A novel 3-miRNA network regulates tumour progression in oral squamous cell carcinoma.

BACKGROUND: Late diagnosis is one of the major confounders in oral squamous cell carcinoma (OSCC). Despite recent advances in molecular diagnostics, no disease-specific biomarkers are clinically available for early risk prediction of OSCC. Therefore, it is important to identify robust biomarkers that are detectable using non-invasive liquid biopsy techniques to facilitate the early diagnosis of oral cancer. This study identified potential salivary exosome-derived miRNA biomarkers and crucial miRNA-mRNA networks/underlying mechanisms responsible for OSCC progression. METHODS: Small RNASeq (n = 23) was performed in order to identify potential miRNA biomarkers in both tissue and salivary exosomes derived from OSCC patients. Further, integrated analysis of The Cancer Genome Atlas (TCGA) datasets (n = 114), qPCR validation on larger patient cohorts (n = 70) and statistical analysis with various clinicopathological parameters was conducted to assess the effectiveness of the identified miRNA signature. miRNA-mRNA networks and pathway analysis was conducted by integrating the transcriptome sequencing and TCGA data. The OECM-1 cell line was transfected with the identified miRNA signature in order to observe its effect on various functional mechanisms such as cell proliferation, cell cycle, apoptosis, invasive as well as migratory potential and the downstream signaling pathways regulated by these miRNA-mRNA networks. RESULTS: Small RNASeq and TCGA data identified 12 differentially expressed miRNAs in OSCC patients compared to controls. On validating these findings in a larger cohort of patients, miR-140-5p, miR-143-5p, and miR-145-5p were found to be significantly downregulated. This 3-miRNA signature demonstrated higher efficacy in predicting disease progression and clinically correlated with poor prognosis (p < 0.05). Transcriptome, TCGA, and miRNA-mRNA network analysis identified HIF1a, CDH1, CD44, EGFR, and CCND1 as hub genes regulated by the miRNA signature. Further, transfection-mediated upregulation of the 3-miRNA signature significantly decreased cell proliferation, induced apoptosis, resulted in G2/M phase cell cycle arrest and reduced the invasive and migratory potential by reversing the EMT process in the OECM-1 cell line. CONCLUSIONS: Thus, this study identifies a 3-miRNA signature that can be utilized as a potential biomarker for predicting disease progression of OSCC and uncovers the underlying mechanisms responsible for converting a normal epithelial cell into a malignant phenotype.

In vitro, in vivo, and in silico analysis of synbiotics as preventive interventions for lipid metabolism in ethanol-induced adipose tissue injury.

The risk of alcoholic liver disease (ALD) is increased by excessive ethanol drinking. For the prevention of ALD, the effects of ethanol on the liver, adipose tissue, and gut are crucial. Interestingly, garlic and a few probiotic strains can protect against ethanol-induced hepatotoxicity. However, the relationship between adipose tissue inflammation, Kyolic aged garlic extract (AGE), and Lactobacillus rhamnosus MTCC1423 in developing ALD is unknown. Therefore, the present study explored the effect of synbiotics (a combination of prebiotics and probiotics) on adipose tissue to prevent ALD. To investigate the efficacy of synbiotics administration on adipose tissue in preventing ALD, in vitro (3T3-L1 cells, N = 3) groups: control, control + LPS (lipopolysaccharide), ethanol, ethanol + LPS, ethanol + synbiotics, ethanol + synbiotics + LPS; in vivo (Wistar male rats, N = 6) groups: control, ethanol, pairfed, ethanol + synbiotics and in silico experiments were conducted. Lactobacillus multiplies in accordance with the growth curve when exposed to AGE. Additionally, Oil red O staining and scanning electron microscopy (SEM) demonstrated that synbiotics therapy maintained the morphology of adipocytes in the alcoholic model. In support of the morphological changes, quantitative real-time PCR demonstrated overexpression of adiponectin and downregulation of leptin, resistin, PPARγ, CYP2E1, iNOS, IL-6, and TNF-α after administration of synbiotics compared to the ethanol group. In addition, MDA estimation by high-performance liquid chromatography (HPLC) indicated that the synbiotics treatment reduced oxidative stress in rat adipose tissue. Consequently, the in-silico analysis revealed that AGE inhibited the C-D-T networks as PPARγ acting as the main target protein. The current study demonstrates that using synbiotics improves adipose tissue metabolism in ALD.

