Elsevier

Matrix Biology

Volume 28, Issue 8, October 2009, Pages 470-479
Matrix Biology

ADAM17 co-purifies with TIMP-3 and modulates endothelial invasion responses in three-dimensional collagen matrices

https://doi.org/10.1016/j.matbio.2009.07.007Get rights and content

Abstract

In this study, we investigated potential mechanisms through which the known anti-angiogenic factor, tissue inhibitor of metalloproteinase-3 (TIMP-3) blocks angiogenesis. As a strategy to identify TIMP-3 binding proteins, we used tandem affinity purification, employing recombinant adenoviruses constructed to deliver TIMP-3 fused to C-terminal S and His tags (TIMP-3-S-His) or TIMP-1-S-His control to endothelial cells prior to extraction. Western blotting of final eluates revealed robust binding of A Disintegrin and Metalloproteinase (ADAM) 17 and a slight association of ADAM15 to TIMP-3, but not TIMP-1 control. To confirm a functional requirement for ADAM15 and 17 in mediating angiogenic events, a model of endothelial cell invasion was utilized. Silencing of ADAM17, but not ADAM15, expression using small interfering RNA (siRNA) interfered with invasion, resulting in decreased density of invading cells and decreased invasion distance. Stable EC lines expressing short hairpin RNA directed to ADAM17 were similarly inhibited. To confirm these results, dominant negative mutants (ΔMPs) of ADAM10, ADAM15 or ADAM17 were delivered using recombinant lentiviruses. Expression of ADAM17 ΔMP, but not ADAM10 or ADAM15 ΔMP, decreased invasion density and distance. Further, time-lapse analyses revealed ADAM17 ΔMP cells exhibited far greater numbers of protruding sprouts compared to control, suggesting an inability of extended processes to retract properly. Immunofluorescence analyses revealed ADAM17 localized to bifurcations in invading sprouts. These data jointly indicate a role for ADAM17 in modulating endothelial sprouting events during angiogenesis.

Introduction

Angiogenesis is the development of new blood vessels from pre-existing structures and required for embryonic development, wound healing and pathological events (Carmeliet, 2003a, Carmeliet, 2003b, Carmeliet, 2004, Davis et al., 2002, Folkman and D'Amore, 1996). Successful angiogenesis requires proteolysis of the extracellular matrix (ECM), proliferation and migration of endothelial cells, as well as synthesis of new matrix components (Carmeliet et al., 1999, Folkman, 1999, Stetler et al., 1999, Werb et al., 1999). Endoproteases, including metalloproteinases and others, play an important role in the processes that regulate angiogenesis, including the remodeling of the ECM, cell migration and invasion, and the liberation and modification of growth factors (van Hinsbergh et al., 2006), and are collectively able to degrade all protein components of the extracellular matrix (Handsley and Edwards, 2005).

MMPs are zinc-dependent enzymes that mediate tissue morphogenesis (Curry and Osteen, 2003, Kheradmand and Werb, 2002, Nagase and Woessner, 1999, Werb et al., 1996) and vascularization (Lafleur et al., 2003, Werb et al., 1999) by regulating many biological processes including liberation of growth factors (Kheradmand and Werb, 2002, Nagase and Woessner, 1999). Various studies have linked MMPs and their endogenous inhibitors to regulating angiogenic sprouting and invasion events (Anand-Apte et al., 1997, Bayless and Davis, 2003, Curry and Osteen, 2001, Curry and Osteen, 2003, Davis et al., 2002, Itoh et al., 1998, Koivunen et al., 1999, Kokorine et al., 1996, Lafleur et al., 2003, Marbaix et al., 1996a, Marbaix et al., 1996b, Qi et al., 2003, Seo et al., 2003, Spurbeck et al., 2002, Spurbeck et al., 2003, Sympson et al., 1994, Talhouk et al., 1992, Vu et al., 1998, Werb et al., 1996). The membrane-type matrix metalloproteinases (MT–MMPs), have been shown to play a critical role in invasion of three-dimensional matrices by degrading ECM proteins at the cell surface-ECM interface (Bayless and Davis, 2003, Hotary et al., 2000, Hotary et al., 2002, Saunders et al., 2006). MT–MMPs allow highly-regulated proteolysis to occur at the cell surface, while maintaining the integrity of the supporting ECM scaffold (Chun et al., 2004, Hotary et al., 2000, Hotary et al., 2002, Sabeh et al., 2004). ADAMs are membrane-associated proteinases that contain N-terminal zinc-dependent metalloproteinase, disintegrin, transmembrane and cytoplasmic tail domains. Immobilization at the plasma membrane combined with catalytic activity renders these molecules perfectly poised to mediate shedding events. ADAM17 was the first sheddase to be identified and is also known as tumor necrosis factor (TNF)-alpha-converting-enzyme, or TACE, because of its ability to mediate TNF-α release (Black et al., 1997). Other identified substrates for ADAMs include adhesion molecules, surface receptors and members of epidermal growth factor family (Blobel, 2000, Blobel, 2005). ADAMs also co-localize with integrins (Evans, 2001). Functional ADAM17 appears to be required for normal embryonic development and most transgenic animals lacking the ADAM17 metalloproteinase domain (taceΔZn/ΔZn) die between embryonic day 17.5 (E17.5) and birth. Those few that did survive exhibited open eyelids, lacked a conjunctival sac and had attenuated corneas, low birth weight, perturbed hair coats and irregular pigmentation patterns. Also, taceΔZn/ΔZn placentas displayed a mild attenuation of the spongiotrophoblast layer associated with a disorganized and discontinuous plate of giant trophoblast cells (Peschon et al., 1998). ADAM15 is required for endocardial and bone development; mice lacking expression of ADAM15 exhibit reduced neovascularization responses in a model of retinopathy of prematurity (Horiuchi et al., 2003). ADAM10 null mice die at day 9.5 of embryogenesis, a critical time at which the vasculature is forming (Hartmann et al., 2002). Thus, MT–MMPs and ADAMs have been implicated in vasculogenesis and angiogenesis, but due to lethality associated with transgenic animals lacking ADAMs, a definitive role for this class of molecules has not been documented.

