ADAM17 mRNA Research Articles

Complex patterns of ADAM12 mRNA and protein splice variants in the human placenta

Trophoblast fusion in the placenta is prerequisite to successful pregnancy and the pathological conditions related to it. The presence of syncytin-1, is not sufficient to explain the complete event and ADAM12 is a major co-player candidate. Via differential splicing, the ADAM12 gene produces a short and a long form, being the ADAM12-S and the ADAM12-L respectively. We investigated the localisation of both variants in the human placenta using whole mount in situ hybridisation, immunohistochemistry and Northern blotting in 1st (n=8) and 3rd (n=8) trimester placentae and in the case of NB in several cell lines. In Northern blotting, 1st and 3rd trimester placentae were positive for the ADAM12-S and Bewo, 293HEK, JAR, leucocytes, macrophages, 1st and 3rd trimester placentae were positive for ADAM12-L. In whole mount in situ hybridisation, the 1st and 3rd trimester placental syncytium was positive for both variants. In immunohistochemistry, ADAM12-L localised in the cytotrophoblast of both 1st and 3rd trimester placentae, while ADAM12-S localised in the complete syncytium, often including the cytotrophoblast. The different localisation of ADAM12-S and ADAM12-L indicates a possible different role making ADAM12-L a candidate for the fusion event, while the syncytial localisation of the ADAM12-S makes it a candidate for cell-cell and cell-matrix interactions between the placental syncytium and the maternal interface.

Etoposide induces apoptosis and upregulation of TACE/ADAM17 and ADAM10 in an in vitro male germ cell line model

Etoposide is a widely used anticancer drug in the treatment of different tumors. Etoposide is known to activate a wide range of intracellular signals, which may in turn induce cellular responses other than apoptosis. ADAM10 and TACE/ADAM17 belong to a family of transmembrane extracellular metalloproteinases involved in paracrine/juxtacrine regulation of many signaling pathways. The aim of this work was to evaluate if etoposide induces upregulation of ADAM10 or TACE/ADAM17 in two cell lines (GC-1 and GC-2) derived from male germ cells. Results showed that etoposide induced apoptosis in a dose–response manner in both GC-1 and GC-2 cells. Apoptosis started to increase 6 h after etoposide addition in GC-2 cells, whereas the same was observed 18 h after addition to the GC-1 cells. Protein and mRNA levels of ADAM10 and TACE/ADAM17 increased 18 h after etoposide was removed from the GC-1 cells. In GC-2 cells, the protein levels of both proteins increased 12 h after etoposide was removed. ADAM10 mRNA increased after 3 h and then steadily decreased up to 12 h after removal, whereas TACE/ADAM17 mRNA decreased after etoposide removal. Finally, apoptosis was prevented in GC-1 and GC-2 cells by the addition of pharmacological inhibitors of ADAM10 and TACE/ADAM17 to the culture medium of etoposide-treated cells. Our results show for the first time that etoposide upregulates ADAM10 and TACE/ADAM17 mRNA and protein levels. In addition, we also show that ADAM10 and TACE/ADAM17 have a role in etoposide-induced apoptosis.

Taiwan cobra phospholipase A2 elicits posttranscriptional up‐regulation of ADAM17 in human neuroblastoma SK‐N‐SH cells

Taiwan cobra phospholipase A(2) (PLA(2)) treatment promoted proADAM17 processing into mature ADAM17 in human neuroblastoma SK-N-SH cells. The abolishment of catalytic activity caused a drastic drop in the PLA(2) ability to induce ADAM17 maturation, and lysophosphatidylcholine treatment mimicked the effect of PLA(2). ADAM17 activity measurement, ADAM17 cell surface levels, TNFR2 ectodomain shedding, and ADAM17 mRNA transcription supported that posttranscriptional up-regulation of ADAM17 occurred in PLA(2)-treated SK-N-SH cells. PLA(2) treatment induced p38 MAPK activation and ERK inactivation. p38 MAPK activation suppression by SB202190 (p38 MAPK inhibitor) abolished posttranscriptional up-regulation of ADAM17 in PLA(2)-treated cells, while treatment with U0126 (MEK1 and MEK2 inhibitor) increased ADAM17 maturation in SK-N-SH cells. Constitutively active MEK1 expression abrogated PLA(2)-induced ADAM17 maturation. Taken together, our data indicate that PLA(2)-evoked p38 MAPK activation and ERK inactivation are involved in ADAM17 posttranscriptional up-regulation, and suggest that the action of PLA(2) is catalytic activity-dependent.

