© 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

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(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. As shown in Fig. 1F, ADAM17 KO MEFs transfected Src associates with ADAM17 in response to hormonal stimulation with the catalytically inactive mutant (with expression shown in the (Zhang et al., 2006) and was shown to phosphorylate ADAM17 at inset) had neither an increase in basal activity, nor did they show Y702 in response to mechanical stress (Niu et al., 2013, 2015). Src increased activity in response to HG, indicating that both the is known to mediate ADAM17 activation after long-term HG C-terminus and ADAM17 catalytic activity are required for exposure (24–48 h) (Taniguchi et al., 2013). To determine whether ADAM17 activation by HG. acute activation of ADAM17 is mediated by Src, the effect of its We previously showed that ADAM17 activation was required for inhibitors on ADAM17 activity after 1 h of HG was assessed. Two HG-induced upregulation of the profibrotic cytokine TGFβ1 structurally distinct inhibitors, SU6656 and PP2, prevented early (Uttarwar et al., 2011), a key pathogenic factor in diabetic ADAM17 activation (Fig. 2A). HG increased the interaction nephropathy (Li et al., 2003). To confirm the importance of the between ADAM17 and Src as assessed by coimmunoprecipitation ADAM17 C-terminus in this response, we used ADAM17 KO (Fig. 2B). Phosphorylation of ADAM17 at Y702 in response MEFs transfected with WT or ΔC ADAM17. Only in cells to HG was observed in a similar timeframe (Fig. 2C). This

Fig. 2. Src phosphorylation of the ADAM17 C-terminus is required for its activation by HG. (A) Src inhibitors SU6656 or PP2 blocked HG (1 h)-induced ADAM17 activation (n=7, ‡P<0.001 versus all others). (B) Mesangial cells were treated with HG for the indicated times. ADAM17 was then immunoprecipitated. Immunoblotting shows HG induces ADAM17–Src association (n=3). (C) HG induces a time-dependent increase in ADAM17 Y702 phosphorylation (pADAM17 Y702) (n=2). (D) This was blocked by the Src inhibitors PP2 and SU6656 after 1 h of HG (n=6, *P<0.05 versus all others). (E) ADAM17 KO MEFs were transfected with WT or ΔC ADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. HG-induced Src/ADAM17 association was seen only in cells expressing WT ADAM17 (n=3, †P<0.01 versus all others). Note that the HA blot is the same as that used in Fig. 3I as this originates from the same experiment, with reprobing done to detect FAK and Src. (F) ADAM17 KO MEFs were transfected with empty vector pcDNA, WT ADAM17 or Y702A ADAM17. HG † ‡ induced ADAM17 activation only in cells expressing WT ADAM17 (n=4; *P<0.05, P<0.01, P<0.001, #P<0.001 versus both pcDNA groups). Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629 phosphorylation was confirmed to be mediated by Src (Fig. 2D). FAK mediates Src phosphorylation and activation of Antibody specificity was confirmed in ADAM17 KO MEFs ADAM17 transfected with WT or Y702A ADAM17 (Fig. S1). We next sought to determine the upstream factors mediating Src– Tyrosine 702 is located in the C-terminus of ADAM17 (aa 695– ADAM17 activation by HG. Activation of the nonreceptor tyrosine 824). Using ADAM17 KO MEFs transfected with either WT or ΔC kinase FAK was seen in diabetic kidney lysates (Regoli and ADAM17, a requirement for the C-terminus in the association Bendayan, 1999), but whether FAK is activated by HG in mesangial between ADAM17 and Src was confirmed (Fig. 2E). To determine cells or regulates ADAM17 activation is as yet unknown. In other whether Y702 phosphorylation is required for ADAM17 activation settings, activated FAK is known to recruit Src, enabling its by HG, ADAM17 KO MEFs were transfected with either WT or activation (Bolos et al., 2010). We first assessed whether HG nonphosphorylatable Y702A ADAM17. Fig. 2F shows that although activates FAK in mesangial cells. Fig. 3A shows that HG induced the baseline activity remains higher in Y702A-transfected cells, early activation of FAK, determined by immunoblotting for its consistent with that seen with expression of ΔC ADAM17, HG fails autophosphorylation at Y397. HG induced the association of FAK to induce ADAM17 activation. Together, these data show that Src with Src, as assessed by coimmunoprecipitation (Fig. 3B). Using interacts with and phosphorylates ADAM17 at its C-terminal Y702, the FAK inhibitor PF573228, Fig. 3C shows that FAK is required and this is required for ADAM17 activation by HG. for ADAM17 activation by HG. FAK activation by HG is also

Fig. 3. FAK is required for ADAM17 activation and Src enables FAK–ADAM17 interaction in response to HG. (A) Mesangial cells were treated for the indicated times with HG. FAK activation, assessed by its phosphorylation on Y397 (pFAK), was increased by HG (n=2). (B) FAK was immunoprecipitated from whole cell lysate after treatment with HG. Immunoblotting shows Src association with FAK increases in response to HG (n=3). (C) ADAM17 activation by HG (1 h) is prevented by the FAK inhibitor PF573228 (n=6, ‡P<0.001 versus others). (D) Association of ADAM17 and Src in response to HG (1 h) was prevented by PF573228, as assessed by coimmunoprecipitation (n=2). (E) ADAM17 phosphorylation at Y702 was also prevented by FAK inhibition with PF573228 (n=2). (F) ADAM17 was immunoprecipitated from whole cell lysate after treatment with HG for the indicated times. Immunoblotting shows FAK association with ADAM17 increases in response to HG (n=4). (G) Inhibition of FAK activity with PF573228 prevents ADAM17/FAK association in response to HG (1 h) (n=4). (H) ADAM17 interaction with FAK in response to HG requires Src activity, since this was prevented by the two Src inhibitors SU6656 and PP2 (n=3). (I) ADAM17 KO MEFs were transfected with WT or ΔC ADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. HG-induced FAK–ADAM17 association was seen only in cells expressing WT ADAM17 (n=3, *P<0.05 versus other groups). The HA blot is the same as that used in Fig. 2E as this originates from the same experiment, with reprobing done to detect FAK. (J) ADAM17 KO MEFs were transfected with WT or Y702A ADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. No HG-induced association between ADAM17 and FAK was seen in cells expressing Y702A (n=3).

