Transcriptional regulation of SARS-CoV-2 receptor ACE2 by SP1.

Angiotensin-converting enzyme 2 (ACE2) is a major cell entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The induction of ACE2 expression may serve as a strategy by SARS-CoV-2 to facilitate its propagation. However, the regulatory mechanisms of ACE2 expression after viral infection remain largely unknown. Using 45 different luciferase reporters, the transcription factors SP1 and HNF4α were found to positively and negatively regulate ACE2 expression, respectively, at the transcriptional level in human lung epithelial cells (HPAEpiCs). SARS-CoV-2 infection increased the transcriptional activity of SP1 while inhibiting that of HNF4α. The PI3K/AKT signaling pathway, activated by SARS-CoV-2 infection, served as a crucial regulatory node, inducing ACE2 expression by enhancing SP1 phosphorylation-a marker of its activity-and reducing the nuclear localization of HNF4α. However, colchicine treatment inhibited the PI3K/AKT signaling pathway, thereby suppressing ACE2 expression. In Syrian hamsters (Mesocricetus auratus) infected with SARS-CoV-2, inhibition of SP1 by either mithramycin A or colchicine resulted in reduced viral replication and tissue injury. In summary, our study uncovers a novel function of SP1 in the regulation of ACE2 expression and identifies SP1 as a potential target to reduce SARS-CoV-2 infection.

Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter.

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Specificity protein 1 (Sp1) and glucocorticoid receptor (GR) stimulate bovine alphaherpesvirus 1 (BoHV-1) replication and cooperatively transactivate the immediate early transcription unit 1 promoter.

Bovine alphaherpesvirus 1 (BoHV-1) infections cause respiratory tract disorders and suppress immune responses, which can culminate in bacterial pneumonia. Following acute infection, BoHV-1 establishes lifelong latency in sensory neurons present in trigeminal ganglia (TG) and unknown cells in pharyngeal tonsil. Latently infected calves consistently reactivate from latency after an intravenous injection of the synthetic corticosteroid dexamethasone (DEX), which mimics the effects of stress. The immediate early transcription unit 1 (IEtu1) promoter drives expression of infected cell protein 0 (bICP0) and bICP4, two key viral transcriptional regulators. The IEtu1 promoter contains two functional glucocorticoid receptor (GR) response elements (GREs), and this promoter is transactivated by GR, DEX, and certain Krüppel transcription factors that interact with GC-rich motifs, including consensus specificity protein 1 (Sp1) binding sites. Based on these observations, we hypothesized that Sp1 stimulates productive infection and transactivates key BoHV-1 promoters. DEX treatment of latently infected calves increased the number of Sp1+ TG neurons and cells in pharyngeal tonsil indicating that Sp1 expression is induced by stress. Silencing Sp1 protein expression with siRNA or mithramycin A, a drug that preferentially binds GC-rich DNA, significantly reduced BoHV-1 replication. Moreover, BoHV-1 infection of permissive cells increased Sp1 steady-state protein levels. In transient transfection studies, GR and Sp1 cooperatively transactivate IEtu1 promoter activity unless both GREs are mutated. Co-immunoprecipitation studies revealed that GR and Sp1 interact in mouse neuroblastoma cells (Neuro-2A) suggesting this interaction stimulates IEtu1 promoter activity. Collectively, these studies suggested that the cellular transcription factor Sp1 enhances productive infection and stress-induced BoHV-1 reactivation from latency.IMPORTANCEFollowing acute infection, bovine alphaherpesvirus 1 (BoHV-1) establishes lifelong latency in sensory neurons in trigeminal ganglia (TG) and pharyngeal tonsil. The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. The number of TG neurons and cells in pharyngeal tonsil expressing the cellular transcription factor specificity protein 1 (Sp1) protein increases during early stages of dexamethasone-induced reactivation from latency. Silencing Sp1 expression impairs BoHV-1 replication in permissive cells. Interestingly, mithramycin A, a neuroprotective antibiotic that preferentially binds GC-rich DNA, impairs Sp1 functions and reduces BoHV-1 replication suggesting that it is a potential antiviral drug. The glucocorticoid receptor (GR) and Sp1 cooperatively transactivate the BoHV-1 immediate early transcript unit 1 (IEtu1) promoter, which drives expression of infected cell protein 0 (bICP0) and bICP4. Mithramycin A also reduced Sp1- and GR-mediated transactivation of the IEtu1 promoter. These studies revealed that GR and Sp1 trigger viral gene expression and replication following stressful stimuli.

