Posttranslational modifications by ADAM10 shape myeloid antigen-presenting cell homeostasis in the splenic marginal zone.

The spleen contains phenotypically and functionally distinct conventional dendritic cell (cDC) subpopulations, termed cDC1 and cDC2, which each can be divided into several smaller and less well-characterized subsets. Despite advances in understanding the complexity of cDC ontogeny by transcriptional programming, the significance of posttranslational modifications in controlling tissue-specific cDC subset immunobiology remains elusive. Here, we identified the cell-surface-expressed A-disintegrin-and-metalloproteinase 10 (ADAM10) as an essential regulator of cDC1 and cDC2 homeostasis in the splenic marginal zone (MZ). Mice with a CD11c-specific deletion of ADAM10 (ADAM10ΔCD11c) exhibited a complete loss of splenic ESAMhi cDC2A because ADAM10 regulated the commitment, differentiation, and survival of these cells. The major pathways controlled by ADAM10 in ESAMhi cDC2A are Notch, signaling pathways involved in cell proliferation and survival (e.g., mTOR, PI3K/AKT, and EIF2 signaling), and EBI2-mediated localization within the MZ. In addition, we discovered that ADAM10 is a molecular switch regulating cDC2 subset heterogeneity in the spleen, as the disappearance of ESAMhi cDC2A in ADAM10ΔCD11c mice was compensated for by the emergence of a Clec12a+ cDC2B subset closely resembling cDC2 generally found in peripheral lymph nodes. Moreover, in ADAM10ΔCD11c mice, terminal differentiation of cDC1 was abrogated, resulting in severely reduced splenic Langerin+ cDC1 numbers. Next to the disturbed splenic cDC compartment, ADAM10 deficiency on CD11c+ cells led to an increase in marginal metallophilic macrophage (MMM) numbers. In conclusion, our data identify ADAM10 as a molecular hub on both cDC and MMM regulating their transcriptional programming, turnover, homeostasis, and ability to shape the anatomical niche of the MZ.

Regulation of ADAM10 by the TspanC8 Family of Tetraspanins and Their Therapeutic Potential.

The ubiquitously expressed transmembrane protein a disintegrin and metalloproteinase 10 (ADAM10) functions as a "molecular scissor", by cleaving the extracellular regions from its membrane protein substrates in a process termed ectodomain shedding. ADAM10 is known to have over 100 substrates including Notch, amyloid precursor protein, cadherins, and growth factors, and is important in health and implicated in diseases such as cancer and Alzheimer's. The tetraspanins are a superfamily of membrane proteins that interact with specific partner proteins to regulate their intracellular trafficking, lateral mobility, and clustering at the cell surface. We and others have shown that ADAM10 interacts with a subgroup of six tetraspanins, termed the TspanC8 subgroup, which are closely related by protein sequence and comprise Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33. Recent evidence suggests that different TspanC8/ADAM10 complexes have distinct substrates and that ADAM10 should not be regarded as a single scissor, but as six different TspanC8/ADAM10 scissor complexes. This review discusses the published evidence for this "six scissor" hypothesis and the therapeutic potential this offers.

ADAM10 and ADAM17 regulate EGFR, c-Met and TNF RI signalling in liver regeneration and fibrosis.

ADAM10 and ADAM17 are proteases that affect multiple signalling pathways by releasing molecules from the cell surface. As their substrate specificities partially overlaps, we investigated their concurrent role in liver regeneration and fibrosis, using three liver-specific deficient mouse lines: ADAM10- and ADAM17-deficient lines, and a line deficient for both proteases. In the model of partial hepatectomy, double deficient mice exhibited decreased AKT phosphorylation, decreased release of EGFR activating factors and lower shedding of HGF receptor c-Met. Thus, simultaneous ablation of ADAM10 and ADAM17 resulted in inhibited EGFR signalling, while HGF/c-Met signalling pathway was enhanced. In contrast, antagonistic effects of ADAM10 and ADAM17 were observed in the model of chronic CCl4 intoxication. While ADAM10-deficient mice develop more severe fibrosis manifested by high ALT, AST, ALP and higher collagen deposition, combined deficiency of ADAM10 and ADAM17 surprisingly results in comparable degree of liver damage as in control littermates. Therefore, ADAM17 deficiency is not protective in fibrosis development per se, but can ameliorate the damaging effect of ADAM10 deficiency on liver fibrosis development. Furthermore, we show that while ablation of ADAM17 resulted in decreased shedding of TNF RI, ADAM10 deficiency leads to increased levels of soluble TNF RI in serum. In conclusion, hepatocyte-derived ADAM10 and ADAM17 are important regulators of growth receptor signalling and TNF RI release, and pathological roles of these proteases are dependent on the cellular context.