IgG control immunoprecipitation of HG-treated cells with WT ADAM17 shows some nonspecific pull-down of FAK. HC, antibody heavy chain. Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629 required for Src association with ADAM17 (Fig. 3D), as well as enabling its activation (Cantley, 2002). To assess whether PI3K is HG-induced phosphorylation of ADAM17 at Y702 by Src required for HG-induced ADAM17 activation, we used two distinct (Fig. 3E). Thus, FAK is required as an upstream effector of Src- PI3K inhibitors, wortmannin and LY294002. Fig. 4A shows that mediated ADAM17 phosphorylation and ADAM17 activation. PI3K inhibition prevented HG-induced ADAM17 activation. As Activated Src is known to enable the full catalytic activity of FAK expected, since FAK is known to recruit PI3K, FAK activation through phosphorylation at additional tyrosine residues on FAK. This (assessed by its Y397 autophosphorylation) was unaffected by expands the ability of FAK to interact with other proteins, further PI3K inhibitors (Fig. 4B). Src activation by HG was similarly enhancing its scaffolding function (Bolos et al., 2010). As shown in unaffected by PI3K inhibitors (Fig. 4C). However, PI3K activity Fig. 3F, we found that FAK associated with ADAM17 in response to was required for FAK–ADAM17 association in response to HG HG. This was not only dependent on FAK activity, as it was blocked (Fig. 4D). These data suggest that FAK and Src form a complex that by the inhibitor PF573228 (Fig. 3G), but also required Src activity associates with ADAM17. PI3K activity is required for this, as part since it was prevented by two independent Src inhibitors (Fig. 3H). of the complex and/or by facilitating complex formation through its To determine whether the C-terminus of ADAM17 is needed for generation of signaling intermediates. FAK–ADAM17 interaction, ADAM17 KO MEFs were transfected with either WT or ΔC ADAM17. After HG treatment for 1 h, FAK ADAM17 phosphorylation at T735 is required for its associated with only WT ADAM17, showing the requirement of the activation by HG C-terminus of ADAM17 for this interaction (Fig. 3I). Interestingly, PI3K was shown to mediate the serine/threonine phosphorylation expression of Y702A ADAM17 in ADAM17 KO MEFs also of ADAM17 through PDK1 in cancer cells, although the prevented the HG-induced association between FAK and ADAM17 phosphorylated residue was not identified (Zhang et al., 2006). (Fig. 3J). Here, IgG control immunoprecipitates of cells transfected Several serine/threonine residues of the ADAM17 C-terminus are with WT ADAM17 showed minor nonspecific pull-down of FAK known to be phosphorylated, with phosphorylation at T735 (Fig. 3J). Taken together, FAK-induced Src activation and important for ADAM17 activation and/or trafficking and subsequent phosphorylation of ADAM17 at Y702 promotes maturation in response to specific stimuli (Diaz-Rodriguez et al., interaction between FAK and ADAM17. 2002; Soond et al., 2005; Xu and Derynck, 2010). We first determined whether HG induces ADAM17 phosphorylation on PI3K mediates FAK–ADAM17 association and ADAM17 T735. Fig. 5A shows phosphorylation at this site within 30 min of activation by HG HG exposure. The mitogen-activated protein kinases p38 and Erk We previously showed an important role for PI3K–Akt signaling in are known to phosphorylate ADAM17 at T735 in different settings, HG-induced matrix upregulation (Wu et al., 2007, 2009). and PI3K–PDK1 can also regulate Erk activation (Ha et al., 2013). Interestingly, PI3K was shown to associate with both ADAM17 We thus first tested the effects of p38 or Erk inhibition on ADAM17 and Src in cancer cells (Zhang et al., 2006). Furthermore, after its activation by HG. Fig. 5B,C shows that Erk inhibition using the full activation, FAK is able to recruit PI3K by binding to its SH2 MEK inhibitor U0126, but not p38 inhibition, prevented HG- domain (Bolos et al., 2010). This enables a conformational change induced ADAM17 activation. We next assessed the requirement of that releases the catalytic from the inhibitory subunit of PI3K, PI3K and Erk for T735 phosphorylation. Fig. 5D shows that

Fig. 4. PI3K mediates FAK–ADAM17 association and ADAM17 activation by HG. (A) PI3K inhibitors LY294002 or wortmannin inhibited HG (1 h)-induced ADAM17 activation (n=4, ‡P<0.001 versus other groups, †P<0.01, #P<0.01 versus wort). (B) FAK activation, as assessed by its Y397 autophosphorylation, was unaffected by PI3K inhibitors (n=3). (C) Similarly, Src activation, assessed by its Y416 phosphorylation, was also unaffected by PI3K inhibitors (n=3). (D) Mesangial cells were treated with both PI3K inhibitors prior to HG for 1 h, after which ADAM17 was immunoprecipitated. FAK–ADAM17 association was prevented by both inhibitors (n=3). Journal of Cell Science

