Aging-related defects in macrophage function are driven by MYC and USF1 transcriptional programs.

Macrophages are central innate immune cells whose function declines with age. The molecular mechanisms underlying age-related changes remain poorly understood, particularly in human macrophages. We report a substantial reduction in phagocytosis, migration, and chemotaxis in human monocyte-derived macrophages (MDMs) from older (>50 years old) compared with younger (18-30 years old) donors, alongside downregulation of transcription factors MYC and USF1. In MDMs from young donors, knockdown of MYC or USF1 decreases phagocytosis and chemotaxis and alters the expression of associated genes, alongside adhesion and extracellular matrix remodeling. A concordant dysregulation of MYC and USF1 target genes is also seen in MDMs from older donors. Furthermore, older age and loss of either MYC or USF1 in MDMs leads to an increased cell size, altered morphology, and reduced actin content. Together, these results define MYC and USF1 as key drivers of MDM age-related functional decline and identify downstream targets to improve macrophage function in aging.

Systematic analysis reveals distinct roles of USF family proteins in various cancer types.

BACKGROUND: Upstream stimulatory factors (USFs) are members of the basic helix-loop-helix leucine zipper transcription factor family, including USF1, USF2, and USF3. The first two members have been well studied compared to the third member, USF3, which has received scarce attention in cancer research to date. Despite a recently reported association of its alteration with thyroid carcinoma, its expression has not been previously analyzed. METHODS: We comprehensively analyzed differential levels of USFs expression, genomic alteration, DNA methylation, and their prognostic value across different cancer types and the possible correlation with tumor-infiltrating immune cells and drug response by using different bioinformatics tools. RESULTS: Our findings established that USFs play an important role in cancers related to the urinary system and justify the necessity for further investigation. We implemented and offer a useful ShinyApp to facilitate researchers' efforts to inquire about any other gene of interest and to perform the analysis of drug response in a user-friendly fashion at http://zzdlab.com:3838/Drugdiscovery/.

Long non-coding RNA X-Inactive Specific Transcript (XIST) interacting with USF2 promotes osteogenic differentiation of periodontal ligament stem cells through regulation of WDR72 transcription.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) are the most potential cells in periodontal tissue regeneration and bone tissue regeneration. Our prior work had revealed that WD repeat-containing protein 72 (WDR72) was crucial for osteogenic differentiation of PDLSCs. Here, we further elucidated its underlying mechanism in PDLSC osteogenic differentiation. METHODS: Human PDLSCs, isolated and identified by flow cytometry, were prepared for osteogenic differentiation induction. Levels of WDR72, long non-coding RNA X-Inactive Specific Transcript (XIST), upstream stimulatory factor 2 (USF2), and osteogenic marker genes (Runx2, Osteocalcin, and Collagen I) in human PDLSCs and clinical specimens were detected by RT-qPCR. Protein expressions of WDR72, Runx2, Osteocalcin, and Colla1 were tested by Western blot. The interactions among the molecules were verified by RIP, RNA pull-down, ChIP, and luciferase reporter assays. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red staining (ARS). RESULTS: WDR72 was decreased in periodontal tissues of periodontitis patients, and overexpression reversed TNF-α-mediated suppressive effects on PDLSC osteogenic differentiation. Mechanically, XIST recruited the enrichment of USF2 to the WDR72 promoter region, thereby positively regulating WDR72. WDR72 silencing overturned XIST-mediated biological effects in PDLSCs. CONCLUSION: WDR72, regulated by the XIST/USF2 axis, enhances osteogenic differentiation of PDLSCs, implying a novel strategy for alleviating periodontitis.

Syntaxin-6 promotes the progression of hepatocellular carcinoma and alters its sensitivity to chemotherapies by activating the USF2/LC3B axis.