The intervention of epithelial-mesenchymal transition in homeostasis of human retinal pigment epithelial cells: a review.

The health and activity of photoreceptors and Bruch's membrane are promoted by the retinal pigment epithelium (RPE), which is essential for normal vision. Age-related macular degeneration (AMD), diabetic retinopathy (DR), and proliferative vitreoretinopathy (PVR) are examples of retinopathies that result in vision loss. Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells transform into mesenchymal cells as a result of a faulty microenvironment, and it is associated with the oculopathies stated above. Cell differentiation, autophagy, growth factors (GFs), the blood-retinal barrier (BRB), and other complicated signaling pathways all contribute to proper morphology, and their disruption by harmful compounds has an impact on RPE function. The inducer and suppressor of EMT in RPE, on the other hand, are unknown. The current article reviews the experimental research investigations, suggested that certain modulators like glucosamine (Glc-N) and bradykinin (BK) suppress the TGFß signaling pathway and that other variables like oxidative stress triggered EMT, which is not found in normal RPE homeostasis. Finding molecular targets and treatments to prevent and restore RPE function, as well as understanding how EMT regulators affect RPE degeneration, are therefore crucial.

Reckoning apigenin and kaempferol as a potential multi-targeted inhibitor of EGFR/HER2-MEK pathway of metastatic colorectal cancer identified using rigorous computational workflow.

In the past two decades, the treatment of metastatic colorectal cancer (mCRC) has been revolutionized as multiple cytotoxic, biological, and targeted drugs are being approved. Unfortunately, tumors treated with single targeted agents or therapeutics usually develop resistance. According to pathway-oriented screens, mCRC cells evade EGFR inhibition by HER2 amplification and/or activating Kras-MEK downstream signaling. Therefore, treating mCRC patients with dual EGFR/HER2 inhibitors, MEK inhibitors, or the combination of the two drugs envisaged to prevent the resistance development which eventually improves the overall survival rate. In the present study, we aimed to screen potential phytochemical lead compounds that could multi-target EGFR, HER2, and MEK1 (Mitogen-activated protein kinase kinase) using a computer-aided drug design approach that includes molecular docking, endpoint binding free energy calculation using MM-GBSA, ADMET, and molecular dynamics (MD) simulations. Docking studies revealed that, unlike all other ligands, apigenin and kaempferol exhibit the highest docking score against all three targets. Details of ADMET analysis, MM/GBSA, and MD simulations helped us to conclusively determine apigenin and kaempferol as potentially an inhibitor of EGFR, HER2, and MEK1 apigenin and kaempferol against mCRC at a systemic level. Additionally, both apigenin and kaempferol elicited antiangiogenic properties in a dose-dependent manner. Collectively, these findings provide the rationale for drug development aimed at preventing CRC rather than intercepting resistance.

Systemic study of selected histone deacetylase inhibitors in cardiac complications associated with cancer cachexia.

Cancer cachexia is mainly characterized by wasting of skeletal muscles and fat and body weight loss, along with severe complications of major organs like liver, heart, brain and bone. There can be diminishing performance of these major organs as cancer cachexia progresses, one such drastic effect on the cardiac system. In the present study, differential effect of histone deacetylase inhibitors (HDACi) on cardiac complications associated with cancer cachexia is studied. Two models were used to induce cancer cachexia: B16F1 induced metastatic cancer cachexia and Lewis lung carcinoma cell - induced cancer cachexia. Potential of Class I HDACi entinostat, Class II HDACi MC1568, and nonspecific HDACi sodium butyrate on cardiac complications were evaluated using the cardiac hypertrophy markers, hemodynamic markers, and cardiac markers along with histopathological evaluation of heart sections by Periodic acid-Schiff staining, Masson's trichrome staining, Picro-sirius red staining, and haematoxylin and eosin staining. Immunohistochemistry evaluation by vimentin and caspase 3 protein expression was evaluated. Entinostat showed promising results by attenuating the cardiac complications, and MC1568 treatment further exacerbated the cardiac complications, while non-conclusive effect were recorded after treatment with sodium butyrate. This study will be helpful in evaluating other HDACi for potential in cardiac complications associated with cancer cachexia.

