ADAM17 mRNA Research Articles

Abstract 13466: In Experimental Pad miR29a Modulates Perfusion Recovery and ADAM12 Expression Through Direct Interaction With ADAM12 mRNA

Peripheral artery disease (PAD) is caused by atherosclerotic occlusion of vessels outside the heart and most commonly affects vessels of the lower extremities. Angiogenesis is a part of the post ischemic adaption involved in restoring blood flow in PAD. Previously, in a murine hind limb ischemia (HLI) model of PAD, we identified ADAM12 as a key genetic modifier of post-ischemic perfusion recovery. However, less is known about ADAM12 regulation in ischemia. MiRNAs are a class of small, non-coding, single-stranded RNAs that regulate gene expression primarily through transcriptional repression of mRNA. We showed miR29a modulates ADAM12 expression in the setting of type 1 DM and ischemia. However, how miR29a modulates ADAM12 was not known. Moreover, the physiological effects of miR29a modulation in a non-diabetic setting was not known. Here, we demonstrate that AAV-mediated ectopic overexpression of miR29a in ischemic mouse hind limbs impairs post ischemic perfusion recovery and angiogenesis. We further demonstrate that miR29a regulates ADAM12 through direct interaction with ADAM12 mRNA. Treatment of ischemic mouse hind limbs with AAV9 particles containing miR29a (pAV-miR29a) increased miR29a expression (pAV-control vs pAV-miR29a: 3.22 ± 0.36, vs 1.0 ± 0.09, p<0.05, n=5-6,) and decreased ADAM12 mRNA expression (1.0 ± 0.31 vs 0.30 ± 0.08, p<0.05, n=7-10) resulting in decreased angiogenesis (1.0 ± 0.07 vs 0.66 ± 0.04, p<0.05, n=5), and impairment perfusion (week 3 post HLI perfusion, 101.9±5.55 vs 66.13±12.39, p<0.05, n=5-6,).AGO2 miRNA/RNA-immunoprecipitation studies showed levels of miR29a and ADAM12 mRNA in the complex decreased in samples treated with antagomiR29a (4, 40, 400nM) in a dose dependent manner (miR29a: 0.87, 0.68, 0.44, and ADAM12: 0.89, 0.78, 0.67, fold) but was unchanged by control antagomir (miR29a: 0.84, 0.84, 0.87, fold, and ADAM12: 1.11, 1.12, 1.10, fold). Taken together the data shows miR29a suppresses ADAM12 expression by directly binding to its mRNA resulting in impaired angiogenesis and poor perfusion. Hence, elevated levels of miR29a as seen in diabetes likely contributes to vascular pathology and lowering miR29a could be a therapeutic target.

OOCYTE SECRETED FACTORS REGULATE THE EXPRESSION OF INDUCERS OF THE OVULATORY CASCADE IN CUMULUS CELLS

Considering that oocyte secreted factors (OSFs) seem to slow the passe of oocyte nuclear maturation by stimulating the production and delivery of cyclic guanosine monophosphate (cGMP) to the oocyte via increased expression of natriuretic peptide C (NPPC) in cumulus cells, we hypothesized that, as part of the same regulatory effort, OSFs would also inhibit the expression of the main inducers of the ovulatory cascade in cumulus cells, namely amphiregulin (AREG) and epiregulin (EREG), as well as of a metalloproteinase crucial for autocrine signal amplification (disintegrin and metalloprotease 17; ADAM17). The relative mRNA abundance of AREG, EREG and ADAM17 in cumulus cell was compared among 3 treatment-groups: (1) Intact COC subjected to in vitro maturation (IVM); (2) Oocytectomized COC (OOX) subjected to IVM and (3) OOX subjected to IVM with the addition of denuded oocytes (OOX+DO; 1 DO/μL). Four culture replicates including all three treatment-groups were performedCOC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by aspiration of the ooplasm with a micromanipulator while preserving the structure of the COC. COC and OOX underwent IVM in pools of 20 for 22 hours. IVM was performed in 100 μL of TCM199 with Earl’s salts supplemented with 1μg/mL FSH, 0.4% BSA, 22 μg/mL sodium pyruvate, 75 μg/mL of amikacin at 38,5°C and 5.5% CO2 in humid atmosphere. Abundance of mRNA encoding the target genes in cumulus cells was assessed by real-time RT-PCR using Power SybrGreen (LifeTech®) and RPL15 (ribosomal protein 15) as the reference gene. Effects of treatments were tested by ANOVA and groups were compared with the Tukey test. Statistically significant differences were those with p<0.05. Oocyctectomy increased AREG, EREG and ADAM17 mRNA levels (p<0.02), while the addition of secreting DO reversed the effects of oocytectomy on all genes. Relative mRNA levels (target gene/RPL15 mRNA abundance; mean ± SEM) in COC, OOX and OOX+DO, respectively, were: 0.91 ± 0.14, 7.53 ± 3.36 and 1.53 ± 0.64 for AREG; 0.87 ± 0.13, 3.15 ± 1.03 and 1.14 ± 0,08 for EREG; 1.22 ± 0.08, 1.64 ± 0.06 and 1,01 ± 0,12 for ADAM17. Oocyte secreted factors appear to reduce the expression of genes triggering the ovulatory cascade and final oocyte maturation. We speculate that this is part of an effort of the oocyte to prevent precocious meiotic resumption thus favouring nuclear and cytoplasmic maturation synchrony and developmental competence.