ADAM10 is Expressed by Ameloblasts, Cleaves the RELT TNF Receptor Extracellular Domain and Facilitates Enamel Development.

MMP20 cleaves cadherins and may facilitate cell movement, however MMP20 is not known to cleave tight junction or desmosome proteins. Ameloblasts had not previously been screened for membrane anchored proteases that could contribute to cell movement. Here we performed a PCR screen for proteolyticlly active A Disintegrin And Metalloproteinase (ADAM) family members. These proteinases are termed sheddases because they have a transmembrane domain and their catalytic domain on the cell surface can function to release anchored proteins. Significantly, ADAMs can be targeted to specific substrates on the cell membrane through their interaction with tetraspanins. Six ADAMs (ADAM8, 9, 10, 15, 17, 19) were expressed in mouse enamel organs. We show that Adam10 expression begins in the apical loop, continues through the secretory stage and abruptly ends at the transition stage when ameloblast migration ceases. ADAM10 cleaves cadherins and tight junction plus desmosome proteins and is well characterized for its role in cell movement. ADAM10 facilitated LS8 cell migration/invasion through a Matrigel coated membrane and we demonstrate that ADAM10, but not ADAM17 cleaves the RELT extracellular domain. This striking result is significant because RELT mutations cause amelogenesis imperfecta (AI) and this directly links ADAM10 to an important role in enamel development.

Sensory lesioning induces microglial synapse elimination via ADAM10 and fractalkine signaling.

Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remain a key open question. Here we show that whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. This synapse elimination is dependent on signaling by CX3CR1, the receptor for microglial fractalkine (also known as CXCL1), but not complement receptor 3. Furthermore, mice deficient in CX3CL1 have profound defects in synapse elimination. Single-cell RNA sequencing revealed that Cx3cl1 is derived from cortical neurons, and ADAM10, a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and in microglia following whisker lesioning. Finally, inhibition of ADAM10 phenocopies Cx3cr1-/- and Cx3cl1-/- synapse elimination defects. Together, these results identify neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal that context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.

ADAM10 controls the differentiation of the coronary arterial endothelium.

The coronary vasculature is crucial for normal heart function, yet much remains to be learned about its development, especially the maturation of coronary arterial endothelium. Here, we show that endothelial inactivation of ADAM10, a key regulator of Notch signaling, leads to defects in coronary arterial differentiation, as evidenced by dysregulated genes related to Notch signaling and arterial identity. Moreover, transcriptome analysis indicated reduced EGFR signaling in A10ΔEC coronary endothelium. Further analysis revealed that A10ΔEC mice have enlarged dysfunctional hearts with abnormal myocardial compaction, and increased expression of venous and immature endothelium markers. These findings provide the first evidence for a potential role for endothelial ADAM10 in cardioprotective homeostatic EGFR signaling and implicate ADAM10/Notch signaling in coronary arterial cell specification, which is vital for normal heart development and function. The ADAM10/Notch signaling pathway thus emerges as a potential therapeutic target for improving the regenerative capacity and maturation of the coronary vasculature.

Sphingosine 1-phosphate stimulates eyelid closure in the developing rat by stimulating EGFR signaling.

In many mammals, the eyelids migrate over the eye and fuse during embryogenesis to protect the cornea from damage during birth and early life. Loss-of-function mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway cause an eyes-open-at-birth (EOB) phenotype in rodents. We identified an insertional mutation in Spinster homolog 2 (Spns2) in a strain of transgenic rats exhibiting the EOB phenotype. Spns2, a sphingosine 1-phosphate (S1P) transporter that releases S1P from cells, was enriched at the tip of developing eyelids in wild-type rat embryos. Spns2 expression or treatment with S1P or any one of several EGFR ligands rescued the EOB Spns2 mutant phenotype in vivo and in tissue explants in vitro and rescued the formation of stress fibers in primary keratinocytes from mutants. S1P signaled through the receptors S1PR1, S1PR2, and S1PR3 to activate extracellular signal-regulated kinase (ERK) and EGFR-dependent mitogen-activated protein kinase kinase kinase 1 (MEKK1)-c-Jun signaling. S1P also induced the nuclear translocation of the transcription factor MAL in a manner dependent on EGFR signaling. MAL and c-Jun stimulated the expression of the microRNAs miR-21 and miR-222, both of which target the metalloprotease inhibitor TIMP3, thus promoting metalloprotease activity. The metalloproteases ADAM10 and ADAM17 stimulated EGFR signaling by cleaving a membrane-anchored form of EGF to release the ligand. Our results outline a network by which S1P transactivates EGFR signaling through a complex mechanism involving feedback between several intra- and extracellular molecules to promote eyelid fusion in the developing rat.