Network pharmacology-based evaluation of natural compounds with paclitaxel for the treatment of metastatic breast cancer.

Metastatic breast cancer is a prevalent life-threatening disease. Paclitaxel (PTX) is widely used in metastatic breast cancer therapy, but the side effects limit its chemotherapeutic application. Multidrug strategies have recently been used to maximize potency and decrease the toxicity of a particular drug by reducing its dosage. Therefore, we have evaluated the combined anti-cancerous effect of PTX with tested natural compounds (andrographolide (AND), silibinin (SIL), mimosine (MIM) and trans-anethole (TA)) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, trypan blue dye exclusion assay, proliferating cell nuclear antigen (PCNA) staining, network pharmacology, molecular docking, molecular dynamics (MD) and in vivo chick chorioallantoic membrane (CAM) angiogenesis assay. We observed a reduction in the IC50 value of PTX with tested natural compounds. Further, the network pharmacology-based analysis of compound-disease-target (C-D-T) network showed that PTX, AND, SIL, MIM and TA targeted 55, 61, 56, 31 and 18 proteins of metastatic breast cancer, respectively. Molecular docking results indicated that AND and SIL inhibited the C-D-T network's core target kinase insert domain receptor (KDR) protein more effectively than others. While MD showed that the binding of AND with KDR was stronger and more stable than others. In trypan blue dye exclusion assay and PCNA staining, AND and SIL along with PTX were found to be more effective than PTX alone. CAM assay results suggested that AND, SIL and TA increase the anti-angiogenic potential of PTX. Thus, natural compounds can be used to improve the anti-cancer potential of PTX.

Dichloroacetate prevents TGFß-induced epithelial-mesenchymal transition of retinal pigment epithelial cells.

Proliferative retinopathies are associated with formation of fibrous epiretinal membranes. At present, there is no pharmacological intervention for the treatment of retinopathies. Cytokines such as TGFß are elevated in the vitreous humor of the patients with proliferative vitro-retinopathy, diabetic retinopathy and age-related macular degeneration. TGFß isoforms lead to epithelial-mesenchymal transition (EMT) or trans-differentiation of the retinal pigment epithelial (RPE) cells. PI3K/Akt and MAPK/Erk pathways play important roles in the EMT of RPE cells. Therefore, inhibition of EMT by pharmacological agents is an important therapeutic strategy in retinopathy. Dichloroacetate (DCA) is shown to prevent proliferation and EMT of cancer cell lines but its effects are not explored on the prevention of EMT of RPE cells. In the present study, we have investigated the role of DCA in preventing TGFß2 induced EMT of RPE cell line, ARPE-19. A wound-healing assay was utilized to detect the anti-EMT effect of DCA. The expressions of EMT and cell adhesion markers were carried out by immunofluorescence, western blotting, and quantitative real-time PCR. The expression of MAPK/Erk and PI3K/Akt pathway members was carried out using western blotting. We found that TGFß2 exposure leads to an increase in the wound healing response, expression of EMT markers (Fibronectin, Collagen I, N-cadherin, MMP9, S100A4, α-SMA, Snai1, Slug) and a decrease in the expression of cell adhesion/epithelial markers (ZO-1, Connexin 43, E-cadherin). These changes were accompanied by the activation of PI3K/Akt and MAPK/Erk pathways. Simultaneous exposure of DCA along with TGFß2 significantly inhibited wound healing response, expression of EMT markers and cell adhesion/epithelial markers. Furthermore, DCA and TGFß2 effectively attenuated the activation of MAPK/Erk/JNK and PI3K/Akt/GSK3ß pathways. Our results demonstrate that DCA has a strong anti-EMT effect on the ARPE-19 cells and hence can be utilized as a therapeutic agent in the prevention of proliferative retinopathies.