Consistent with a role for proteinases in mediating angiogenic events, the endogenous inhibitors of metalloproteinases, tissue inhibitor of metalloproteinases (TIMPs), exhibit anti-angiogenic activity (Qi et al., 2003, Seo et al., 2003). Four mammalian TIMPs (TIMP-1 to -4) have been cloned, purified and characterized. TIMP-1 inhibits the majority of the soluble MMPs with the exception of MMP-19, but exhibits no ability to inhibit membrane-type (MT)–MMPs (Hernandez-Barrantes et al., 2002, Stracke et al., 2000, Will et al., 1996). TIMP-2 and -4 likewise inhibit soluble MMPs and can also block MT–MMP activity (Baker et al., 2002). TIMP-3 inhibits ADAM-10, ADAM-12, ADAM-17, and the aggrecanases ADAMTS-4 and ADAMTS-5 (Amour et al., 2000, Amour et al., 1998, Kashiwagi et al., 2001). TIMP-3 is uniquely sequestered to the ECM by binding to heparan-sulphate-containing proteoglycans and possibly chondroitin sulphate-containing proteoglycans (Yu and Woessner, 2000). TIMP-3 inhibits chemotaxis of vascular endothelial cells toward VEGF and bFGF (Anand-Apte et al., 1997), collagen gel invasion and capillary morphogenesis in vitro (Bayless and Davis, 2003), and bFGF-induced angiogenesis in the CAM assay in vivo (Anand-Apte et al., 1997). Spurbeck et al. (2003) successfully blocked tumor-induced angiogenesis and tumor growth by expressing TIMP-3 in tumor cells. Consistent with these data, ablation of TIMP-3 in the host stroma led to enhanced tumor growth and angiogenesis (Cruz-Munoz et al., 2006). Because TIMP-3 has the ability to block angiogenesis and also interact with ADAMs metalloproteinases, we investigated here which ADAMs in primary human endothelial cells (ECs) bound TIMP-3 directly, and of these, which modulate angiogenic responses.

Section snippets

Tandem affinity purification of TIMP-1 and TIMP-3

To investigate potential endothelial binding partners for TIMP-3, we performed tandem affinity purification (TAP). TIMP-3 fused to C-terminal S-tag and His-tag epitopes were expressed in primary human ECs using recombinant adenoviruses, and an identical TIMP-1–S–His control was tested. Detergent extracts were prepared and incubated with Ni-NTA resin prior to elution. Silver staining of eluates is shown in Fig. 1, with the majority of the eluate containing TIMP-1–S–His and TIMP-3–S–His,

Discussion

Our data demonstrate that endothelial-derived ADAM17 binds TIMP-3 directly. Lee et al. (2001) reported that full-length recombinant TIMP-3 and its N-terminal truncated domain tightly bind to ADAM17. Consistent with the ability of ADAM17 to liberate TNF-α, studies of TIMP-3 knockout mice showed a higher TNF activity in the liver associated with chronic hepatic inflammation (Mohammed et al., 2004). In the present study, we found that TIMP-3, but not TIMP-1, binds directly to ADAM17 and to a

Cell culture

Primary human umbilical vein endothelial cells (Lonza) were cultured on tissue culture flasks (Corning-Costar, Fisher Scientific) pre-coated with 1 mg/ml gelatin. Growth medium consisted of M199 (Invitrogen) containing 0.1 mg/mL heparin (Sigma-Aldrich, St. Louis, MO), 0.4 mg/ mL lyophilized bovine brain extract (ECGM, Pel-Freeze Biologicals) (Maciag et al., 1979), 15% FBS (Lonza), as previously described (Su et al., 2008). 293AD cells (Stratagene) were cultured on flasks coated with 20 μg/ml

Acknowledgements

The authors would like to thank Dr. George Davis for initial helpful discussions and Dr. Steve Maxwell for reading the manuscript. This project was funded by American Heart Association Scientist Development Grant to Dr. Kayla Bayless (#0530020N).

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