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Fig. 5. Phosphorylation of ADAM17 at T735 is required for its activation by HG. (A) HG induces a time-dependent increase in ADAM17 T735 phosphorylation (pADAM17 T735) (n=6; *P<0.05 versus control, †P<0.01 versus control). (B) The MEK inhibitor U0126, which inhibits Erk activation, prevented ADAM17 activation by HG (1 h) (n=4, †P<0.01 versus others). (C) The p38 inhibitor SB203580 had no effect on ADAM17 activation by HG (n=3, †P<0.01 versus control). (D) HG (1 h)-induced ADAM17 T735 phosphorylation was inhibited by the PI3K inhibitors LY294002 (LY) and wortmannin (wort), and by the MEK inhibitors PD98059 (PD) and U0126 (n=3, †P<0.01 versus control and LY, and *P<0.05 versus wortmannin, PD98059 and U0126). (E) PI3K inhibitors LY294002 and wortmannin, and the FAK inhibitor PF5732008 (PF) inhibit HG (1 h)-induced Erk activation (n=3, †P<0.01 versus other groups). (F) ADAM17 KO MEFs were transfected with empty vector pcDNA, WT ADAM17 or T735A ADAM17. HG induced ADAM17 activation only in cells expressing WT ADAM17 (n=4, *P<0.05, †P<0.01, #P<0.001 versus both pcDNA groups). (G) ADAM17 KO MEFs were transfected with WT or T735A ADAM17, treated with HG for 1 h, then ADAM17 immunoprecipitated using its HA tag. No HG-induced association between ADAM17 and FAK was seen in cells expressing T735A (n=3, †P<0.01 versus other groups). IgG control immunoprecipitation of HG-treated cells with WT ADAM17 shows some nonspecific pull-down of FAK. LC, antibody light chain. inhibitors of either PI3K or MEK, the immediate upstream activator phosphorylation in this interaction. Using ADAM17 KO MEFs of Erk, prevented HG-induced T735 phosphorylation. To confirm transfected with either WT or T735A ADAM17, Fig. 5G shows that that PI3K functions upstream of the MEK–Erk signaling pathway, expression of ADAM17 T735A prevented HG-induced association we determined whether PI3K inhibition could block Erk activation between FAK and ADAM17. These data suggest that PI3K–MEK– by HG. Fig. 5E shows that both PI3K inhibitors prevented HG- Erk induction of T735 phosphorylation is required for HG-induced induced Erk activation. Inhibition of FAK, upstream of PI3K, also ADAM17 association with FAK and ADAM17 activation. prevented HG-induced Erk activation. None of the inhibitors affected basal Erk activity (Fig. S2). HG-induced increase of mature ADAM17 at the cell surface is To verify the importance of T735 phosphorylation in ADAM17 regulated by T735 and Y702 phosphorylation activation by HG, ADAM17 KO MEFs were transfected with either Presence of ADAM17 at the cell surface has been correlated in some WT ADAM17 or the nonphosphorylatable mutant T735A. Fig. 5F cases with ligand cleavage (Lorenzen et al., 2016). We first assessed shows that compared with empty vector, both WT and T735A whether HG increases cell surface presence of the mature enzyme ADAM17 demonstrated basal activity, but the HG response was using biotinylation and pull down of cell surface proteins. Fig. 6A observed only in cells expressing WT ADAM17. Finally, since we shows a time-dependent increase in the mature form of ADAM17 had observed that PI3K inhibition prevented ADAM17–FAK (∼100 kDa) in response to HG. PDGFR served as the loading association (Fig. 4D), we assessed the role of T735 control for cell surface proteins. In contrast, the pro-form of Journal of Cell Science

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Fig. 6. HG increases mature ADAM17 at the cell surface through furin-mediated processing. (A) Mesangial cells were treated with HG for the indicated times. Cell surface proteins were biotinylated and immunoprecipitated, and ADAM17 identified by immunoblotting. Two bands are seen in the input (cell lysate), at around 120 kDa and 100 kDa, representing the proform of ADAM17 (pro) at the higher molecular weight, and the mature form at the lower molecular weight. Only the mature form appears at the cell surface, which is increased by HG (n=4). PDGFR serves as the loading control for cell surface proteins. (B) Mesangial cells were treated with the furin inhibitors hexa-D-arginine (hexa) or decanoyl-RVKR-CMK (CMK) prior to HG for 1 h. Cell surface proteins were biotinylated and pulled down, and ADAM17 assessed by immunoblotting. Both inhibitors decrease the HG-induced increase in mature ADAM17 at the cell surface, with a concomitant increase in the pro-form (n=4; †P<0.01, *P<0.05). (C,D) Mesangial cells were incubated with increasing concentrations of either hexa-D-arginine or decanoyl-RVKR-CMK. There was a dose-dependent decrease in ADAM17 activation by HG (1 h) with both inhibitors (for both, n=4; †P<0.01, ‡P<0.001).