Epigenetically dysregulated genes and pathways implicated in the pathogenesis of non-syndromic high myopia.

Myopia, commonly referred to as nearsightedness, is one of the most common causes of visual disability throughout the world. It affects more people worldwide than any other chronic visual impairment condition. Although the prevalence varies among various ethnic groups, the incidence of myopia is increasing in all populations across globe. Thus, it is considered a pressing public health problem. Both genetics and environment play a role in development of myopia. To elucidate the epigenetic mechanism(s) underlying the pathophysiology of high-myopia, we conducted methylation profiling in 18 cases and 18 matched controls (aged 4-12 years), using Illumina MethylationEPIC BeadChips array. The degree of myopia was variable among subjects, ranging from -6 to -15D. We identified 1541 hypermethylated CpGs, representing 1745 genes (2.0-fold or higher) (false discovery rate (FDR) p ≤ 0.05), multiple CpGs were p < 5 × 10-8 with a receiver operating characteristic area under the curve (ROC-AUC) ≥ 0.75 in high-myopia subjects compared to controls. Among these, 48 CpGs had excellent correlation (AUC ≥ 0.90). Herein, we present the first genome-wide DNA methylation analysis in a unique high-myopia cohort, showing extensive and discrete methylation changes relative to controls. The genes we identified hold significant potential as targets for novel therapeutic intervention either alone, or in combination.

A Prospective Evaluation of Posterior Capsule Opacification in Eyes With Posterior Capsule Plaque-A Case-Control Study.

PURPOSE: To compare the development of posterior capsule opacification (PCO) between eyes with and without posterior capsule plaque after single-piece hydrophobic acrylic intraocular lens (IOL) implantation 5 years postoperatively. DESIGN: A prospective observational case-control study. METHODS: One hundred one consecutive eyes with posterior capsule plaque (cases) were compared with the same number of cataractous eyes without posterior capsule plaque (controls). A detailed preoperative evaluation was done to detect the presence of posterior capsule plaque. Histomorphology of posterior capsule plaque was evaluated. Postoperatively, digital retroillumination photographic documentation was performed at 1 month and 1, 2, 3, and 5 years and analyzed for PCO using the Evaluation of Posterior Capsule Opacification (EPCO) software; EPCO scores and areas were calculated. The development of PCO and the influence of the anterior capsule cover (total and partial) on the IOL optic were compared. RESULTS: Posterior capsule plaque on histomorphology showed a large amount of collagenous, fibrous extracellular matrix, and immunofluorescence staining was positive for alpha smooth muscle actin. In the development of PCO, there was no difference between cases and controls at 1 month and 1, 2, 3, and 5 years. Between the 2 groups, there was no difference in the development of PCO within total cover and within partial cover of the anterior capsule on the IOL up to 5 years. CONCLUSIONS: The presence of posterior capsule plaque did not increase the incidence of PCO at 5 years.

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.

Andrographolide suppresses epithelial mesenchymal transition by inhibition of MAPK signalling pathway in lens epithelial cells.

Epithelial mesenchymal transition (EMT) of lens epithelial cells (LECs) may contribute to the development of posterior capsular opacification (PCO), which leads to visual impairment. Andrographolide has been shown to have therapeutic potential against various cancers. However, its effect on human LECs is still unknown. The purpose of this study is to evaluate the effect of andrographolide on EMT induced by growth factors in the fetal human lens epithelial cell line (FHL 124). Initially the LECs were treated with growth factors (TGF-beta 2 and bFGF) to induce EMT. Subsequently these EMT-induced cells were treated with andrographolide at 100 and 500 nM concentrations for 24 h. Our results showed that FHL 124 cells treated with growth factors had a significant decrease in protein and m-RNA levels of epithelial markers pax6 and E-Cadherin. After administering andrographolide, these levels significantly increased. It was noticed that EMT markers alpha-SMA, fibronectin and collagen IV significantly decreased after treatment with andrographolide when compared to the other group. Treatment with andrographolide significantly inhibited phosphorylation of ERK and JNK. Cell cycle analysis showed that andrographolide did not arrest cells at G0/G1 or G2/M at tested concentrations. Our findings suggest that andrographolide helps sustain epithelial characteristics by modulating EMT markers and inhibiting the mitogen-activated protein kinase (MAPK) signalling pathway in LECs. Hence it can prove to be useful in curbing EMT-mediated PCO.

