ITIH5 inhibits proliferation, adipogenic differentiation, and secretion of inflammatory cytokines of human adipose stem cells-A new key in treating obesity?

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is widely expressed in the human body, and it is detected to be particularly abundant in adipose tissue. ITIH5 expression is increased in people with obesity compared to lean persons and is decreased by diet-induced weight loss. This suggests that ITIH5 may be involved in the development of adiposity and clinical metabolic variables, although its exact function remains unknown. We measured the protein concentration of ITIH5 in adipose samples from patients undergoing abdominoplasty and tested for correlation with the subjects' BMI as well as inflammatory mediators. We stimulated human adipose stem cells (ASCs) with recombinant (r)ITIH5 protein and tested for an effect on proliferation, differentiation, and immunosuppressive properties when the cells were exposed to an artificial inflammatory environment. We found positive correlations between ITIH5 levels and the BMI (p < .001) as well as concentrations of inflammatory cytokines (TNF-α, IL-6, and MCP-1) in adipose tissue (p < .01). Application of the rITIH5 protein inhibited both proliferation (p < .001) and differentiation of ASCs. Especially, the development of mature adipocytes was reduced by over 50%. Moreover, rITIH5 decreased the release of IL-6 and MCP-1 when the cells were exposed to TNF-α and IL-1ß (p < .001). Our data suggest that ITIH5 is an adipokine that is increasingly released during human adipose tissue development, acting as a regulator that inhibits proliferation and adipogenic differentiation of ASCs. ITIH5 thus presents itself as a positive regulator of adipose tissue homeostasis, possibly protecting against both hyperplasia and hypertrophy of adipose tissue and the associated chronic inflammation.

ITIH5, as a predictor of prognosis and immunotherapy response for P53-like bladder cancer, is related to cell proliferation and invasion.

p53-like bladder cancer (BLCA) is a bladder cancer subtype that is resistant to cisplatin-based chemotherapy. The ideal treatment modality for such tumors remains poorly defined, and immunotherapy seems to be a potential approach. Therefore, it is significant to understand the risk stratification of p53-like BLCA and identify novel therapeutic targets. ITIH5 is a member of the inter-α-trypsin inhibitory (ITI) gene family, and the effect of ITIH5 on p53-like BLCA remains elusive. In this study, TCGA data and in vitro experiments were used to explore the prognostic value of ITIH5 for p53-like BLCA and its effect on tumor cell proliferation, migration, and invasion. The impact of ITIH5 on the level of immune cell infiltration was explored using seven different algorithms, and the predictive value of ITIH5 on the efficacy of immunotherapy for p53-like BLCA was explored in combination with an independent immunotherapy cohort. The results showed that patients with high ITIH5 expression had a better prognosis, and overexpression of ITIH5 could inhibit the proliferation, migration, and invasion of tumor cells. Two or more algorithms consistently showed that ITIH5 promoted the infiltration of antitumor immune cells, such as B cells, CD4+ T cells, and CD8+ T cells. In addition, ITIH5 expression was positively correlated with the expression levels of many immune checkpoints, and the high ITIH5 expression group showed better response rates to PD-L1 and CTLA-4 therapies. In short, ITIH5 is a predictor of prognosis and the immunotherapy response for p53-like BLCA and is correlated with tumor immunity.

Secretory leukocyte protease inhibitor modulates FcεRI-dependent but not Mrgprb2-dependent mastocyte function in psoriasis.

Psoriasis, which involves mast cells, is a chronic inflammatory skin disorder whose pathophysiology is still not fully understood. We investigated the role of secretory leukocyte protease inhibitor (SLPI), a potential inhibitor of mastocyte serine proteases, on mast cell-dependent processes of relevance to the skin barrier defense in psoriasis. Here, we demonstrate that the dermal mast cells of patients with psoriasis express SLPI but not those of healthy donors. Moreover, SLPI transcripts were found to be markedly upregulated in murine mast cells by mediators derived from psoriasis skin explant cultures. Using mast cells from SLPI-deficient mice and their SLPI+ wild-type controls, we show that SLPI inhibits the activity of serine protease chymase in mastocytes. SLPI was also found to enhance the degranulation of mast cells activated via anti-IgE Abs but not Mrgprb2 ligands. Finally, we demonstrate that the expression and function of Mrgprb2 in mast cells are suppressed by a normal and, to a larger extent, psoriatic skin environment. Together, these findings reveal mechanisms underlying FcεRI- and Mrgprb2-dependent mast cell function that have not been described previously.