SARS‐CoV‐2 Viral Entry Proteins in Hyperandrogenemic Female Mice: Implications for Women with Polycystic Ovary Syndrome (PCOS) and COVID‐19

BackgroundSARS‐CoV‐2, the causative agent of COVID‐19, infects host cells using the angiotensin I converting enzyme 2 (ACE2) as its receptor after priming by host proteases, including the transmembrane serine protease TMPRSS2. Androgen action in target tissues may partially mediate entry of SAR‐CoV‐2 and male patients have been shown to suffer increased severity and mortality compared with females. Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in reproductive‐age women and is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. PCOS is associated with obesity and cardiometabolic dysfunctions, both being risk factors associated with severe COVID‐19 pathology. The purpose of this study is to investigate the role of hyperandrogenism in a female mouse model of PCOS and to characterize the differences in host machinery required for SARS‐CoV‐2 viral entry in multiple tissues that are affected by COVID‐19 in humans.MethodsFemale mice were treated with dihydrotestosterone (DHT, 8 mg) s.c. for 90 days. Body composition was measured by EchoMRI. Fasting glucose was determined by an enzymatic method and the oral glucose tolerance test was performed with an oral glucose load (2g glucose per kg lean mass weight). mRNA and protein levels of ACE2, Tmprss2, Cathepsin L, Furin, Tmprss4, and Adam17 were quantified by RT‐qPCR, Western‐blot, and ELISA in tissues, serum, and urine. All differences are considered significant p<0.05.ResultsDHT treatment increased body weight, fat and lean mass. DHT treated females showed altered glucose homeostasis, having increased fasting glucose (201.10 ± 11.11 vs. 152.80 ± 9.23 mg/dL, p<0.05) and an increased area under the curve (209.2 ± 11.0 vs. 160.8 ± 3.5 mg.min/dL, p<0.05) by OGTT. The small intestine was shown to have the greatest relative Ace2 mRNA expression compared with the other tissues. Ace2 mRNA was upregulated in DHT‐treated animals in the lung (1.24‐fold), cecum (4.4‐fold), left ventricle (1.54‐fold), and kidney (2.37‐fold), and was downregulated in DHT‐treated animals in the brain (0.68‐fold) and colon (0.37‐fold). ACE2 protein expression was increased in the small intestine (1.98‐fold), left ventricle (1.30‐fold), and kidney (1.32‐fold). The SARS‐CoV‐2 priming proteases Tmprss2, Cathepsin L, and Furin mRNA were upregulated by DHT in the kidney, and protein TMPRSS2 was increased in the small intestine (1.38‐fold). ACE2 sheddase Adam17 mRNA was upregulated in the kidney by DHT, which corresponded with increased urinary ACE2 in DHT‐treated mice (14.13±3.33 vs. 0.55±0.19 pg/ng creatinine, p<0.05).ConclusionsOur results highlight the potential for increased cardiac, renal, and gastrointestinal dysfunction in PCOS women with COVID‐19. The differences observed in androgen‐treated mice highlight that several mechanisms involved in SARS‐CoV‐2entry may be altered in women with PCOS and translate to the reduced protection of specific target organs known to be susceptible to COVID‐19 injury.

SARS-CoV-2 Viral Entry Proteins in Hyperandrogenemic Female Mice: Implications for Women with PCOS and COVID-19.

SARS-CoV-2, the causative agent of COVID-19, infects host cells using the angiotensin I converting enzyme 2 (ACE2) as its receptor after priming by host proteases, including TMPRSS2. COVID-19 affects multiple organ systems, and male patients suffer increased severity and mortality. Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology. PCOS is associated with obesity and cardiometabolic comorbidities, both being risk factors associated with severe COVID-19 pathology. We hypothesize that elevated androgens in PCOS regulate SARS-CoV-2 entry proteins in multiple tissues increasing the risk for this population. Female mice were treated with dihydrotestosterone (DHT) for 90 days. Body composition was measured by EchoMRI. Fasting glucose was determined by an enzymatic method. mRNA and protein levels of ACE2, Tmprss2, Cathepsin L, Furin, Tmprss4, and Adam17 were quantified by RT-qPCR, Western-blot, or ELISA in tissues, serum, and urine. DHT treatment increased body weight, fat and lean mass, and fasting glucose. Ace2 mRNA was upregulated in the lung, cecum, heart, and kidney, while downregulated in the brain by DHT. ACE2 protein was upregulated by DHT in the small intestine, heart, and kidney. The SARS-CoV-2 priming proteases Tmprss2, Cathepsin L, and Furin mRNA were upregulated by DHT in the kidney. ACE2 sheddase Adam17 mRNA was upregulated by DHT in the kidney, which corresponded with increased urinary ACE2 in DHT treated mice. Our results highlight the potential for increased cardiac, renal, and gastrointestinal dysfunction in PCOS women with COVID-19.

