RESEARCH ARTICLE

Regulation of profibrotic responses by ADAM17 activation in highglucose requires its C-terminus and FAKRenzhong Li, Tony Wang, Khyati Walia, Bo Gao and Joan C. Krepinsky*

ABSTRACTGlomerular matrix accumulation is the hallmark of diabeticnephropathy. The metalloprotease ADAM17 mediates high glucose(HG)-induced matrix production by kidney mesangial cells throughrelease of ligands for the epidermal growth factor receptor. Here,we study the mechanism by which HG activates ADAM17. We findthat the C-terminus is essential for ADAM17 activation and theprofibrotic response to HG. In the C-terminus, Src-mediated Y702phosphorylation and PI3K–MEK–Erk-mediated T735 phosphorylationare crucial for ADAM17 activation, both are also required for theHG-induced increase in cell surface mature ADAM17. The non-receptor tyrosine kinase FAK is a central mediator of these processes.These data not only support a crucial role for the C-terminus inADAM17 activation and downstream profibrotic responses to HG, butalso highlight FAK as a potential alternative therapeutic target fordiabetic nephropathy.

KEY WORDS: ADAM17, Glucose, Fibrosis, Trafficking, Signaling

INTRODUCTIONDiabetic nephropathy is a major complication of diabetes and theleading cause of kidney failure in North America. Patients withdiabetic nephropathy suffer the highest morbidity and mortality ofany kidney failure patient group (Johnson and Spurney, 2015).Prevention and treatment of diabetic nephropathy is thus a majorunmet clinical need. Although pathological changes occur in allkidney compartments, glomerular sclerosis initiated by depositionof extracellular matrix (ECM) protein is the hallmark and earliestmanifestation. Multifactorial interventions including glucosecontrol at best only delay disease progression (Lewis et al., 1993,2001). There is thus a major need to better understand itspathogenesis to enable identification of new therapeutic targets.High glucose (HG) plays a central role in the pathogenesis of

diabetic nephropathy by increasing ECM production in glomerularmesangial cells. The prosclerotic cytokine transforming growthfactor β1 (TGFβ1) is a major mediator of the HG-induced MCfibrotic response (Li et al., 2003).We and others have shown that themetalloprotease A disintegrin and metalloprotease 17 (ADAM17),through release of ligands for the epidermal growth factor receptor(EGFR), is an important mediator of HG-induced TGFβ1upregulation and ECM protein production in kidney cells (Fordet al., 2013; Uttarwar et al., 2011). Of the several EGFR ligands thatcan be cleaved by ADAM17, TGFα, heparin binding (HB)-EGF

and amphiregulin have been implicated in the pathogenesis ofdiabetic kidney disease (Kefaloyianni et al., 2016; Melenhorst et al.,2009; Uttarwar et al., 2011). Cleavage of the renoprotectiveexopeptidase ACE2 by ADAM17 is also thought to contribute todiabetic kidney tubular injury (Salem et al., 2014). In diabetic mice,a nonselective metalloprotease inhibitor decreased glomerularsclerosis, although effects cannot be specifically attributed toinhibition of ADAM17 (Ford et al., 2013). More specific ADAM17inhibitors, however, have been associated with adverse effects,preventing their long-term use in the treatment of diabeticnephropathy (Liu and Kurzrock, 2014; Rossello et al., 2016).Toxicity may be due to the ubiquitous expression of the enzyme andits multitude of substrates including proinflammatory cytokines andadhesion molecules in addition to growth factor receptor ligands(Gooz, 2010). Since ADAM17 activation shows stimulusspecificity, this provides an opportunity for developing targetedcontext-dependent ADAM17 inhibition. We thus focused ourstudies on understanding the key mediators of ADAM17 regulationby HG.

ADAM17 is a transmembrane metalloprotease with an inhibitoryN-terminal prodomain that is cleaved by proprotein convertases,primarily furin, in the Golgi. The mature enzyme comprises ametalloprotease domain followed by a disintegrin domain, acysteine-rich domain important for substrate recognition, an EGF-like domain, a transmembrane domain and a cytoplasmic domain.The transmembrane domain is required for enzyme activation(Maretzky et al., 2011; Rossello et al., 2016).

The role of the cytoplasmic domain (amino acids 695–824) inregulating ADAM17 activation, however, is as yet not fully clear,and appears to depend on the type of stimulus-inducing activity(Gooz, 2010). For example, phorbol esters and several growthfactors can induce early ADAM17 activation in the absence of a C-terminus (Doedens et al., 2003; Hall and Blobel, 2012; Le Gallet al., 2010; Maretzky et al., 2011; Mendelson et al., 2010), whereasangiotensin II requires the C-terminus to be present for induction ofenzyme activity (Elliott et al., 2013). The C-terminus may allowinteraction with other proteins through its SH2 and SH3 bindingdomains, with phosphorylation at both serine/threonine and tyrosineresidues shown to be functionally important in different settings(Gooz, 2010). Whether this is relevant to HG-induced ADAM17activation is as yet undetermined.

Indeed, little is known of the mechanism by which HG enablesactivation ofADAM17.We thus began by investigating the relevanceof the C-terminus in this setting, and found it to be essential foractivation of ADAM17 by HG. We identified two phosphorylationsites in the C-terminus – Src-mediated Y702 phosphorylation andPI3K–MEK–Erk-mediated T735 phosphorylation – to be crucial forthe activation and downstream profibrotic responses of ADAM17.HG also induced increases in the amount of mature ADAM17 at thecell surface, which is dependent on furin activity and phosphorylationat both T735 and Y702. We further identified the non-receptorReceived 17 July 2017; Accepted 28 December 2017

Division of Nephrology, McMaster University, Hamilton, Canada, L8N 4A6.

*Author for correspondence (krepinj@mcmaster.ca)

T.W., 0000-0002-1203-1619; B.G., 0000-0001-8051-1430; J.C.K., 0000-0002-6761-909X

1

© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs208629. doi:10.1242/jcs.208629

Journal

ofCe

llScience

tyrosine kinase FAK as a central upstream regulator of HG-inducedADAM17 activation. These data identify a crucial role for theC-terminus of ADAM17 in HG-induced activation, but alsodemonstrate regulation of ADAM17 by HG at multiple levels.