HAI-1 is required for the novel role of FGFBP1 in maintenance of cell morphology and F-actin rearrangement in human keratinocytes.

Formation and maintenance of skin barrier function require tightly controlled membrane-associated proteolysis, in which the integral membrane Kunitz-type serine protease inhibitor, HAI-1, functions as the primary inhibitor of the membrane-associated serine proteases, matriptase and prostasin. Previously, HAI-1 loss in HaCaT human keratinocytes resulted in an expected increase in prostasin proteolysis but a paradoxical decrease in matriptase proteolysis. The paradoxical decrease in shed active matriptase is further investigated in this study with an unexpected discovery of novel functions of fibroblast growth factor-binding protein 1 (FGFBP1), which acts as an extracellular ligand that can rapidly elicit F-actin rearrangement and subsequently affect the morphology of human keratinocytes. This novel growth factor-like function is in stark contrast to the canonical activity of this protein through interactions with FGFs for its pathophysiological functions. This discovery began with the observation that HAI-1 KO HaCaT cells lose the characteristic cobblestone morphology of the parental cells and exhibit aberrant F-actin formation along with altered subcellular targeting of matriptase and HAI-2. The alterations in cell morphology and F-actin status caused by targeted HAI-1 deletion can be restored by treatment with conditioned medium from parental HaCaT cells, in which FGFBP1 was identified by tandem mass spectrometry. Recombinant FGFBP1 down to 1 ng/ml was able to revert the changes caused by HAI-1 loss. Our study reveals a novel function of FGFBP1 in the maintenance of keratinocyte morphology, which depends on HAI-1.

Degradation of EGFR on lung epithelial cells by neutrophil elastase contributes to the aggravation of pneumococcal pneumonia.

Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia.

Possible role of combined therapy targeting MET and pro-HGF activation for renal cell carcinoma: analysis by human HGF-producing SCID mice.

MET is a high-affinity receptor tyrosine kinase of HGF (hepatocyte growth factor). HGF is secreted as an inactive single-chain precursor (pro-HGF), which requires proteolytic activation for conversion to an active form. HGF activator inhibitor (HAI)-2 is a transmembrane Kunitz-type serine protease inhibitor, which inhibits all pro-HGF-activating enzymes. In RCC, increased expression of MET and decreased expression of HAI-2 were reported to be poor prognostic factors. In the current study, we tried to inhibit the growth of RCC cells by dual inhibition of both MET phosphorylation and pro-HGF-activation using MET inhibitor and HAI-2 overexpression. A transgenic mouse model which expressed human HGF (HGF mouse) was used for in vivo analysis to evaluate the HGF/MET signaling axis accurately. Initially, doxycycline-induced HAI-2 overexpression RCC cells (786-O-HAI2) were prepared. The cells were cultured with pro-HGF, and inhibitory effect of MET inhibitor (SCC244) and HAI-2 was evaluated by phosphorylation of MET and cell proliferation. Next, the cells were subcutaneously implanted to HGF mice and the growth inhibition was determined by SCC244 and HAI-2. Single use of each inhibitor showed significant inhibition in MET phosphorylation, migration and proliferation of 786-O-HAI2 cells; however, the strongest effect was observed by combined use of both inhibitors. Although in vivo analysis also showed apparent downregulation of MET phosphorylation and growth inhibition in combined treatment, statistical significance was not observed compared with single use of MET inhibitor. Combined treatment with MET-TKI and HAI-2 suggested to consider as a candidate for new strong therapy for RCC.

The serine protease matriptase inhibits migration and proliferation in multiple myeloma cells.