Sustained Surface ICAM-1 Expression and Transient PDGF-B Production by Phorbol Myristate Acetate-Activated THP-1 Cells Harboring Blau Syndrome-Associated NOD2 Mutations.

Objectives: Blau syndrome is a distinct class of autoinflammatory syndrome presenting with early-onset systemic granulomatosis. Blau syndrome-causing NOD2 mutations located in the central nucleotide-oligomerization domain induce ligand-independent basal NF-κB activation in an in vitro reporter assay. However, the precise role of this signaling on granuloma formation has not yet been clarified. Methods: Blau syndrome-causing NOD2 mutations were introduced into human monocytic THP-1 cells, and their morphological and molecular changes from parental cells were analyzed. Identified molecules with altered expression were examined in the patient’s lesional skin by immunostaining. Results: Although the production of proinflammatory cytokines was not altered without stimulation, mutant NOD2-expressing THP-1 cells attached persistently to the culture plate after stimulation with phorbol myristate acetate. Sustained surface ICAM-1 expression was observed in association with this phenomenon, but neither persistent ICAM-1 mRNA expression nor impaired ADAM17 mRNA expression was revealed. However, the transient induction of PDGF-B mRNA expression was specifically observed in stimulated THP-1 derivatives. In the granulomatous skin lesion of a Blau syndrome patient, ICAM-1 and PDGF-B were positively immunostained in NOD2-expressing giant cells. Conclusions: Sustained surface ICAM-1 expression and transient PDGF-B production by newly differentiating macrophages harboring mutant NOD2 might play a role in granuloma formation in Blau syndrome.

Involvement of the A disintegrin and metalloproteinase 9 in oral cancer cell invasion

The aims of this study were to determine the functional roles of the transmembrane glycoprotein, Disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), in the phosphorylation of epidermal growth factor receptor (EGFR) and AKT and in the aggressiveness of oral cancer cells. Immunohistochemistry and immunoblotting were conducted to determine expression of ADAM 9 and the levels of EGFR phosphorylated at the tyrosine 1173 residue (p-EGFRtyr1173 ) and AKT phosphorylated at the serine 473 residue (p-AKTser473 ) in oral cancer tissues and in the oral cancer cell lines HN5, HN6, HN15, and HN008. Small interfering RNA (siRNA) was used to inhibit expression of ADAM9 mRNA, and thus production of ADAM9 protein, in oral cancer cells. ADAM9-knockdown cells were examined for p-EGFRtyr1173 and p-AKTser473 levels and used for cell proliferation and invasion assays. A positive correlation among overexpression of ADAM 9, p-EGFRtyr1173 , and p-AKTser473 was found in oral cancer tissues. These biomolecules were also overexpressed in HN6 and HN15 cell lines.Expression of ADAM9 in HN6 and HN15 cells was statistically significantly inhibited by siRNA against ADAM9 mRNA (siADAM9) compared with the negative-control siRNA (scramble). The levels of p-AKTser473 , but not those of p-EGFRtyr1173 , were statistically significantly blocked by siADAM9. Although the proliferation rates of ADAM9 knocked-down HN6 and HN15 cells did not differ from those of cells exposed to scramble, a statistically significant decrease in cell invasion was found in these ADAM9-silenced cells. These results suggest a functional role of the ADAM 9/AKT signaling pathway in oral cancer cell invasion, which may be beneficial as a therapeutic target of oral cancer.

Expression of the Metalloproteinase ADAM8 Is Upregulated in Liver Inflammation Models and Enhances Cytokine Release In Vitro

Acute and chronic liver inflammation is driven by cytokine and chemokine release from various cell types in the liver. Here, we report that the induction of inflammatory mediators is associated with a yet undescribed upregulation of the metalloproteinase ADAM8 in different murine hepatitis models. We further show the importance of ADAM8 expression for the production of inflammatory mediators in cultured liver cells. As a model of acute inflammation, we investigated liver tissue from lipopolysaccharide- (LPS-) treated mice in which ADAM8 expression was markedly upregulated compared to control mice. In vitro, stimulation with LPS enhanced ADAM8 expression in murine and human endothelial and hepatoma cell lines as well as in primary murine hepatocytes. The enhanced ADAM8 expression was associated with an upregulation of TNF-α and IL-6 expression and release. Inhibition studies indicate that the cytokine response of hepatoma cells to LPS depends on the activity of ADAM8 and that signalling by TNF-α can contribute to these ADAM8-dependent effects. The role of ADAM8 was further confirmed with primary hepatocytes from ADAM8 knockout mice in which TNF-α and IL-6 induction and release were considerably attenuated. As a model of chronic liver injury, we studied liver tissue from mice undergoing high-fat diet-induced steatohepatitis and again observed upregulation of ADAM8 mRNA expression compared to healthy controls. In vitro, ADAM8 expression was upregulated in hepatoma, endothelial, and stellate cell lines by various mediators of steatohepatitis including fatty acid (linoleic-oleic acid), IL-1β, TNF-α, IFN-γ, and TGF-β. Upregulation of ADAM8 was associated with the induction and release of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CX3CL1). Finally, knockdown of ADAM8 expression in all tested cell types attenuated the release of these mediators. Thus, ADAM8 is upregulated in acute and chronic liver inflammation and is able to promote inflammation by enhancing expression and release of inflammatory mediators.