Keystone Symposium 2010 Highlights

Unseasonably warm days gave way to some notable nights at this year’s Keystone Symposium, Alzheimer’s Disease Beyond Aβ, held 10–15 January at Copper Mountain, Colorado. One evening offering was a short talk from Gizem Donmez, a postdoctoral fellow in Leonard Guarente’s laboratory at MIT. Donmez reported that SIRT1, the histone deacetylase linked to longevity, might protect against AD by boosting ADAM10 (aka α-secretase) and promoting nonamyloidogenic processing of amyloid-β protein precursor (AβPP). If true, then you might want to eat more carrots because the effect seems to rely on SIRT1 playing vassal to the retinoic acid receptor. SIRT1 is activated by caloric restriction, which protects against brain atrophy in primates. SIRT1 itself also protects against neurodegeneration in mouse models of AD [1], and previous work from Giulio Pasinetti’s laboratory at Mount Sinai School of Medicine, New York, suggested that activation of α-secretase may be responsible [2]. Pasinetti and colleagues attributed the increase in α-secretase to SIRT1 inhibition of the Rho kinase ROCK1, previously linked to suppression of the non-amyloidogenic secretase). But Donmez’s work suggests that there is more to the tale. To explore the relationship between SIRT1 and AD, Donmez and colleagues made mice with either the SIRT1 gene knocked out or overexpressed. For knockouts, Donmez used the cre/lox system driven by a nestin promoter, limiting SIRT1 loss to neurons. For overexpression, she knocked the SIRT1 gene into the β actin locus, getting a mild, twofold overexpression. Donmez tested the effects of the SIRT1 mice on Aβ pathology by crossing them with AβPP/PS1 transgenic animals (AβPPSwe/PS1∆E9). Donmez reported that the AβPP/PS1/SIRT1 knockouts die earlier than control AβPP/PS1 animals, and that the knockouts have increased amyloid plaques and gliosis. The increased pathology in these mice was accompanied by a reduction in α-secretase activity. In contrast, AβPP/PS1 mice overexpressing SIRT1 had reduced levels of Aβ42 compared to controls and increased ADAM10 and ADAM10 mRNA. Levels of Notch intracellular domain, which is produced following α-secretase processing of the transmembrane receptor, were also increased when SIRT1 was overexpressed but not when it was knocked out. The results support the theory that SIRT1 can boost expression of the secretase. Donmez jousted with the ADAM10 promoter using chromatin immunoprecipitation assays to determine exactly how SIRT1 might exert its influence. She reported that the deacetylase attaches to the promoter very close to a binding site for the retinoic acid receptor (RAR)/retinoid X receptor (RXR) heterodimer. Activation of the ADAM10 gene depended on SIRT1 deacetylase activity (an inactive mutant has no effect) and also the presence of retinoic acid. The evidence suggests that SIRT1 deacetylates RAR leading to increased expression of ADAM10, presumably by allowing RAR to bind more tightly to the promoter. In support of this, Donmez found that RARβ is deacetylated in the presence of SIRT1 and that RARβ acetylation is increased in SIRT1 knockout cells. Coming back full circle, she showed that she was able to reverse the reduced production of Aβ in SIRT1-overexpressing cells by knocking down ADAM10 transcripts with RNA interference. Donmez concluded that SIRT1 activators might be worth pursuing as potential therapeutics for AD. Resveratrol, a SIRT1 activator found in miniscule quantities in red wine, is widely promoted in the popular

The Role of SnoN in Transforming Growth Factor β1-induced Expression of Metalloprotease-Disintegrin ADAM12

Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor beta1 (TGFbeta1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFbeta1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFbeta signaling pathway, is a master regulator of ADAM12 expression in response to TGFbeta1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFbeta1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFbeta1-induced expression of ADAM12. In a panel of TGFbeta1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFbeta1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFbeta1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.