BACKGROUND: Multiple myeloma (MM) is an incurable malignancy of plasma cells. The serine protease matriptase is frequently dysregulated in human carcinomas, which facilitates tumor progression and metastatic dissemination. The importance of matriptase in hematological malignancies is yet to be clarified. In this study, we aimed to characterize the role of matriptase in MM. MATERIALS AND METHODS: mRNA expression of matriptase and its inhibitors hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 was studied in primary MM cells from patient samples and human myeloma cell lines (HMCLs). We further investigated the effect of matriptase on migration and proliferation of myeloma cells in vitro. By use of the CoMMpass database, we assessed the clinical relevance of matriptase in MM patients. RESULTS: Matriptase was expressed in 96% of patient samples and all HMCLs tested. Overexpression of matriptase in vitro reduced proliferation, and significantly decreased cytokine-induced migration. Conversely, matriptase knockdown significantly enhanced migration. Mechanistically, overexpression of matriptase inhibited activation of Src kinase. CONCLUSIONS: Our findings may suggest a novel role of matriptase as a tumor suppressor in MM pathogenesis.

LncRNA SPINT1-AS1/miR-433-3p/E2F3 positive feedback loop promotes the KRAS-mutant colorectal cancer cell proliferation, migration and invasion.

Colorectal cancer (CRC) features high prevalence and mortality. Long non-coding RNAs (lncRNAs) exert nonnegligible roles in human cancer development. Nevertheless, the functions of most lncRNAs still remain unexplored. We currently focused on detecting the influence of SPINT1 antisense RNA 1 (SPINT1-AS1) on CRC development and investigating into the potential regulatory mechanism. RT-qPCR analysis first confirmed that SPINT1-AS1 exhibited high expression in KRAS-mutant (KRASMUT) CRC cells. Through series of functional experiments, we observed that knockdown of SPINT1-AS1 weakened KRASMUT CRC cell proliferation, migration and invasion. Afterwards, the implementation of mechanism assays help to verify that SPINT1-AS1 sequestered microRNA-433-3p (miR-433-3p) to regulate the expression of E2F transcription factor 3 (E2F3). Besides, E2F3 was validated to activate the transcription of SPINT1-AS1 in turn. Rescue experiments confirmed the functional influence of SPINT1-AS1/miR-433-3p/E2F3 on CRC cells. In summary, the molecular axis of SPINT1-AS1/miR-433-3p/E2F3 forms a positive loop which might become a potential biomarker in CRC.

Levels of secretory leukocyte protease inhibitor expression in acute wounds.

OBJECTIVE: Even with our best practices, we are frequently unable to prevent slow and stalled wound healing-particularly in people with impaired circulation and conditions such as diabetes. As a result, greater insight into the nature of wound healing and alternative treatment approaches is needed. An avenue that may be of particular promise is increasing understanding of the role of secretory leukocyte protease inhibitor (SLPI) as there is evidence that it enhances wound healing, its expression increases in response to inflammation and infection, and it exhibits anti-protease, anti-inflammatory, antiviral antibacterial and antifungal activities. METHOD: The response of SLPI levels to wounding and skin injury was assessed by taking punch skin biopsies from healthy volunteers and assessing the levels of SLPI at the site of injury at the time of wounding (baseline) as well as one, two, three, four, seven, nine and 12 weeks later. RESULTS: A total of 35 volunteers took part in the study. Significant elevations were found: levels of SLPI were greatly increased, 12 times that at baseline, and remained elevated at three weeks despite re-epithelialisation having occurred. CONCLUSION: These findings not only suggest that levels of SLPI rise rapidly following wounding, but that these elevations are sustained, and continue to increase even when re-epithelialisation has occurred. These results suggest that the role and potential benefits of this protease inhibitor deserve further exploration.

Insufficiency of hepatocyte growth factor activator inhibitor-1 confers lymphatic invasion of tongue carcinoma cells.