Syntaxin-6 (STX6), a protein of the syntaxin family, is located in the trans-Golgi network and is involved in a variety of intracellular membrane transport events. STX6 is overexpressed in different human malignant tumors. However, little is known about its exact function and molecular mechanism in hepatocellular carcinoma (HCC). In this study, we found that the expression of STX6 was significantly increased in HCC tissues and was associated with poor survival. Gain- and loss-of-function experiments showed that STX6 promotes cell proliferation and metastasis of HCC cells both in vitro and in vivo. Mechanistically, STX6 was negatively regulated by the upstream stimulatory factor 2 (USF2). In addition, STX6 facilitates the association of autophagosomes with lysosomes. Importantly, we demonstrated that STX6 overexpression, despite enhanced resistance to lenvatinib, sensitizes HCC cells to the autophagy activator rapamycin. This study revealed that, under the control of USF2, STX6 accelerates the degradation of microtubule-associated protein 1 light chain 3 beta (LC3) by promoting autophagic flux, ultimately promoting HCC progression. Collectively, we suggest that the USF2-STX6-LC3B axis is a potential therapeutic target in liver cancer.

USF1-mediated ALKBH5 stabilizes FLII mRNA in an m6A-YTHDF2-dependent manner to repress glycolytic activity in prostate adenocarcinoma.

Upstream-stimulating factor 1 (USF1) is a ubiquitously expressed transcription factor implicated in multiple cellular processes, including metabolism and proliferation. This study focused on the function of USF1 in glycolysis and the malignant development of prostate adenocarcinoma (PRAD). Bioinformatics predictions suggested that USF1 is poorly expressed in PRAD. The clinical PRAD samples revealed a low level of USF1, which was correlated with an unfavorable prognosis. Artificial upregulation of USF1 significantly repressed glycolytic activity in PRAD cells and reduced cell growth and metastasis in vitro and in vivo. Potential downstream genes of USF1 were probed by integrated bioinformatics analyses. The chromatin immunoprecipitation and luciferase assays indicated that USF1 bound to the α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) promoter for transcription activation. Flightless I (FLII) was identified as the gene showing the highest degree of correlation with ALKBH5. As an m6A demethylase, ALKBH5 enhanced FLII mRNA stability by inducing m6A demethylation in an m6A-YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2)-dependent manner. Either silencing of ALKBH5 or FLII blocked the role of USF1 in PARD cells and restored glycolysis, cell proliferation, and invasion. This study demonstrates that USF1 activates ALKBH5 to stabilize FLII mRNA in an m6A-YTHDF2-dependent manner, thereby repressing glycolysis processes and the progression of PRAD.

A natural variation-based screen in mouse cells reveals USF2 as a regulator of the DNA damage response and cellular senescence.

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-κB, p53, and C/EBPß. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory Mus musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, upstream stimulatory factor 2 (USF2), for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program-shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

WDR3 promotes stem cell-like properties in prostate cancer by inhibiting USF2-mediated transcription of RASSF1A.

BACKGROUND: WD repeat domain 3 (WDR3) is involved in tumor growth and proliferation, but its role in the pathological mechanism of prostate cancer (PCa) is still unclear. METHODS: WDR3 gene expression levels were obtained by analyzing databases and our clinical specimens. The expression levels of genes and proteins were determined by a real-time polymerase chain reaction, western blotting and immunohistochemistry, respectively. Cell-counting kit-8 assays were used to measure the proliferation of PCa cells. Cell transfection was used to investigate the role of WDR3 and USF2 in PCa. Fluorescence reporter and chromatin immunoprecipitation assays were used to detect USF2 binding to the promoter region of RASSF1A. Mouse experiments were used to confirm the mechanism in vivo. RESULTS: By analyzing the database and our clinical specimens, we found that WDR3 expression was significantly increased in PCa tissues. Overexpression of WDR3 enhanced PCa cell proliferation, decreased cell apoptosis rate, increased spherical cell number and increased indicators of stem cell-like properties. However, these effects were reversed by WDR3 knockdown. WDR3 was negatively correlated with USF2, which was degraded by promoting ubiquitination of USF2, and USF2 interacted with promoter region-binding elements of RASSF1A to depress PCa stemness and growth. In vivo studies showed that WDR3 knockdown reduced tumor size and weight, reduced cell proliferation and enhanced cell apoptosis. CONCLUSIONS: WDR3 ubiquitinated USF2 and inhibited its stability, whereas USF2 interacted with promoter region-binding elements of RASSF1A. USF2 transcriptionally activated RASSF1A, which inhibited the carcinogenic effect of WDR3 overexpression.