RESULTSThe C-terminus is required for ADAM17 activation by HG anddownstream profibrotic responsesTo determine the role of the C-terminus in HG responses in MC,either WTADAM17 or ADAM17 with its C-terminus deleted (ΔC)were transfected into mesangial cells, with ADAM17 activityassessed after 1 h of HG (Fig. 1A). The HG-induced ADAM17activation seen in cells transfected with the empty vector pcDNAwas augmented after overexpression of WT ADAM17. Thisaugmentation is similar to the response seen after prolonged HG

exposure (24 h), which results in ADAM17 upregulation (Li et al.,2015a). In contrast, no HG response occurred after transfection withΔC ADAM17. To confirm these results, we next used ADAM17-knockout (KO) mouse embryonic fibroblasts (MEFs). Absenceof ADAM17 in these cells was confirmed by immunoblotting(not shown). We first established that HG increases ADAM17activity in MEF cells (Fig. 1B). In ADAM17 KO cells, baselinevalues measured by the assay were significantly lower, and noHG response was seen. ADAM17 activity was next tested aftertransfection of empty vector, WT ADAM17 or ΔC-ADAM17 intoKO cells. Construct expression was confirmed by immunoblottingfor the HA tag (Fig. 1C). While transfection of either ADAM17construct increased basal activity, only cells transfected with WTADAM17 showed a HG response (Fig. 1D). Since PMA is wellknown 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 wasexpressed in mesangial cells. Absence of the C-terminus prevented HG-induced ADAM17 activation (n=6, ‡P<0.001). (B) ADAM17 activity was tested inADAM17 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, orempty vector, were expressed in ADAM17 KOMEFs. 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 cellsexpressingWTADAM17 (n=4, †P<0.01 versus all others). (E) ADAM17WTor ΔCwas expressed in KOMEFs. PMA induced ADAM17 activation equally with bothconstructs (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 thecatalytically inactivemutant of ADAM17, E406A. This mutant also did not show basal activity, unlike that seenwithWTADAM17 (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 aTGFβ1 promoter luciferase by HG (48 h) was seen only withWTADAM17 (n=6, ‡P<0.001 versus all others). (H) Total TGFβ1 in themediumwas also increased byHG only in cells expressing WT ADAM17 (n=5, †P<0.01 versus all others).

2

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

Journal

ofCe

llScience

(Doedens et al., 2003), we next tested responsiveness of WT or ΔCADAM17 to PMA in KO cells (Fig. 1E). ADAM17 activity wasinduced to the same degree with both constructs, confirmingprevious findings. Finally, to determine whether the catalyticactivity of ADAM17 was required, we determined the HGresponsiveness of a catalytically inactive mutant ADAM17E406A. As shown in Fig. 1F, ADAM17 KO MEFs transfectedwith the catalytically inactive mutant (with expression shown in theinset) had neither an increase in basal activity, nor did they showincreased activity in response to HG, indicating that both theC-terminus and ADAM17 catalytic activity are required forADAM17 activation by HG.We previously showed that ADAM17 activation was required for

HG-induced upregulation of the profibrotic cytokine TGFβ1(Uttarwar et al., 2011), a key pathogenic factor in diabeticnephropathy (Li et al., 2003). To confirm the importance of theADAM17 C-terminus in this response, we used ADAM17 KOMEFs transfected with WT or ΔC ADAM17. Only in cells

expressing WT ADAM17 was HG able to increase TGFβ1promoter luciferase activity (Fig. 1G) and protein secretion intothe medium (Fig. 1H).

Src phosphorylation of the C-terminus is required forADAM17 activation by HGSrc associates with ADAM17 in response to hormonal stimulation(Zhang et al., 2006) and was shown to phosphorylate ADAM17 atY702 in response to mechanical stress (Niu et al., 2013, 2015). Srcis known to mediate ADAM17 activation after long-term HGexposure (24–48 h) (Taniguchi et al., 2013). To determine whetheracute activation of ADAM17 is mediated by Src, the effect of itsinhibitors on ADAM17 activity after 1 h of HG was assessed. Twostructurally distinct inhibitors, SU6656 and PP2, prevented earlyADAM17 activation (Fig. 2A). HG increased the interactionbetween ADAM17 and Src as assessed by coimmunoprecipitation(Fig. 2B). Phosphorylation of ADAM17 at Y702 in responseto 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)-inducedADAM17 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 (pADAM17Y702) (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 KOMEFs were transfectedwithWT or ΔCADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. HG-induced Src/ADAM17 association was seen only incells 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 sameexperiment, with reprobing done to detect FAK and Src. (F) ADAM17 KOMEFs were transfected with empty vector pcDNA,WTADAM17 or Y702A ADAM17. HGinduced 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).

3

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

Journal

ofCe

llScience

phosphorylation was confirmed to be mediated by Src (Fig. 2D).Antibody specificity was confirmed in ADAM17 KO MEFstransfected with WT or Y702A ADAM17 (Fig. S1).Tyrosine 702 is located in the C-terminus of ADAM17 (aa 695–

824). Using ADAM17 KO MEFs transfected with either WT or ΔCADAM17, a requirement for the C-terminus in the associationbetween ADAM17 and Src was confirmed (Fig. 2E). To determinewhether Y702 phosphorylation is required for ADAM17 activationby HG, ADAM17 KO MEFs were transfected with either WT ornonphosphorylatable Y702AADAM17. Fig. 2F shows that althoughthe baseline activity remains higher in Y702A-transfected cells,consistent with that seen with expression of ΔC ADAM17, HG failsto induce ADAM17 activation. Together, these data show that Srcinteracts with and phosphorylates ADAM17 at its C-terminal Y702,and this is required for ADAM17 activation by HG.

FAK mediates Src phosphorylation and activation ofADAM17We next sought to determine the upstream factors mediating Src–ADAM17 activation by HG. Activation of the nonreceptor tyrosinekinase FAK was seen in diabetic kidney lysates (Regoli andBendayan, 1999), but whether FAK is activated by HG in mesangialcells or regulates ADAM17 activation is as yet unknown. In othersettings, activated FAK is known to recruit Src, enabling itsactivation (Bolos et al., 2010). We first assessed whether HGactivates FAK in mesangial cells. Fig. 3A shows that HG inducedearly activation of FAK, determined by immunoblotting for itsautophosphorylation at Y397. HG induced the association of FAKwith Src, as assessed by coimmunoprecipitation (Fig. 3B). Usingthe FAK inhibitor PF573228, Fig. 3C shows that FAK is requiredfor 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 theindicated times with HG. FAK activation, assessed by its phosphorylation on Y397 (pFAK), was increased by HG (n=2). (B) FAK was immunoprecipitated fromwhole 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 byPF573228, as assessed by coimmunoprecipitation (n=2). (E) ADAM17 phosphorylation at Y702 was also prevented by FAK inhibition with PF573228 (n=2).(F) ADAM17was immunoprecipitated fromwhole cell lysate after treatment with HG for the indicated times. Immunoblotting shows FAK association with ADAM17increases 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) ADAM17interaction 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 MEFswere transfected with WT or ΔC ADAM17, treated with HG for 1 h, then ADAM17 was immunoprecipitated using its HA tag. HG-induced FAK–ADAM17association was seen only in cells expressing WTADAM17 (n=3, *P<0.05 versus other groups). The HA blot is the same as that used in Fig. 2E as this originatesfrom 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.