KLF6 activates Sp1-mediated prolidase transcription during TGF-ß1 signaling.

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline-the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor ß1 (TGF-ß1) signaling, since both KLF6 and Sp1 are key players in TGF-ß1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-ß1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-ß1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-ß1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

Magnolin targeting of the JNK/Sp1/MMP15 signaling axis suppresses cervical cancer microenvironment and metastasis via microbiota modulation.

Magnolin (MGL), a compound derived from the magnolia plant, has inhibitory effects on tumor cell invasion and growth. His study aims to explore the antitumor effect and underlying molecular mechanism of MGL against human cervical cancer. We found that MGL inhibited the proliferation, migration, and invasiveness of cervical cancer cells in vitro and in vivo. The underlying mechanism was shown to involve MGL-induced inhibition of JNK/Sp1-mediated MMP15 transcription and translation. Overexpression of JNK/Sp1 resulted in significant restoration of MMP15 expression and the migration and invasion capabilities of MGL-treated cervical cancer cells. MGL modulated the cervical cancer microenvironment by inhibiting cell metastasis via targeting IL-10/IL-10 receptor B (IL-10RB) expression, thereby attenuating JNK/Sp1-mediated MMP15 expression. Analysis of the gut microbiota of mice fed MGL revealed a significant augmentation in Lachnospiraceae bacteria, known for their production of sodium butyrate. In vivo experiments also demonstrated synergistic inhibition of cervical cancer cell metastasis by MGL and sodium butyrate co-administration. Our study provides pioneering evidence of a novel mechanism by which MGL inhibits tumor growth and metastasis through the IL-10/IL-10RB targeting of the JNK/Sp1/MMP15 axis in human cervical cancer cells.

MiR-29b Downregulation by p53/Sp1 Complex Plays a Critical Role in Bleb Scar Formation After Glaucoma Filtration Surgery.

Purpose: To investigate the function and mechanism of tumor protein p53 in pathological scarring after glaucoma filtration surgery (GFS) using human Tenon's fibroblasts (HTFs) and a rabbit GFS model. Methods: The expression of p53 in bleb scarring after GFS and transforming growth factor-ß (TGF-ß)-induced HTFs (myofibroblasts [MFs]) was examined by western blot and immunochemical analysis. The interaction between p53 and specificity protein 1 (Sp1) was investigated by immunoprecipitation. The role of p53 and Sp1 in the accumulation of collagen type I alpha 1 chain (COL1A1) and the migration of MFs was evaluated by western blot, quantitative real-time polymerase chain reaction (qRT-PCR), wound healing, and Transwell assay. The regulatory mechanisms among p53/Sp1 and miR-29b were detected via qRT-PCR, western blot, luciferase reporter assay, and chromatin immunoprecipitation assay. The therapeutic effect of mithramycin A, a specific inhibitor of Sp1, on scarring formation was evaluated in a rabbit GFS model. Results: p53 was upregulated in bleb scar tissue and MFs. p53 and Sp1 form a transcription factor complex that induces the accumulation of COL1A1 and promotes the migration of MFs through downregulation of miR-29b, a known suppressor of COL1A1. The p53/Sp1 axis inhibits miR-29b expression by the direct binding promoter of the miR-29b gene. Mithramycin A treatment attenuated bleb scar formation in vivo. Conclusions: The p53/Sp1/miR-29b signaling pathway plays a critical role in bleb scar formation after GFS. This pathway could be targeted for therapeutic intervention of pathological scarring after GFS. Translational Relevance: Our research indicates that inhibition of p53/Sp1/miR-29b is a promising therapeutic strategy for preventing post-GFS pathological scarring.