Endothelial PTP4A1 mitigates vascular inflammation via USF1/A20 axis-mediated NF-κB inactivation.

AIMS: The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs. METHODS AND RESULTS: In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1ß-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs. CONCLUSION: Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.

USF2-mediated upregulation of TXNRD1 contributes to hepatocellular carcinoma progression by activating Akt/mTOR signaling.

Thioredoxin reductase 1 (TXNRD1) is one of the major redox regulators in mammalian cells, which has been reported to be involved in tumorigenesis. However, its roles and regulatory mechanism underlying the progression of HCC remains poorly understood. In this study, we demonstrated that TXNRD1 was significantly upregulated in HCC tumor tissues and correlated with poor survival in HCC patients. Functional studies indicated TXNRD1 knockdown substantially suppressed HCC cell proliferation and metastasis both in vitro and in vivo, and its overexpression showed opposite effects. Mechanistically, TXNRD1 attenuated the interaction between Trx1 and PTEN which resulting in acceleration of PTEN degradation, thereby activated Akt/mTOR signaling and its target genes which conferred to elevated HCC cell mobility and metastasis. Moreover, USF2 was identified as a transcriptional suppressor of TXNRD1, which directly interacted with two E-box sites in TXNRD1 promoter. USF2 functioned as tumor suppressor through the downstream repression of TXNRD1. Further clinical data revealed negative co-expression correlations between USF2 and TXNRD1. In conclusion, our findings reveal that USF2-mediated upregulation of TXNRD1 contributes to hepatocellular carcinoma progression by activating Akt/mTOR signaling.

SEZ6L2, regulated by USF1, accelerates the growth and metastasis of breast cancer.

Breast cancer (BC) is the second cause of cancer-related mortality in women. Seizure related 6 homolog like 2 (SEZ6L2), a protein presented on cell surface, is involved in tumor development. It was found to be highly expressed in BC, however, its role in BC remains unclear. Herein, we aimed to explore the role of SEZ6L2 in BC. Firstly, the correlationship between SEZ6L2 expression and the clinic pathological characteristics of patients diagnosed with BC was analyzed. Subsequently, the role of SEZ6L2 was further explored using MTT, transwell invasion, flow cytometry, colony formation and wound healing assays. The result showed that the level of SEZ6L2 was remarkably correlated with the TNM stage, HER-2 status and lymph node metastasis of BC. Knockdown of SEZ6L2 significantly suppressed the proliferation of BC cells and induced cell cycle arrest at G1 phase. In addition, SEZ6L2 knockdown repressed their migration and invasion. On the contrary, SEZ6L2 overexpression performed the opposite effects. Furthermore, SEZ6L2 also accelerated the in vivo tumorigenesis of BC cells. Additionally, according to bioinformatics resources, we identified upstream transcription factor 1 (USF1) as a transcriptional factor which bound to the promoter of SEZ6L2 and positively regulated its transcription. In conclusion, this study demonstrated that SEZ6L2 was transcriptionally regulated by USF1 and was involved in the growth and metastasis of BC cells. Revealing the role of SEZ6L2 in BC provides additional knowledge for the pathogenesis of BC, which may benefit to BC therapy.

USF1-ATRAP-PBX3 Axis Promote Breast Cancer Glycolysis and Malignant Phenotype by Activating AKT/mTOR Signaling.

Angiotensin II type 1 receptor-associated protein (ATRAP) is widely expressed in different tissues and organs, although its mechanistic role in breast cancer remains unclear. Here, we show that ATRAP is highly expressed in breast cancer tissues. Its aberrant upregulation promotes breast cancer aggressiveness and is positively correlated with poor prognosis. Functional assays revealed that ATRAP participates in promoting cell growth, metastasis, and aerobic glycolysis, while microarray analysis showed that ATRAP can activate the AKT/mTOR signaling pathway in cancer progression. In addition, ATRAP was revealed to direct Ubiquitin-specific protease 14 (USP14)-mediated deubiquitination and stabilization of Pre-B cell leukemia homeobox 3 (PBX3). Importantly, ATRAP is a direct target of Upstream stimulatory factor 1 (USF1), and that ATRAP overexpression reverses the inhibitory effects of USF1 knockdown. Our study demonstrates the broad contribution of the USF1/ATRAP/PBX3 axis to breast cancer progression and provides a strong potential therapeutic target.