Gαi Signaling Promotes Marginal Zone B Cell Development by Enabling Transitional B Cell ADAM10 Expression.

The follicular (FO) versus marginal zone (MZ) B cell fate decision in the spleen depends upon BCR, BAFF, and Notch2 signaling. Whether or how Gi signaling affects this fate decision is unknown. Here, we show that direct contact with Notch ligand expressing stromal cells (OP9-Delta-like 1) cannot promote normal MZ B cell development when progenitor B cells lack Gαi proteins, or if Gi signaling is disabled. Consistent with faulty ADAM10-dependent Notch2 processing, Gαi-deficient transitional B cells had low ADAM10 membrane expression levels and reduced Notch2 target gene expression. Immunoblotting Gαi-deficient B cell lysates revealed a reduction in mature, processed ADAM10. Suggesting that Gαi signaling promotes ADAM10 membrane expression, stimulating normal transitional B cells with CXCL12 raised it, while inhibiting Gαi nucleotide exchange blocked its upregulation. Surprisingly, inhibiting Gαi nucleotide exchange in transitional B cells also impaired the upregulation of ADAM10 that occurs following antigen receptor crosslinking. These results indicate that Gαi signaling supports ADAM10 maturation and activity in transitional B cells, and ultimately Notch2 signaling to promote MZ B cell development.

[Improvement and the mechanism of cardiac function by knockdown of ADAM10 in adriamycin-induced cardiomyopathy rats].

Objective To study the role of a disintegrin and metalloproteinase10 (ADAM10) in shedding neural cadherin (N-cadherin) and develop an approach to interfere the process of ventricular remodeling in adriamycin-induced cardiomyopathy (ACM) rats. Methods In a rat model of ACM, the effects of intraperitoneal injection of the lentiviral RNAi vector of ADAM10 on the morphology of cardiomyocytes and contractile function were observed by HE staining and color Doppler echocardiography. The expressions of N-cadherin and C-terminal fragment 1 (CTF1) were detected by Western blotting and immunohistochemistry. Results In the in vivo experiment, a large amount of fluorescence was seen in the isolated primary cardiomyocytes, which indicated that the transfection in the rat model was successful. In the treatment group, the morphology of cardiomyocytes and function of the heart were evidently improved, N-cadherin protein expression was remarkably up-regulated and CTF1 protein was obviously down-regulated compared with the model group. Conclusion Knock-down of ADAM10 increases N-cadherin expression and decreases CTF1 expression, thus improves cardiac function in the rat model of ACM.

Semaphorin 4D from CD15+ Granulocytes via ADAM10-Induced Cleavage Contributes to Antibody Production in Bullous Pemphigoid.

Autoreactive B-cell activation and antibody production are critical events for the development of bullous pemphigoid (BP). However, the mechanism that is involved in the modulation of B-cell activation and autoantibody generation has not been fully understood. Semaphorin 4D (Sema4D, or CD100) plays important roles in immune regulation related to B cells, but its implications in BP remain obscure. The aim of our study was to characterize Sema4D and the underlying mechanism contributing to the autoimmune features of BP. We found that soluble Sema4D (sSema4D) levels were elevated and correlated with disease severity and activity in serum and blister fluids from patients with BP. Additionally, Sema4D-expressing cells accumulated in subepidermal blisters of BP lesions. In patient-derived peripheral blood mononuclear cells, by promoting the differentiation of B cells into plasmablasts, sSema4D boosted anti-BP180/anti-BP230 antibody production in a time- and dose-dependent manner, which may be attributed to CD72-mediated activation of Akt/NF-κB phosphorylated (p-)65/ERK cascades in B cells. We determined that a disintegrin and metalloproteinase 10 is a proteolytic enzyme for the cleavage of sSema4D from CD15+ granulocytes instead of T cells, which is probably responsible for the high concentration of sSema4D in BP blister fluid and serum. These findings suggest that Sema4D is a crucial participant in BP pathogenesis.

ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis.

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however, the molecular mediators of myofibroblast activation have yet to be fully identified. Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a disintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression is increased by transforming growth factor (TGF)-ß1, and ADAM10-mediated sEphrin-B2 generation is required for TGF-ß1-induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10-sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases.

Molecular Pathways: Receptor Ectodomain Shedding in Treatment, Resistance, and Monitoring of Cancer.