Pulmonary, cardiac and renal distribution of ACE2, furin, TMPRSS2 and ADAM17 in rats with heart failure: Potential implication for COVID-19 disease.

Congestive heart failure (CHF) is often associated with kidney and pulmonary dysfunction. Activation of the renin‐angiotensin‐aldosterone system (RAAS) contributes to avid sodium retention, cardiac hypertrophy and oedema formation, including lung congestion. While the status of the classic components of RAAS such as renin, angiotensin converting enzyme (ACE), angiotensin II (Ang II) and angiotensin II receptor AT‐1 is well studied in CHF, the expression of angiotensin converting enzyme‐2 (ACE2), a key enzyme of angiotensin 1‐7 (Ang 1‐7) generation in the pulmonary, cardiac and renal systems has not been studied thoroughly in this clinical setting. This issue is of a special interest as Ang 1‐7 counterbalance the vasoconstrictory, pro‐inflammatory and pro‐proliferative actions of Ang II. Furthermore, CHF predisposes to COVID‐19 disease severity, while ACE2 also serves as the binding domain of SARS‐CoV‐2 in human host‐cells, and acts in concert with furin, an important enzyme in the synthesis of BNP in CHF, in permeating viral functionality along TMPRSST2. ADAM17 governs ACE2 shedding from cell membranes. Therefore, the present study was designed to investigate the expression of ACE2, furin, TMPRSS2 and ADAM17 in the lung, heart and kidneys of rats with CHF to understand the exaggerated susceptibility of clinical CHF to COVID‐19 disease. Heart failure was induced in male Sprague Dawley rats by the creation of a surgical aorto‐caval fistula. Sham‐operated rats served as controls. One week after surgery, the animals were subdivided into compensated and decompensated CHF according to urinary sodium excretion. Both groups and their controls were sacrificed, and their hearts, lungs and kidneys were harvested for assessment of tissue remodelling and ACE2, furin, TMPRSS2 and ADAM17 immunoreactivity, expression and immunohistochemical staining. ACE2 immunoreactivity and mRNA levels increased in pulmonary, cardiac and renal tissues of compensated, but not in decompensated CHF. Furin immunoreactivity was increased in both compensated and decompensated CHF in the pulmonary, cardiac tissues and renal cortex but not in the medulla. Interestingly, both the expression and abundance of pulmonary, cardiac and renal TMPRSS2 decreased in CHF in correlation with the severity of the disease. Pulmonary, cardiac and renal ADAM17 mRNA levels were also downregulated in decompensated CHF. Circulating furin levels increased in proportion to CHF severity, whereas plasma ACE2 remained unchanged. In summary, ACE2 and furin are overexpressed in the pulmonary, cardiac and renal tissues of compensated and to a lesser extent of decompensated CHF as compared with their sham controls. The increased expression of the ACE2 in heart failure may serve as a compensatory mechanism, counterbalancing the over‐activity of the deleterious isoform, ACE. Downregulated ADAM17 might enhance membranal ACE2 in COVID‐19 disease, whereas the suppression of TMPRSS2 in CHF argues against its involvement in the exaggerated susceptibility of CHF patients to SARS‐CoV2.

MiR-708-3p Alleviates Inflammation and Myocardial Injury After Myocardial Infarction by Suppressing ADAM17 Expression.

MicroRNAs (miRNAs) emerge as important regulators for myocardial infarction (MI). However, the function of miR-708-3p during MI is unclear. H9c2 cells were cultured in a hypoxic environment and Sprague-Dawley rats experienced surgical ligation of the left anterior descending coronary artery to establish MI models. qPCR was used to measure the expression level of miR-708-3p and ADAM17 mRNA. ELISA was used to detect inflammatory cytokines TNF-α, IL-6, and IL-1β, and myocardial injury markers LDH, CK-MB, and cTnI. Cell apoptosis and viability were monitored by flow cytometry analysis and MTT assay. ADAM17 expression was detected by Western blot. Dual-luciferase reporter gene experiments were carried out to identify binding sites between miR-708-3p and ADAM17 3'UTR. In vivo, left ventricle functions and myocardial remodeling of the rats were measured by echocardiography. MiR-708-3p was found to be significantly decreased in H9c2 cells after hypoxia induction and in heart tissues of rats with MI or serum samples of patients with MI, while ADAM17 was upregulated. Overexpression of miR-708-3p inhibited inflammation and injury of H9c2 cells cultured in hypoxia and the heart of the rats with MI. ADAM17 was verified as a direct target of miR-708-3p and restoration of ADAM17 reversed the effects of miR-708-3p. MiR-708-3p alleviated the inflammation and injury of cardiomyocytes via targeting ADAM17.