USF1/CD90 signaling in maintaining glioblastoma stem cells and tumor-associated macrophages adhesion.

BACKGROUND: Glioblastoma stem cells (GSCs) and their interplay with tumor-associated macrophages (TAMs) are responsible for malignant growth and tumor recurrence of glioblastoma multiforme (GBM), but the underlying mechanisms are largely unknown. METHODS: Cell viability, stemness, migration, and invasion were measured in GSCs after the knockdown of upstream stimulating factor 1 (USF1). Luciferase assay and chromatin immunoprecipitation qPCR were performed to determine the regulation of CD90 by USF1. Immunohistochemistry and immunofluorescent staining were used to examine the expression of USF1 and GSC markers, as well as the crosstalk between GSCs and TAMs. In addition, the interaction between GSCs and TAMs was confirmed using in vivo GBM models. RESULTS: We show that USF1 promotes malignant glioblastoma phenotypes and GSCs-TAMs physical interaction by inducing CD90 expression. USF1 predicts a poor prognosis for glioma patients and is upregulated in patient-derived GSCs and glioblastoma cell lines. USF1 overexpression increases the proliferation, invasion, and neurosphere formation of GSCs and glioblastoma cell lines, while USF1 knockdown exerts an opposite effect. Further mechanistic studies reveal that USF1 promotes GSC stemness by directly regulating CD90 expression. Importantly, CD90 of GSCs functions as an anchor for physical interaction with macrophages. Additionally, the USF1/CD90 signaling axis supports the GSCs and TAMs adhesion and immunosuppressive feature of TAMs, which in turn enhance the stemness of GSCs. Moreover, the overexpression of CD90 restores the stemness property in USF1 knockdown GSCs and its immunosuppressive microenvironment. CONCLUSIONS: Our findings indicate that the USF1/CD90 axis might be a potential therapeutic target for the treatment of glioblastoma.

hsa-miR-875-5p inhibits tumorigenesis and suppresses TGF-ß signalling by targeting USF2 in gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common malignancies, and an increasing number of studies have shown that its pathogenesis is regulated by various miRNAs. In this study, we investigated the role of miR-875-5p in GC. METHODS: The expression of miR-875-5p was detected in human GC specimens and cell lines by miRNA qRT-PCR. The effect of miR-875-5p on GC proliferation was determined by Cell Counting Kit-8 (CCK-8) proliferation and 5-ethynyl-2'-deoxyuridine (EdU) assays. Migration and invasion were examined by transwell migration and invasion assays as well as wound healing assays. The interaction between miR-875-5p and its target gene upstream stimulatory factor 2(USF2) was verified by dual luciferase reporter assays. The effects of miR-875-5p in vivo were studied in xenograft nude mouse models. Related proteins were detected by western blot. RESULTS: The results showed that miR-875-5p inhibited the proliferation, migration and invasion of GC cells in vitro and inhibited tumorigenesis in vivo. USF2 was proved to be a direct target of miR-875-5p. Knockdown of USF2 partially counteracted the effects of miR-875-5p inhibitor. Overexpression of miR-875-5p could inhibit proliferation, migration and invasion and suppress the TGF-ß signalling pathway by downregulating USF2. CONCLUSIONS: MiR-875-5p can inhibit the progression of GC by directly targeting USF2. And in the future, miR-875-5p is expected to be a potential target for GC diagnosis and treatment.

Upstream stimulatory factor 2 (USF2) induced upregulation of triggering receptor expressed on myeloid cells 1 (TREM1) promotes endometritis by regulating toll-like receptor (TLR) 2/4-nuclear factor-kappaB (NF-κB) signaling pathway.