ADAM17 was not seen at the cell surface. The increase in the and transfected ADAM17 identified by its HA tag. As seen in mature form of ADAM17 at the cell surface suggests that cleavage Fig. 7A, HG increased cell surface presence of only WT ADAM17. of its prodomain in response to HG is important for its activation. Both Src and Erk have been shown to regulate inducible trafficking We thus used two inhibitors of furin, decanoyl-RVKR-CMK and of ADAM17 to the cell surface (Soond et al., 2005; Taniguchi et al., the more specific inhibitor hexa-D-arginine, to assess their effects 2013). Having identified phosphorylation of Y702 by Src and T735 on ADAM17 maturation and activity in HG. Mesangial cells were by Erk as important to ADAM17 activation by HG, we next first treated with inhibitors to assess their effect on cell surface determined if phosphorylation at these sites regulated the cell ADAM17 after 1 h of HG. As seen in Fig. 6B, both inhibitors surface increase of ADAM17. We used ADAM17 KO MEFs prevented the increase of mature ADAM17 at the cell surface. We expressing WT, T735A or Y702A ADAM17 to assess their next determined whether this increase in mature ADAM17 was effect on cell surface localization in response to HG. As seen in required for activity in response to HG. As seen in Fig. 6C,D, there Fig. 7A, only WT ADAM17 trafficked to the cell surface, while this was a dose-dependent decrease in HG-induced ADAM17 activation was completely prevented in the absence of T735 or Y702 with both inhibitors, confirming that the increased level of mature phosphorylation. ADAM17 is functionally relevant. Since ADAM17 T735 phosphorylation is mediated by PI3K–Erk Since we showed that the C-terminus is required for ADAM17 (Fig. 5), we next tested the effect of PI3K inhibition on the increase activation by HG, we next determined whether it was necessary for in cell surface mature ADAM17 seen with HG. Fig. 7B shows that the increased cell surface levels. ADAM17 KO MEFs were both PI3K inhibitors LY294002 and wortmannin effectively transfected with either WT ADAM17 or ΔC ADAM17, treated prevented this. Src inhibitors SU6656 and PP2 also prevented the with HG for 1 h, cell surface proteins biotinylated and pulled down cell surface increase of mature ADAM17 in response to HG Journal of Cell Science

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Fig. 7. HG-induced increase of mature ADAM17 at the cell surface is differentially regulated by T735 and Y702 phosphorylation. (A) ADAM17 KO MEFs were transfected with WT ADAM17, T735A ADAM17 or Y702A ADAM17. After HG for 1 h, cell surface proteins were isolated and constructs identified by their HA tag. HG-induced ADAM17 cell surface localization required both T735 and Y702 phosphorylation (n=4, ‡P<0.001 versus its control). (B) PI3K inhibitors LY294002 (LY) and wortmannin (wort) prevented cell surface localization of mature ADAM17 (n=6, †P<0.01 versus other groups). (C) Src inhibitors SU6656 and PP2 also prevented cell surface localization of mature ADAM17 (n=4; †P<0.01 versus control, *P<0.05 versus HG).

(Fig. 7C). These data suggest that both PI3K and Src mediate mature however, has not been identified. We propose the following model for ADAM17 cell surface localization in response to HG, with their ADAM17activationbyHGinkidneymesangialcells:FAKisan downstream phosphorylation of ADAM17 at T735 and Y702 upstream key mediator of ADAM17 activation through its recruitment respectively being required. However, the precise mechanism of both Src and PI3K, with subsequent phosphorylation of ADAM17 underlying this increase is as yet unknown. at two sites in its C-terminus. Y702 is phosphorylated by Src and T735 by PI3K–MEK-activated Erk. Phosphorylation at both sites enhances Phosphorylation at T735 and Y702 are both required for association of ADAM17 with FAK and is required for downstream ADAM17-mediated TGFβ1 upregulation in response to HG profibrotic effects. HG also leads to increased furin-mediated Since we identified the necessity of both T735 and Y702 processing of ADAM17 to its mature form and increased phosphorylation for ADAM17 activation by HG (Fig. 2F, translocation of mature ADAM17 to the membrane. These studies Fig. 4F), and we previously showed that ADAM17 is required for underscore a crucial role for the C-terminus of ADAM17 in its ability HG-induced TGFβ1 upregulation (Uttarwar et al., 2011), we tested to interact with upstream signaling mediators, and highlight the central the effects of these two mutants on TGFβ1 production. Either WT role of FAK in ADAM17 activation by HG. Inhibition of ADAM17 ADAM17 or one of the mutants was transfected into ADAM17 KO activation through targeting HG-specific activators such as FAK, or MEFs, and TGFβ1 promoter upregulation and secretion into the their interaction with ADAM17, may offer an alternative novel medium were assessed. Fig. 8 shows that phosphorylation at both approach to the treatment of diabetic nephropathy. sites is required for this profibrotic response. The importance of the cytoplasmic domain of ADAM17 to its activation has been controversial. It is well established that this DISCUSSION region is not required for constitutive activity (Le Gall et al., 2010), ADAM17 is becoming increasingly recognized as important to the and our data confirm this since there was no difference between the pathogenesis of diabetic nephropathy. How it is activated by HG, basal activity of WT and ΔC ADAM17. We further confirmed that Journal of Cell Science