Hepatocyte growth factor (HGF) activator inhibitor type-1 (HAI-1), encoded by the SPINT1 gene, is a transmembrane protease inhibitor that regulates membrane-anchored serine proteases, particularly matriptase. Here, we explored the role of HAI-1 in tongue squamous cell carcinoma (TSCC) cells. An immunohistochemical study of HAI-1 in surgically resected TSCC revealed the cell surface immunoreactivity of HAI-1 in the main portion of the tumor. The immunoreactivity decreased in the infiltrative front, and this decrease correlated with enhanced lymphatic invasion as judged by podoplanin immunostaining. In vitro homozygous deletion of SPINT1 (HAI-1KO) in TSCC cell lines (HSC3 and SAS) suppressed the cell growth rate but significantly enhanced invasion in vitro. The loss of HAI-1 resulted in enhanced pericellular activities of proteases, such as matriptase and urokinase-type plasminogen activator, which induced activation of HGF/MET signaling in the co-culture with pro-HGF-expressing fibroblasts and plasminogen-dependent plasmin generation, respectively. The enhanced plasminogen-dependent plasmin generation was abrogated partly by matriptase silencing. Culture supernatants of HAI-1KO cells had enhanced potency for converting the proform of vascular endothelial growth factor-C (VEGF-C), a lymphangiogenesis factor, into the mature form in a plasminogen-dependent manner. Furthermore, HGF significantly stimulated VEGF-C expression in TSCC cells. Orthotopic xenotransplantation into nude mouse tongue revealed enhanced lymphatic invasion of HAI-1KO TSCC cells compared to control cells. Our results suggest that HAI-1 insufficiency leads to dysregulated pericellular protease activity, which eventually orchestrates robust activation of protease-dependent growth factors, such as HGF and VEGF-C, in a tumor microenvironment to contribute to TSCC progression.

Understanding HAIs: Ally proteins in the fight against cancer.

Understanding how HAI-1 and HAI-2 regulate the epithelial serine protease matriptase may hold the key to curing epithelial-derived cancer. HAIs are serine protease inhibitors that inhibit matriptase and have a poorly understood effect on the presence of matriptase protein in cells. In this issue of The FEBS Journal, Yamashita et al. provide much-needed new insights into this effect, describing it as a 'chaperone-like function' of HAI-1. However, several observations suggest that matriptase folds correctly without HAIs and that HAIs are not chaperones. We introduce the concept of 'ally proteins' to categorize the poorly understood function of HAIs, distinguishing them from chaperones. Comment on: https://doi.org/10.1111/febs.16348.

Role of the polycystic kidney disease domain in matriptase chaperone activity and localization of hepatocyte growth factor activator inhibitor-1.

Hepatocyte growth factor activator inhibitor-1 (HAI-1, also known as SPINT1) is an inhibitor of matriptase, a type-2 transmembrane protease widely expressed in epithelial cells. HAI-1 also functions as a chaperone to maintain the processing and localization of matriptase required for epithelial integrity. However, mechanisms underpinning the chaperone function remain to be elucidated. Here, we show that the first Kunitz domain (KD1) and the adjacent polycystic kidney disease (PKD) domain-like internal domain of HAI-1 are essential for the chaperone function. In HEK293T cells, which do not express endogenous HAI-1 or matriptase, forced matriptase overexpression was unsuccessful unless sufficient HAI-1 was co-expressed. Among mutant HAI-1 constructs, HAI-1 with inactivation mutation in KD1 (HAI-1mKD1) or HAI-1 lacking the PKD domain (HAI-1dPKD) was unable to support matriptase expression, and neither mutant formed a complex with activated matriptase. Matriptase did not localize to the cell surface when co-expressed with HAI-1dPKD. Moreover, HAI-1dPKD accumulated in the cytoplasm of HEK293T and HaCaT cells rather than localizing to the cell surface, presumably due to misfolding as judged by altered antibody recognition. On the other hand, activationlocked and activity-incompetent matriptase were stable and readily overexpressed and localized to the cell surface without HAI-1. Therefore, the observed matriptase instability was caused by its own catalytic activity in the absence of inhibitory HAI-1. The matriptase chaperone function of HAI-1 is thus mediated primarily by the inhibition of undesired intracellular matriptase activity, and the PKD domain is essential for the proper folding and trafficking of inhibitory HAI-1 and its chaperone function.