Triggering receptor expressed on myeloid cells 1 (TREM1) participates in the development of endometritis. This study aims at identifying the effects and interaction of TREM1 and upstream stimulatory factor 2 (USF2) in endometritis by using a model of lipopolysaccharide (LPS)-induced human endometrial epithelial cells (HEnEpCs). ELISA was performed to determine the levels of interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF-α) after LPS stimulation. TREM1 and USF2 expression was examined with RT-qPCR and Western blot. The JASPAR database was employed to predict the binding site between USF2 and TREM1, which was confirmed by luciferase reporter and chromatin immunoprecipitation assays. After TREM1 overexpression, IL-6, IL-1ß, and TNF-α expression was detected by ELISA. Next, the binding of TREM1 to toll-like receptor (TLR) 2/4 was examined with co-immunoprecipitation. Then, proteins in TLR2/4-nuclear factor-kappaB (NF-κB) signaling in HEnEpCs under LPS condition were assessed by Western blot or immunofluorescence before and after TREM1 knockdown. Finally, TLR2 or TLR4 was silenced to explore whether intervene TLR2/4-NF-κB signaling pathway could rescue TREM1-overexpression-induced inflammation in LPS-induced HEnEpCs. Results revealed that upregulated TREM1 was observed in LPS-challenged HEnEpCs. Next, USF2 was found to have transcriptionally active TREM1 expression. Additionally, USF2 knockdown decreased the levels of IL-6, IL-1ß, and TNF-α, whereas this effect was rescued after TREM1 overexpression. Besides, TREM1 could bind to TLR2/4 to regulate NF-κB signaling. Moreover, the intervention of TLR2/4-NF-κB signaling pathway rescued TREM1-overexpression-induced inflammation in LPS-stimulated HEnEpCs. Collectively, USF2 promotes endometritis by upregulating TREM1, thereby activating TLR2/4-NF-κB pathway.

Targeting radiation-induced upstream stimulatory factor-1 by histone deacetylase inhibitors to reverse radioresistance in prostate cancer.

BACKGROUND: Ionizing radiation (IR) is a standard modality for the management of solid tumors. Apart from its killing effects, IR can induce pro-survival factors leading to radioresistance of cancer. Mechanistic understanding of radiation resistance is warranted to overcome the pro-survival effects of IR. AIM: The aim of this study was to investigate the role of upstream stimulatory factor-1 (USF-1) in the induction of radioresistance in prostate cancer and its targeting by histone deacetylase (HDAC) inhibitors to reverse resistance. METHODS AND RESULTS: This study reports here that USF-1 is a marker for radioresistance in PC-3 cells. Using protein-DNA array analysis, it was documented that DNA binding activity of USF-1 was elevated following IR in PC-3 cells. Novel HDAC inhibitors downregulated USF-1 binding either alone or in combination with IR. A 5 Gy dose of IR induced the expression of target genes of USF-1 (human telomerase reverse transcriptase [hTERT], IGF2R, CyclinB1, and Cdk1), however, HDAC inhibitors alone or in combination with IR reduced their expression as measured by real time RT PCR analysis. Furthermore, immunofluorescence analysis revealed that while USF-1 localized primarily in the nucleus following IR, it localized in the cytoplasm when treated with HDAC inhibitors/combination. Maximum effects of modulation of USF-1 expression (overexpression or suppression) were observed on hTERT activity as determined by dual-luciferase reporter assay. To further confirm the role of USF-1 in radioresistance, cell growth was analyzed using the real-time cell electronic sensing (RT-CES) system. This study found that USF-1-transfected cells proliferated faster than the vector-transfected cells with or without treatments with HDAC inhibitors/IR/combination. Colony forming assay also confirmed that USF-1 overexpression led to increased survival following IR. Importantly, colony-forming assay demonstrated that HDAC inhibitors reversed the radioresistance in both PC-3 and DU-145 cells. CONCLUSION: These studies demonstrate that HDAC inhibitors reverse the radioresistance in prostate cancer through down-modulation of USF-1-mediated transactivation of target genes involved in cell proliferation and cell cycle.

Identification of KLF6/PSGs and NPY-Related USF2/CEACAM Transcriptional Regulatory Networks via Spinal Cord Bulk and Single-Cell RNA-Seq Analysis.