Expression of A disintegrin and metalloprotease 10 in pancreatic carcinoma

The protease ADAM10 influences progression and metastasis of cancer cells and is overexpressed in various malignancies. Therefore, the aim of our study was to evaluate the expression and potential function of ADAM10 in the pathophysiology of pancreatic cancer (PDAC). ADAM10 expression in normal pancreatic (NP), chronic pancreatitis (CP), PDAC tissues, as well as PDAC cell lines was determined. To evaluate whether rhADAM10 or ADAM10 silencing influences cancer cell viability, MTT assay was used. Matrigel invasion and wound healing assays were performed to observe influence on invasion and migration. ADAM10 mRNA was expressed in all samples of NP, CP and PDAC tissue and cell lines. Western blotting and immunohistochemistry revealed stronger ADAM10 expression in PDAC than in NP. ADAM10 silencing or rhADAM10 had no effect on cell viability. ADAM10 silencing markedly reduced invasiveness and migration of cancer cells. These findings establish ADAM10 as a contributing factor in PDAC invasion and metastasis.

P75 Neurotrophin Receptor-mediated Apoptosis in Sympathetic Neurons Involves a Biphasic Activation of JNK and Up-regulation of Tumor Necrosis Factor-α-converting Enzyme/ADAM17

During the development of the sympathetic nervous system, the p75 neurotrophin receptor (p75NTR) has a dual function: promoting survival together with TrkA in response to NGF, but inducing cell death upon binding pro or mature brain-derived neurotrophic factor (BDNF). Apoptotic signaling through p75NTR requires activation of the stress kinase, JNK. However, the receptor also undergoes regulated proteolysis, first by a metalloprotease, and then by gamma-secretase, in response to pro-apoptotic ligands and this is necessary for receptor mediated neuronal death (Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L., and Carter, B. D. (2006) Neuron 50, 219-232). Hence, the relationship between JNK activation and receptor proteolysis remains to be defined. Here, we report that JNK3 activation is necessary for p75NTR cleavage; however, following release of the intracellular domain, there is a secondary activation of JNK3 that is cleavage dependent. Receptor proteolysis and apoptosis were prevented in sympathetic neurons from jnk3(-/-) mice, while activation of JNK by ectopic expression of MEKK1 induced p75NTR cleavage and cell death. Proteolysis of the receptor was not detected until 6 h after BDNF treatment, suggesting that JNK3 promotes cleavage through a transcriptional mechanism. In support of this hypothesis, BDNF up-regulated tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17 mRNA and protein in wild-type, but not jnk3(-/-) sympathetic neurons. Down-regulation of TACE by RNA interference blocked BDNF-induced p75NTR cleavage and apoptosis, indicating that this metalloprotease is responsible for the initial processing of the receptor. Together, these results demonstrate that p75NTR-mediated activation of JNK3 is required for up-regulation of TACE, which promotes receptor proteolysis, leading to prolonged activation of JNK3 and subsequent apoptosis in sympathetic neurons.

Elevation of ADAM10, ADAM17, MMP-2 and MMP-9 expression with media degeneration features CaCl 2-induced thoracic aortic aneurysm in a rat model