4

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

Journal

ofCe

llScience

required for Src association with ADAM17 (Fig. 3D), as well asHG-induced phosphorylation of ADAM17 at Y702 by Src(Fig. 3E). Thus, FAK is required as an upstream effector of Src-mediated ADAM17 phosphorylation and ADAM17 activation.Activated Src is known to enable the full catalytic activity of FAK

through phosphorylation at additional tyrosine residues on FAK. Thisexpands the ability of FAK to interact with other proteins, furtherenhancing its scaffolding function (Bolos et al., 2010). As shown inFig. 3F, we found that FAK associated with ADAM17 in response toHG. This was not only dependent on FAK activity, as it was blockedby the inhibitor PF573228 (Fig. 3G), but also required Src activitysince it was prevented by two independent Src inhibitors (Fig. 3H).To determine whether the C-terminus of ADAM17 is needed forFAK–ADAM17 interaction, ADAM17 KO MEFs were transfectedwith either WT or ΔC ADAM17. After HG treatment for 1 h, FAKassociated with only WTADAM17, showing the requirement of theC-terminus of ADAM17 for this interaction (Fig. 3I). Interestingly,expression of Y702A ADAM17 in ADAM17 KO MEFs alsoprevented the HG-induced association between FAK and ADAM17(Fig. 3J). Here, IgG control immunoprecipitates of cells transfectedwith WT ADAM17 showed minor nonspecific pull-down of FAK(Fig. 3J). Taken together, FAK-induced Src activation andsubsequent phosphorylation of ADAM17 at Y702 promotesinteraction between FAK and ADAM17.

PI3K mediates FAK–ADAM17 association and ADAM17activation by HGWe previously showed an important role for PI3K–Akt signaling inHG-induced matrix upregulation (Wu et al., 2007, 2009).Interestingly, PI3K was shown to associate with both ADAM17and Src in cancer cells (Zhang et al., 2006). Furthermore, after itsfull activation, FAK is able to recruit PI3K by binding to its SH2domain (Bolos et al., 2010). This enables a conformational changethat releases the catalytic from the inhibitory subunit of PI3K,

enabling its activation (Cantley, 2002). To assess whether PI3K isrequired for HG-induced ADAM17 activation, we used two distinctPI3K inhibitors, wortmannin and LY294002. Fig. 4A shows thatPI3K inhibition prevented HG-induced ADAM17 activation. Asexpected, since FAK is known to recruit PI3K, FAK activation(assessed by its Y397 autophosphorylation) was unaffected byPI3K inhibitors (Fig. 4B). Src activation by HG was similarlyunaffected by PI3K inhibitors (Fig. 4C). However, PI3K activitywas required for FAK–ADAM17 association in response to HG(Fig. 4D). These data suggest that FAK and Src form a complex thatassociates with ADAM17. PI3K activity is required for this, as partof the complex and/or by facilitating complex formation through itsgeneration of signaling intermediates.

ADAM17 phosphorylation at T735 is required for itsactivation by HGPI3K was shown to mediate the serine/threonine phosphorylationof ADAM17 through PDK1 in cancer cells, although thephosphorylated residue was not identified (Zhang et al., 2006).Several serine/threonine residues of the ADAM17 C-terminus areknown to be phosphorylated, with phosphorylation at T735important for ADAM17 activation and/or trafficking andmaturation in response to specific stimuli (Diaz-Rodriguez et al.,2002; Soond et al., 2005; Xu and Derynck, 2010). We firstdetermined whether HG induces ADAM17 phosphorylation onT735. Fig. 5A shows phosphorylation at this site within 30 min ofHG exposure. The mitogen-activated protein kinases p38 and Erkare known to phosphorylate ADAM17 at T735 in different settings,and PI3K–PDK1 can also regulate Erk activation (Ha et al., 2013).We thus first tested the effects of p38 or Erk inhibition on ADAM17activation by HG. Fig. 5B,C shows that Erk inhibition using theMEK inhibitor U0126, but not p38 inhibition, prevented HG-induced ADAM17 activation. We next assessed the requirement ofPI3K 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)-inducedADAM17 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 associationwas prevented by both inhibitors (n=3).

5

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

Journal

ofCe

llScience

inhibitors of either PI3K or MEK, the immediate upstream activatorof Erk, prevented HG-induced T735 phosphorylation. To confirmthat PI3K functions upstream of the MEK–Erk signaling pathway,we determined whether PI3K inhibition could block Erk activationby HG. Fig. 5E shows that both PI3K inhibitors prevented HG-induced Erk activation. Inhibition of FAK, upstream of PI3K, alsoprevented HG-induced Erk activation. None of the inhibitorsaffected basal Erk activity (Fig. S2).To verify the importance of T735 phosphorylation in ADAM17

activation by HG, ADAM17 KOMEFs were transfected with eitherWT ADAM17 or the nonphosphorylatable mutant T735A. Fig. 5Fshows that compared with empty vector, both WT and T735AADAM17 demonstrated basal activity, but the HG response wasobserved only in cells expressing WT ADAM17. Finally, since wehad observed that PI3K inhibition prevented ADAM17–FAKassociation (Fig. 4D), we assessed the role of T735

phosphorylation in this interaction. Using ADAM17 KO MEFstransfected with either WT or T735A ADAM17, Fig. 5G shows thatexpression of ADAM17 T735A prevented HG-induced associationbetween FAK and ADAM17. These data suggest that PI3K–MEK–Erk induction of T735 phosphorylation is required for HG-inducedADAM17 association with FAK and ADAM17 activation.

HG-induced increase ofmature ADAM17at the cell surface isregulated by T735 and Y702 phosphorylationPresence of ADAM17 at the cell surface has been correlated in somecases with ligand cleavage (Lorenzen et al., 2016). We first assessedwhether HG increases cell surface presence of the mature enzymeusing biotinylation and pull down of cell surface proteins. Fig. 6Ashows a time-dependent increase in the mature form of ADAM17(∼100 kDa) in response to HG. PDGFR served as the loadingcontrol for cell surface proteins. In contrast, the pro-form of

Fig. 5. Phosphorylation of ADAM17 at T735 is required for its activation byHG. (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 ADAM17activation 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 inhibitorsPD98059 (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 andwortmannin, 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 weretransfected 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 ADAM17immunoprecipitated using its HA tag. No HG-induced association between ADAM17 and FAK was seen in cells expressing T735A (n=3, †P<0.01 versusother groups). IgG control immunoprecipitation of HG-treated cells with WT ADAM17 shows some nonspecific pull-down of FAK. LC, antibody light chain.

6

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

Journal

ofCe

llScience

ADAM17 was not seen at the cell surface. The increase in themature form of ADAM17 at the cell surface suggests that cleavageof its prodomain in response to HG is important for its activation.We thus used two inhibitors of furin, decanoyl-RVKR-CMK andthe more specific inhibitor hexa-D-arginine, to assess their effectson ADAM17 maturation and activity in HG. Mesangial cells werefirst treated with inhibitors to assess their effect on cell surfaceADAM17 after 1 h of HG. As seen in Fig. 6B, both inhibitorsprevented the increase of mature ADAM17 at the cell surface. Wenext determined whether this increase in mature ADAM17 wasrequired for activity in response to HG. As seen in Fig. 6C,D, therewas a dose-dependent decrease in HG-induced ADAM17 activationwith both inhibitors, confirming that the increased level of matureADAM17 is functionally relevant.Since we showed that the C-terminus is required for ADAM17

activation by HG, we next determined whether it was necessary forthe increased cell surface levels. ADAM17 KO MEFs weretransfected with either WT ADAM17 or ΔC ADAM17, treatedwith HG for 1 h, cell surface proteins biotinylated and pulled down

and transfected ADAM17 identified by its HA tag. As seen inFig. 7A, HG increased cell surface presence of only WTADAM17.Both Src and Erk have been shown to regulate inducible traffickingof ADAM17 to the cell surface (Soond et al., 2005; Taniguchi et al.,2013). Having identified phosphorylation of Y702 by Src and T735by Erk as important to ADAM17 activation by HG, we nextdetermined if phosphorylation at these sites regulated the cellsurface increase of ADAM17. We used ADAM17 KO MEFsexpressing WT, T735A or Y702A ADAM17 to assess theireffect on cell surface localization in response to HG. As seen inFig. 7A, only WTADAM17 trafficked to the cell surface, while thiswas completely prevented in the absence of T735 or Y702phosphorylation.