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Fig. 8. Phosphorylation at T735 and Y702 are both required for ADAM17-mediated TGFβ1 upregulation in response to HG. ADAM17 KO MEFs were transfected with WT ADAM17 or T735A ADAM17 (A,C) and Y702A ADAM17 (B,D). (A,B) Activation of a TGFβ1 promoter luciferase by HG (24 h) was seen only with WT ADAM17 (A, n=6; ‡P<0.001 versus other groups; B, n=6, *P<0.05 versus WT controls). (C,D) Total TGFβ1 secretion into the medium, as assessed by ELISA, was increased only by WT ADAM17 (C, n=6; †P<0.01 versus other groups; D, n=6, ‡P<0.001 versus other groups). phorbol esters do not require the C-terminus for induction of coimmunoprecipitation studies is likely to be indirect. Interestingly, ADAM17 activity. However, the requirement for the C-terminus in however, phosphorylation of ADAM17 at both its Y702 and T735 ADAM17 activation appears to be stimulus specific. For example, appear to be required for mediating interaction with FAK. The while phorbol esters, IL-1, thrombin, EGF, LPA, TNFα, FGF and precise nature of the molecular interactions in this complex remain PDGF can activate ADAM17 independently of its C-terminus to be more fully defined. (Doedens et al., 2003; Hall and Blobel, 2012; Le Gall et al., 2010; Several phosphorylation sites have been identified in ADAM17, Maretzky et al., 2011; Mendelson et al., 2010), activation by including T735, S791, S819 and Y702. Phosphorylation at T735 is angiotensin II requires the presence of the C-terminus (Elliott et al., the most studied and is induced by either Erk or p38, depending on 2013). The cytoplasmic domain of ADAM17 is also required for the stimulus (Diaz-Rodriguez et al., 2002; Rousseau et al., 2008; Xu shedding of TNFα (Schwarz et al., 2013), although interestingly, it and Derynck, 2010). Here, we showed that phosphorylation on has been suggested that only the six most membrane-proximal T735 is essential for ADAM17 activation by HG, and that Erk, but amino acids of the cytoplasmic tail are required (Schwarz et al., not p38, was required for this. How phosphorylation at this site 2013). We now show that HG-induced ADAM17 activation is also enables ADAM17 activation is, as yet, not fully understood. dependent on an intact C-terminus, and confirm similar dependence Interestingly, however, T735 phosphorylation has recently been on the C-terminus of HG-induced shedding of the membrane- shown to prevent the dimerization of ADAM17, releasing it from anchored substrate HB-EGF (Fig. S3). This is likely to be due to a inhibition by TIMP3 and thereby allowing activation (Xu et al., dependence on interaction of this region with upstream signaling 2012). It has also been shown to regulate ADAM17 maturation and mediators, namely a FAK–PI3K–Src complex. Indeed, this region trafficking to the cell surface in COS-7 cells (Soond et al., 2005; Xu of ADAM17 has both a phosphotyrosine site which can bind to SH2 and Derynck, 2010), potentially increasing the pool of available domains, as well as a proline-rich region which can bind to SH3 mature ADAM17 for activation. Our data support a role for T735 domains, enabling interaction with various signaling molecules phosphorylation in increasing membrane-localized mature (Arribas and Esselens, 2009; Kleino et al., 2015). Thus, Src and ADAM17 in response to HG. Interestingly, phosphorylation at PI3K can directly interact with the ADAM17 C-terminus through this site is also important in allowing interaction with FAK, their SH2/SH3 and SH2 domains, respectively. While FAK does suggesting multiple roles in enabling ADAM17 activation. not possess either of these domains, it is able to bind both Src and Although Src is known to contribute to ADAM17 activation in

PI3K. Thus, the interaction between FAK and ADAM17 seen in our various settings, its phosphorylation of ADAM17 at Y702 has been Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629 appreciated more recently. Mechanical stress in myoblasts and An important role for phosphatidylserine exposure at the outer cardiomyocytes induces Src-mediated ADAM17 phosphorylation leaflet of the cell membrane is now proposed as a key common event at Y702, mediating myogenesis and TNFα shedding, respectively in ADAM17 activation by diverse stimuli (Sommer et al., 2016). (Niu et al., 2013, 2015). Y702 phosphorylation is also needed for Phosphatidylserine binds the membrane-proximal domain of angiotensin-II-mediated hypertrophy in vascular smooth muscle ADAM17, facilitating ADAM17 membrane binding through its cells (Elliott et al., 2013). We now