Purpose This study was designed to establish a rat model of thoracic aortic aneurysm (TAA) by calcium chloride (CaCl 2)-induced arterial injury and to explore the potential role of a disintegrin and metalloproteinase (ADAM), matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in TAA formation. Methods Thoracic aorta of male Sprague–Dawley rats was exposed to 0.5 M CaCl 2 or normal saline (NaCl). After 12 weeks, animals were euthanized, and CaCl 2-treated, CaCl 2-untreated ( n = 12) and NaCl-treated aortic segments ( n = 12) were collected for histological and molecular assessments. MMP-TIMP and ADAM mRNAs were semi-quantitatively analyzed and protein expressions were determined by immunohistochemistry. Results Despite similar external diameters among CaCl 2-treated, non-CaCl 2-treated and NaCl-treated segments, aneurymal alteration ( n = 6, 50%), media degeneration with regional disruption, fragmentation of elastic fiber, and increased collagen deposition ( n = 12, 100%) were demonstrated in CaCl 2-treated segments. MMP-2, MMP-9, ADAM-10 and ADAM-17 mRNA levels were increased in CaCl 2-treated segments (all p < 0.01), with trends of elevation in CaCl 2-untreated segments, as compared with NaCl-treated segments. Immunohistochemistry displayed significantly increased expressions of MMP-2, MMP-9, ADAM-10 and ADAM-17 (all p < 0.01) in intima and media for CaCl 2-treated segments. TIMP mRNA and tissue levels did not differ obviously among the three aortic segments. Conclusion This study establishes a TAA model by periarterial CaCl 2 exposure in rats, and demonstrates a significant elevation of expression of MMP-2, MMP-9, ADAM10 and ADAM17 in the pathogenesis of vascular remodeling.

Abstract 416: ADAM12 long and short isoforms promote breast tumor growth and metastasis via distinct mechanisms

Abstract Human ADAM12 is expressed as two alternatively spliced forms, a transmembrane form (ADAM12-L), and a secreted form (ADAM12-S). ADAM12 transcript and protein levels are elevated in several types of cancers. We have previously reported that urinary ADAM12 is predictive of disease status in breast cancer patients and that ADAM12 protein levels in urine increase with progression of disease. In this study, our goal was to elucidate the contribution of the isoforms of ADAM12 in breast tumor growth and progression. We engineered stable MCF-7 clones that overexpress ADAM12-L or ADAM12-S and characterized their properties in vitro as well as in an orthotopic breast tumor model. Overexpression of both the transmembrane ADAM12-L and the secreted ADAM12-S isoforms in breast tumor cells resulted in a significantly higher rate of tumor take and increased tumor size. ADAM12-S-expressing tumors also displayed a significantly higher incidence of lymph node and lung metastasis as compared to ADAM12-L and WT MCF-7 tumors respectively. ADAM12-S expression enhanced the ability of tumor cells to migrate and invade in vitro based on its catalytic function, whereas both isoforms conferred estrogen-independent growth capabilities to hormone responsive MCF-7 cells. In ADAM12-L-expressing breast tumor cells, this enhanced proliferation was a direct result of increased EGFR expression and activation and downstream MAPK signaling. This was confirmed when treatment with EGFR inhibitors AG1478 or PD15035 or the MAPK inhibitor U0126 resulted in complete abrogation of estrogen-independent growth in these cells. However, for ADAM12-S-expressing cells, the increased activation of MAPK and proliferation may be due to an alternate mechanism. We have also determined that ADAM12 protein and mRNA expression is elevated in malignant tissue as compared to normal breast tissue via analysis of human breast tissue arrays. Interestingly, there appears to be a differential expression pattern of the transmembrane and secreted isoforms during breast tumor progression. Taken together, our results suggest that ADAM12-S, via its catalytic function enhances the ability of malignant cells to locally invade and form distant metastasis. Expression of both ADAM12 isoforms may confer a proliferative advantage to breast tumor cells in the absence of estrogen stimulation, although via distinct mechanisms, resulting in increased tumor take, tumor size and metastasis in vivo. These findings highlight the therapeutic potential of targeting ADAM12, given that hormone independence is a hallmark of aggressive breast cancer. [The authors acknowledge the support of NIH PO1 CA45548, The JoAnn Webb Fund for Angiogenesis Research and The Fortin Foundation] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 416.

