Loss of ADAM9 expression impairs beta 1 integrin endocytosis, focal adhesion formation and cancer cell migration

The transmembrane protease ADAM9 is frequently upregulated in human cancers, and it promotes tumour progression in mice. In vitro, ADAM9 regulates cancer cell adhesion and migration by interacting with integrins. However, how ADAM9 modulates integrin functions is not known. We here show that ADAM9 knockdown increases beta 1 integrin levels through mechanisms that are independent of its protease activity, in ADAM9-silenced cells, adhesion to collagen and fibronectin is reduced, suggesting an altered function of the accumulated integrins. Mechanistically, ADAM9 co-immunoprecipitates with beta 1 integrin, and both internalization and subsequent degradation of beta 1 integrin are significantly decreased in ADAM9-silenced cells, with no effect on beta 1 integrin recycling. Accordingly, the formation of focal adhesions and actin stress fibres in ADAM9-silenced cells is altered, possibly explaining the reduction in cell adhesion and migration in these cells. Taken together, our data provide mechanistic insight into the ADAM9-integrin interaction, demonstrating that ADAM9 regulates beta 1 integrin endocytosis. Moreover, our findings indicate that the reduced migration of ADAM9-silenced cells is, at least in part, caused by the accumulation and altered activity of beta 1 integrin at the cell surface

ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function

Changes in cell shape are a morphological hallmark of differentiation. In this study we report that the expression of ADAM12, a disintegrin and metalloprotease, dramatically affects cell morphology in preadipocytes, changing them from a flattened, fibroblastic appearance to a more rounded shape. We showed that the highest levels of ADAM12 mRNA were detected in preadipocytes at the critical stage when preadipocytes become permissive for adipogenic differentiation. Furthermore, as assessed by immunostaining, ADAM12 was transiently expressed at the cell surface concomitant with the reduced activity of beta1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/beta1 integrin complexes in these preadipocytes. Overexpression of ADAM12 at the cell surface of 3T3-L1 preadipocytes achieved by transient transfection or retroviral transduction led to the disappearance of the extensive network of actin stress fibers that are characteristic of these cells, and its reorganization into a cortical network located beneath the cell membrane. The cells became more rounded, exhibited fewer vinculin-positive focal adhesions, and adhered less efficiently to fibronectin in attachment assays. Moreover, ADAM12-expressing cells were more prone to apoptosis, which could be prevented by treating the cells with beta1-activating antibodies. A reduced and re-organized fibronectin-rich extracellular matrix accompanied these changes. In addition, beta1 integrin was more readily extracted with Triton X-100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary for early adipocyte differentiation

Functional Analysis of a Breast Cancer-Associated Mutation in the Intracellular Domain of the Metalloprotease ADAM12

A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated, but is potentially significant given its location within a di-leucine motif, which is recognized as a potential cellular trafficking signal. The present study was motivated both by the potential relevance of this documented mutation to cancer, as well as for determining the role of the di-leucine motif in ADAM12 trafficking. Expression of ADAM12 p.L792F in mammalian cells demonstrated quantitatively similar expression levels and zymogen maturation as wild-type (WT) ADAM12, as well as comparable cellular localizations. A cell surface biotinylation assay demonstrated that cell surface levels of ADAM12 WT and ADAM12 p.L792F were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 p.L792F mutation is unlikely to be a driver (cancer causing)-mutation in breast cancer

PKCα and PKCδ Regulate ADAM17-Mediated Ectodomain Shedding of Heparin Binding-EGF through Separate Pathways

Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction

Effect of local TGF-β1 and IGF-1 release on implant fixation: comparison with hydroxyapatite coating: A paired study in dogs

Background and purpose Hydroxyapatite (HA) coating stimulates the osseointegration of cementless orthopedic implants. Recently, locally released osteogenic growth factors have also been shown experimentally to stimulate osseointegration so that bone fills gaps around orthopedic implants. Here, we have compared the effect of local release of TGF-β 1 and IGF-1 with that of hydroxyapatite coating on implant fixation

Effects of Heparin and Enoxaparin on APP Processing and Aβ Production in Primary Cortical Neurons from Tg2576 Mice

Alzheimer's disease (AD) is caused by accumulation of Aβ, which is produced through sequential cleavage of β-amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE1) and γ-secretase. Enoxaparin, a low molecular weight form of the glycosaminoglycan (GAG) heparin, has been reported to lower Aβ plaque deposition and improve cognitive function in AD transgenic mice

Thrombospondin-2 and SPARC/osteonectin are critical regulators of bone remodeling

Thrombospondin-2 (TSP2) and osteonectin/BM-40/SPARC are matricellular proteins that are highly expressed by bone cells. Mice deficient in either of these proteins show phenotypic alterations in the skeleton, and these phenotypes are most pronounced under conditions of altered bone remodeling. For example, TSP2-null mice have higher cortical bone volume and are resistant to bone loss associated with ovariectomy, whereas SPARC-null mice have decreased trabecular bone volume and fail to demonstrate an increase in bone mineral density in response to a bone-anabolic parathyroid hormone treatment regimen. In vitro, marrow stromal cell (MSC) osteoprogenitors from TSP2-null mice have increased proliferation but delayed formation of mineralized matrix. Similarly, in cultures of SPARC-null MSCs, osteoblastic differentiation and mineralized matrix formation are decreased. Overall, both TSP2 and SPARC positively influence osteoblastic differentiation. Intriguingly, both of these matricellular proteins appear to impact MSC fate through mechanisms that could involve the Notch signaling system. This review provides an overview of the role of TSP2 and SPARC in regulating bone structure, function, and remodeling, as determined by both in vitro and in vivo studies