N-terminal domain of classical swine fever virus Npro induces proteasomal degradation of specificity protein 1 with reduced HDAC1 expression to evade from innate immune responses.

IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.

SGCE promotes breast cancer stemness by promoting the transcription of FGF-BP1 by Sp1.

Breast cancer stem cells are mainly responsible for poor prognosis, especially in triple-negative breast cancer (TNBC). In a previous study, we demonstrated that ε-Sarcoglycan (SGCE), a type Ⅰ single-transmembrane protein, is a potential oncogene that promotes TNBC stemness by stabilizing EGFR. Here, we further found that SGCE depletion reduces breast cancer stem cells, partially through inhibiting the transcription of FGF-BP1, a secreted oncoprotein. Mechanistically, we demonstrate that SGCE could interact with the specific protein 1 transcription factor and translocate into the nucleus, which leads to an increase in the transcription of FGF-BP1, and the secreted FBF-BP1 activates FGF-FGFR signaling to promote cancer cell stemness. The novel SGCE-Sp1-FGF-BP1 axis provides novel potential candidate diagnostic markers and therapeutic targets for TNBC.

Sp1 mediated the inhibitory effect of glutamate on pulmonary surfactant synthesis.

BACKGROUND: Studies have shown that the release of endogenous glutamate (Glu) participates in lung injury by activating N-methyl-D-aspartate receptor (NMDAR), but the mechanism is still unclear. This study was to investigate the effects and related mechanisms of Glu on the lipid synthesis of pulmonary surfactant (PS) in isolated rat lung tissues. METHODS: The cultured lung tissues of adult SD rats were treated with Glu. The amount of [3H]-choline incorporation into phosphatidylcholine (PC) was detected. RT-PCR and Western blot were used to detect the changes of mRNA and protein expression of cytidine triphosphate: phosphocholine cytidylyltransferase alpha (CCTα), a key regulatory enzyme in PC biosynthesis. Western blot was used to detect the expression of NMDAR1, which is a functional subunit of NMDAR. Specific protein 1 (Sp1) expression plasmids were used. After transfected with Sp1 expression plasmids, the mRNA and protein levels of CCTα were detected by RT-PCR and Western blot in A549 cells. After treated with NMDA and MK-801, the mRNA and protein levels of Sp1 were detected by RT-PCR and Western blot in A549 cells. RESULTS: Glu decreased the incorporation of [3H]-choline into PC in a concentration- and time- dependent manner. Glu treatment significantly reduced the mRNA and protein levels of CCTα in lungs. Glu treatment up-regulated NMDAR1 protein expression, and the NMDAR blocker MK-801 could partially reverse the reduction of [3H]-choline incorporation induced by Glu (10-4 mol/L) in lungs. After transfected with Sp1 plasmid for 30 h, the mRNA and protein expression levels of CCTα were increased and the protein expression of Sp1 was also up-regulated. After A549 cells were treated with NMDA, the level of Sp1 mRNA did not change significantly, but the expression of nucleus protein in Sp1 was significantly decreased, while the expression of cytoplasmic protein was significantly increased. However, MK-801could reverse these changes. CONCLUSIONS: Glu reduced the biosynthesis of the main lipid PC in PS and inhibited CCTα expression by activating NMDAR, which were mediated by the inhibition of the nuclear translocation of Sp1 and the promoter activity of CCTα. In conclusion, NMDAR-mediated Glu toxicity leading to impaired PS synthesis may be a potential pathogenesis of lung injury.

Sp1 Upregulation Bolsters the Radioresistance of Glioblastoma Cells by Promoting Double Strand Breaks Repair.