Cleavage of CD23 by ADAM proteins is regulated by beta-2 adrenergic receptor stimulation on B cells (91.14)

Abstract The level of IgE is often associated with the severity of allergic asthma. Beta-2 adrenergic receptor (β2AR) agonists alleviate bronchoconstriction by targeting the β2AR on bronchiolar smooth muscle cells, triggering airway relaxation. The β2AR is also expressed on B lymphocytes. In vitro data from our laboratory showed that β2AR stimulation on a CD40L/IL-4 activated B cell increases the level of IgE produced per cell, without affecting class switch recombination. We also found that surface CD23 expression remained constant, while CD23 mRNA, total CD23 protein, and CD23 cleavage increased. We know that soluble CD23 positively regulates IgE production. Therefore, β2AR stimulation may worsen bronchoconstriction over time, while relieving it in the short term. We hypothesized that the primary sheddase of CD23, ADAM10, was regulated by β2AR stimulation on the activated B cell. However, we found that β2AR stimulation was unable to affect the level of ADAM10 protein or mRNA in an activated B cell as compared to activated cells alone. Our recent data indicated that ADAM8 and ADAM28 might instead be involved in the β2AR-associated shedding of CD23. The differential β2AR-associated ADAM protein cleavage of CD23 may provide a potential molecular target to prevent a β2AR agonist-induced increase in IgE, which may counteract the therapeutic effect to relieve bronchoconstriction. Funded by NIH-AI-37326 and T32-AI55411

Mast Cells Expression of the ADAM-10 Protease is Inhibited BY IL-10 and TGFb1 (91.13)

Abstract Asthma is an inflammatory lung disease that is caused in part by mast cell activation via IgE and antigen. B cell-mediated IgE secretion is suppressed by the low affinity IgE receptor, CD23. Recent data have demonstrated that CD23 function can be blocked by its cleavage with the protease ADAM-10. We demonstrate that mouse mast cells abundantly express surface ADAM-10 in vitro and in vivo. ADAM-10 surface expression is suppressed by the cytokines IL-10 and TGFb1, with approximately 50% inhibition after 3 days of culture. IL-10-mediated effects were Stat3-dependent and correlated with reduced ADAM-10 mRNA. These data suggest that IL-10 and TGFb1 may suppress allergic disease in part by reducing the effects of ADAM-10, allowing for CD23-mediated negative feedback. Supported by NIH grants 1R01 AI59638 and U19A1077435.

Glutamate signaling through the kainate receptor is critical for human B cell IgE production (91.9)

Abstract Glutamate, the major excitatory neurotransmitter, has recently been implicated in a variety of diseases. Patients with HIV, certain cancers, and autoimmune illnesses have elevated levels of glutamate in the periphery. While glutamate receptor signaling has been examined in T cells, there are currently no published observations on the effects of glutamatergic stimuli on B cells. Human B cells were first examined for the presence of glutamate receptors via RT-PCR and Western Blot. The kainate receptor (KAR), a specific subtype of ionotropic glutamate receptor was found on human B cells. We found that KAR activation increased ADAM10 mRNA which is in agreement with receptor activity in the CNS. A significant corresponding increase in CD23 released into the supernatant was seen as ADAM10 has been identified as the protease responsible for CD23’s ectodomain shedding. Remarkably, we also detect a strong increase in cellular proliferation and IgE synthesis. Thus, we report for the first time the unique presence of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for enhancing B cell activation. This enhancement has implications for allergic and autoimmune diseases.

ADAM17 upregulation in human renal disease: a role in modulating TGF-α availability?