Comprehensive analysis of maillard protein modifications in human lenses: effect of age and cataract.

In human lens proteins, advanced glycation endproducts (AGEs) originate from the reaction of glycating agents, e.g., vitamin C and glucose. AGEs have been considered to play a significant role in lens aging and cataract formation. Although several AGEs have been detected in the human lens, the contribution of individual glycating agents to their formation remains unclear. A highly sensitive liquid chromatography-tandem mass spectrometry multimethod was developed that allowed us to quantitate 21 protein modifications in normal and cataractous lenses, respectively. N(6)-Carboxymethyl lysine, N(6)-carboxyethyl lysine, N(7)-carboxyethyl arginine, methylglyoxal hydroimidazolone 1, and N(6)-lactoyl lysine were found to be the major Maillard protein modifications among these AGEs. The novel vitamin C specific amide AGEs, N(6)-xylonyl and N(6)-lyxonyl lysine, but also AGEs from glyoxal were detected, albeit in minor quantities. Among the 21 modifications, AGEs from the Amadori product (derived from the reaction of glucose and lysine) and methylglyoxal were dominant.

Nuclear respiratory factor 2 regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

Neuronal activity is highly dependent on energy metabolism. Nuclear respiratory factor 2 (NRF-2) tightly couples neuronal activity and energy metabolism by transcriptionally co-regulating all 13 subunits of an important energy-generating enzyme, cytochrome c oxidase (COX), as well as critical subunits of excitatory NMDA receptors. AMPA receptors are another major class of excitatory glutamatergic receptors that mediate most of the fast excitatory synaptic transmission in the brain. They are heterotetrameric proteins composed of various combinations of GluA1-4 subunits, with GluA2 being the most common one. We have previously shown that GluA2 (Gria2) is transcriptionally regulated by nuclear respiratory factor 1 (NRF-1) and specificity protein 4 (Sp4), which also regulate all subunits of COX. However, it was not known if NRF-2 also couples neuronal activity and energy metabolism by regulating subunits of the AMPA receptors. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate the expression of Gria2, but not of Gria1, Gria3, or Gria4 genes in neurons. By regulating the GluA2 subunit of the AMPA receptor, NRF-2 couples energy metabolism and neuronal activity at the transcriptional level through a concurrent and parallel mechanism with NRF-1 and Sp4.

Andrographolide reduces proliferation and migration of lens epithelial cells by modulating PI3K/Akt pathway.

Lens epithelial cell proliferation, migration, and transdifferentiation are involved in the development of subcapsular cataracts and postoperative capsular opacification (PCO). PI3K/Akt pathway is involved in the proliferation and migration of lens epithelial cells. Andrographolide is the main bioactive component of Andrographis paniculata and is known to possess anti-proliferative and anti-migratory activities. The purpose of this study is to evaluate the effect of andrographolide on proliferation and migration induced by growth factors (TGF-ß and bFGF) in the lens epithelial cell line, FHL 124. We have also evaluated the role of the PI3K/Akt pathway and its alteration by andrographolide during proliferation and migration of lens epithelial cells. The results showed that andrographolide significantly inhibited proliferation in a dose and time dependent manner. The growth factors, TGF-ß and bFGF, induced migration of lens epithelial cells, which was lowered by andrographolide. The growth factors also up regulated phosphorylated Akt (Ser473) and Akt (Thr308), which was abolished by simultaneous treatment of andrographolide. Similar changes were also observed with the PI3K inhibitor, LY290042. Our findings suggest that andrographolide reduces proliferation, migration, and phosphorylated Akt levels in lens epithelial cells. Hence andrographolide can be utilized for the prevention of PCO.

UVA light-excited kynurenines oxidize ascorbate and modify lens proteins through the formation of advanced glycation end products: implications for human lens aging and cataract formation.

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320-400 nm, 100 milliwatts/cm(2), 45 min to 2 h), young human lenses (20-36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.