show that HG induces Src- short juxtamembrane segment CANDIS. This brings the catalytic mediated ADAM17 Y702 phosphorylation. While not affecting site in close proximity to the cleavage site of membrane-bound basal ADAM17 activity, phosphorylation at this site is essential to HG- substrates, enabling substrate cleavage. Whether this process also induced ADAM17 activation and downstream matrix upregulation. occurs in response to HG requires confirmation. Interestingly, Y702 phosphorylation is also required for HG-induced In an effort to prevent adverse effects seen with compounds which association between ADAM17 and FAK, likely through intermediates more broadly inhibit metalloproteases, ongoing efforts are underway including Src and/or PI3K. Like T735 phosphorylation, Y702 for the development of inhibitors with increased specificity for phosphorylation is also required for increased membrane localization ADAM17. Yet even ADAM17-specific inhibitors are associated of ADAM17 in response to HG. These data are consistent with other with unwanted adverse effects, probably because of the multiple studies in which Src was found to be required for ADAM17 substrates and ubiquitous nature of the enzyme (Rossello et al., translocation to the membrane after prolonged (48 h) exposure to HG 2016). An alternative approach to ADAM17 inhibition is the in mesangial cells (Taniguchi et al., 2013). We confirmed a identification of stimulus-specific regulation of its activation. Our requirement for Src also in early (1 h) increases in the level of cell data provide novel insight into the mechanism of HG-induced surface mature ADAM17 in response to HG. These data suggest an ADAM17 activation, showing the importance of C-terminus important role for Src-induced Y702 phosphorylation in mature interaction with a FAK–PI3K–Src complex and phosphorylation ADAM17 membrane translocation and activation. The precise of ADAM17 at both Y702 and T735 in regulating ADAM17 mechanism by which this occurs has yet to be determined. activation through complementary mechanisms. Thus, targeting one Our data show a novel key role for the focal adhesion protein or a combination of these upstream regulators can be explored as FAK in ADAM17 activation. FAK is a non-receptor tyrosine kinase novel approaches to the treatment of diabetic nephropathy. Whether known to link integrin stimulation to intracellular signals. Upon this may be more broadly extended to include other complications of integrin engagement, FAK is activated by autophosphorylation on diabetes bears investigation. Y397 (pY397), enabling its interaction with, and activation of, Src. Phosphorylation at additional sites by Src leads to the full catalytic MATERIALS AND METHODS activity of FAK and enables interaction with additional kinases, Cell culture including PI3K (Bolos et al., 2010). FAK thus appears to provide a Primary rat mesangial cells (passages 6–15) were isolated from Sprague– scaffold for the assembly of a Src–PI3K–ADAM17 complex, as Dawley rats. They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal calf serum (Invitrogen), discussed above. Of relevance, ADAM17 has also been shown to μ associate with integrin α5β1, but whether this interaction is direct or streptomycin (100 g/ml) and penicillin (100 units/ml) at 37°C in 95% air, 5% CO2. These are periodically checked for mycoplasma contamination. mediated by FAK is unknown. Functionally, this integrin either SV40-immortalized MEFs isolated from ADAM17 KO and WT mice were inhibits ADAM17 activation in unstimulated cells (Gooz et al., generously provided by Carl Blobel (Cornell University, NY, USA), and 2012; Saha et al., 2010), improves cell adhesion (Bax et al., 2004) or their expression of ADAM17 was confirmed by immunoblotting. They were inhibits cell migration (Gooz, 2010). Integrin β1 was shown to be cultured in DMEM supplemented with 10% fetal calf serum. Medium for activated by HG to regulate ECM assembly in mesangial cells with both cell types contained 5.6 mM glucose. Glucose at 24.4 mM (final longer term HG exposure (Miller et al., 2014). Whether it is also concentration 30 mM, Sigma) was added for high glucose (HG) conditions. required for early FAK-mediated activation of ADAM17 will be Cells were made quiescent by serum deprivation for 24 h prior to treatment. addressed in future studies. Pharmacological inhibitors were added prior to glucose as follows: LY294002 (10 μM, 30 min, Sigma), wortmannin (100 nM, 1 h, Sigma), ADAM17 is produced as a proenzyme synthesized in the μ μ endoplasmic reticulum. This intracellular pool can undergo rapid PF573228 (1 M, 1 h, Tocris), SU6656 (10 M, 30 min, Calbiochem), PP2 (10 μM, 30 min, Calbiochem), PD98059 (10 μM, 30 min, Alexis), U0126 processing to the mature form, increasing the availability of mature (10 μM, 30 min, Promega), SB203580 (50 μM, 30 min, Sigma), PMA enzyme at the membrane (Lorenzen et al., 2016). While some (500 ng/ml, 1.5 h, Sigma), decanoyl-RVKR-CMK (100 μM, 24 h, Tocris) stimuli do not increase the availability of membrane