Shedding of the Urinary Biomarker Kidney Injury Molecule-1 (KIM-1) Is Regulated by MAP Kinases and Juxtamembrane Region
Zhiwei Zhang,
Benjamin D. Humphreys and
Joseph V. Bonventre
Renal Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, and Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Boston, Massachusetts
Correspondence: Dr. Joseph V. Bonventre, Harvard Institute of Medicine, 4 Blackfan Circle, Boston, MA 02115. Phone: 617-525-5960; Fax: 617-525-5965; E-mail: joseph_bonventre{at}hms.harvard.edu
Received for publication April 7, 2007.
Accepted for publication June 10, 2007.
Kidney injury molecule-1 (KIM-1) is markedly upregulated inrenal proximal tubule cells by stimuli that promote dedifferentiation,including ischemic or toxic injury, as well as in cases of tubulointerstitialdisease, polycystic kidney disease, and renal cell carcinoma.Structurally, KIM-1 possesses a single transmembrane domainand undergoes membrane-proximal cleavage, which leads to therelease of soluble KIM-1 ectodomain into the urine. UrinaryKIM-1 ectodomain is a promising sensitive and specific biomarkerfor acute kidney injury in humans, and therefore it is importantto determine what regulates KIM-1 shedding. We found that constitutivecleavage of KIM-1 is mediated by ERK activation, and that cleavageis accelerated by p38 MAP kinase activation. After cleavage,a 14-kD membrane-bound fragment of KIM-1, which contains twohighly conserved tyrosine residues, was tyrosine-phosphorylated.Mutagenesis studies demonstrated that the juxtamembrane secondarystructure, not the primary amino acid sequence, was criticalto the cleavage of KIM-1.
Kidney injury molecule-1 (KIM-1) is a type 1 membrane proteinthat is not expressed in normal kidney but is markedly upregulatedin the injured proximal tubular epithelial cells of the humanand rodent kidney in ischemic1 and toxic2 acute kidney injury.KIM-1, also known as T cell Ig mucin (TIM-1)3 and hepatitisA virus cellular receptor-1,4 is also expressed in other conditionswhere proximal tubules are dedifferentiated, including renalcell carcinoma,5 chronic cyclosporine nephrotoxicity,6 a protein-overloadmodel of tubulointerstitial disease,7 and polycystic kidneydisease.8 Extrarenal functions for KIM-1 are described in theimmune system, where the mouse kim-1 gene3 is a susceptibilitylocus for experimental allergic asthma,9 and human KIM-1 (TIM-1)has been implicated in the regulation of TH2 cytokine production.KIM-1 contains a six-cysteine Ig-like domain and a mucin domainin its extracellular region and three tyrosine residues includinga predicted tyrosine kinase phosphorylation motif in the cytosolicdomain. Two splice variants of the cytoplasmic domain are expressed.10The KIM-1a variant is mainly expressed by human liver and lacksthe tyrosine-kinase phosphorylation motif, whereas the KIM-1bvariant (referred to as KIM-1 hereafter) is mainly expressedby human kidney and contains two conserved tyrosine residues,including a predicted tyrosine kinase phosphorylation motif,QAEDNIY. A ligand for mouse TIM-1 has been reported to be TIM-4.11The regulated shedding of KIM-1 ectodomain should contributeto regulation of ligand binding during the reparative responseof the injured proximal tubule and potentially during the TH2immune response as well.10
Many transmembrane proteins undergo proteolytic cleavage, releasingsoluble extracellular domain (ectodomain), which may have autocrineand paracrine signaling functions.12,13 Shedding is generallyenhanced by phorbol ester (PMA)-mediated activation of proteinkinase C, which occurs in close proximity to the cell membraneand is blocked by hydroxamate-based metalloproteinase inhibitors.12,13There is no apparent sequence similarity at the cleavage siteof different proteins, and no minimal consensus shedding sequencehas been identified.14,15 It has been hypothesized that structuralchanges may allow access of a protease to a membrane-proximalregion of cleaved proteins.16
KIM-1 is shed constitutively into the culture medium of celllines expressing endogenous or recombinant KIM-1 by membrane-proximalcleavage in a metalloproteinase-dependent manner.10 SolubleKIM-1 is a very sensitive urinary biomarker of human tubularinjury17,18 and is under investigation as a tissue and urinarybiomarker of renal cell carcinoma.5,19 Despite the mountingevidence of its clinical utility, the regulation and underlyingmechanism for KIM-1 cleavage are poorly characterized. We undertookthis study to understand the molecular mechanisms that regulateurinary levels of this important biomarker. Here, we demonstratethat KIM-1 shedding is enhanced dramatically by pervanadate,a potent inhibitor of protein tyrosine phosphatases. We showthat separate mitogen-activated protein kinases (MAPKs) regulateconstitutive and pervanadate-induced shedding and investigatethe structural requirements for KIM-1 cleavage. We also showthat a 14-kDa tyrosine-phosphorylated cell membrane–associatedfragment of KIM-1 can be detected after cleavage of the KIM-1ectodomain.