Increased ADAM12 Expression Predicts Poor Prognosis in Cervical Cancer Patients before General Anesthesia.

The current study aims to investigate the expression and diagnostic value of ADAM12 in patients with cervical cancer before general anesthesia. Seventy-eight cases of cervical cancer patients were included in the present study. RT-PCR and western blot were used to detect the expression of ADAM12 in cervical cancer tissues and adjacent tissues. Meanwhile, the expression of secretory ADAM12 in serum of cervical cancer patients and healthy people was detected by ELISA. The relationship between ADAM12 expression and prognosis of cervical cancer patients was analyzed. ROC analysis was carried out to explore the diagnostic value of ADAM12. Our data showed that the expression of ADAM12 mRNA and protein in cervical cancer tissues was significantly up-regulated compared with the adjacent tissues. ELISA assay showed that the content of ADAM12 in serum of cervical cancer patients was significantly higher than that of healthy people. Furthermore, ADAM12 expression was closely related to tumor invasion, TNM stage, lymph node metastasis and tumor differentiation. Kaplan-Meier survival analysis showed that the overall survival rate of patients with high ADAM12 was significantly lower than that of patients with low ADAM12 expression. The AUC of ADAM12, CEA, CA125, and SCC for cervical cancer was 0.893, 0.510, 0.769 and 0.550, respectively, while the highest value of AUC was 0.946 by the combination of the four indexes. In summary, increased expression of ADAM12 in cervical cancer patients can be used as an independent prognostic marker.

Depression-Associated Gene Negr1-Fgfr2 Pathway Is Altered by Antidepressant Treatment.

The Negr1 gene has been significantly associated with major depression in genetic studies. Negr1 encodes for a cell adhesion molecule cleaved by the protease Adam10, thus activating Fgfr2 and promoting neuronal spine plasticity. We investigated whether antidepressants modulate the expression of genes belonging to Negr1-Fgfr2 pathway in Flinders sensitive line (FSL) rats, in a corticosterone-treated mouse model of depression, and in mouse primary neurons. Negr1 and Adam10 were the genes mostly affected by antidepressant treatment, and in opposite directions. Negr1 was down-regulated by escitalopram in the hypothalamus of FSL rats, by fluoxetine in the hippocampal dentate gyrus of corticosterone-treated mice, and by nortriptyline in hippocampal primary neurons. Adam10 mRNA was increased by nortriptyline administration in the hypothalamus, by escitalopram in the hippocampus of FSL rats, and by fluoxetine in mouse dorsal dentate gyrus. Similarly, nortriptyline increased Adam10 expression in hippocampal cultures. Fgfr2 expression was increased by nortriptyline in the hypothalamus of FSL rats and in hippocampal neurons. Lsamp, another IgLON family protein, increased in mouse dentate gyrus after fluoxetine treatment. These findings suggest that Negr1-Fgfr2 pathway plays a role in the modulation of synaptic plasticity induced by antidepressant treatment to promote therapeutic efficacy by rearranging connectivity in corticolimbic circuits impaired in depression.

FMRP Regulates the Nuclear Export of Adam9 and Psen1 mRNAs: Secondary Analysis of an N6-Methyladenosine Dataset

Fragile X mental retardation protein (FMRP) binds to and regulates the translation of amyloid-β protein precursor (App) mRNA, but the detailed mechanism remains to be determined. Differential methylation of App mRNA could underlie FMRP binding, message localization and translation efficiency. We sought to determine the role of FMRP and N6-methyladeonsine (m6A) on nuclear export of App mRNA. We utilized the m6A dataset by Hsu and colleagues to identify m6A sites in App mRNA and to determine if the abundance of message in the cytoplasm relative to the nucleus is altered in Fmr1 knockout mouse brain cortex. Given that processing of APP to Aβ and soluble APP alpha (sAPPα) contributes to disease phenotypes, we also investigated whether Fmr1KO associates with nuclear export of the mRNAs for APP protein processing enzymes, including β-site amyloid cleaving enzyme (Bace1), A disintegrin and metalloproteinases (Adams), and presenilins (Psen). Fmr1KO did not alter the nuclear/cytoplasmic abundance of App mRNA. Of 36 validated FMRP targets, 35 messages contained m6A peaks but only Agap2 mRNA was selectively enriched in Fmr1KO nucleus. The abundance of the APP processing enzymes Adam9 and Psen1 mRNA, which code for a minor alpha-secretase and gamma-secretase, respectively, were selectively enriched in wild type cytoplasm.

Metalloprotease ADAM10 inhibition mitigates acute liver injury via repression of intrahepatic inflammation.