Proteases known as sheddases cleave the extracellular domains of their substrates from the cell surface. The A Disintegrin and Metalloproteinases ADAM10 and ADAM17 are among the most prominent sheddases, being widely expressed in many tissues, frequently overexpressed in cancer, and promiscuously cleaving diverse substrates. It is increasingly clear that the proteolytic shedding of transmembrane receptors impacts pathophysiology and drug response. Receptor substrates of sheddases include the cytokine receptors TNFR1 and IL6R; the Notch receptors; type-I and -III TGFß receptors; receptor tyrosine kinases (RTK) such as HER2, HER4, and VEGFR2; and, in particular, MET and TAM-family RTKs AXL and Mer (MerTK). Activation of receptor shedding by mechanical cues, hypoxia, radiation, and phosphosignaling offers insight into mechanisms of drug resistance. This particularly holds for kinase inhibitors targeting BRAF (such as vemurafenib and dabrafenib) and MEK (such as trametinib and cobimetinib), along with direct sheddase inhibitors. Receptor proteolysis can be detected in patient fluids and is especially relevant in melanoma, glioblastoma, lung cancer, and triple-negative breast cancer where RTK substrates, MAPK signaling, and ADAMs are frequently dysregulated. Translatable strategies to exploit receptor shedding include combination kinase inhibitor regimens, recombinant decoy receptors based on endogenous counterparts, and, potentially, immunotherapy. Clin Cancer Res; 23(3); 623-9. ©2016 AACR.

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth.

The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance.

ADAM10-Dependent Signaling Through Notch1 and Notch4 Controls Development of Organ-Specific Vascular Beds.

RATIONALE: Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10. OBJECTIVE: The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells. METHODS AND RESULTS: Mice lacking ADAM10 and ADAM17 in endothelial cells (Adam10/Adam17ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10ΔEC mice. In contrast, Adam10ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch (Adam10ΔEC(Flv)) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10ΔEC(Flv) mice than in the previously described Adam10ΔEC mice. Finally, mice lacking endothelial Notch1 (Notch1ΔEC) share some organ-specific vascular defects with Adam10ΔEC mice, whereas Notch4(-/-) mice lacking endothelial Notch1 (Notch1ΔEC/Notch4(-/-)) had defects in all vascular beds affected in Adam10ΔEC mice. CONCLUSIONS: Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds.

ADAM10 mediates the cell invasion and metastasis of human esophageal squamous cell carcinoma via regulation of E-cadherin activity.

A disintegrin and metalloprotease 10 (ADAM10) is involved in the tumorigenesis, invasion and metastasis of several types of solid tumors. However, the potential role of ADAM10 in human esophageal squamous cell carcinoma (ESCC) is not yet well understood. The present study showed that ADAM10 was overexpressed in human ESCC tissues in vivo, and positively associated with depth of tumor invasion, lymph node metastasis and TNM stage, contributing to tumor carcinogenesis, invasion and metastasis. Additionally, ADAM10 was overexpressed in 3 types of ESCC cell lines in vitro, as compared to that in normal esophageal epithelial cells (NEECs); and moreover, ESCC cells with high ADAM10 expression obtained enhanced invasion and migration ability. Subsequently, ADAM10 silencing by small interfering (si) RNA in ESCC cell line, EC-1, reduced cell invasion, migration and proliferation in vitro. Finally, ADAM10 negatively regulated E-cadherin in ESCC in vivo and in vitro. In conclusion, active ADAM10 promotes the carcinogenesis, invasion, metastasis and proliferation of ESCC and controls invasion and metastasis at least in part through the shedding of E-cadherin activity, which makes it a potential biomarker and a useful therapeutic target for ESCC.

miR-448 suppressed gastric cancer proliferation and invasion by regulating ADAM10.

MicroRNAs (miRNAs) are a class of short, noncoding RNAs that act a crucial role in tumor development. Previous studies showed that miR-448 expression was deregulated in many tumors. However, the role of miR-448 in gastric cancer (GC) remains unknown. In our study, we demonstrated that miR-448 expression was downregulated in GC tissues compared with the corresponding nontumor tissues. We also showed that miR-448 expression was downregulated in GC cell lines. Ectopic expression of miR-448 suppressed GC cell proliferation, colony formation, and invasion. Moreover, we identified A Disintegrin And Metalloproteinases 10 (ADAM10) as a direct target gene of miR-448 in GC cell. ADAM10 expression was upregulated in GC tissues and cells. Furthermore, the expression level of miR-448 was negatively correlated with the expression level of ADAM10 in GC tissues. Moreover, ADAM10 overexpression rescued the effect of miR-448-mediated GC cell proliferation, colony formation, and invasion. These results demonstrated that miR-448 might play as a tumor suppressor miRNA partly through targeting ADAM10 expression.