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Dipeptidyl peptidase-4 (DPP4) modulates inflammation by enzymatic cleavage of immunoregulatory peptides and through its soluble form (sDPP4) that directly engages immune cells. Here we examine whether reduction of DPP4 activity alters inflammation. Prolonged DPP4 inhibition increases plasma levels of sDPP4, and induces sDPP4 expression in lymphocyte-enriched organs in mice. Bone marrow transplantation experiments identify hematopoietic cells as the predominant source of plasma sDPP4 following catalytic DPP4 inhibition. Surprisingly, systemic DPP4 inhibition increases plasma levels of inflammatory markers in regular chow-fed but not in high fat-fed mice. Plasma levels of sDPP4 and biomarkers of inflammation are lower in metformin-treated subjects with type 2 diabetes (T2D) and cardiovascular disease, yet exhibit considerable inter-individual variation. Sitagliptin therapy for 12 months reduces DPP4 activity yet does not increase markers of inflammation or levels of sDPP4. Collectively our findings dissociate levels of DPP4 enzyme activity, sDPP4 and biomarkers of inflammation in mice and humans. DPP4 inhibitors are used for the treatment of diabetes, but the impact of DPP4 activity and soluble DPP4 on development of diabetes-associated inflammation remains uncertain. Here the authors study whether DPP4 inhibition controls sDPP4 and inflammatory biomarkers, and demonstrate that DPP4 inhibition is dissociated from changes in inflammation in mice and humans.

Vasopressin possesses potent anti-inflammatory capacity. Phosphoinositide 3-kinase (PI3K) and its downstream activator Akt contribute to endogenous anti-inflammation capacity. We sought to elucidate whether PI3K is involved in mediating the anti-inflammation effects of vasopressin. Macrophages (RAW264.7 cells) were randomized to receive endotoxin, endotoxin plus vasopressin, or endotoxin plus vasopressin plus the nonselective PI3K inhibitor (LY294002) or the selective isoform inhibitor of PI3Kα (PIK-75), PI3Kβ (TGX-221), PI3Kδ (IC-87114), or PI3Kγ (AS-252424). Compared to macrophages treated with endotoxin, the concentrations of cytokines (tumor necrosis factor-α, interleukin-6) and chemokine (macrophage inflammatory protein-2) in macrophages treated with endotoxin plus vasopressin were significantly lower (all P  < 0.05). The concentrations of phosphorylated nuclear factor-κB p65 (p-NF-κB p65) in nuclear extracts and phosphorylated inhibitor-κBα (p-I-κBα) in cytosolic extracts as well as NF-κB-DNA binding activity were also lower (all P  < 0.05). Of note, except for macrophages treated with endotoxin plus vasopressin plus PIK-75, the concentrations of cytokines, chemokine, p-NF-κB p65, and p-I-κBα as well as NF-κB-DNA binding activity in macrophages treated with endotoxin plus vasopressin plus LY294002, TGX-221, IC-87114, or AS-252424 were significantly higher than those in macrophages treated with endotoxin plus vasopressin (all P  < 0.05). In contrast, the phosphorylated Akt concentration in macrophages treated with endotoxin plus vasopressin was significantly higher than that in macrophages treated with endotoxin or in macrophages treated with endotoxin plus vasopressin plus LY294002, TGX-221, IC-87114, or AS-252424, but not PIK-75. These data confirmed that PI3K, especially the isoforms of PI3Kβ, PI3Kδ, and PI3Kγ, is involved in mediating the anti-inflammatory effects of vasopressin.

CD44 is a marker of cancer stem cell (CSC) in many types of tumors. Alternative splicing of its 20 exons generates various CD44 isoforms that have different tissue specific expression and functions, including the CD44 standard isoform (CD44s) encoded by the constant exons and the CD44 variant isoforms (CD44v) with variant exon insertions. Switching between the CD44v and CD44s isoforms plays pivotal roles in tumor progression. Here we reported a novel mechanism of CD44 alternative splicing induced by TGF-β1 and its connection to enhanced epithelial-to-mesenchymal transition (EMT) and stemness in human prostate cancer cells. TGF-β1 treatment increased the expression of CD44s and N-cadherin while decreased the expression of CD44v and E-cadherin in DU-145 prostate cancer cells. Other EMT markers and cancer stem cell markers were also upregulated after TGF-β1 treatment. RNAi knockdown of CD44 reversed the phenotype, which could be rescued by overexpressing CD44s but not CD44v, indicating the alternatively spliced isoform CD44s mediated the activity of TGF-β1 treatment. Mechanistically, TGF-β1 treatment induced the phosphorylation, poly-ubiquitination, and degradation of PCBP1, a well-characterized RNA binding protein known to regulate CD44 splicing. RNAi knockdown of PCBP1 was able to mimic TGF-β1 treatment to increase the expression of CD44s, as well as the EMT and cancer stem cell markers. In vitro and in vivo experiments were performed to show that CD44s promoted prostate cancer cell migration, invasion, and tumor initiation. Taken together, we defined a mechanism by which TGF-β1 induces CD44 alternative splicing and promotes prostate cancer progression.