Acute liver injury (ALI) is associated with the occurrence and progress of intrahepatic inflammation. Recent studies have shown that ADAM10, a significant member of metalloproteinase family, has modulated the inflammation level in various neurologic diseases. However, it is elusive whether ADAM10 regulation exert a hepatic protective effect on ALI by the suppression of inflammation level. The study aimed to explore the regulated function of ADAM10 on acute liver injury. C57BL/6J mice (eight-week-old male) were carried out intraperitoneal injection of tetrachloromethane (CTC) to provoke ALI. ADAM10 loaded in adeno-associated viral vectors (AAV-ADAM10) or short hairpin RNA loaded in lentivirus aimed at murine ADAM10 mRNA (sh-RNA-ADAM10) were respectively delivered to mice via tail intravenous injection to achieve overexpression or silence of ADAM10. Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunohistochemical (IHC) and hematoxylin and eosin (HE) staining were conducted to measure ADAM10 alteration, inflammation level, histology and liver function. We show that the expression of ADAM10 markedly increases in CTC-induced injured liver tissues. Moreover, we demonstrate that the knockdown of ADAM10 attenuates the intrahepatic inflammation and protects hepatic histology and function in ALI mice; however, the overexpression of ADAM10 aggravates inflammation and liver lesion. The above suggested that the inhibition of ADAM10 ameliorates ALI through inhibiting inflammation. Our research provides novel view on the ADAM10 modulation of process of ALI by the inflammation aspect and verify a potential target for the therapy of ALI in the future.

MiR-21-3p regulates AGE/RAGE signalling and improves diabetic atherosclerosis.

To explore the effects of miR-21-3p on diabetic atherosclerosis. Using enzyme-linked immunosorbent assay (ELISA), we also detected the levels of soluble receptor for advanced glycation endproducts RAGE (sRAGE) in the cellular supernatant of vascular endothelial cells after transfecting them with adenovirus vector having miR-21-3p mimic or inhibitor. We found decrease in the expression levels of miR-21-3p in vascular endothelial cells (VECs) induced by high-concentration glucose. We also observed that the introduction of miR-21-3p mimic significantly increased the expression of ADAM10 in the VECs. Similarly, significantly higher levels of sRAGE were found in the cultured supernatant after administration of miR-21-3p mimic in human vein endothelial cells. The production of reactive oxygen species and expression of inflammatory cytokines in VECs induced by LPS and high-concentration glucose were significantly decreased after administration of miR-21-3p. in vivo studies revealed that intravenous injection of miR-21-3p at regular intervals would reduce the area of atherosclerotic lesion and elevate the serum levels of sRAGE in atherosclerotic diabetic mice. miR-21-3p may be beneficial in diabetic atherosclerosis by promoting the cleaved form of sRAGE and inhibition of RAGE/NADPH oxidase signalling depending on the increased expression of ADAM10. SIGNIFICANCE OF THE STUDY: We identified a novel microRNA, miR-21-3p, which is characteristically at elevated levels in serum derived from diabetic patients and responsible for target degradation of ADAM10 mRNA. Further, we show that miR-21-3p aggravates the atherosclerotic lesion via dysfunction of the ectodomain shedding of molecular binding RAGE in the diabetic atherosclerotic mice.

MiR-126 inhibits cell migration and invasion by targeting ADAM9 in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC), the most frequent head and neck cancer, has a high potential for metastasis. MiR-126 plays an important role in the tumorigenesis of many tumors; however, there were little studies in OSCC. The purpose of our study was to explore how miR-126 and ADAM9 worked on migration and invasion in OSCC. The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was applied to detect the mRNA level of miR-126 and ADAM9. The transwell assay was utilized to calculate the migratory and invasive capacities in the OSCC cells. The luciferase report assay was utilized to verify that ADAM9 was a direct target of miR-126. MicroR-126 was downregulated in OSCC tissues and cell lines SCC25 and HSC3. ADAM9 was predicted to be a direct target of miR-126 and was upregulated in the OSCC cells. In addition, miR-126 suppressed the migratory and invasive ability via mediating the expression of ADAM9 by directly targeting its mRNA 3'-noncoding region (UTR), whose partial functions was reversed by ADAM9. MiR-126 inhibited the migratory and invasive capacities of OSCC by directly targeting the 3'-UTR of ADAM9 mRNA. It is suggested that miR-126/ADAM9 axis may play an essential role in inhibiting the abilities of migration and invasion in oral squamous cell carcinoma cells.

