole-cell patch-clamp method. The pre-incubation with IFN-γ increased the frequency but not the mean amplitude, rise time or decay time of both sIPSCs and mIPSCs in hippocampal CA1 pyramidal neurons, suggesting a presynaptic effect of IFN-γ. Moreover, the GABA-activated tonic currents were enhanced by IFN-γ. In conclusion, the potentiation of GABAergic currents in hippocampal neurons by IFN-γ may contribute to the disturbed neuronal excitability and cognitive dysfunction during neuroinflammation.

Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

Inhibition of the proteasome leads to proteotoxic stress, which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor Nrf1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes, resulting in the restoration of proteasome activity. Further understanding of the Nrf1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cell-killing effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of Nrf1, we performed an RNAi screen in an engineered cell line, reporting on Nrf1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA+ ATPase RUVBL1 is necessary for Nrf1's transcriptional activity. Given that RUVBL1 is part of different multisubunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin-regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60-Nrf1 axis to alter proteasome function in various human diseases, including cancer.

SIN3B promotes integrin αV subunit gene transcription and cell migration of hepatocellular carcinoma.

Paired amphipathic helix protein (SIN3B) is a transcription corepressor for many genes. Here we show a different regulation mechanism of integrin αV gene expression by SIN3B in human hepatocellular carcinoma (HCC). We first observed a close relationship between Integrin αV and SIN3B expressions in HCC patients and tumor cell lines with different metastatic potentials. Overexpression of SIN3B significantly accelerated the cell migration rate of SMMC-7721, but failed when integrin αV expression was silenced. Interestingly, SIN3B stimulated integrin αV subunit promoter activity only in the presence of sulfatide. Importantly, SIN3B was identified in the complex with sulfatide by mass spectrometry. Fat blot assay indicated that SIN3B specifically interacted with sulfatide. Molecular modeling suggested that sulfatide induced the conformational change of SIN3B from compacted α-helices to a relaxed β-sheet in PAH2 domain. The data of immunoprecipitation and ChIP assay indicated that altered SIN3B lost the binding affinity with MAD1 and HDAC2, which reduced the recruitment of HDAC2 on integrin αV gene promoter and prevented the deacetylation of the histone 3. In conclusion, this study demonstrated that SIN3B promoted the transcriptional activation of the integrin αV subunit gene promoter by reducing interaction with HDAC2.

Pyrethroid Insecticide Cypermethrin Modulates Gonadotropin Synthesis via Calcium Homeostasis and ERK1/2 Signaling in LβT2 Mouse Pituitary Cells.

Pyrethroids are a class of widely used insecticides. Cypermethrin (CP) is one of most commonly used pyrethroid insecticides and its residue has been frequently detected in environmental media. Our recent animal study reported that early postnatal exposure to CP induced an increase in serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as well as the expression of gonadotropin subunit genes (chorionic gonadotropin α [CGα], LHβ and FSHβ) in pituitary tissues. In this study, we further investigated the precise mechanism by which CP at concentrations of 1-100 nM affected the synthesis of gonadotropins using a murine pituitary gonadotropic cell line LβT2. We found that calcium (Ca2+)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) activity was required for CP-regulated transcription of CGαs, LHβ and FSHβ. We provided the novel evidence that CP caused both influx of extracellular Ca2+ through L-type voltage-gated calcium channels (VGCCs) and release of intracellular Ca2+ from endoplasmic reticulum (ER) via inhibition of Ca2+-ATPase. Our results showed that CP disrupted Ca2+ homeostasis via these two separate and independent pathways, thus resulting in the activation of protein kinase C /c-Raf/ERK1/2/immediate-early genes pathways and subsequent increase in the transcription of gonadotropin subunit genes. Our findings would have important implications for understanding the underlying mechanisms of the disrupting effects of some pyrethroids (such as CP) on the synthesis of pituitary gonadotropins.

Transcriptional regulation of voltage-gated Ca2+ channels.

The transcriptional regulation of voltage-gated Ca2+ (CaV ) channels is an emerging research area that promises to improve our understanding of how many relevant physiological events are shaped in the central nervous system, the skeletal muscle and other tissues. Interestingly, a picture of how transcription of CaV channel subunit genes is controlled is evolving with the identification of the promoter regions required for tissue-specific expression and the identification of transcription factors that control their expression. These promoters share several characteristics that include multiple transcriptional start sites, lack of a TATA box and the presence of elements conferring tissue-selective expression. Likewise, changes in CaV channel expression occur throughout development, following ischaemia, seizures or chronic drug administration. This review focuses on insights achieved regarding the control of CaV channel gene expression. To further understand the complexities of expression and to increase the possibilities of detecting CaV channel alterations causing human disease, a deeper knowledge on the structure of the 5' upstream regions of the genes encoding these remarkable proteins will be necessary.