BACKGROUND: To further understand the development of the spinal cord, an exploration of the patterns and transcriptional features of spinal cord development in newborn mice at the cellular transcriptome level was carried out. METHODS: The mouse single-cell sequencing (scRNA-seq) dataset was downloaded from the GSE108788 dataset. Single-cell RNA-Seq (scRNA-Seq) was conducted on cervical and lumbar spinal V2a interneurons from 2 P0 neonates. Single-cell analysis using the Seurat package was completed, and marker mRNAs were identified for each cluster. Then, pseudotemporal analysis was used to analyze the transcription changes of marker mRNAs in different clusters over time. Finally, the functions of these marker mRNAs were assessed by enrichment analysis and protein-protein interaction (PPI) networks. A transcriptional regulatory network was then constructed using the TRRUST dataset. RESULTS: A total of 949 cells were screened. Single-cell analysis was conducted based on marker mRNAs of each cluster, which revealed the heterogeneity of neonatal mouse spinal cord neuronal cells. Functional analysis of pseudotemporal trajectory-related marker mRNAs suggested that pregnancy-specific glycoproteins (PSGs) and carcinoembryonic antigen cell adhesion molecules (CEACAMs) were the core mRNAs in cluster 3. GSVA analysis then demonstrated that the different clusters had differences in pathway activity. By constructing a transcriptional regulatory network, USF2 was identified to be a transcriptional regulator of CEACAM1 and CEACAM5, while KLF6 was identified to be a transcriptional regulator of PSG3 and PSG5. This conclusion was then validated using the Genotype-Tissue Expression (GTEx) spinal cord transcriptome dataset. CONCLUSIONS: This study completed an integrated analysis of a single-cell dataset with the utilization of marker mRNAs. USF2/CEACAM1&5 and KLF6/PSG3&5 transcriptional regulatory networks were identified by spinal cord single-cell analysis.

Hematopoietic upstream stimulating factor 1 deficiency is associated with increased atherosclerosis susceptibility in LDL receptor knockout mice.

Total body upstream stimulatory factor 1 (USF1) deficiency in mice is associated with brown adipose tissue activation and a marked protection against the development of obesity and atherosclerotic lesions. Functional expression of USF1 has also been detected in monocytes and monocyte-derived macrophages. In the current study we therefore tested whether selective hematopoietic USF1 deficiency can also beneficially impact the development of atherosclerosis. For this purpose, LDL receptor knockout mice were transplanted with bone marrow from USF1 knockout mice or their wild-type littermate controls and subsequently fed a Western-type diet for 20 weeks to stimulate atherosclerotic lesion development. Strikingly, absence of USF1 function in bone marrow-derived cells was associated with exacerbated blood leukocyte (+ 100%; P < 0.01) and peritoneal leukocyte (+ 50%; P < 0.05) lipid loading and an increased atherosclerosis susceptibility (+ 31%; P < 0.05). These effects could be attributed to aggravated hyperlipidemia, i.e. higher plasma free cholesterol (+ 33%; P < 0.001) and cholesteryl esters (+ 39%; P < 0.001), and the development of hepatosteatosis. In conclusion, we have shown that hematopoietic USF1 deficiency is associated with an increased atherosclerosis susceptibility in LDL receptor knockout mice. These findings argue against a contribution of macrophage-specific USF1 deficiency to the previously described beneficial effect of total body USF1 deficiency on atherosclerosis susceptibility in mice.

[USF1 Suppresses Expression of Fibrillar Type I, II, and III Collagen and pNP Adamts-3 in Osteosarcoma Cells].