Since ADAM17 T735 phosphorylation is mediated by PI3K–Erk(Fig. 5), we next tested the effect of PI3K inhibition on the increasein cell surface mature ADAM17 seen with HG. Fig. 7B shows thatboth PI3K inhibitors LY294002 and wortmannin effectivelyprevented this. Src inhibitors SU6656 and PP2 also prevented thecell surface increase of mature ADAM17 in response to HG

Fig. 6. HG increases mature ADAM17 at the cell surface through furin-mediated processing. (A) Mesangial cells were treated with HG for the indicatedtimes. 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) Mesangialcells 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 biotinylatedand pulled down, and ADAM17 assessed by immunoblotting. Both inhibitors decrease the HG-induced increase in mature ADAM17 at the cell surface, with aconcomitant 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-arginineor 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).

7

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

Journal

ofCe

llScience

(Fig. 7C). These data suggest that both PI3K and Src mediate matureADAM17 cell surface localization in response to HG, with theirdownstream phosphorylation of ADAM17 at T735 and Y702respectively being required. However, the precise mechanismunderlying this increase is as yet unknown.

Phosphorylation at T735 and Y702 are both required forADAM17-mediated TGFβ1 upregulation in response to HGSince we identified the necessity of both T735 and Y702phosphorylation for ADAM17 activation by HG (Fig. 2F,Fig. 4F), and we previously showed that ADAM17 is required forHG-induced TGFβ1 upregulation (Uttarwar et al., 2011), we testedthe effects of these two mutants on TGFβ1 production. Either WTADAM17 or one of the mutants was transfected into ADAM17 KOMEFs, and TGFβ1 promoter upregulation and secretion into themedium were assessed. Fig. 8 shows that phosphorylation at bothsites is required for this profibrotic response.

DISCUSSIONADAM17 is becoming increasingly recognized as important to thepathogenesis of diabetic nephropathy. How it is activated by HG,

however, has not been identified. We propose the following model forADAM17 activation by HG in kidney mesangial cells: FAK is anupstream key mediator of ADAM17 activation through its recruitmentof both Src and PI3K, with subsequent phosphorylation of ADAM17at two sites in its C-terminus. Y702 is phosphorylated by Src and T735by PI3K–MEK-activated Erk. Phosphorylation at both sites enhancesassociation of ADAM17 with FAK and is required for downstreamprofibrotic effects. HG also leads to increased furin-mediatedprocessing of ADAM17 to its mature form and increasedtranslocation of mature ADAM17 to the membrane. These studiesunderscore a crucial role for the C-terminus of ADAM17 in its abilityto interact with upstream signaling mediators, and highlight the centralrole of FAK in ADAM17 activation by HG. Inhibition of ADAM17activation through targeting HG-specific activators such as FAK, ortheir interaction with ADAM17, may offer an alternative novelapproach to the treatment of diabetic nephropathy.

The importance of the cytoplasmic domain of ADAM17 to itsactivation has been controversial. It is well established that thisregion is not required for constitutive activity (Le Gall et al., 2010),and our data confirm this since there was no difference between thebasal activity of WT and ΔC ADAM17. We further confirmed that

Fig. 7. HG-induced increase of mature ADAM17 at the cell surface is differentially regulated by T735 and Y702 phosphorylation. (A) ADAM17 KOMEFswere transfected withWTADAM17, T735A ADAM17 or Y702A ADAM17. After HG for 1 h, cell surface proteins were isolated and constructs identified by their HAtag. 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 alsoprevented cell surface localization of mature ADAM17 (n=4; †P<0.01 versus control, *P<0.05 versus HG).

8

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

Journal

ofCe

llScience

phorbol esters do not require the C-terminus for induction ofADAM17 activity. However, the requirement for the C-terminus inADAM17 activation appears to be stimulus specific. For example,while phorbol esters, IL-1, thrombin, EGF, LPA, TNFα, FGF andPDGF can activate ADAM17 independently of its C-terminus(Doedens et al., 2003; Hall and Blobel, 2012; Le Gall et al., 2010;Maretzky et al., 2011; Mendelson et al., 2010), activation byangiotensin II requires the presence of the C-terminus (Elliott et al.,2013). The cytoplasmic domain of ADAM17 is also required forshedding of TNFα (Schwarz et al., 2013), although interestingly, ithas been suggested that only the six most membrane-proximalamino acids of the cytoplasmic tail are required (Schwarz et al.,2013). We now show that HG-induced ADAM17 activation is alsodependent on an intact C-terminus, and confirm similar dependenceon the C-terminus of HG-induced shedding of the membrane-anchored substrate HB-EGF (Fig. S3). This is likely to be due to adependence on interaction of this region with upstream signalingmediators, namely a FAK–PI3K–Src complex. Indeed, this regionof ADAM17 has both a phosphotyrosine site which can bind to SH2domains, as well as a proline-rich region which can bind to SH3domains, enabling interaction with various signaling molecules(Arribas and Esselens, 2009; Kleino et al., 2015). Thus, Src andPI3K can directly interact with the ADAM17 C-terminus throughtheir SH2/SH3 and SH2 domains, respectively. While FAK doesnot possess either of these domains, it is able to bind both Src andPI3K. Thus, the interaction between FAK and ADAM17 seen in our

coimmunoprecipitation studies is likely to be indirect. Interestingly,however, phosphorylation of ADAM17 at both its Y702 and T735appear to be required for mediating interaction with FAK. Theprecise nature of the molecular interactions in this complex remainto be more fully defined.

Several phosphorylation sites have been identified in ADAM17,including T735, S791, S819 and Y702. Phosphorylation at T735 isthe most studied and is induced by either Erk or p38, depending onthe stimulus (Diaz-Rodriguez et al., 2002; Rousseau et al., 2008; Xuand Derynck, 2010). Here, we showed that phosphorylation onT735 is essential for ADAM17 activation by HG, and that Erk, butnot p38, was required for this. How phosphorylation at this siteenables ADAM17 activation is, as yet, not fully understood.Interestingly, however, T735 phosphorylation has recently beenshown to prevent the dimerization of ADAM17, releasing it frominhibition by TIMP3 and thereby allowing activation (Xu et al.,2012). It has also been shown to regulate ADAM17 maturation andtrafficking to the cell surface in COS-7 cells (Soond et al., 2005; Xuand Derynck, 2010), potentially increasing the pool of availablemature ADAM17 for activation. Our data support a role for T735phosphorylation in increasing membrane-localized matureADAM17 in response to HG. Interestingly, phosphorylation atthis site is also important in allowing interaction with FAK,suggesting multiple roles in enabling ADAM17 activation.