Radioresistance remains a critical obstacle in the clinical management of glioblastoma (GBM) by radiotherapy. Therefore, it is necessary to explore the molecular mechanisms underlying radioresistance to improve patient response to radiotherapy and increase the treatment efficacy. The present study aimed to elucidate the role of specificity protein 1 (Sp1) in the radioresistance of GBM cells. Different human GBM cell lines and tumor-bearing mice were exposed to ionizing radiation (IR). Cell survival was determined by the colony formation assay. The expression of genes and proteins in the cells and tissues was analyzed by RT-PCR and western blotting, respectively. The γ-H2AX, p-Sp1 and dependent protein kinase catalytic subunit (DNA-PKcs phospho S2056) foci were analyzed by immunofluorescence. Apoptotic rates were measured by flow cytometry. Sp1 was upregulated after IR in vitro and in vivo and knocking down Sp1-sensitized GBM cells to IR. Sp1 activated the DNA-PKcs promoter and increased its expression and activity. Furthermore, the loss of Sp1 delayed double-strand breaks (DSB) repair and increased IR-induced apoptosis of GBM cells. Taken together, IR upregulates Sp1 expression in GBM cells, enhancing the activity of DNA-PKcs and promoting IR-induced DSB repair, thereby leading to increased radioresistance.

Immune checkpoint B7-H3 is a therapeutic vulnerability in prostate cancer harboring PTEN and TP53 deficiencies.

Checkpoint immunotherapy has yielded meaningful responses across many cancers but has shown modest efficacy in advanced prostate cancer. B7 homolog 3 protein (B7-H3/CD276) is an immune checkpoint molecule and has emerged as a promising therapeutic target. However, much remains to be understood regarding B7-H3's role in cancer progression, predictive biomarkers for B7-H3-targeted therapy, and combinatorial strategies. Our multi-omics analyses identified B7-H3 as one of the most abundant immune checkpoints in prostate tumors containing PTEN and TP53 genetic inactivation. Here, we sought in vivo genetic evidence for, and mechanistic understanding of, the role of B7-H3 in PTEN/TP53-deficient prostate cancer. We found that loss of PTEN and TP53 induced B7-H3 expression by activating transcriptional factor Sp1. Prostate-specific deletion of Cd276 resulted in delayed tumor progression and reversed the suppression of tumor-infiltrating T cells and NK cells in Pten/Trp53 genetically engineered mouse models. Furthermore, we tested the efficacy of the B7-H3 inhibitor in preclinical models of castration-resistant prostate cancer (CRPC). We demonstrated that enriched regulatory T cells and elevated programmed cell death ligand 1 (PD-L1) in myeloid cells hinder the therapeutic efficacy of B7-H3 inhibition in prostate tumors. Last, we showed that B7-H3 inhibition combined with blockade of PD-L1 or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) achieved durable antitumor effects and had curative potential in a PTEN/TP53-deficient CRPC model. Given that B7-H3-targeted therapies have been evaluated in early clinical trials, our studies provide insights into the potential of biomarker-driven combinatorial immunotherapy targeting B7-H3 in prostate cancer, among other malignancies.

Astragalus polysaccharide ameliorates steroid-induced osteonecrosis of the femoral head by regulating miR-200b-3p-mediated Wnt/ß-catenin signaling pathway via inhibiting SP1 expression : Astragalus polysaccharide regulates SONFH via SP1.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is the necrosis of the femur bone caused by prolonged and massive use of corticosteroids. The present study probed into the significance of Astragalus polysaccharide (APS) in SONFH progression. METHODS: SONFH cell model was constructed using murine long bone osteocyte Y4 (MLO-Y4) cells and then treated with APS. mRNA microarray analysis selected differentially expressed genes between control group and SONFH group. RT-qPCR determined SP1 and miR-200b-3p expression. Levels of SP1, ß-catenin, autophagy-related proteins (LC3II/LC3I, Beclin1, p62) and apoptosis-related proteins (Bax, C-caspase3, C-caspase9, Bcl-2) were tested by Western blot. ChIP and luciferase reporter assays confirmed relationship between SP1 and miR-200b-3p. Fluorescence intensity of LC3 in cells was detected by immunofluorescence. Flow cytometry assessed cell apoptosis. Osteonecrosis tissues from SONFH mice were examined by HE and TRAP staining. RESULTS: APS induced autophagy and suppressed apoptosis in SONFH cell model. APS inhibited SP1 expression and SP1 overexpression reversed effects of APS on SONFH cell model. Mechanistically, SP1 targeted miR-200b-3p to inhibit Wnt/ß-catenin pathway. MiR-200b-3p depletion rescued the promoting effect of SP1 on SONFH cell model by activating Wnt/ß-catenin pathway. HE staining showed that APS treatment reduced the empty lacunae and alleviated inflammation in trabecular bone of SONFH mice. TRAP staining revealed decreased osteoclasts number in SONFH mice after APS treatment. CONCLUSION: APS regulated osteocyte autophagy and apoptosis via SP1/miR-200b-3p axis and activated Wnt/ß-catenin signaling, thereby alleviating SONFH, shedding new insights for therapy of SONFH.