MiR-124 inhibits the migration and invasion of human hepatocellular carcinoma cells by suppressing integrin \u03b1V expression

Tumor metastasis is the major cause of cancer-related death especially in human hepatocellular carcinoma (HCC). Although microRNAs have been implicated in tumor development, the roles of miR-124 in HCC metastasis are still not well understood. We conducted functional analysis in this study to investigate miR-124. We observed that miR-124 significantly retarded the wound healing and migration of HCC SMMC-7721 and BEL-7404 cells. Further analysis indicated miR-124 directly targeting the transcriptional factor Sp1 which is an important transcription factor for the integrin αV subunit gene transcription. Co-transfection of miR-124 with the luciferase reporter containing Sp1 3′ untranslated region (UTR) significantly suppressed the luciferase activities. While mutation of the binding site of miR-124 in Sp1 mRNA 3′UTR completely abrogated the suppression of miR-124. Overexpression of miR-124 resulted in robust downregulation of Sp1 and integrin αV expression at either mRNA or protein level. Ectopic expression of miR-124 in HCC dramatically repressed the wound healing and migration in vitro and tumor metastasis in mouse experiments. Our findings demonstrated that miR-124 played as an important role in regulation of integrin αV expression in HCC, and reintroduction of miR-124 might be an alternative therapeutic strategy for controlling integrin αV expression in HCC.

Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1.

Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies.

Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits.

Transcription of follicle-stimulating hormone subunit genes is modulated by porcine LIM homeobox transcription factors, LHX2 and LHX3.

The LIM-homeobox transcription factors LHX2 and LHX3s (LHX3a and LHX3b) are thought to be involved in regulating the pituitary glycoprotein hormone subunit genes Cga and Fshβ. These two factors show considerable differences in their amino acid sequences for DNA binding and protein-protein interactions and in their vital function in pituitary development. Hence, we compared the DNA binding properties and transcriptional activities of Cga and Fshβ between LHX2 and LHX3s. A gel mobility shift assay for approximately 1.1 kb upstream of Cga and 2.0 kb upstream of Fshβ varied in binding profiles between LHX2 and LHX3s. DNase I footprinting revealed DNA binding sites in 8 regions of the Cga promoter for LHX2 and LHX3s with small differences in the binding range and strength. In the Fshβ promoter, 14 binding sites were identified for LHX2 and LHX3, respectively. There were alternative binding sites to either gene in addition to similar differences observed in the Cga promoter. The transcriptional activities of LHX2 and LHX3s according to a reporter assay showed cell-type dependent activity with repression in the pituitary gonadotrope lineage LβT2 cells and stimulation in Chinese hamster ovary lineage CHO cells. Reactivity of LHX2 and LHX3s was observed in all regions, and differences were observed in the 5'-upstream region of Fshβ. However, immunohistochemistry showed that LHX2 resides in a small number of gonadotropes in contrast to LHX3. Thus, LHX3 mainly controls Cga and Fshβ expression.

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production.

Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.

Specificity protein 4 (Sp4) transcriptionally regulates inhibitory GABAergic receptors in neurons

Previous studies in our laboratory have shown that the neuron-specific specificity protein 4 (Sp4) transcriptionally regulates many excitatory neurotransmitter receptor subunit genes, such as those for GluN1, GluN2A, and GluN2B of N-methyl-d-aspartate (NMDA) receptors and Gria2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It also regulates Atp1a1 and Atp1b1 subunit genes of Na+/K+-ATPase, a major energy-consuming enzyme, as well as all 13 subunits of cytochrome c oxidase (COX), an important energy-generating enzyme. Thus, there is a tight coupling between energy consumption, energy production, and excitatory neuronal activity at the transcriptional level in neurons. The question is whether inhibitory neurotransmitter receptors are also regulated by Sp4. In the present study, we tested our hypothesis that Sp4 regulates receptor subunit genes of a major inhibitory neurotransmitter, GABA, specifically GABAA receptors. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, real-time quantitative PCR, chromatin immunoprecipitation, promoter mutational analysis, over-expression and shRNA of Sp4, functional assays, and western blots, we found that Sp4 functionally regulates the transcription of Gabra1 (GABAA α1) and Gabra2 (GABAA α2), but not Gabra3 (GABAA α3) subunit genes. The binding sites of Sp4 are conserved among rats, humans, and mice. Thus, our results substantiate our hypothesis that Sp4 plays a key role in regulating the transcription of GABAA receptor subunit genes. They also indicate that Sp4 is in a position to transcriptionally regulate the balance between excitatory and inhibitory neurochemical expressions in neurons.