Although Src is known to contribute to ADAM17 activation invarious settings, its phosphorylation of ADAM17 at Y702 has been

Fig. 8. Phosphorylation at T735 and Y702 are both required for ADAM17-mediated TGFβ1 upregulation in response to HG. ADAM17 KO MEFs weretransfected with WTADAM17 or T735A ADAM17 (A,C) and Y702A ADAM17 (B,D). (A,B) Activation of a TGFβ1 promoter luciferase by HG (24 h) was seen onlywith 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 byELISA, was increased only by WT ADAM17 (C, n=6; †P<0.01 versus other groups; D, n=6, ‡P<0.001 versus other groups).

9

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

Journal

ofCe

llScience

appreciated more recently. Mechanical stress in myoblasts andcardiomyocytes induces Src-mediated ADAM17 phosphorylationat Y702, mediating myogenesis and TNFα shedding, respectively(Niu et al., 2013, 2015). Y702 phosphorylation is also needed forangiotensin-II-mediated hypertrophy in vascular smooth musclecells (Elliott et al., 2013). We now show that HG induces Src-mediated ADAM17 Y702 phosphorylation. While not affectingbasal ADAM17 activity, phosphorylation at this site is essential toHG-induced ADAM17 activation and downstream matrix upregulation.Interestingly, Y702 phosphorylation is also required for HG-inducedassociation between ADAM17 and FAK, likely through intermediatesincluding Src and/or PI3K. Like T735 phosphorylation, Y702phosphorylation is also required for increased membrane localizationof ADAM17 in response to HG. These data are consistent with otherstudies in which Src was found to be required for ADAM17translocation to the membrane after prolonged (48 h) exposure to HGin mesangial cells (Taniguchi et al., 2013). We confirmed arequirement for Src also in early (1 h) increases in the level of cellsurface mature ADAM17 in response to HG. These data suggest animportant role for Src-induced Y702 phosphorylation in matureADAM17 membrane translocation and activation. The precisemechanism by which this occurs has yet to be determined.Our data show a novel key role for the focal adhesion protein

FAK in ADAM17 activation. FAK is a non-receptor tyrosine kinaseknown to link integrin stimulation to intracellular signals. Uponintegrin engagement, FAK is activated by autophosphorylation onY397 (pY397), enabling its interaction with, and activation of, Src.Phosphorylation at additional sites by Src leads to the full catalyticactivity of FAK and enables interaction with additional kinases,including PI3K (Bolos et al., 2010). FAK thus appears to provide ascaffold for the assembly of a Src–PI3K–ADAM17 complex, asdiscussed above. Of relevance, ADAM17 has also been shown toassociate with integrin α5β1, but whether this interaction is direct ormediated by FAK is unknown. Functionally, this integrin eitherinhibits ADAM17 activation in unstimulated cells (Gooz et al.,2012; Saha et al., 2010), improves cell adhesion (Bax et al., 2004) orinhibits cell migration (Gooz, 2010). Integrin β1 was shown to beactivated by HG to regulate ECM assembly in mesangial cells withlonger term HG exposure (Miller et al., 2014). Whether it is alsorequired for early FAK-mediated activation of ADAM17 will beaddressed in future studies.ADAM17 is produced as a proenzyme synthesized in the

endoplasmic reticulum. This intracellular pool can undergo rapidprocessing to the mature form, increasing the availability of matureenzyme at the membrane (Lorenzen et al., 2016). While somestimuli do not increase the availability of membrane matureADAM17 (Le Gall et al., 2010; Lorenzen et al., 2016), ourstudies show its induction in response to HG in a short time frame(1 h) which was required for its activation. Proprotein convertases,primarily furin, cleave the N-terminal prodomain of ADAM17 inthe Golgi prior to transport to the cell surface (Srour et al., 2003).Our data show that HG increases furin-mediated cleavage ofADAM17, and this is an important step in the increase in levels ofthe mature form at the cell surface and in HG-induced increases inADAM17 activity. Recently, the endoplasmic reticulum-residentproteins iRhoms1 and 2 were shown to be important chaperones forescort of ADAM17 both to the Golgi for processing and to the cellmembrane (Adrain et al., 2012; Christova et al., 2013; Li et al.,2015b; McIlwain et al., 2012). PACS-2 is another newly describedregulator of ADAM17 trafficking to the cell membrane(Dombernowsky et al., 2015). Whether HG regulates iRhom and/or PACS2 function is as yet unknown.

An important role for phosphatidylserine exposure at the outerleaflet of the cell membrane is now proposed as a key common eventin ADAM17 activation by diverse stimuli (Sommer et al., 2016).Phosphatidylserine binds the membrane-proximal domain ofADAM17, facilitating ADAM17 membrane binding through itsshort juxtamembrane segment CANDIS. This brings the catalyticsite in close proximity to the cleavage site of membrane-boundsubstrates, enabling substrate cleavage. Whether this process alsooccurs in response to HG requires confirmation.

In an effort to prevent adverse effects seen with compounds whichmore broadly inhibit metalloproteases, ongoing efforts are underwayfor the development of inhibitors with increased specificity forADAM17. Yet even ADAM17-specific inhibitors are associatedwith unwanted adverse effects, probably because of the multiplesubstrates and ubiquitous nature of the enzyme (Rossello et al.,2016). An alternative approach to ADAM17 inhibition is theidentification of stimulus-specific regulation of its activation. Ourdata provide novel insight into the mechanism of HG-inducedADAM17 activation, showing the importance of C-terminusinteraction with a FAK–PI3K–Src complex and phosphorylationof ADAM17 at both Y702 and T735 in regulating ADAM17activation through complementary mechanisms. Thus, targeting oneor a combination of these upstream regulators can be explored asnovel approaches to the treatment of diabetic nephropathy. Whetherthis may be more broadly extended to include other complications ofdiabetes bears investigation.

MATERIALS AND METHODSCell culturePrimary rat mesangial cells (passages 6–15) were isolated from Sprague–Dawley rats. They were cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 20% fetal calf serum (Invitrogen),streptomycin (100 μg/ml) and penicillin (100 units/ml) at 37°C in 95% air,5% CO2. These are periodically checked for mycoplasma contamination.SV40-immortalized MEFs isolated from ADAM17 KO and WT mice weregenerously provided by Carl Blobel (Cornell University, NY, USA), andtheir expression of ADAM17 was confirmed by immunoblotting. They werecultured in DMEM supplemented with 10% fetal calf serum. Medium forboth cell types contained 5.6 mM glucose. Glucose at 24.4 mM (finalconcentration 30 mM, Sigma) was added for high glucose (HG) conditions.Cells were made quiescent by serum deprivation for 24 h prior to treatment.