Sp1 promotes tumour progression by remodelling the mitochondrial network in cervical cancer.

BACKGROUND: Cervical cancer remains one of the most prevalent cancers worldwide. Accumulating evidence suggests that specificity protein 1 (Sp1) plays a pivotal role in tumour progression. The underlying role and mechanism of Sp1 in tumour progression remain unclear. METHODS: The protein level of Sp1 in tumour tissues was determined by immunohistochemistry. The effect of Sp1 expression on the biological characteristics of cervical cancer cells was assessed by colony, wound healing, transwell formation, EdU, and TUNEL assays. Finally, the underlying mechanisms and effects of Sp1 on the mitochondrial network and metabolism of cervical cancer were analysed both in vitro and in vivo. RESULTS: Sp1 expression was upregulated in cervical cancer. Sp1 knockdown suppressed cell proliferation both in vitro and in vivo, while overexpression of Sp1 had the opposite effects. Mechanistically, Sp1 facilitated mitochondrial remodelling by regulating mitofusin 1/2 (Mfn1/2), OPA1 mitochondrial dynamin-like GTPase (Opa1), and dynamin 1-like (Drp1). Additionally, the Sp1-mediated reprogramming of glucose metabolism played a critical role in the progression of cervical cancer cells. CONCLUSIONS: Our study demonstrates that Sp1 plays a vital role in cervical tumorigenesis by regulating the mitochondrial network and reprogramming glucose metabolism. Targeting Sp1 could be an effective strategy for the treatment of cervical cancer.

JWA inhibits nicotine-induced lung cancer stemness and progression through CHRNA5/AKT-mediated JWA/SP1/CD44 axis.

Cigarette smoking is an independent risk factor for lung cancer. Nicotine, as an addictive substance in tobacco and e-cigarettes, is known to promote tumor progression and metastasis despite being a non-carcinogen. As a tumor suppressor gene, JWA is widely involved in the inhibition of tumor growth and metastasis and the maintenance of cellular homeostasis, including in non-small cell lung cancer (NSCLC). However, the role of JWA in nicotine-induced tumor progression remains unclear. Here, we reported for the first time that JWA was significantly downregulated in smoking-related lung cancer and associated with overall survival. Nicotine exposure reduced JWA expression in a dose-dependent manner. Gene Set Enrichment Analysis (GSEA) analysis showed the tumor stemness pathway was enriched in smoking-related lung cancer, and JWA was negatively associated with stemness molecules CD44, SOX2, and CD133. JWA also inhibited nicotine-enhanced colony formation, spheroid formation, and EDU incorporation in lung cancer cells. Mechanically, nicotine downregulated JWA expression via the CHRNA5-mediated AKT pathway. Lower JWA expression enhanced CD44 expression through inhibition of ubiquitination-mediated degradation of Specificity Protein 1 (SP1). The in vivo data indicated that JAC4 through the JWA/SP1/CD44 axis inhibited nicotine-triggered lung cancer progression and stemness. In conclusion, JWA via down-regulating CD44 inhibited nicotine-triggered lung cancer cell stemness and progression. Our study may provide new insights to develop JAC4 for the therapy of nicotine-related cancers.

Copper induces liver lipotoxicity disease by up-regulating Nrf2 expression via the activation of MTF-1 and inhibition of SP1/Fyn pathway.