Tumor ADAM10/ADAM17-Mediated PD-L1 Loss May Predict Poor Outcomes in Diffuse Large B Cell Lymphoma

Introduction Tumor surface matrix metalloproteases ADAM10 and ADAM17 are associated with poor outcomes in multiple malignancies. We previously showed that these proteases shed PD-L1 from lymphoma cell line Karpas-299 to produce soluble PD-L1 (sPD-L1) that induces CD8+ T cell apoptosis (1). While higher levels of soluble PD-L1 are associated with worse prognosis in patients with non-Hodgkin lymphoma treated with standard chemoimmunotherapy (cutoff of 1.52 ng/ml, 3-year OS of 76% versus 89%) (2), it is unknown whether the loss of PD-L1 on the surface of B cell lymphoma cells affects clinical outcomes. We hypothesized that tumors expressing low PD-L1 levels despite high PD-L1 mRNA levels would (1) produce higher ADAM10 or ADAM17 transcript levels and (2) predict poorer prognosis in diffuse large B cell lymphomas. Methods We queried the cancer genome atlas (TCGA) public database for cases of diffuse large B cell lymphoma (DLBCL) for which PD-L1 (CD274) protein levels are reported by reverse phase protein array (RPPA). We then queried TCGA for RNA-Seq data for CD274, ADAM10, and ADAM17 expressed as transcripts per million (FPKM). We re-normalized RPPA data and calculated a PD-L1 protein-to-mRNA ratio for each tumor sample. We then compared groups of high and low PD-L1 protein-to-mRNA ratios for ADAM10 and ADAM17 mRNA expression. We further compared groups of high and low PD-L1 protein-to-mRNA ratios for survival (adjusted for age at diagnosis). All statistical analyses were performed using R Statistical Software (R Foundation). Unpaired student's t-test assessed statistical differences in experimental groups except where otherwise indicated. Figures comprise box plots showing quartile values and individual data points. P&lt;0.05 was considered statistically significant. In figures, p values are denoted &lt;0.05 with *, &lt;0.01 with **, and &lt;0.001 with ***. Results Data were available for a total of 33 diffuse large B cell (DLBC) lymphomas. 18 of those samples were categorized as having low PD-L1 protein-to-mRNA ratios (cutoff 6.38e-6) with the remaining categorized as high ratio. DLBCL tumors demonstrating low PD-L1 protein-to-mRNA ratios expressed significantly higher ADAM10 (p=0.0237) and ADAM17 (p&lt;0.0001) transcripts than tumors expressing low PD-L1 protein-to-mRNA ratios (Figure 1). Furthermore, patients with tumors demonstrating low PD-L1 protein-to-mRNA ratios experienced significantly poorer survival according to likelihood ratio (6.6, p=0.01) and log rank (4.67, p=0.03) tests (Figure 2). Conclusions Our findings may explain a crucial phenomenon seen in diffuse large B cell lymphoma, namely that many purportedly PD-L1-positive tumors appear to express PD-L1 but do not respond to PD-(L)1 inhibitor therapy (3,4). This may be caused by the activity of ADAM10 and ADAM17 to cleave PD-L1, which we previously showed in lymphoma cell line Karpas-299 (1). This posits pre-treatment ADAM10/ADAM17 inhibition to "boost" PD-(L)1 inhibitor therapy in lymphoma. Our results contrast with previous findings that PD-L1-positive DLBCL progression free survival is inferior to PD-L1-negative DLBCL (5). This may be due to different methods of protein detection (immunohistochemistry versus RPPA) and the lack of localization of PD-L1 expression in tumor cells versus the microenvironment. Notably, this study is only correlative with a limited case number. Larger prospective studies will be needed to further elucidate this relationship. Acknowledgments The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

MiR-23b Negatively Regulates Sepsis-Induced Inflammatory Responses by Targeting ADAM10 in Human THP-1 Monocytes

Background Previous studies have demonstrated pivotal roles of disintegrin and metalloproteinase 10 (ADAM10) in the pathogenesis of sepsis. MicroRNA- (miR-) 23b has emerged as an anti-inflammatory factor that prevents multiple autoimmune diseases. However, the underlying mechanisms of miR-23b in the regulation of ADAM10 and sepsis remain uncharacterized. Methods The expression levels of ADAM10 and miR-23b were detected by quantitative RT-PCR and western blot analysis. Cytokine production and THP-1 cell apoptosis were measured by enzyme-linked immunosorbent and annexin V apoptosis assays. Bioinformatics analyses and qRT-PCR, western blot, and luciferase reporter assays were performed to identify ADAM10 as the target gene of miR-23b. Results miR-23b expression was downregulated in the peripheral blood mononuclear cells of sepsis patients and LPS-induced THP-1 cells and was negatively correlated with the expression of ADAM10 and inflammatory cytokines. miR-23b regulated ADAM10 expression by directly binding to the 3′-UTR of ADAM10 mRNA. The overexpression of miR-23b alleviated the LPS-stimulated production of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and apoptosis by targeting ADAM10 in THP-1 cells. The inhibitor or knockdown of ADAM10 elicited effects similar to those of miR-23b on THP-1 cells upon LPS stimulation. Conclusions The present study demonstrated that miR-23b negatively regulated LPS-induced inflammatory responses by targeting ADAM10. The molecular regulatory mechanism of miR-23b in ADAM10 expression and sepsis-induced inflammatory consequences may provide potential therapeutic targets for sepsis.