Collagens are the main components of human tissues. Various regulatory factors and cytokines may influence expression levels for collagen-encoding genes, and, therefore, contrubite to some collagen-associated pathologies. In this study, we demonstrate regulatory effects of USF1 on expression of genes encoding fibrillar collagen types I, II, and III in osteoblastic Saos-2 and MG-63 cells. An ectopic expression of the human USF1 led to a decrease in both mRNA and protein expression levels of the collagen-encoding genes mentioned above. ADAMTS-3 is a proteinase primarily responsible for the amino-terminal cleavage of type I and type II collagen precursors. The ADAMTS-3 promoter region contains potential binding sites for USF1. Here we show that an overexpression of USF1 lead to a decrease in ADAMTS-3 mRNA and protein expression levels. In co-transfection studies, USF1 negatively regulated ADAMTS-3 promoter activity. Further, in EMSA studies, we showed that USF1 binds to the ADAMTS-3 promoter region. In conclusion, it seems that ADAMTS-3 and USF1 contribute to the regulation of collagen encoding genes in osteosarcoma.

S100A8 promotes epithelial-mesenchymal transition and metastasis under TGF-ß/USF2 axis in colorectal cancer.

BACKGROUND: The transforming growth factor-ß (TGF-ß) pathway plays a pivotal role in inducing epithelial-mesenchymal transition (EMT), which is a key step in cancer invasion and metastasis. However, the regulatory mechanism of TGF-ß in inducing EMT in colorectal cancer (CRC) has not been fully elucidated. In previous studies, it was found that S100A8 may regulate EMT. This study aimed to clarify the role of S100A8 in TGF-ß-induced EMT and explore the underlying mechanism in CRC. METHODS: S100A8 and upstream transcription factor 2 (USF2) expression was detected by immunohistochemistry in 412 CRC tissues. Kaplan-Meier survival analysis was performed. In vitro, Western blot, and migration and invasion assays were performed to investigate the effects of S100A8 and USF2 on TGF-ß-induced EMT. Mouse metastasis models were used to determine in vivo metastasis ability. Luciferase reporter and chromatin immunoprecipitation assay were used to explore the role of USF2 on S100A8 transcription. RESULTS: During TGF-ß-induced EMT in CRC cells, S100A8 and the transcription factor USF2 were upregulated. S100A8 promoted cell migration and invasion and EMT. USF2 transcriptionally regulated S100A8 expression by directly binding to its promoter region. Furthermore, TGF-ß enhanced the USF2/S100A8 signaling axis of CRC cells whereas extracellular S100A8 inhibited the USF2/S100A8 axis of CRC cells. S100A8 expression in tumor cells was associated with poor overall survival in CRC. USF2 expression was positively related to S100A8 expression in tumor cells but negatively related to S100A8-positive stromal cells. CONCLUSIONS: TGF-ß was found to promote EMT and metastasis through the USF2/S100A8 axis in CRC while extracellular S100A8 suppressed the USF2/S100A8 axis. USF2 was identified as an important switch on the intracellular and extracellular S100A8 feedback loop.

Aberrant expression of USF2 in refractory rheumatoid arthritis and its regulation of proinflammatory cytokines in Th17 cells.

IL-17-producing Th17 cells are implicated in the pathogenesis of rheumatoid arthritis (RA) and TNF-α, a proinflammatory cytokine in the rheumatoid joint, facilitates Th17 differentiation. Anti-TNF therapy ameliorates disease in many patients with rheumatoid arthritis (RA). However, a significant proportion of patients do not respond to this therapy. The impact of anti-TNF therapy on Th17 responses in RA is not well understood. We conducted high-throughput gene expression analysis of Th17-enriched CCR6+CXCR3-CD45RA- CD4+ T (CCR6+ T) cells isolated from anti-TNF-treated RA patients classified as responders or nonresponders to therapy. CCR6+ T cells from responders and nonresponders had distinct gene expression profiles. Proinflammatory signaling was elevated in the CCR6+ T cells of nonresponders, and pathogenic Th17 signature genes were up-regulated in these cells. Gene set enrichment analysis on these signature genes identified transcription factor USF2 as their upstream regulator, which was also increased in nonresponders. Importantly, short hairpin RNA targeting USF2 in pathogenic Th17 cells led to reduced expression of proinflammatory cytokines IL-17A, IFN-γ, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as transcription factor T-bet. Together, our results revealed inadequate suppression of Th17 responses by anti-TNF in nonresponders, and direct targeting of the USF2-signaling pathway may be a potential therapeutic approach in the anti-TNF refractory RA.