Excessive copper (Cu) intake leads to hepatic lipotoxicity disease, which has adverse effects on health, but the underlying mechanism is unclear. We found that Cu increased lipotoxicity by promoting Nrf2 recruitment to the ARE site in the promoters of five lipogenic genes (g6pd, 6pgd, me, icdh and pparγ). We also found that Cu affected the Nrf2 expression via different pathways: metal regulatory transcription factor 1 (MTF-1) mediated the Cu-induced Nrf2 transcriptional activation; Cu also enhanced the expression of Nrf2 by inhibiting the SP1 expression, which was achieved by inhibiting the negative regulator Fyn of Nrf2. These promoted the enrichment of Nrf2 in the nucleus and ultimately affected lipotoxicity. Thus, for the first time, we elucidated that Cu induced liver lipotoxicity disease by up-regulating Nrf2 expression via the MTF-1 activation and the inhibition of SP1/Fyn pathway. Our study elucidates the Cu-associated obesity and NAFLD for fish and possibly humans.

Sp1-like protein KLF13 acts as a negative feedback regulator of TGF-ß signaling and fibrosis.

Transforming growth factor ß (TGF-ß) is the primary factor that drives fibrosis in most forms of chronic kidney disease. The aim of this study was to identify endogenous regulators of TGF-ß signaling and fibrosis. Here, we show that tubulointerstitial fibrosis is aggravated by global deletion of KLF13 and attenuated by adeno-associated virus-mediated KLF13 overexpression in renal tubular epithelial cells. KLF13 recruits a repressor complex comprising SIN3A and histone deacetylase 1 (HDAC1) to the TGF-ß target genes, limiting the profibrotic effects of TGF-ß. Temporary upregulation of TGF-ß induces KLF13 expression, creating a negative feedback loop that triggers the anti-fibrotic effect of KLF13. However, persistent activation of TGF-ß signaling reduces KLF13 levels through FBXW7-mediated ubiquitination degradation and HDAC-dependent mechanisms to inhibit KLF13 transcription and offset the anti-fibrotic effect of KLF13. Collectively, our data demonstrate a role of KLF13 in regulating TGF-ß signaling and fibrosis.

Regulation of USP25 by SP1 Associates with Amyloidogenesis.

BACKGROUND: Trisomy 21, an extra copy of human chromosome 21 (HSA21), causes most Down's syndrome (DS) cases. Individuals with DS inevitably develop Alzheimer's disease (AD) neuropathological phenotypes after middle age including amyloid plaques and tau neurofibrillary tangles. Ubiquitin Specific Peptidase 25 (USP25), encoding by USP25 gene located on HSA21, is a deubiquitinating enzyme, which plays an important role in both DS and AD pathogenesis. However, the regulation of USP25 remains unclear. OBJECTIVE: We aimed to determine the regulation of USP25 by specificity protein 1 (SP1) in neuronal cells and its potential role in amyloidogenesis. METHODS: The transcription start site and promoter activity was identified by SMART-RACE and Dual-luciferase assay. Functional SP1-responsive elements were examined by EMSA. USP25 expression was examined by RT-PCR and immunoblotting. Student's t-test or one-way ANOVA were applied or statistical analysis. RESULTS: The transcription start site of human USP25 gene was identified. Three functional SP1 responsive elements in human USP25 gene were revealed. SP1 promotes USP25 transcription and subsequent USP25 protein expression, while SP1 inhibition significantly reduces USP25 expression in both non-neuronal and neuronal cells. Moreover, SP1 inhibition dramatically reduces amyloidogenesis. CONCLUSION: We demonstrates that transcription factor SP1 regulates USP25 gene expression, which associates with amyloidogenesis. It suggests that SP1 signaling may play an important role in USP25 regulation and contribute to USP25-mediated DS and AD pathogenesis.

X-ray inhibits FUT4-mediated proliferation in A549 cells by downregulating SP1 expression.