Specificity protein 4 (Sp4) regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are important glutamatergic receptors mediating fast excitatory synaptic transmission in the brain. The regulation of the four subunits of AMPA receptors, GluA1-4, is poorly understood. Excitatory synaptic transmission is highly energy-demanding, and this energy is derived mainly from the oxidative pathway. Recently, we found that specificity factor regulates all subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme. COX is also regulated by nuclear respiratory factor 1 (NRF-1), which transcriptionally controls the Gria2 (GluA2) gene of AMPA receptors. The goal of the present study was to test our hypothesis that Sp-factors (Sp1, Sp3, and/or Sp4) also regulate AMPA subunit genes. If so, we wish to determine if Sp-factors and NRF-1 function via a complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel mechanism. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, we found that Sp4, but not Sp1 or Sp3, regulates the Gria2, but not Gria1, 3, or 4, subunit gene of the AMPA receptor in a concurrent and parallel manner with NRF-1. Thus, Sp4 and NRF-1 both mediate the tight coupling between neuronal activity and energy metabolism at the transcriptional level.

Regulation of Na(+)/K(+)-ATPase by neuron-specific transcription factor Sp4: implication in the tight coupling of energy production, neuronal activity and energy consumption in neurons.

A major source of energy demand in neurons is the Na(+)/K(+)-ATPase pump that restores the ionic gradient across the plasma membrane subsequent to depolarizing neuronal activity. The energy comes primarily from mitochondrial oxidative metabolism, of which cytochrome c oxidase (COX) is a key enzyme. Recently, we found that all 13 subunits of COX are regulated by specificity (Sp) factors, and that the neuron-specific Sp4, but not Sp1 or Sp3, regulates the expression of key glutamatergic receptor subunits as well. The present study sought to test our hypothesis that Sp4 also regulates Na(+)/K(+)-ATPase subunit genes in neurons. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutational analysis, over-expression, and RNA interference studies, we found that Sp4, with minor contributions from Sp1 and Sp3, functionally regulate the Atp1a1, Atp1a3, and Atp1b1 subunit genes of Na(+)/K(+)-ATPase in neurons. Transcripts of all three genes were up-regulated by depolarizing KCl stimulation and down-regulated by the impulse blocker tetrodotoxin (TTX), indicating that their expression was activity-dependent. Silencing of Sp4 blocked the up-regulation of these genes induced by KCl, whereas over-expression of Sp4 rescued them from TTX-induced suppression. The effect of silencing or over-expressing Sp4 on primary neurons was much greater than those of Sp1 or Sp3. The binding sites of Sp factors on these genes are conserved among mice, rats and humans. Thus, Sp4 plays an important role in the transcriptional coupling of energy generation and energy consumption in neurons.

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

Specificity protein 4 functionally regulates the transcription of NMDA receptor subunits GluN1, GluN2A, and GluN2B.

N-Methyl-d-aspartate (NMDA) receptors are major glutamatergic receptors involved in most excitatory neurotransmission in the brain. The transcriptional regulation of NMDA receptors is not fully understood. Previously, we found that the GluN1 and GluN2B subunits of the NMDA receptor are regulated by nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2). NRF-1 and NRF-2 also regulate all 13 subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme, thereby coupling neuronal activity and energy metabolism at the transcriptional level. Specificity protein (Sp) is a family of transcription factors that bind to GC-rich regions, with Sp1, Sp3, and Sp4 all binding to the same cis- motifs. Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 expression is restricted to neurons and testicular cells. Recently, we found that the Sp1 factor regulates all subunits of COX. The goal of the present study was to test our hypothesis that the Sp factors also regulate specific subunits of NMDA receptors, and that they function with NRF-1 and NRF-2 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches we found that Sp4 functionally regulated GluN1, GluN2A, and GluN2B, but not GluN2C. On the other hand, Sp1 and Sp3 did not regulate these subunits as previously thought. Our data suggest that Sp4 operates in a complementary and concurrent/parallel manner with NRF-1 and NRF-2 to mediate the tight coupling between energy metabolism and neuronal activity at the molecular level.