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Regulation of Profibrotic Responses by ADAM17 Activation in High Glucose Requires Its C-Terminus and FAK Renzhong Li, Tony Wang, Khyati Walia, Bo Gao and Joan C

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Regulation of Profibrotic Responses by ADAM17 Activation in High Glucose Requires Its C-Terminus and FAK Renzhong Li, Tony Wang, Khyati Walia, Bo Gao and Joan C © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629 RESEARCH ARTICLE Regulation of profibrotic responses by ADAM17 activation in high glucose requires its C-terminus and FAK Renzhong Li, Tony Wang, Khyati Walia, Bo Gao and Joan C. Krepinsky* ABSTRACT and amphiregulin have been implicated in the pathogenesis of Glomerular matrix accumulation is the hallmark of diabetic diabetic kidney disease (Kefaloyianni et al., 2016; Melenhorst et al., nephropathy. The metalloprotease ADAM17 mediates high glucose 2009; Uttarwar et al., 2011). Cleavage of the renoprotective (HG)-induced matrix production by kidney mesangial cells through exopeptidase ACE2 by ADAM17 is also thought to contribute to release of ligands for the epidermal growth factor receptor. Here, diabetic kidney tubular injury (Salem et al., 2014). In diabetic mice, we study the mechanism by which HG activates ADAM17. We find a nonselective metalloprotease inhibitor decreased glomerular that the C-terminus is essential for ADAM17 activation and the sclerosis, although effects cannot be specifically attributed to profibrotic response to HG. In the C-terminus, Src-mediated Y702 inhibition of ADAM17 (Ford et al., 2013). More specific ADAM17 phosphorylation and PI3K–MEK–Erk-mediated T735 phosphorylation inhibitors, however, have been associated with adverse effects, are crucial for ADAM17 activation, both are also required for the preventing their long-term use in the treatment of diabetic HG-induced increase in cell surface mature ADAM17. The non- nephropathy (Liu and Kurzrock, 2014; Rossello et al., 2016). receptor tyrosine kinase FAK is a central mediator of these processes. Toxicity may be due to the ubiquitous expression of the enzyme and These data not only support a crucial role for the C-terminus in its multitude of substrates including proinflammatory cytokines and ADAM17 activation and downstream profibrotic responses to HG, but adhesion molecules in addition to growth factor receptor ligands also highlight FAK as a potential alternative therapeutic target for (Gooz, 2010). Since ADAM17 activation shows stimulus diabetic nephropathy. specificity, this provides an opportunity for developing targeted context-dependent ADAM17 inhibition. We thus focused our KEY WORDS: ADAM17, Glucose, Fibrosis, Trafficking, Signaling studies on understanding the key mediators of ADAM17 regulation by HG. INTRODUCTION ADAM17 is a transmembrane metalloprotease with an inhibitory Diabetic nephropathy is a major complication of diabetes and the N-terminal prodomain that is cleaved by proprotein convertases, leading cause of kidney failure in North America. Patients with primarily furin, in the Golgi. The mature enzyme comprises a diabetic nephropathy suffer the highest morbidity and mortality of metalloprotease domain followed by a disintegrin domain, a any kidney failure patient group (Johnson and Spurney, 2015). cysteine-rich domain important for substrate recognition, an EGF- Prevention and treatment of diabetic nephropathy is thus a major like domain, a transmembrane domain and a cytoplasmic domain. unmet clinical need. Although pathological changes occur in all The transmembrane domain is required for enzyme activation kidney compartments, glomerular sclerosis initiated by deposition (Maretzky et al., 2011; Rossello et al., 2016). of extracellular matrix (ECM) protein is the hallmark and earliest The role of the cytoplasmic domain (amino acids 695–824) in manifestation. Multifactorial interventions including glucose regulating ADAM17 activation, however, is as yet not fully clear, control at best only delay disease progression (Lewis et al., 1993, and appears to depend on the type of stimulus-inducing activity 2001). There is thus a major need to better understand its (Gooz, 2010). For example, phorbol esters and several growth pathogenesis to enable identification of new therapeutic targets. factors can induce early ADAM17 activation in the absence of a C- High glucose (HG) plays a central role in the pathogenesis of terminus (Doedens et al., 2003; Hall and Blobel, 2012; Le Gall diabetic nephropathy by increasing ECM production in glomerular et al., 2010; Maretzky et al., 2011; Mendelson et al., 2010), whereas mesangial cells. The prosclerotic cytokine transforming growth angiotensin II requires the C-terminus to be present for induction of factor β1 (TGFβ1) is a major mediator of the HG-induced MC enzyme activity (Elliott et al., 2013). The C-terminus may allow fibrotic response (Li et al., 2003). We and others have shown that the interaction with other proteins through its SH2 and SH3 binding metalloprotease A disintegrin and metalloprotease 17 (ADAM17), domains, with phosphorylation at both serine/threonine and tyrosine through release of ligands for the epidermal growth factor receptor residues shown to be functionally