Objective: To identify the mechanism of down-regulation of Lewis Y antigen caused by X-ray irradiation. METHODS: The present original research study was conducted at Zhejiang University City College, Hangzhou, Republic of China, from 2020 to 2022. Western blotting, Co-immunoprecipitation (CO-IP), electrophoretic mobility shift assay and Cell Counting Kit-8 (CCK8) were performed to confirm the effect of X-ray irradiation on A549 cell proliferation and its mechanism. Data was analysed using Statistical Package for Social Sciences (SPSS) 11.5. RESULTS: The expressions of fucosyltransferase IV and Lewis Y were decreased after X-ray irradiation, thus inhibiting the proliferation of A549 lung cancer cells. Deoxyribonucleic acid damage caused by the irradiation caused higher level of poly- adenosinediphosphate-ribosylated Specific Protein 1(SP1), and translocation of SP1 from the nucleus, decreasing the expression of fucosyltransferase IV and Lewis Y. Conclusion: There was a significant role of glycosylation in radiation therapy for lung cancer.

Specificity Proteins (Sp) and Cancer.

The specificity protein (Sp) transcription factors (TFs) Sp1, Sp2, Sp3 and Sp4 exhibit structural and functional similarities in cancer cells and extensive studies of Sp1 show that it is a negative prognostic factor for patients with multiple tumor types. In this review, the role of Sp1, Sp3 and Sp4 in the development of cancer and their regulation of pro-oncogenic factors and pathways is reviewed. In addition, interactions with non-coding RNAs and the development of agents that target Sp transcription factors are also discussed. Studies on normal cell transformation into cancer cell lines show that this transformation process is accompanied by increased levels of Sp1 in most cell models, and in the transformation of muscle cells into rhabdomyosarcoma, both Sp1 and Sp3, but not Sp4, are increased. The pro-oncogenic functions of Sp1, Sp3 and Sp4 in cancer cell lines were studied in knockdown studies where silencing of each individual Sp TF decreased cancer growth, invasion and induced apoptosis. Silencing of an individual Sp TF was not compensated for by the other two and it was concluded that Sp1, Sp3 and Sp4 are examples of non-oncogene addicted genes. This conclusion was strengthened by the results of Sp TF interactions with non-coding microRNAs and long non-coding RNAs where Sp1 contributed to pro-oncogenic functions of Sp/non-coding RNAs. There are now many examples of anticancer agents and pharmaceuticals that induce downregulation/degradation of Sp1, Sp3 and Sp4, yet clinical applications of drugs specifically targeting Sp TFs are not being used. The application of agents targeting Sp TFs in combination therapies should be considered for their potential to enhance treatment efficacy and decrease toxic side effects.

Specificity protein 1-mediated ACSL4 transcription promoted the osteoarthritis progression through suppressing the ferroptosis of chondrocytes.

BACKGROUND: Chondrocytes are the main cell damage type involved in the occurrence and development of osteoarthritis (OA). Ferroptosis has been confirmed to be related to many degenerative diseases. This research aimed to explore the role of Sp1 and ACSL4 in ferroptosis in the IL-1ß-treated human chondrocyte cells line (HCCs). METHODS: The cell viability was detected with CCK8 assay. The ROS, MDA, GSH, and Fe2+ levels were assessed with corresponding detecting kits. The Col2a1, Acan, Mmp13, Gpx4 and Tfr1 levels were determined by RT-qPCR assay. Western blot was conducted to evaluate the Acsl4 and Sp1 levels. PI staining was carried out to analyze the cell death. The double luciferase report was conducted to verify the interaction between Acsl4 and Sp1. RESULTS: The results showed that IL-1ß stimulation elevated the LDH release, cell viability, ROS, MDA and Fe2+ levels and declined the GSH levels in the HCCs. Additionally, the mRNA levels of Col2a1, Acan, and Gpx4 were prominently decreased, while Mmp13 and Tfr1 were prominently elevated in the IL-1ß stimulated HCCs. Furthermore, Acsl4 protein levels were upregulated in the IL-1ß-stimulated HCCs. Both Acsl4 knockdown and ferrostatin-1 treatment neutralized the role of IL-1ß in the HCCs. What's more, Acsl4 was transcriptionally regulated by Specificity protein 1 (Sp1). Sp1 overexpression enhanced the Acsl4 levels and Sp1 knockdown declined it. CONCLUSION: Upregulation of Sp1 activates Ascl4 transcription and thus mediates the occurrence of ferroptosis. Hence, Acsl4 may be a therapeutic target for intervention of OA.