Pharmacological inhibitors were added prior to glucose as follows:LY294002 (10 μM, 30 min, Sigma), wortmannin (100 nM, 1 h, Sigma),PF573228 (1 μM, 1 h, Tocris), SU6656 (10 μM, 30 min, Calbiochem), PP2(10 μM, 30 min, Calbiochem), PD98059 (10 μM, 30 min, Alexis), U0126(10 μM, 30 min, Promega), SB203580 (50 μM, 30 min, Sigma), PMA(500 ng/ml, 1.5 h, Sigma), decanoyl-RVKR-CMK (100 μM, 24 h, Tocris)and hexa-D-arginine (100μM, 24 h, Tocris).

Transfection and constructsConstructs for WTADAM17 or ADAM17 with the C-terminus deleted (ΔC)and containing a 3′ HA tag were kindly provided by Carl Blobel (CornellUniversity), with both originally provided by Gillian Murphy, University ofCambridge, UK). A 3′ HA tag was added to the WT ADAM17 construct.ADAM17 T735A (3′HA) and ADAM17 E406A (5′HA) were also providedby Carl Blobel, and ADAM17 Y702A (5′ HA) was provided by Yi-Ping Li(University of Texas, TX, USA). All constructs were based on mouseADAM17.

ADAM17 activity assayWhere indicated, ADAM17 KO MEFs or mesangial cells were transfectedwith 7.5 μg of ADAM17 construct in 6-well plates, coated with poly-D-lysine for MEFs, at 40% confluence using X-fect (Clontech). Cells wereserum-deprived for 24 h, then treated with HG for 1 h. Protein was extractedin activity assay buffer (50 mM Tris-HCl, pH 7.4, 25 mM NaCl, 4%

10

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

Journal

ofCe

llScience

glycerol, 10 mM ZnCl2). ADAM17 activity was measured in duplicate foreach sample using 20 μg protein and the TACE Substrate IV (Calbiochem).Cleavage of this substrate was measured in a fluorometer at 420 nm.

HB-EGF cleavage was assessed in cells transfected with pRC/CMV-HBEGF-AP, kindly provided by Michael Freeman (Harvard University,Cambridge, MA, USA). ADAM17 KO MEFs were transfected with 3.5 μgof this vector in conjunction with 3.5 μg ADAM17 construct as above. Afterserum deprivation, they were incubated in 1 ml of 0% FBSmedium to assessbaseline shedding. This was replaced with 1 ml of new medium with orwithout HG which was collected after 1 h. Shedding was assessed using analkaline phosphatase activity (ALP) assay (AnaSpec). Shedding induced bytreatment was assessed by comparing to baseline shedding for each well.

Luciferase assayADAM17 KO MEFs plated to 30% confluence were transfected with 2.5 μgin 12-well plates with the ADAM17 construct as indicated using X-fect(Clontech), followed the next day by transfection with 0.5 μg of TGFβ1promoter-luciferase construct (kindly provided by Naoya Kato, University ofTokyo, Japan) and 0.05 μg pCMV-β-galactosidase (β-gal) (Clontech) usingLipofectamine (Qiagen). Cells were serum-deprived for 24 h aftertransfection, then exposed to HG for 48 h. Lysis was achieved withReporter Lysis Buffer (Promega) using one freeze–thaw cycle, and luciferaseand β-gal activities measured on clarified lysate using specific kits (Promega)with a Berthold luminometer and a plate reader (420 nm), respectively. β-galactivity was used to adjust for transfection efficiency.

TGFβ1 ELISAADAM17 KO MEFs were transfected with 2.5 μg ADAM17 construct asabove in 12-well plates. After HG (48 h), medium was harvested and debrisremoved by centrifugation at 4°C, 4000 rpm. Medium was stored at −80°Cuntil processing. Total TGFβ1 was measured after acid activation of samplesaccording to the manufacturer’s instructions (R&D Systems).

Protein extraction and analysisCells were lysed and protein extracted as we described previously (Krepinskyet al., 2003), with the addition of BB-94 (1 μM, Tocris) to the lysis buffer.Cell lysates were centrifuged at 4°C, 14,000 rpm for 10 min to pellet celldebris. Supernatant (50 g) was separated on SDS-PAGE and western blottingwas performed. Antibodies were goat ADAM17 (1:1000, Santa Cruz, sc-6416), rabbit pADAM17Y702, kindly provided byYi-Ping Li (University ofTexas) (Niu et al., 2013) (1:1000), rabbit pADAM17 T735 (1:1000, Sigma,SAB4504073), mouse HA (1:1000, Abcam, Ab18181), rabbit Src (1:1000,Cell Signaling, 2108), rabbit pSrc Y416 (1:1000, Cell Signaling, 2101), rabbitFAK (1:1000, Santa Cruz, sc-558), rabbit pFAK Y397 (1:1000, Upstate, 07-012), mouse pErk T202/Y204 (1:1000, Cell Signaling, 9106), total Erk(1:1000, Cell Signaling, 9102) and mouse tubulin (1:10,000, Sigma, T6074).

For immunoprecipitation experiments, cells were lysed, clarified andequal amounts of lysate incubated overnight with 2 μg primary antibodyrotating at 4°C, followed by 25 μl of protein-G–agarose slurry for 1.5 h at4°C. Immunoprecipitates were extensively washed, resuspended in 2×sample buffer, boiled and analyzed by immunoblotting.

Biotinylation for isolation of cell surface proteinsCells plated in 100 mm plates were washed with ice-cold PBS and incubatedwith EZ-link Sulfo-NHSLC-Biotin (0.5 mg/ml in PBS, Fisher) for 20 min.Biotinylation was stopped with 0.1 M glycine in PBS. Cells were lysed in IPlysis buffer (PBS pH 7.4, 5 mM EDTA, 5 mM EGTA, 10 mM sodiumpyrophosphate, 50 mM NaF, 1 mM NaVO3, 1% Triton X-100, 1 μM BB-94, protease inhibitors). Biotinylated proteins were precipitated with 50%neutravidin slurry (Fisher) overnight, after which the beads were washed,boiled in PSB and proteins assessed by immunoblotting. When used,ADAM17 KOMEFs were transfected with 30 μg of the indicated ADAM17plasmid as above prior to treatment.

Statistical analysesStatistical analyses were performed with SPSS 22 (IBM) forWindows usingone-wayANOVA, with Tukey’s HSD for post hoc analysis. For experiments

with two conditions being analysed, a t-test was used and a test for equalvariances used to interpret significance. A P-value <0.05 (two-tailed) wasconsidered significant. Data are presented as the mean±s.e.m. The numberof experimental repetitions (n) is indicated.

AcknowledgementsJ.C.K. gratefully acknowledges the support of St Joseph’s Healthcare for nephrologyresearch. We thank Naoya Kato (University of Tokyo, Japan) for providing theTGFβ1 promoter luciferase construct, Carl Blobel (Cornell University) for providingthe ADAM17 KO MEFs, ADAM17 T735A and ADAM17 E406A; Carl Blobel andGillian Murphy (University of Cambridge, UK) for providing WT ADAM17 and ΔCADAM17; Dr Yi-Ping Li (University of Texas) for providing ADAM17 Y702A; andMichael Freeman (Harvard University) for providing pRC/CMV-HBEGF-AP.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: J.C.K.; Methodology: R.L.; Formal analysis: J.C.K.; Datacuration: 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.