The metalloproteinase ADAM15 is upregulated by shear stress and promotes survival of endothelial cells

Reduced shear stress resulting from disturbed blood flow can impair endothelial integrity and drive the development of vascular inflammatory lesions. Metalloproteinases of the ADAM family have been implicated in the regulation of cell survival and inflammatory responses. Here we investigate the mechanism and function of ADAM15 upregulation in primary flow cultured endothelial cells.Transcriptomic analysis indicated that within the ADAM family ADAM15 mRNA is most prominently upregulated (4-fold) when endothelial cells are exposed to physiologic shear stress. This induction was confirmed in venous, arterial and microvascular endothelial cells and is associated with increased presence of ADAM15 protein in the cell lysates (5.6-fold) and on the surface (3.1-fold). The ADAM15 promoter contains several consensus sites for the transcription factor KLF2 which is also upregulated by shear stress. Induction of endothelial KLF2 by simvastatin treatment is associated with ADAM15 upregulation (1.8-fold) which is suppressed by counteracting simvastatin with geranylgeranyl pyrophosphate. KLF2 overexpression promotes ADAM15 expression (2.1-fold) under static conditions whereas KLF2 siRNA knockdown prevents ADAM15 induction by shear stress. Functionally, ADAM15 promotes survival of endothelial cells challenged by growth factor depletion or TNF stimulation as shown by ADAM15 shRNA knockdown (1.6-fold). Exposure to shear stress increases endothelial survival while additional knockdown of ADAM15 reduces survival (6.7-fold) under flow conditions.Thus, physiologic shear stress resulting from laminar flow promotes KLF2 induced ADAM15 expression which contributes to endothelial survival. The absence of ADAM15 at low shear stress or static conditions may therefore lead to increased endothelial damage and promote vascular inflammation.

Molecular profiling of the tumor microenvironment in glioblastoma patients: correlation of microglia/macrophage polarization state with metalloprotease expression profiles and survival.

Due to poor prognosis of glioblastoma (GBM), there is an urgent need to develop new therapeutic strategies. Besides eliminating GBM tumor cells and stem cells, a novel therapeutic approach aims to target Glioma-associated microglia/macrophages (GAMs). We investigated the molecular profile of GAMs correlated with patient prognosis by exploiting M1/M2-like polarization markers in a cohort of 20 GBM patients. Using quantitative PCR (qPCR), the markers CXCL10 (M1) and CCL13 (M2) were validated in human macrophages and applied to a global analysis of GBM tissue. Furthermore, proteinase genes, known to be associated with GBM progression (ADAM8, MMP9, MMP14, ADAM10, ADAM17), were analyzed in correlation to M1/M2 markers. Notably, expression levels of ADAM10 and ADAM17 are significantly correlated with an M1-like phenotype and are positively associated to patient survival. Whilst ADAM8 mRNA expression was equally correlated with M1- and M2-like markers, genes for MMP9 and MMP14 are significantly associated with an M2-like phenotype and association to impaired prognosis in the GBM patient cohort. Thus, we provide a robust and reliable combination of qPCR markers to characterize global microglia/macrophage status and the associated proteinase profiles in GBM patients that can be used to analyze the tumor microenvironment, the patients’ prognosis and preselect those GBM patients for which targeting the microglia/macrophage population by repolarization might be beneficial.

Chronological Molecular Changes in Neuronal Communication in Androgen-Deficient Rats.

We investigated the early onset of molecular changes in the hippocampus of orchidectomy (ODX)-induced androgen-deficient rats. Transcript levels of the genes associated with loss of synaptic plasticity (Bdnf, Syn, GluN1, α7-nAChR, and M1-mAChR), formation of neurofibrillary tangles (Tau4 and Tau3), and amyloid plaques (App, Adam10, and Bace1), in the hippocampus of rats at 0, 1, 3, 6, and 9days after ODX (D0, D1, D3, D6 and D9, respectively) were determined. Primarily, the sudden loss of androgen, as confirmed by the decreased serum testosterone levels and accessory sex organ weights, induced a chronological reduction in Syn (at D1), and increase in GluN1 (at D3), α7-nAChR, and M1-mAChR (at D6) and a decrease in Bdnf (at D9) transcript levels. Tau4 and Tau3 mRNA levels were increased at D6 and D9, respectively. No changes in App, Adam10, and Bace1 mRNA levels were detected within the 9-day study period. To confirm those changes were caused by androgen deprivation and not increasing age, the mRNA expression levels of those genes in 9-day orchidectomized rats (ODX-D9) were compared with age-matched intact rats. All changes of mRNA expression levels of the ODX-D9 rats were aligned with the D9 rats, except for GluN1 that was decreased in the ODX-D9 rats. Moreover, the total and phosphorylated tau protein levels were increased in the ODX-D9 rats. These results denote that androgen deficiency induces the early onset of neurodegeneration, while the loss of synaptic plasticity together with the formation of neurofibrillary tangles could be used as markers for neurodegenerative prediction.