The Expression Profile of Protease Inhibitors in the Airway Epithelial Cells after Allergen (Der p 1) Stimulation.

BACKGROUND: Airway epithelial cells are constantly exposed to intracellular and extracellular proteases that play a pivotal role in several airway diseases. Dermatophagoides pteronyssinus (Der p) 1 derived from house dust mite has protease activity that causes epithelial barrier defect and inflammatory response. Protease inhibitors released against proteases are involved in the maintenance of homeostasis. A disruption of the balance between proteases and protease inhibitors can lead to distortion of the cellular structures and cellular activities and thus culminate in disease processes. Although the effects of Der p 1 allergen on epithelial barrier integrity and inflammatory response are well-established, its contribution to protease inhibitor production is highly limited. OBJECTIVE: This study aimed to determine the profile of the protease inhibitor response to Der p 1 allergen in human airway epithelial cells, A549 and BEAS-2B. METHODS: Differentiated cells by the air-liquid interface were exposed to Der p 1 with or without Th2 type cytokines (IL-4 and IL-13). Gene expression of protease inhibitors was determined by qPCR at 2 different time points. RESULTS: We found that the effect of allergen exposure on the protease inhibitor profile can vary depending on the antigen concentration, treatment duration, and the presence or absence of type 2 cytokines. Gene expressions of serine protease inhibitor (SERPIN)B3 and SERPINB4 were increased following Th2 cytokine stimulation in both cell types at both time points, whereas SERPINB2 and TFPI-2 expressions were induced by 24-h Der p 1 stimulation in both cells. CONCLUSIONS: Our study suggests that Der p 1 exposure of the airway epithelium may have consequences related to its protease activity in the presence as well as in the absence of Th2 cytokines in the microenvironment.

BST-1 as a serum protein biomarker involved in neutrophil infiltration in schizophrenia.

OBJECTIVES: Schizophrenia is a serious mental illness. The serum protein biomarkers of schizophrenia were explored using isobaric tags for relative and absolute quantitation (iTRAQ) technology. The underlying function of the identified protein biomarker was also investigated. METHODS: We first collected serum samples from 12 schizophrenia patients and 12 healthy control (HC) subjects, followed by global screening with iTRAQ and tandem mass spectrometry (LC-MS/MS). In total, 691 serum proteins were detected and eight proteins, including ZYX, OSCAR, TPM4, SDPR, BST1, ARGHDB, ITIH5 and SH3BGRL3, were selected for further specific validation with enzyme-linked immunosorbent assay (ELISA) on the serum samples from 52 schizophrenia patients and 50 HC subjects. RESULTS: Schizophrenia patients had significantly lower serum level of BST1 and higher ITIH5 level than the HC subjects did. Using the levels of BST1, ITIH5 and OSCAR combined with machine learning algorithm, we developed a prediction model of schizophrenia with an auROC value 0.78. Moreover, in vitro cell assay confirmed that BST1 significantly repressed neutrophil infiltration through endothelial layer, highlighted the anti-inflammation nature of BST1. CONCLUSIONS: Four novel protein markers (BST1, ITIH5, SDPR, and OSCAR) of schizophrenia were identified, and BST-1 could serve as a serum protein biomarker involved in neutrophil infiltration in schizophrenia.

The difference in the intracellular Arg/Lys-rich and EHLVY motifs contributes to distinct subcellular distribution of HAI-1 versus HAI-2.