important in different settings (EGFR), is an important mediator of HG-induced TGFβ1 (Gooz, 2010). Whether this is relevant to HG-induced ADAM17 upregulation and ECM protein production in kidney cells (Ford activation is as yet undetermined. et al., 2013; Uttarwar et al., 2011). Of the several EGFR ligands that Indeed, little is known of the mechanism by which HG enables can be cleaved by ADAM17, TGFα, heparin binding (HB)-EGF activation of ADAM17. We thus began by investigating the relevance of the C-terminus in this setting, and found it to be essential for activation of ADAM17 by HG. We identified two phosphorylation Division of Nephrology, McMaster University, Hamilton, Canada, L8N 4A6. sites in the C-terminus – Src-mediated Y702 phosphorylation and *Author for correspondence ([email protected]) PI3K–MEK–Erk-mediated T735 phosphorylation – to be crucial for the activation and downstream profibrotic responses of ADAM17. T.W., 0000-0002-1203-1619; B.G., 0000-0001-8051-1430; J.C.K., 0000-0002- 6761-909X HG also induced increases in the amount of mature ADAM17 at the cell surface, which is dependent on furin activity and phosphorylation Received 17 July 2017; Accepted 28 December 2017 at both T735 and Y702. We further identified the non-receptor Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629 tyrosine kinase FAK as a central upstream regulator of HG-induced exposure (24 h), which results in ADAM17 upregulation (Li et al., ADAM17 activation. These data identify a crucial role for the 2015a). In contrast, no HG response occurred after transfection with C-terminus of ADAM17 in HG-induced activation, but also ΔC ADAM17. To confirm these results, we next used ADAM17- demonstrate regulation of ADAM17 by HG at multiple levels. knockout (KO) mouse embryonic fibroblasts (MEFs). Absence of ADAM17 in these cells was confirmed by immunoblotting RESULTS (not shown). We first established that HG increases ADAM17 The C-terminus is required for ADAM17 activation by HG and activity in MEF cells (Fig. 1B). In ADAM17 KO cells, baseline downstream profibrotic responses values measured by the assay were significantly lower, and no To determine the role of the C-terminus in HG responses in MC, HG response was seen. ADAM17 activity was next tested after either WT ADAM17 or ADAM17 with its C-terminus deleted (ΔC) transfection of empty vector, WT ADAM17 or ΔC-ADAM17 into were transfected into mesangial cells, with ADAM17 activity KO cells. Construct expression was confirmed by immunoblotting assessed after 1 h of HG (Fig. 1A). The HG-induced ADAM17 for the HA tag (Fig. 1C). While transfection of either ADAM17 activation seen in cells transfected with the empty vector pcDNA construct increased basal activity, only cells transfected with WT was augmented after overexpression of WT ADAM17. This ADAM17 showed a HG response (Fig. 1D). Since PMA is well augmentation is similar to the response seen after prolonged HG known to induce ADAM17 activity independently of its C-terminus Fig. 1. ADAM17 C-terminus is required for its activation by HG and for the profibrotic response to HG. (A) ADAM17 (WT or ΔC) or empty vector was expressed in mesangial cells. Absence of the C-terminus prevented HG-induced ADAM17 activation (n=6, ‡P<0.001). (B) ADAM17 activity was tested in ADAM17 KO MEFs. As expected, no response to HG was observed (n=6, ‡P<0.001 versus all others; *P<0.001 versus WT control). (C) ADAM17 WT or ΔC, or empty vector, were expressed in ADAM17 KO MEFs. Immunoblotting for HA confirmed construct expression. It should be noted that with overexposure of the film, two bands can also be seen, although that of the pro-form predominates (not shown). (D) ADAM17 activation was induced by HG only in KO MEFs cells expressing WT ADAM17 (n=4, †P<0.01 versus all others). (E) ADAM17 WT or ΔC was expressed in KO MEFs. PMA induced ADAM17 activation equally with both constructs (n=2, *P<0.05 versus either control). (F) ADAM17 activation was induced by HG only in KO MEFs cells expressing WT ADAM17, but not the catalytically inactive mutant of ADAM17, E406A. This mutant also did not show basal activity, unlike that seen with WT ADAM17 (n=4, ‡P<0.001 versus all others). The inset shows successful overexpression of ADAM17 E460A. (G,H) ADAM17 KO MEFs were transfected with either WT or ΔC ADAM17. (G) Activation of a TGFβ1 promoter luciferase by HG (48 h) was seen only with WTADAM17 (n=6, ‡P<0.001 versus all others). (H) Total TGFβ1 in the medium was also increased by † HG only in cells expressing WT ADAM17 (n=5, P<0.01 versus all others). Journal of Cell Science 2 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629 (Doedens et al., 2003), we next tested responsiveness of WT or ΔC expressing WT ADAM17 was HG able to increase TGFβ1 ADAM17 to PMA in KO cells (Fig. 1E). ADAM17 activity was promoter luciferase activity (Fig. 1G) and protein secretion into induced to the same degree with both constructs, confirming the medium (Fig. 1H). previous findings. Finally, to determine whether the catalytic activity of ADAM17 was required, we determined the HG Src phosphorylation of the C-terminus is required for responsiveness of a catalytically inactive mutant ADAM17 ADAM17 activation by HG E406A.
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