mature and hexa-D-arginine (100μM, 24 h, Tocris). ADAM17 (Le Gall et al., 2010; Lorenzen et al., 2016), our studies show its induction in response to HG in a short time frame Transfection and constructs (1 h) which was required for its activation. Proprotein convertases, Constructs for WT ADAM17 or ADAM17 with the C-terminus deleted (ΔC) primarily furin, cleave the N-terminal prodomain of ADAM17 in and containing a 3′ HA tag were kindly provided by Carl Blobel (Cornell the Golgi prior to transport to the cell surface (Srour et al., 2003). University), with both originally provided by Gillian Murphy, University of Our data show that HG increases furin-mediated cleavage of Cambridge, UK). A 3′ HA tag was added to the WT ADAM17 construct. ′ ′ ADAM17, and this is an important step in the increase in levels of ADAM17 T735A (3 HA) and ADAM17 E406A (5 HA) were also provided ′ the mature form at the cell surface and in HG-induced increases in by Carl Blobel, and ADAM17 Y702A (5 HA) was provided by Yi-Ping Li (University of Texas, TX, USA). All constructs were based on mouse ADAM17 activity. Recently, the endoplasmic reticulum-resident ADAM17. proteins iRhoms1 and 2 were shown to be important chaperones for escort of ADAM17 both to the Golgi for processing and to the cell ADAM17 activity assay membrane (Adrain et al., 2012; Christova et al., 2013; Li et al., Where indicated, ADAM17 KO MEFs or mesangial cells were transfected 2015b; McIlwain et al., 2012). PACS-2 is another newly described with 7.5 μg of ADAM17 construct in 6-well plates, coated with poly-D- regulator of ADAM17 trafficking to the cell membrane lysine for MEFs, at 40% confluence using X-fect (Clontech). Cells were (Dombernowsky et al., 2015). Whether HG regulates iRhom and/ serum-deprived for 24 h, then treated with HG for 1 h. Protein was extracted or PACS2 function is as yet unknown. in activity assay buffer (50 mM Tris-HCl, pH 7.4, 25 mM NaCl, 4% Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629

glycerol, 10 mM ZnCl2). ADAM17 activity was measured in duplicate for with two conditions being analysed, a t-test was used and a test for equal each sample using 20 μg protein and the TACE Substrate IV (Calbiochem). variances used to interpret significance. A P-value <0.05 (two-tailed) was Cleavage of this substrate was measured in a fluorometer at 420 nm. considered significant. Data are presented as the mean±s.e.m. The number HB-EGF cleavage was assessed in cells transfected with pRC/CMV- of experimental repetitions (n) is indicated. HBEGF-AP, kindly provided by Michael Freeman (Harvard University, Cambridge, MA, USA). ADAM17 KO MEFs were transfected with 3.5 μg Acknowledgements of this vector in conjunction with 3.5 μg ADAM17 construct as above. After J.C.K. gratefully acknowledges the support of St Joseph’s Healthcare for nephrology serum deprivation, they were incubated in 1 ml of 0% FBS medium to assess research. We thank Naoya Kato (University of Tokyo, Japan) for providing the β baseline shedding. This was replaced with 1 ml of new medium with or TGF 1 promoter luciferase construct, Carl Blobel (Cornell University) for providing without HG which was collected after 1 h. Shedding was assessed using an the ADAM17 KO MEFs, ADAM17 T735A and ADAM17 E406A; Carl Blobel and Gillian Murphy (University of Cambridge, UK) for providing WT ADAM17 and ΔC alkaline phosphatase activity (ALP) assay (AnaSpec). Shedding induced by ADAM17; Dr Yi-Ping Li (University of Texas) for providing ADAM17 Y702A; and treatment was assessed by comparing to baseline shedding for each well. Michael Freeman (Harvard University) for providing pRC/CMV-HBEGF-AP.

Luciferase assay Competing interests ADAM17 KO MEFs plated to 30% confluence were transfected with 2.5 μg The authors declare no competing or financial interests. in 12-well plates with the ADAM17 construct as indicated using X-fect (Clontech), followed the next day by transfection with 0.5 μgofTGFβ1 Author contributions promoter-luciferase construct (kindly provided by Naoya Kato, University of Conceptualization: J.C.K.; Methodology: R.L.; Formal analysis: J.C.K.; Data Tokyo, Japan) and 0.05 μg pCMV-β-galactosidase (β-gal) (Clontech) using curation: R.L., T.W., K.W., B.G.; Writing - original draft: J.C.K.; Writing - review & editing: J.C.K.; Supervision: J.C.K.; Project administration: J.C.K. Lipofectamine (Qiagen). Cells were serum-deprived for 24 h after transfection, then exposed to HG for 48 h. Lysis was achieved with Funding Reporter Lysis Buffer (Promega) using one freeze–thaw cycle, and luciferase β This work was supported by the Institute of Nutrition, Metabolism and Diabetes of the and -gal activities measured on clarified lysate using specific kits (Promega) Canadian Institutes of Health Research (CIHR) (MOP119308 to J.C.K.). with a Berthold luminometer and a plate reader (420 nm), respectively. β-gal activity was used to adjust for transfection efficiency. Supplementary information Supplementary information available online at TGFβ1 ELISA http://jcs.biologists.org/lookup/doi/10.1242/jcs.208629.supplemental ADAM17 KO MEFs were transfected with 2.5 μg ADAM17 construct as above in 12-well plates. After HG (48 h), medium was harvested and debris References removed by centrifugation at 4°C, 4000 rpm. Medium was stored at −80°C Adrain, C., Zettl, M., Christova, Y., Taylor, N. and Freeman, M. (2012). Tumor necrosis factor signaling requires iRhom2 to promote trafficking and activation of until processing. Total TGFβ1 was measured after acid activation of samples ’ TACE. Science 335, 225-228. according to the manufacturer s instructions (R&D Systems). Arribas, J. and Esselens, C. (2009). ADAM17 as a therapeutic target in multiple diseases. Curr. Pharm. Des 15, 2319-2335. Protein extraction and analysis Bax, D. V., Messent, A. J., Tart, J., van Hoang, M., Kott, J., Maciewicz, R. A. and Cells were lysed and protein extracted as we described previously (Krepinsky Humphries, M. J. (2004). Integrin alpha5beta1 and ADAM-17 interact in vitro and μ co-localize in migrating HeLa cells. J. Biol. Chem. 279, 22377-22386. et al., 2003), with the addition of BB-94 (1 M, Tocris) to the lysis buffer. Bolos, V., Gasent, J. M., Lopez-Tarruella, S. and Grande, E. (2010). The dual Cell lysates were centrifuged at 4°C, 14,000 rpm for 10 min to pellet cell kinase complex FAK-Src as a promising therapeutic target in cancer. Onco. debris. Supernatant (50 g) was separated on SDS-PAGE and western blotting Targets. Ther. 3, 83-97. was performed. Antibodies were goat ADAM17 (1:1000, Santa Cruz, sc- Cantley, L. C. (2002). The phosphoinositide 3-kinase pathway. Science 296, 6416), rabbit pADAM17 Y702, kindly provided by Yi-Ping Li (University of 1655-1657. Texas) (Niu et al., 2013) (1:1000), rabbit pADAM17 T735 (1:1000, Sigma, Christova, Y., Adrain, C., Bambrough, P., Ibrahim, A. and Freeman, M. (2013). SAB4504073), mouse HA (1:1000, Abcam, Ab18181), rabbit Src (1:1000, Mammalian iRhoms have distinct physiological functions including an essential Cell Signaling, 2108), rabbit pSrc Y416 (1:1000, Cell Signaling, 2101), rabbit role in TACE regulation. EMBO Rep. 14, 884-890. Diaz-Rodriguez, E., Montero, J. C., Esparis-Ogando, A., Yuste, L. and FAK (1:1000, Santa Cruz, sc-558), rabbit pFAK Y397 (1:1000, Upstate, 07- Pandiella, A. (2002). Extracellular signal-regulated kinase phosphorylates 012), mouse pErk T202/Y204 (1:1000, Cell Signaling, 9106), total Erk tumor necrosis factor alpha-converting enzyme at threonine 735: a potential (1:1000, Cell Signaling, 9102) and mouse tubulin (1:10,000, Sigma, T6074). role in regulated shedding. Mol. Biol. Cell 13, 2031-2044. For immunoprecipitation experiments, cells were lysed, clarified and Doedens, J. R., Mahimkar, R. M. and Black, R. A. (2003). TACE/ADAM-17 equal amounts of lysate incubated overnight with 2 μg primary antibody enzymatic activity is increased in response to cellular stimulation. Biochem. rotating at 4°C, followed by 25 μl of protein-G–agarose slurry for 1.5 h at Biophys. Res. Commun. 308, 331-338. 4°C. Immunoprecipitates were extensively washed, resuspended in 2× Dombernowsky, S. L., Samsøe-Petersen, J., Petersen, C. H., Instrell, R., sample buffer, boiled and analyzed by immunoblotting. Hedegaard, A. M. B., Thomas, L., Atkins, K. M., Auclair, S., Albrechtsen, R., Mygind, K. J. et al. (2015). The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17. Nat. Commun. 6, 7518. Biotinylation for isolation of cell surface proteins Elliott, K. J., Bourne, A. M., Takayanagi, T., Takaguri, A., Kobayashi, T., Eguchi, Cells plated in 100 mm plates were washed with ice-cold PBS and incubated K. and Eguchi, S. (2013). ADAM17 silencing by adenovirus encoding miRNA- with EZ-link Sulfo-NHSLC-Biotin (0.5 mg/ml in PBS, Fisher) for 20 min. embedded siRNA revealed essential signal transduction by angiotensin II in Biotinylation was stopped with 0.1 M glycine in PBS. Cells were lysed in IP vascular smooth muscle cells. J. Mol. Cell Cardiol. 62C, 1-7. ̈} lysis buffer (PBS pH 7.4, 5 mM EDTA, 5 mM EGTA, 10 mM sodium Ford, B. M., Eid, A. A., Gooz, M., Barnes, J. L., Gorin, Y. C. and Abboud, H. E. (2013). ADAM17 mediates Nox4 expression and NADPH oxidase activity in the pyrophosphate, 50 mM NaF, 1 mM NaVO , 1% Triton X-100, 1 μM BB- 3 kidney cortex of OVE26 mice. Am. J. Physiol. Renal Physiol. 305, F323-F332. 94, protease inhibitors). Biotinylated proteins were precipitated with 50% Gooz, M. (2010). ADAM-17: the enzyme that does it all. Crit Rev. Biochem. Mol. Biol. neutravidin slurry (Fisher) overnight, after which the beads were washed, 45, 146-169. boiled in PSB and proteins assessed by immunoblotting. When used, Gooz, P., Dang, Y., Higashiyama, S., Twal, W. O., Haycraft, C. J. and Gooz, M. ADAM17 KO MEFs were transfected with 30 μg of the indicated ADAM17 (2012). A disintegrin and metalloenzyme (ADAM) 17 activation is regulated by plasmid as above prior to treatment. alpha5beta1 integrin in kidney mesangial cells. PLoS One 7, e33350. Ha, J. S., Kwon, K. S. and Park, S. S. (2013). 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