FundingThis work was supported by the Institute of Nutrition, Metabolism andDiabetes of theCanadian Institutes of Health Research (CIHR) (MOP119308 to J.C.K.).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.208629.supplemental

ReferencesAdrain, C., Zettl, M., Christova, Y., Taylor, N. and Freeman, M. (2012). Tumor

necrosis factor signaling requires iRhom2 to promote trafficking and activation ofTACE. Science 335, 225-228.

Arribas, J. and Esselens, C. (2009). ADAM17 as a therapeutic target in multiplediseases. Curr. Pharm. Des 15, 2319-2335.

Bax, D. V., Messent, A. J., Tart, J., van Hoang, M., Kott, J., Maciewicz, R. A. andHumphries, M. J. (2004). Integrin alpha5beta1 and ADAM-17 interact in vitro andco-localize in migrating HeLa cells. J. Biol. Chem. 279, 22377-22386.

Bolos, V., Gasent, J. M., Lopez-Tarruella, S. and Grande, E. (2010). The dualkinase complex FAK-Src as a promising therapeutic target in cancer. Onco.Targets. Ther. 3, 83-97.

Cantley, L. C. (2002). The phosphoinositide 3-kinase pathway. Science 296,1655-1657.

Christova, Y., Adrain, C., Bambrough, P., Ibrahim, A. and Freeman, M. (2013).Mammalian iRhoms have distinct physiological functions including an essentialrole in TACE regulation. EMBO Rep. 14, 884-890.

Diaz-Rodriguez, E., Montero, J. C., Esparis-Ogando, A., Yuste, L. andPandiella, A. (2002). Extracellular signal-regulated kinase phosphorylatestumor necrosis factor alpha-converting enzyme at threonine 735: a potentialrole in regulated shedding. Mol. Biol. Cell 13, 2031-2044.

Doedens, J. R., Mahimkar, R. M. and Black, R. A. (2003). TACE/ADAM-17enzymatic activity is increased in response to cellular stimulation. Biochem.Biophys. Res. Commun. 308, 331-338.

Dombernowsky, S. L., Samsøe-Petersen, J., Petersen, C. H., Instrell, R.,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 signallingby regulating recycling of the metalloproteinase ADAM17.Nat. Commun. 6, 7518.

Elliott, K. J., Bourne, A. M., Takayanagi, T., Takaguri, A., Kobayashi, T., Eguchi,K. and Eguchi, S. (2013). ADAM17 silencing by adenovirus encoding miRNA-embedded siRNA revealed essential signal transduction by angiotensin II invascular smooth muscle cells. J. Mol. Cell Cardiol. 62C, 1-7.

Ford, B. M., Eid, A. A., Go}oz, M., Barnes, J. L., Gorin, Y. C. and Abboud, H. E.(2013). ADAM17 mediates Nox4 expression and NADPH oxidase activity in thekidney cortex of OVE26 mice. Am. J. Physiol. Renal Physiol. 305, F323-F332.

Gooz, M. (2010). ADAM-17: the enzyme that does it all.Crit Rev. Biochem.Mol. Biol.45, 146-169.

Gooz, P., Dang, Y., Higashiyama, S., Twal, W. O., Haycraft, C. J. and Gooz, M.(2012). A disintegrin and metalloenzyme (ADAM) 17 activation is regulated byalpha5beta1 integrin in kidney mesangial cells. PLoS One 7, e33350.

Ha, J. S., Kwon, K. S. and Park, S. S. (2013). PI3Kgamma contributes to MEK1/2activation in oxidative glutamate toxicity via PDK1. J. Neurochem. 127, 139-148.

Hall, K. C. and Blobel, C. P. (2012). Interleukin-1 stimulates ADAM17 through amechanism independent of its cytoplasmic domain or phosphorylation atthreonine 735. PLoS One 7, e31600.

11

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

Journal

ofCe

llScience

Johnson, S. A. and Spurney, R. F. (2015). Twenty years after ACEIs and ARBs:emerging treatment strategies for diabetic nephropathy. Am. J. Physiol. RenalPhysiol. 309, F807-F820.

Kefaloyianni, E., Muthu, M. L., Kaeppler, J., Sun, X., Sabbisetti, V., Chalaris, A.,Rose-John, S., Wong, E., Sagi, I., Waikar, S. S. et al. (2016). ADAM17 substraterelease in proximal tubule drives kidney fibrosis. JCI. Insight. 1, e87023.

Kleino, I., Jarviluoma, A., Hepojoki, J., Huovila, A. P. and Saksela, K. (2015).Preferred SH3 domain partners of ADAM metalloproteases include shared andADAM-specific SH3 interactions. PLoS One 10, e0121301.

Krepinsky, J. C., Ingram, A. J., Tang, D., Wu, D., Liu, L. and Scholey, J. W.(2003). Nitric oxide inhibits stretch-induced MAPK activation in mesangial cellsthrough RhoA inactivation. J. Am. Soc. Nephrol. 14, 2790-2800.

Le Gall, S. M., Maretzky, T., Issuree, P. D. A., Niu, X.-D., Reiss, K., Saftig, P.,Khokha, R., Lundell, D. and Blobel, C. P. (2010). ADAM17 is regulated by arapid and reversiblemechanism that controls access to its catalytic site. J. Cell Sci.123, 3913-3922.

Lewis, E. J., Hunsicker, L. G., Bain, R. P. and Rohde, R. D. (1993). The effect ofangiotensin-converting-enzyme inhibition on diabetic nephropathy. TheCollaborative Study Group. N. Engl. J. Med. 329, 1456-1462.

Lewis, E. J., Hunsicker, L. G., Clarke, W. R., Berl, T., Pohl, M. A., Lewis, J. B.,Ritz, E., Atkins, R. C., Rohde, R. and Raz, I. (2001). Renoprotective effect of theangiotensin-receptor antagonist irbesartan in patients with nephropathy due totype 2 diabetes. N. Engl. J. Med. 345, 851-860.

Li, J. H., Huang, X. R., Zhu, H.-J., Johnson, R. and Lan, H. Y. (2003). Role of TGF-beta signaling in extracellular matrix production under high glucose conditions.Kidney Int. 63, 2010-2019.

Li, R., Uttarwar, L., Gao, B., Charbonneau, M., Shi, Y., Chan, J. S. D., Dubois,C. M. and Krepinsky, J. C. (2015a). High glucose up-regulates ADAM17 throughHIF-1alpha in mesangial cells. J. Biol. Chem. 290, 21603-21614.

Li, X., Maretzky, T., Weskamp, G., Monette, S., Qing, X., Issuree, P. D. A.,Crawford, H. C., McIlwain, D. R., Mak, T. W., Salmon, J. E. et al. (2015b).iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFRsignaling. Proc. Natl. Acad. Sci. USA 112, 6080-6085.

Liu, S. and Kurzrock, R. (2014). Toxicity of targeted therapy: Implications forresponse and impact of genetic polymorphisms. Cancer Treat. Rev. 40, 883-891.