Signal transducer and activator of transcription 3 (STAT3) is one of seven STAT family members involved with the regulation of cellular growth, differentiation and survival. STAT proteins are conserved among eukaryotes and are important for biological functions of embryogenesis, immunity, haematopoiesis and cell migration. STAT3 is widely expressed and located in the cytoplasm in an inactive form. STAT3 is rapidly and transiently activated by tyrosine phosphorylation by a range of signalling pathways, including cytokines from the IL‐6 family and growth factors, such as EGF and PDGF. STAT3 activation and subsequent dimer formation initiates nuclear translocation of STAT3 for the regulation of target gene transcription. Four STAT3 isoforms have been identified, which have distinct biological functions. STAT3 is considered a proto‐oncogene and constitutive activation of STAT3 is implicated in the development of various cancers, including multiple myeloma, leukaemia and lymphomas. In this review, we focus on recent progress on STAT3 and osteosarcoma (OS). Notably, STAT3 is overexpressed and associated with the poor prognosis of OS. Constitutive activation of STAT3 in OS appears to upregulate the expression of target oncogenes, leading to OS cell transformation, proliferation, tumour formation, invasion, metastasis, immune evasion and drug resistance. Taken together, STAT3 is a target for cancer therapy, and STAT3 inhibitors represent potential therapeutic candidates for the treatment of OS. Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family, vitally important for eukaryotic cells. We review the molecular structure and function of STAT3 and its isoforms, highlighting signalling pathways for the regulation of gene transcription. A critical appraisal of STAT3 in cancers, such as osteosarcoma, is provided emphasizing potential therapeutic approaches targeting STAT3 and its inhibitors

The Bromodomain and Extra-Terminal Domain (BET) family of proteins is characterized by the presence of two tandem bromodomains and an extra-terminal domain. The mammalian BET family of proteins comprises BRD2, BRD3, BRD4, and BRDT, which are encoded by paralogous genes that may have been generated by repeated duplication of an ancestral gene during evolution. Bromodomains that can specifically bind acetylated lysine residues in histones serve as chromatin-targeting modules that decipher the histone acetylation code. BET proteins play a crucial role in regulating gene transcription through epigenetic interactions between bromodomains and acetylated histones during cellular proliferation and differentiation processes. On the other hand, BET proteins have been reported to mediate latent viral infection in host cells and be involved in oncogenesis. Human BRD4 is involved in multiple processes of the DNA virus life cycle, including viral replication, genome maintenance, and gene transcription through interaction with viral proteins. Aberrant BRD4 expression contributes to carcinogenesis by mediating hyperacetylation of the chromatin containing the cell proliferation-promoting genes. BET bromodomain blockade using small-molecule inhibitors gives rise to selective repression of the transcriptional network driven by c-MYC These inhibitors are expected to be potential therapeutic drugs for a wide range of cancers. This review presents an overview of the basic roles of BET proteins and highlights the pathological functions of BET and the recent developments in cancer therapy targeting BET proteins in animal models.

Hypoxia-inducible factor (HIF), an αβ dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αβ dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin ( EPO ) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO . Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53 β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53 β , which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53 β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro , showed decreased Δ133p53 and increased p53 β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.

Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH. Parathyroid hormone (PTH) is an endogenous hormone and osteoporosis therapeutic that suppresses sclerostin activity. Here the authors develop SIK inhibitors as potential therapeutic tools and use them to show that PTH-cAMP signalling in osteocytes inhibits SIK2 from driving Hdac4/5 nuclear shuttling to suppress sclerostin.

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxini isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.

Protein arginine methyltransferases (PRMTs) catalyze the methyl transfer to the arginine residues of protein substrates and are classified into three major types based on the final form of the methylated arginine. Recent studies have shown a strong correlation between PRMT expression level and the prognosis of cancer patients. Currently, crystal structures of eight PRMT members have been determined. Kinetic and structural studies have shown that all PRMTs share similar, but unique catalytic and substrate recognition mechanism. In this review, we discuss the structural similarities and differences of different PRMT members, focusing on their overall structure, S-adenosyl-l-methionine-binding pocket, substrate arginine recognition and catalytic mechanisms. Since PRMTs are valuable targets for drug discovery, we also rationally classify the known PRMT inhibitors into five classes and discuss their mechanisms of action at the atomic level.