The integral membrane, Kunitz-type, serine protease inhibitors, HAI-1 and HAI-2, closely resemble one another structurally and with regard to their specificity and potency against proteases. Structural complementarity between the Kunitz domains and serine protease domains renders the membrane-associated serine proteases, matriptase and prostasin, the primary target proteases of the HAIs. The shared biochemical enzyme-inhibitor relationships are, however, at odds with their behavior at the cellular level, where HAI-1 appears to be the default inhibitor of these proteases and HAI-2 a cell-type-selective inhibitor, even though they are widely co-expressed. The limited motility of these proteins caused by their membrane anchorages may require their co-localization within a certain distance to allow the establishment of a cellular level functional relationship between the proteases and the inhibitors. The differences in their subcellular localization with HAI-1 both inside the cell and on the cell surface, compared to HAI-2 predominately in intracellular granules has, therefore, been implicated in the differential manner of their control of matriptase and prostasin proteolysis. The targeting signals present in the intracellular domains of the HAIs are systematically investigated herein. Studies involving domain swap and point mutation, in combination with immunocytochemistry and cell surface biotinylation/avidin depletion, reveal that the different subcellular localization between the HAIs can largely be attributed to differences in the intracellular Arg/Lys-rich and EHLVY motifs. These intrinsic differences in the targeting signal render the HAIs as two independent rather than redundant proteolysis regulators.

Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development.

EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.

Alternative poly-adenylation modulates α1-antitrypsin expression in chronic obstructive pulmonary disease.

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3' end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3' UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αß-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

Proteomics changes after negative pressure wound therapy in diabetic foot ulcers.

Label­free quantitative mass spectrometry was used to analyze the differences in the granulation tissue protein expression profiles of patients with diabetic foot ulcers (DFUs) before and after negative­pressure wound therapy (NPWT) to understand how NPWT promotes the healing of diabetic foot wounds. A total of three patients with DFUs hospitalized for Wagner grade 3 were enrolled. The patients received NPWT for one week. The granulation tissue samples of the patients prior to and following NPWT for one week were collected. The protein expression profiles were analyzed with label­free quantitative mass spectrometry and the differentially expressed proteins (DEPs) in the DFU patients prior to and following NPWT for one week were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to annotate the DEPs and DEP­associated signaling pathways. Western blotting and ELISA were performed to validate the results. By comparing the differences in the protein profiles of granulation tissue samples prior to and following NPWT for one week, 36 proteins with significant differences were identified (P<0.05); 33 of these proteins were upregulated and three proteins were downregulated. NPWT altered proteins mainly associated with antioxidation and detoxification, the cytoskeleton, regulation of the inflammatory response, complement and coagulation cascades and lipid metabolism. The functional validation of the DEPs demonstrated that the levels of cathepsin S in peripheral blood and granulation tissue were significantly lower than those prior to NPWT (P<0.05), while the levels of protein S isoform 1, inter α­trypsin inhibitor heavy chain H4 and peroxiredoxin­2 in peripheral blood and granulation tissue were significantly higher than those prior to NPWT (P<0.05). The present study identified multiple novel proteins altered by NPWT and laid a foundation for further studies investigating the mechanism of action of NPWT.

Comprehensive Analysis of the Expression and Prognostic Value of SPINT1/2 in Breast Carcinoma.

Background: Hepatocyte growth factor (HGF) signaling plays a plethora of roles in tumorigenesis and progression in many cancer types. As HGF activator inhibitors, serine protease inhibitor, Kunitz types 1 and 2 (SPINT1 and SPINT2) have been reported to be differentially expressed in breast cancer, but their prognostic significance and functioning mechanism remain unclear. Methods: In our study, multiple databases and bioinformatics tools were used to investigate SPINT1/2 expression profiles, prognostic significance, genetic alteration, methylation, and regulatory network in breast carcinoma. Results: SPINT1/2 expression was upregulated in breast cancer, and was relatively higher in human epidermal growth factor receptor 2 (HER2) and node positive patients. Elevated SPINT1/2 expression was significantly correlated with a poorer prognosis. Genetic alterations and SPINT1/2 hypomethylation were observed. In breast carcinoma, SPINT1/2 were reciprocally correlated and shared common co-expressed genes. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that their common co-expressed genes were primarily involved in regulating cell attachment and migration. Conclusions: Our study identified the expression profiles, prognostic significance and potential roles of SPINT1/2 in breast carcinoma. These study results showed that the SPINT1/2 were potential prognostic biomarker for patients with breast cancer.