Lorenzen, I., Lokau, J., Korpys, Y., Oldefest, M., Flynn, C. M., Kunzel, U.,Garbers, C., Freeman, M., Grotzinger, J. and Dusterhoft, S. (2016). Control ofADAM17 activity by regulation of its cellular localisation. Sci. Rep. 6, 35067.

Maretzky, T., Evers, A., Zhou, W., Swendeman, S. L., Wong, P.-M., Rafii, S.,Reiss, K. andBlobel, C. P. (2011). Migration of growth factor-stimulated epithelialand endothelial cells depends on EGFR transactivation by ADAM17. Nat.Commun. 2, 229.

McIlwain, D. R., Lang, P. A., Maretzky, T., Hamada, K., Ohishi, K., Maney, S. K.,Berger, T., Murthy, A., Duncan, G., Xu, H. C. et al. (2012). iRhom2 regulation ofTACE controls TNF-mediated protection against Listeria and responses to LPS.Science 335, 229-232.

Melenhorst, W. B., Visser, L., Timmer, A., van den Heuvel, M. C., Stegeman,C. A. and van Goor, H. (2009). ADAM17 upregulation in human renal disease: arole in modulating TGF-alpha availability? Am. J. Physiol. Renal Physiol. 297,F781-F790.

Mendelson, K., Swendeman, S., Saftig, P. and Blobel, C. P. (2010). Stimulationof platelet-derived growth factor receptor {beta} (PDGFR{beta}) activatesADAM17 and promotes metalloproteinase-dependent cross-talk between thePDGFR{beta} and epidermal growth factor receptor (EGFR) signaling pathways.J. Biol. Chem. 285, 25024-25032.

Miller, C. G., Pozzi, A., Zent, R. and Schwarzbauer, J. E. (2014). Effects of highglucose on integrin activity and fibronectin matrix assembly by mesangial cells.Mol. Biol. Cell 25, 2342-2350.

Niu, A., Wen, Y., Liu, H., Zhan, M., Jin, B. and Li, Y.-P. (2013). Src mediates themechanical activation of myogenesis by activating TNFalpha-converting enzyme.J. Cell Sci. 126, 4349-4357.

Niu, A.,Wang, B. and Li, Y.-P. (2015). TNFalpha shedding in mechanically stressedcardiomyocytes is mediated by Src activation of TACE. J. Cell Biochem. 116,559-565.

Regoli, M. and Bendayan, M. (1999). Expression of beta1 integrins in glomerulartissue of Streptozotocin-induced diabetic rats. Biochem. Cell Biol. 77, 71-78.

Rossello, A., Nuti, E., Ferrini, S. and Fabbi, M. (2016). Targeting ADAM17sheddase activity in cancer. Curr. Drug Targets. 17, 1908-1927.

Rousseau, S., Papoutsopoulou, M., Symons, A., Cook, D., Lucocq, J. M.,Prescott, A. R., O’Garra, A., Ley, S. C. and Cohen, P. (2008). TPL2-mediatedactivation of ERK1 and ERK2 regulates the processing of pre-TNF alpha in LPS-stimulated macrophages. J. Cell Sci. 121, 149-154.

Saha, A., Backert, S., Hammond, C. E., Gooz, M. and Smolka, A. J. (2010).Helicobacter pylori CagL activates ADAM17 to induce repression of the gastric H,K-ATPase alpha subunit. Gastroenterology 139, 239-248.

Salem, E. S. B., Grobe, N. and Elased, K. M. (2014). Insulin treatment attenuatesrenal ADAM17 and ACE2 shedding in diabetic Akita mice. Am. J. Physiol. RenalPhysiol. 306, F629-F639.

Schwarz, J., Broder, C., Helmstetter, A., Schmidt, S., Yan, I., Muller, M.,Schmidt-Arras, D., Becker-Pauly, C., Koch-Nolte, F., Mittrucker, H.-W. et al.(2013). Short-term TNFalpha shedding is independent of cytoplasmicphosphorylation or furin cleavage of ADAM17. Biochim. Biophys. Acta 1833,3355-3367.

Sommer, A., Kordowski, F., Buch, J., Maretzky, T., Evers, A., Andra, J.,Dusterhoft, S., Michalek, M., Lorenzen, I., Somasundaram, P. et al. (2016).Phosphatidylserine exposure is required for ADAM17 sheddase function. Nat.Commun. 7, 11523.

Soond, S. M., Everson, B., Riches, D. W. H. and Murphy, G. (2005). ERK-mediated phosphorylation of Thr735 in TNFalpha-converting enzyme and itspotential role in TACE protein trafficking. J. Cell Sci. 118, 2371-2380.

Srour, N., Lebel, A., McMahon, S., Fournier, I., Fugere, M., Day, R. and Dubois,C. M. (2003). TACE/ADAM-17 maturation and activation of sheddase activityrequire proprotein convertase activity. FEBS Lett. 554, 275-283.

Taniguchi, K., Xia, L., Goldberg, H. J., Lee, K. W. K., Shah, A., Stavar, L.,Masson, E. A. Y., Momen, A., Shikatani, E. A., John, R. et al. (2013). Inhibitionof Src kinase blocks high glucose-induced EGFR transactivation and collagensynthesis in mesangial cells and prevents diabetic nephropathy in mice. Diabetes62, 3874-3886.

Uttarwar, L., Peng, F., Wu, D., Kumar, S., Gao, B., Ingram, A. J. and Krepinsky,J. C. (2011). HB-EGF release mediates glucose-induced activation of theepidermal growth factor receptor inmesangial cells.Am. J. Physiol. Renal Physiol.300, F921-F931.

Wu, D., Peng, F., Zhang, B., Ingram, A. J., Gao, B. and Krepinsky, J. C. (2007).Collagen I induction by high glucose levels is mediated by epidermal growth factorreceptor and phosphoinositide 3-kinase/Akt signalling in mesangial cells.Diabetologia 50, 2008-2018.

Wu, D., Peng, F., Zhang, B., Ingram, A. J., Kelly, D. J., Gilbert, R. E., Gao, B. andKrepinsky, J. C. (2009). PKC-beta1mediates glucose-induced Akt activation andTGF-beta1 upregulation in mesangial cells. J. Am. Soc. Nephrol. 20, 554-566.

Xu, P. and Derynck, R. (2010). Direct activation of TACE-mediated ectodomainshedding by p38MAP kinase regulates EGF receptor-dependent cell proliferation.Mol. Cell 37, 551-566.

Xu, P., Liu, J., Sakaki-Yumoto, M. and Derynck, R. (2012). TACE activation byMAPK-mediated regulation of cell surface dimerization and TIMP3 association.Sci. Signal. 5, ra34.

Zhang, Q., Thomas, S. M., Lui, V.W. Y., Xi, S., Siegfried, J. M., Fan, H., Smithgall,T. E., Mills, G. B. and Grandis, J. R. (2006). Phosphorylation of TNF-alphaconverting enzyme by gastrin-releasing peptide induces amphiregulin releaseand EGF receptor activation. Proc. Natl. Acad. Sci. USA 103, 6901-6906.

12

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

Journal

ofCe

llScience