A disintegrin and metalloproteinase (ADAM)17 sheds growth factors from the cell membrane, including epidermal growth factor receptor (EGFR) ligand transforming growth factor (TGF)-alpha. In mice, angiotensin II infusion induces renal fibrosis via ADAM17-mediated TGF-alpha shedding and subsequent EGFR activation. Pharmacological ADAM17 inhibition reduced renal fibrotic lesions and improved renal function, positioning ADAM17 as a promising target of intervention in renal disease. We studied ADAM17 expression in the human kidney. ADAM17 mRNA was constitutively expressed in normal adult kidneys, with highest expression in distal tubules. In human renal disease, ADAM17 was de novo expressed in proximal tubules, peritubular capillaries, and glomerular mesangium and upregulated in podocytes. Glomerular mesangial and endothelial ADAM17 were associated with mesangial matrix expansion, focal glomerulosclerosis, and glomerular macrophage infiltration (P < 0.01). Peritubular capillary and proximal tubular ADAM17 were associated with interstitial fibrosis and interstitial macrophage infiltration (P < 0.05). Both glomerular and interstitial ADAM17 were associated with decreased renal function (P < 0.05). In renal fibrosis, ADAM17 colocalized with TGF-alpha. Moreover, in cultured human podocytes and proximal tubular cells, pharmacological ADAM17 inhibition reduced constitutive TGF-alpha shedding by 78% (P < 0.005) and 100% (P < 0.05), respectively, and phorbol ester-induced TGF-alpha shedding by 84% (P < 0.005) and 92% (P = 0.005), respectively. Finally, ADAM17 inhibition reduced cellular proliferation. In conclusion, the ADAM17 expression pattern and its role in shedding TGF-alpha from cultured human kidney cells suggest a role in the development of fibrosis. Since EGFR signaling is implicated in renal fibrosis, targeting ADAM17 to reduce availability of EGFR ligand TGF-alpha may represent a promising way of intervention in human renal disease.

Exogenous nitric oxide inhibits shedding of ADAM17 substrates.

Both ADAM17, the secretase responsible for the shedding of ectodomains of numerous membrane proteins including TNF and its receptors, as well as nitric oxide synthesized by inducible nitric oxide synthase play regulatory roles in inflammation and tumor progression. We analyzed the effect of endogenous and exogenous nitric oxide on the expression and activity of ADAM17 in murine endothelial cells and a monocyte/macrophage cell line. We found that endogenous nitric oxide influenced neither ADAM17 mRNA level nor the shedding of two ADAM17 substrates, TNF and TNFR1. Exogenous NO significantly diminished the release of TNF and TNFR1 without affecting the ADAM17 transcript level. Our data seem contrary to a previous report that showed the activation of ADAM17 by nitric oxide (Zhang et al., 2000, J Biol Chem 275: 15839-15844). We discuss potential mechanisms of NO-mediated inhibition of ectodomain shedding and possible reasons of discrepancy between our results and the previous report.

ADAM10 Is the Constitutive Functional Sheddase of CD44 in Human Melanoma Cells

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.

Expression and activation of ADAM15 in bladder cancer

e16010 Background: ADAM15 is a member of the disintegrin family of metalloproteinases. These molecules function as membrane-bound proteases, and, when activated, cleave important membrane anchored growth factors such as heparin binding epidermal growth factor and cell adhesion molecules such as E-cadherin. This proteolysis may contribute to bladder cancer growth through release of growth factors and progression by altering cell adherence. We evaluated ADAM15 expression in bladder cancer cell lines and tissues. Methods: ADAM15 expression was assessed in human bladder cell lines, normal bladder and bladder cancer tissues. A datamining program (Oncomine) was used to evaluate previous array studies in bladder cancer. ADAM15 activation results in the release of a prodomain fragment. Expression and activation of ADAM15 in bladder cancer cell lines were monitored by enzyme-linked immunosorbent assay and western blotting using ADAM15 antibody (Millipore). Staining was performed on formalin-fixed, paraffin-embedded human tissues using ADAM15 antibody (Millipore) and the peroxidase- avidin-biotin complex (ABC) method. Results: Of 7 bladder cancer cell lines, 6 demonstrated expression of full length (100 kD) and “active” (90kD) ADAM15 molecules. Four of these cell lines showed release of the ADAM15 prodomain fragment, and 5 cell lines provided evidence for proteolytic activity, indicated by release of 80kD soluble E-cadherin molecule. Immunostaining of a bladder cancer tissue microarray revealed that normal urothelium expressed low levels of ADAM15, but focal overexpression of ADAM15 was observed in 17/29 bladder cancer specimens. Datamining of independent microarray analysis revealed that ADAM15 mRNA is upregulated in bladder cancer. Conclusions: ADAM15 is expressed in the majority of human bladder cancer specimens. Cell line studies indicate that ADAM15 can be activated, as indicated by release of the ADAM15 prodomain fragment. These results suggest that ADAM15 may be a therapeutic target to modulate bladder cancer growth and progression. Future studies will continue evaluation of the biology of ADAM15 in bladder cancer and evaluate if the soluble ADAM15 prodomain can be detected in plasma, serum or urine from patients with bladder cancer. No significant financial relationships to disclose.