Pervanadate Enhances Ectodomain Shedding of KIM-1
769-P cells (human renal cell adenocarcinoma cell line) expresshigh levels of endogenous KIM-1.10 Pervanadate, a potent inhibitorof protein tyrosine phosphatases, enhances tyrosine phosphorylationof cellular proteins whose steady state of phosphorylation isnormally reduced by phosphatases. Pervanadate was freshly preparedfor each experiment by mixing H2O2 (0.5 M) and orthovanadate(0.5 M) and was used within 20 min of preparation.20 Pervanadatetreatment of 769-P cells results in the rapid appearance ofa large amount of a 90-kDa molecule in the culture medium thatis immunoreactive with antibody directed to the ectodomain ofKIM-1, AKG7 (Figure 1A). Induction of KIM-1 shedding does notoccur when cells are exposed to similar concentrations of eitherorthovanadate or H2O2 alone (Figure 1A). Pervanadate-inducedectodomain shedding of KIM-1 is time dependent, with increasedamounts in the culture medium apparent within 15 min of exposureand reaching a peak at 30 min of incubation (Figure 1B, top).In a dosage-response study, 50 µM pervanadate achieveda maximal effect (Figure 1B, bottom).
Figure 1. Pervanadate enhances ectodomain shedding of KIM-1. (A) Confluent 769-P cells were incubated in serum-free medium in the presence of either 50 µM H2O2 or orthovanadate (Na3VO4) or a combination of both (pervanadate) for 30 min. Shed KIM-1 was detected in conditioned medium using an antibody against the ectodomain of KIM-1 (AKG7). (B) Cells were incubated in serum-free medium in the presence of 50 µM pervanadate for various times (top) or incubated for 30 min with various concentrations of pervanadate (bottom), and conditioned media were subjected to Western blot analysis for assessment of shed KIM-1 using AKG7.
Pervanadate-Induced Ectodomain Shedding of KIM-1 Is Inhibited by an Oxygen Free Radical Scavenger and a Metalloproteinase Inhibitor
The underlying molecular mechanism for the inhibitory actionof pervanadate on protein tyrosine phosphatases is thought toinvolve oxidation of an essential thiol at the active site ofthe enzyme, explaining why the oxygen radical scavenger pyrrolidinedithiocarbamate blocks the effect of pervanadate on tyrosinephosphorylation of cellular proteins.21 Pyrrolidine dithiocarbamatepretreatment of 769-P cells prevented pervanadate-induced ectodomainshedding of KIM-1 in a dose-dependent manner (Figure 2A), indicatingthat oxidation is required for pervanadate-stimulated KIM-1shedding and supporting the conclusion that pervanadate regulatesKIM-1 shedding via inhibition of protein phosphatase activity.In our previous study, we reported that GM6001, a hydroxamate-basedbroad-spectrum metalloproteinase inhibitor, completely blockedthe constitutive shedding of KIM-1 ectodomain in 769-P cells.10We found that GM6001 also inhibited the accelerated sheddingstimulated by pervanadate in a dose-dependent manner (Figure 2B).Thus, the accelerated shedding induced by pervanadate is alsomediated by a metalloproteinase.
Figure 2. Pervanadate-induced ectodomain shedding of KIM-1 is inhibited by an oxygen free radical scavenger and a metalloproteinase inhibitor. Confluent 769-P cells were preincubated in serum-free medium with various concentrations of pyrrolidine dithiocarbamate (PDTC), an oxygen radical scavenger (A), or GM6001, a metalloproteinase inhibitor (B), for 30 min before the addition of pervanadate (50 µM final concentration for 30 min). Shed KIM-1 was detected as in Figure 1.
p-Aminophenylmercuric Acid Induces Ectodomain Shedding of KIM-1
Because KIM-1 shedding is mediated by a metalloproteinase(s),we tested whether direct metalloproteinase activation couldstimulate KIM-1 shedding. Incubation of 769-P cells with p-aminophenylmercuricacid (APMA), a metalloproteinase activator,22 significantlyincreased the amount of shed KIM-1 present in the culture mediumboth time (Figure 3A, left) and dosage (Figure 3A, right) dependently.GM6001 attenuated APMA-induced ectodomain shedding (Figure 3B),supporting the conclusion that the shedding of KIM-1 ectodomainwas induced by APMA via activation of a member of the metalloproteinasefamily.
Figure 3. Activation of metalloproteinase-induced ectodomain shedding of KIM-1. (A) Confluent 769-P cells were incubated in serum-free medium in the presence of the metalloproteinase activator APMA (50 µM) for various times (left) or incubated for 30 min with various concentrations of APMA (right), and conditioned media were collected. (B) Effect of metalloproteinase inhibitor on APMA-induced ectodomain shedding of KIM-1. 769-P cells were preincubated in serum-free medium with 10 µM of GM6001 or vehicle (DMSO) for 30 min and then exposed to various concentrations of APMA for an additional 30 min. Shed KIM-1 was detected as in Figure 1.
A KIM-1 Splice Variant Is Also Shed
As an initial step toward defining the structural requirementsof shedding, the splice variant KIM-1a, which encodes a KIM-1polypeptide with identical extracellular and transmembrane domainsto KIM-1 but a different cytosolic domain (Figure 4A), was expressedin LLC-PK1 cells (porcine renal tubular epithelial cell line)that do not express detectable endogenous KIM-1 (B.D.H. andJ.V.B., unpublished observations, 2002). Cells were infectedwith adenovirus encoding either LacZ (AdLacZ) or KIM-1a (AdKIM-1a).The ectodomain of KIM-1a was constitutively shed into the culturemedium (Figure 4B) and pervanadate accelerated the sheddingpathway (Figure 4C). