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Gastroenterology Nov 2022The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use...
BACKGROUND & AIMS
The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism.
METHODS
We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites.
RESULTS
Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells.
CONCLUSIONS
Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.
Topics: Humans; Mice; Animals; Circadian Rhythm; Gastrointestinal Microbiome; Histone Deacetylases; Caco-2 Cells; ARNTL Transcription Factors; Propionates; Fatty Acids, Volatile; Butyrates; Histone Deacetylase Inhibitors; Luciferases
PubMed: 35934064
DOI: 10.1053/j.gastro.2022.07.051 -
Gene Oct 2022The rabbitfish Siganus canaliculatus is the first marine teleost found to have the biosynthetic ability of long-chain polyunsaturated fatty acids (LC-PUFA) from C...
The rabbitfish Siganus canaliculatus is the first marine teleost found to have the biosynthetic ability of long-chain polyunsaturated fatty acids (LC-PUFA) from C precursors catalyzed by fatty acyl desaturases (Δ6/Δ5 Fads, Δ4 Fads) and elongases of very long chain fatty acids (Elovls). Previously, we predicted the existence of insulin (INS) response elements (IREs) including nuclear factor Y (NF-Y) and sterol regulatory element (SRE) in the core promoter region of rabbitfish Δ6/Δ5 fads and Δ4 fads. To clarify the potential regulatory effect and mechanism of INS in LC-PUFA biosynthesis, INS responding region was identified at -456 bp to + 51 bp of Δ6/Δ5 fads core promoter, but not in Δ4 fads promoter. Moreover, a unique stimulatory protein 1 (Sp1) element was predicted in the INS responding region of Δ6/Δ5 fads. Subsequently, SRE, NF-Y and Sp1 elements were proved as IREs in Δ6/Δ5 fads promoter. The up-regulation of INS on gene expression of Srebp-1c, Sp1, Δ6/Δ5 fads and elovl5 as well as the LC-PUFA biosynthesis was further demonstrated in S. canaliculatus hepatocyte line (SCHL) cells, but no influence was detected on Δ4 fads. Besides, inhibitors of transcription factors Srebp-1c (Fatostatin, PF-429242) and Sp1 (Mithramycin) could inhibit the gene expression of Srebp-1c, Δ6/Δ5 fads and elovl5, and abolish the up-regulation of INS on these genes' expression and LC-PUFA biosynthesis. These results indicated that INS could up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and Sp1 in rabbitfish S. canaliculatus, which is the first report in teleost.
Topics: Animals; Fish Proteins; Fishes; Insulin; Lipogenesis; Sterol Regulatory Element Binding Protein 1
PubMed: 35905852
DOI: 10.1016/j.gene.2022.146755 -
Frontiers in Pharmacology 2022Irinotecan (CPT-11) in combination with 5-fluorouracil and leucovorin is a first-line chemotherapy regimen for the treatment of colorectal cancer; however, its clinical...
Irinotecan (CPT-11) in combination with 5-fluorouracil and leucovorin is a first-line chemotherapy regimen for the treatment of colorectal cancer; however, its clinical application is limited by the dose-limiting gastrointestinal toxicity of colitis. In our previous studies, several bile acids (BAs) were found significantly elevated in the colon of the CPT-11-induced rat colitis model. On the other hand, NLRP3 inflammasome has been reported to play important roles in mediating colitis. Interestingly, BA was stated to activate the NLRP3 inflammasome in some studies, while in some other reports, it showed an inhibitory effect. We assumed that the inflammatory status in different circumstances might have contributed to the controversial findings. In this study, we first discovered, under non-inflammatory conditions, that supplementing BA could activate the NLRP3 inflammasome in THP-1-differentiated macrophages and promote inflammation. In lipopolysaccharide (LPS)-induced inflammatory macrophages, however, BA inhibited the NLRP3 inflammasome and reduced inflammation. Further experiments demonstrated that Takeda G protein-coupled receptor 5 (TGR5) is essential in mediating the inhibitory effect of BA, while phospho-SP1 (p-SP1) is key to the activation. Furthermore, we applied the above findings to ameliorate CPT-11-caused colitis in rats by inhibiting SP1 with mithramycin A (MitA) or activating TGR5 using oleanolic acid (OA). Our findings may shed light on the discovery of effective interventions for reducing dose-limiting chemotherapy-induced colitis.
PubMed: 35712724
DOI: 10.3389/fphar.2022.677738 -
Antibiotics (Basel, Switzerland) May 2022bacteria produce a wide number of antibiotics and antitumor compounds that have attracted the attention of pharmaceutical and biotech companies. In this study, we...
bacteria produce a wide number of antibiotics and antitumor compounds that have attracted the attention of pharmaceutical and biotech companies. In this study, we provide evidence showing that the xylem sap from grapevines has a positive effect on the production of different antibiotics by several species, including ATCC 23877 and ATCC 12596 among others. The production of several already known compounds was induced: actinomycin D, chromomycin A3, fungichromin B, mithramycin A, etc., and four compounds with molecular formulas not included in the Dictionary of Natural Products (DNP v28.2) were also produced. The molecules present in the xylem sap that acts as elicitors were smaller than 3 kDa and soluble in water and insoluble in ether, ethyl acetate, or methanol. A combination of potassium citrate and di-D-fructose dianhydrides (related to levanbiose or inulobiose) seemed to be the main effectors identified from the active fraction. However, the level of induction obtained in the presence of these compounds mix was weaker and delayed with respect to the one got when using the whole xylem sap or the 3 kDa sap fraction, suggesting that another, not identified, elicitor must be also implied in this induction.
PubMed: 35625316
DOI: 10.3390/antibiotics11050672 -
Biological Trace Element Research Apr 2023The sphingosine-1-phosphate (S1P) transporter spinster homolog 2 (SPNS2) promotes tumor progression by modulating tumor immunity and enhancing tumor cells migration and...
The sphingosine-1-phosphate (S1P) transporter spinster homolog 2 (SPNS2) promotes tumor progression by modulating tumor immunity and enhancing tumor cells migration and invasion. Previously we found that iron deficiency in hepatocellular carcinoma upregulated SPNS2 expression to increase tumor metastasis. The present study aimed to identify the underlying mechanism of SPNS2 upregulation. Since the mRNA of SPNS2 was significantly increased, we used a transcription factor activity microarray to find the transcription factor responsible for this. The results showed that iron deprivation in hepatoma cells increased the transcriptional activities of 14 transcription factors while only 2 were decreased. Among these, 3 transcription factors, HIF1α, SP1, and YY1, were predicted to bind with the transcription promoter region of SPNS2. But only HIF1α and SP1 transcriptional activities on SPNS2 were increased by iron deficiency, and the increase of SP1 transcriptional activity was stronger than HIF1α. The protein level of HIF1α was increased by iron deficiency, while SP1 was not changed at the protein level but the phosphorylation level was increased. The inhibitor of HIF1α, PX478, and the inhibitor of SP1, Mithramycin A, reversed the increased mRNA and protein expressions of SPNS2 by iron deficiency, with a more significant effect by Mithramycin A. These results provided a comprehensive view of changes in transcriptional activities by iron deficiency and identified that SP1 was the main regulator of iron deficiency-inducing SPNS2 expression in hepatoma cells.
Topics: Humans; Carcinoma, Hepatocellular; Phosphorylation; Liver Neoplasms; Iron Deficiencies; Transcription Factors; RNA, Messenger; Sp1 Transcription Factor; Anion Transport Proteins
PubMed: 35614326
DOI: 10.1007/s12011-022-03296-2 -
Biomedicine & Pharmacotherapy =... Jul 2022Glutathione S-transferases (GSTs) belong to one class of phase 2 detoxification enzymes which are important in metabolism and/or detoxification of various electrophilic...
Glutathione S-transferases (GSTs) belong to one class of phase 2 detoxification enzymes which are important in metabolism and/or detoxification of various electrophilic endogenous metabolites and xenobiotics. From the available database, we found that GSTM2 gene expression is lower in high stages of bladder urothelial carcinoma than in stage 1 and normal bladder tissue. GSTM2 overexpression retards invasion, migration and tumor sphere formation of bladder cancer cells. Analysis of GSTM2 promoter activity shows that one SP1 site located at - 48 to - 40 bp is important for GSTM2 gene expression in BFTC 905 cells. An SP1 inhibitor, mithramycin A, inhibits GSTM2 promoter activity and protein expression. SP1 overexpression also increases GSTM2 expression in BFTC 905 and 5637 cells. Eight potential phytochemicals were analyzed for GSTM2 promoter activation, and results indicated that baicalein, berberrubine, chalcone, curcumin, resveratrol, and wogonin can increase promoter activity. In endogenous GSTM2 expression, berberrubine and resveratrol activated GSTM2 mRNA and protein expression the most. A DNA methylation inhibitor, 5-aza-deoxycytidine, can decrease GSTM2 gene methylation level and then increase its gene expression; 50 μM berberrubine decreased the GSTM2 gene methylation level, providing a mechanism for activating GSTM2 gene expression. Berberrubine and resveratrol also increased SP1 protein expression as one of the mechanisms for GSTM2 gene expression. In summary, berberrubine and resveratrol activates GSTM2 expression which inhibits cell proliferation, migration, and invasion of bladder cancer cells. The GSTM2 expression mechanism is partially via SP1 activation, and the effect of berberrubine is also partly via DNA CpG demethylation.
Topics: Carcinoma, Transitional Cell; DNA Methylation; Glutathione Transferase; Humans; Phytochemicals; Resveratrol; Sp1 Transcription Factor; Urinary Bladder Neoplasms
PubMed: 35594716
DOI: 10.1016/j.biopha.2022.113102 -
Antioxidants (Basel, Switzerland) Apr 2022Heme oxygenase-1 (HO-1) has been shown to exert antioxidant, anti-inflammatory, and anti-apoptotic effects in various types of cells. Therefore, the induction of HO-1 is...
Induction of Heme Oxygenase-1 by 15d-Prostaglandin J Mediated via a ROS-Dependent Sp1 and AP-1 Cascade Suppresses Lipopolysaccharide-Triggered Interleukin-6 Expression in Mouse Brain Microvascular Endothelial Cells.
Heme oxygenase-1 (HO-1) has been shown to exert antioxidant, anti-inflammatory, and anti-apoptotic effects in various types of cells. Therefore, the induction of HO-1 is an excellent rationale for the development of protective drugs. 15-Deoxy-Δ-prostaglandin J (15d-PGJ) can modulate the expression of antioxidant defense proteins and be beneficial for neuroinflammation. Brain endothelial cells play an important role in the pathophysiology of brain disorders. Whether 15d-PGJ can induce HO-1 expression and protect against the inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells remains unclear. Here, we reveal that 15d-PGJ stimulated HO-1 protein and mRNA expression in a time- and concentration-dependent manner in bEnd.3 cells, which was attenuated by diphenyleneiodonium chloride (DPI) and MitoTempo. Thus, activation of NADPH oxidase (NOX)- and mitochondria-derived reactive oxygen species (ROS) mediated 15d-PGJ-induced HO-1 expression. ROS generation could cause phosphorylation of protein kinase C (PKC)δ, leading to HO-1 expression, which was suppressed by Rottlerin (selective inhibitor PKCδ), DPI, and MitoTempo. We further demonstrated that phosphorylation of c-Jun N-terminal kinase (JNK)1/2 participated in 15d-PGJ-upregulated HO-1 expression, which was blocked by SP600125 or Rottlerin. Moreover, 15d-PGJ-induced HO-1 expression was mediated through the activation of c-Jun (a subunit of activator protein 1 (AP-1)) and specificity protein 1 (Sp1), leading to their interaction with the HO-1 promoter, revealed by chromatin immunoprecipitation assay, which was attenuated by SP600125, Mithramycin A, or Tanshinone II A. We further verified the anti-inflammatory effect of HO-1 expression. Our results showed that 15d-PGJ-induced HO-1 could mitigate the lipopolysaccharide-triggered interleukin-6 expression and secretion, as measured by an ELISA assay kit. These results suggest that 15d-PGJ-induced HO-1 expression is mediated through the activation of NOX- and mitochondria-derived ROS-dependent PKCδ/JNK1/2/Sp1 and the AP-1 signaling pathway and protects against inflammatory responses in bEnd.3 cells.
PubMed: 35453404
DOI: 10.3390/antiox11040719 -
Oxidative Medicine and Cellular... 2022Recently, we found that 5,8-dihydroxy-4',7-dimethoxyflavone (DDF) upregulated the expression of heme oxygenase (HO)-1 via p38 mitogen-activated protein kinase/nuclear...
5,8-Dihydroxy-4 ', 7-dimethoxyflavone Attenuates TNF--Induced Expression of Vascular Cell Adhesion Molecule-1 through EGFR/PKC/PI3K/Akt/Sp1-Dependent Induction of Heme Oxygenase-1 in Human Cardiac Fibroblasts.
Recently, we found that 5,8-dihydroxy-4',7-dimethoxyflavone (DDF) upregulated the expression of heme oxygenase (HO)-1 via p38 mitogen-activated protein kinase/nuclear factor-erythroid factor 2-related factor 2 (MAPK/Nrf2) pathway in human cardiac fibroblasts (HCFs). However, the alternative processes by which DDF induces the upregulation of HO-1 expression are unknown. Activation of epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), and protein kinase C (PKC) may initiate specificity protein (Sp)1 activity, which has been reported to induce expression of antioxidant molecules. Thus, we explored whether these components are engaged in DDF-induced HO-1 upregulation in HCFs. Western blotting, promoter-reporter analyses, and real-time polymerase chain reactions were adopted to measure HO-1 and vascular cell adhesion molecule (VCAM)-1 expressions in HCFs. Respective small interfering (si)RNAs and pharmacological inhibitors were employed to investigate the signaling components engaged in DDF-induced HO-1 upregulation. The chromatin immunoprecipitation assay was conducted to detect the binding interaction of Sp1 and antioxidant response elements (ARE) on the promoter of HO-1. An adhesion assay of THP-1 monocyte was undertaken to examine the functional effect of HO-1 on tumor necrosis factor (TNF)--induced VCAM-1 expression. DDF stimulated the EGFR/PKC/PI3K/Akt pathway leading to activation of Sp1 in HCFs. The roles of these protein kinases in HO-1 induction were ensured by transfection with their respective siRNAs. Chromatin immunoprecipitation assays revealed the interaction between Sp1 and the binding site of proximal ARE on the HO-1 promoter, which was abolished by glutathione, AG1478, Gö6976, LY294002, or mithramycin A. HO-1 expression enhanced by DDF abolished the monocyte adherence to HCFs and VCAM-1 expression induced by TNF-. Pretreatment with an inhibitor of HO-1: zinc protoporphyrin IX reversed these inhibitory effects of HO-1. We concluded that DDF-induced HO-1 expression was mediated via an EGFR/PKC/PI3K/Akt-dependent Sp1 pathway and attenuated the responses of inflammation in HCFs.
Topics: ErbB Receptors; Fibroblasts; Heme Oxygenase-1; Humans; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Transfection; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1
PubMed: 35281466
DOI: 10.1155/2022/1372958 -
International Journal of Molecular... Jan 2022Sp1 transcription factor regulates genes involved in various phenomena of tumor progression. Vasculogenic mimicry (VM) is the alternative neovascularization by...
Sp1 transcription factor regulates genes involved in various phenomena of tumor progression. Vasculogenic mimicry (VM) is the alternative neovascularization by aggressive tumor cells. However, there is no evidence of the relationship between Sp1 and VM. This study investigated whether and how Sp1 plays a crucial role in the process of VM in human prostate cancer (PCa) cell lines, PC-3 and DU145. A cell viability assay and three-dimensional culture VM tube formation assay were performed. Protein and mRNA expression levels were detected by Western blot and reverse transcriptase-polymerase chain reaction, respectively. The nuclear twist expression was observed by immunofluorescence assay. A co-immunoprecipitation assay was performed. Mithramycin A (MiA) and Sp1 siRNA significantly decreased serum-induced VM, whereas Sp1 overexpression caused a significant induction of VM. Serum-upregulated vascular endothelial cadherin (VE-cadherin) protein and mRNA expression levels were decreased after MiA treatment or Sp1 silencing. The protein expression and the nuclear localization of twist were increased by serum, which was effectively inhibited after MiA treatment or Sp1 silencing. The interaction between Sp1 and twist was reduced by MiA. On the contrary, Sp1 overexpression enhanced VE-cadherin and twist expressions. Serum phosphorylated AKT and raised matrix metalloproteinase-2 (MMP-2) and laminin subunit 5 gamma-2 (LAMC2) expressions. MiA or Sp1 silencing impaired these effects. However, Sp1 overexpression upregulated phosphor-AKT, MMP-2 and LAMC2 expressions. Serum-upregulated Sp1 was significantly reduced by an AKT inhibitor, wortmannin. These results demonstrate that Sp1 mediates VM formation through interacting with the twist/VE-cadherin/AKT pathway in human PCa cells.
Topics: Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Male; Neovascularization, Pathologic; PC-3 Cells; Prostatic Neoplasms; RNA, Messenger; Sp1 Transcription Factor; Up-Regulation
PubMed: 35163245
DOI: 10.3390/ijms23031321 -
Biochemical Pharmacology Mar 2022Mithramycin A (MIT) has reacquired extensive research attention due to its anti-solid tumor activity and improved pharmacological production. Mechanismly, MIT was...
Mithramycin A (MIT) has reacquired extensive research attention due to its anti-solid tumor activity and improved pharmacological production. Mechanismly, MIT was broadly used as a c-Myc inhibitor, and c-Myc regulated CD47 and PD-L1 expression which has been demonstrated. However, how MIT affects immune check-point molecules remains unknown. In this study, we found CD47 expression was higher in melanoma of pan-tissue array. MIT inhibited CD47 expression both in mRNA and protein level in melanoma cells (SK-MEL-28 and B16). MIT inhibited c-Myc, Sp-1 and CD47 expression in a concentration-dependent way. MIT inhibited the surface CD47 expression and promoted the phagocytosis of SK-MEL-28 cells by THP-1 cells. We found MIT inhibited tumor growth in melanoma allograft mice and CD47 expression in tumor mass. We also found MIT upregulated PD-L1 expression in cancer cells possibly via inhibiting PD-L1 ubiquitination, increasing ROS and IFN-γ. Combination of MIT and anti-PD-1 antibody showed enhanced antitumor activity compared to MIT and anti-PD-1 antibody alone in MC38 allograft mice. Using immune checkpoint array we found MIT inhibited expression of FasL and Galectin3. These results suggest that MIT inhibits CD47 expression, while improves PD-L1 expression. Furthermore, the combination of MIT and anti-PD-1 antibody exerts potent antitumor effect.
Topics: Animals; Antibiotics, Antineoplastic; B7-H1 Antigen; CD47 Antigen; Dose-Response Relationship, Drug; Female; Gene Expression; Humans; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Plicamycin; THP-1 Cells; Xenograft Model Antitumor Assays
PubMed: 34968486
DOI: 10.1016/j.bcp.2021.114894