Metalloproteinase-Dependent Shedding of Low-Density Lipoprotein Receptor-Related Protein-1 Ectodomain Decreases Endocytic Clearance of Endometrial Matrix Metalloproteinase-2 and -9 at Menstruation

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

Kainate receptor activation may serve as a novel mechanism of B cell activation in autoimmunity (99.47)

Abstract Glutamate, the major excitatory neurotransmitter in the CNS, has recently been implicated in a variety of diseases. For example, it has been shown that patients with HIV, certain cancers, epilepsy, and autoimmune illnesses such as SLE all have elevated levels of glutamate in the periphery. While glutamate receptor signaling has been examined in T cells, there are currently no published observations on the effects of glutaminergic stimuli on B cells. Therefore, we performed a novel study to examine the effects of glutamate on B cells and thus humoral immunity. Both human B cell lines as well as primary B cells were first examined for the presence of glutamate receptors via RT-PCR and Western Blot. We identified the presence of the kainate receptor (KAR), a specific subtype of ionotropic glutamate receptor thought to be exclusively resident in the CNS. We found that KAR activation increased ADAM10 mRNA which is in agreement with receptor activity in the CNS. We also observed a significant corresponding increase in CD23 released into the supernatant as ADAM10 has been identified as the protease responsible for CD23's ectodomain shedding. Remarkably, we also detect a significant increase in cellular proliferation and Ig synthesis. Thus, we report for the first time the unique presence of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for B cell activation in autoimmunity.

The role of the disintegrin metalloproteinase ADAM15 in prostate cancer progression

The metalloproteinase ADAM15 is a multi-domain disintegrin protease that is upregulated in a variety of human cancers. ADAM15 mRNA and protein levels are increased in prostate cancer and its expression is significantly increased during metastatic progression. It is likely that ADAM15 supports disease progression differentially through the action of its various functional domains. ADAM15 may downregulate adhesion of tumor cells to the extracellular matrix, reduce cell-cell adhesion, and promote metastasis through the activity of its disintegrin and metalloproteinase domains. Additionally, ADAM15 can influence cell signaling by shedding membrane-bound growth factors and other proteins that interact with receptor tyrosine kinases, leading to receptor activation. There is also evidence supporting a role for ADAM15 in angiogenesis and angioinvasion of tumor cells, which are critical for unrestrained tumor growth and metastatic spread. Given its diverse functions, ADAM15 may represent a pivotal regulatory component of tumor progression, an important target for therapeutic intervention, or emerge as a biomarker of disease progression.

A strategy for identifying circulating placental RNA markers for fetal growth assessment

To evaluate whether circulating placental mRNAs in maternal plasma could serve as markers for the assessment of fetal growth or intrauterine growth restriction (IUGR). From a panel of placental transcripts detectable in maternal plasma identified by microarray previously, we chose growth-related transcripts, namely CSH1, GH2, KISS1, and ADAM12, as potential growth markers. Relationships between the maternal plasma mRNA concentrations with several fetal growth indicators were studied. Maternal plasma mRNA concentrations from IUGR pregnancies with or without pre-eclampsia (PET) were compared with gestational age matched controls cross-sectionally and longitudinally. The four transcripts were quantified by one-step real-time RT-PCR. Maternal plasma GH2 mRNA significantly correlated with birth weight and fetal biometric measurements. Maternal plasma ADAM12 mRNA concentration was significantly higher in IUGR with PET than normal pregnancies in the cross-sectional comparison. No significant difference was observed for all markers between IUGR without PET and controls in both the cross-sectional and longitudinal comparisons. This study presents a potential strategy in identifying surrogate markers for the study of fetal growth. Circulating GH2 mRNA in maternal plasma appeared to be associated with fetal growth. The utility of this strategy and the currently assessed markers could be explored in further studies.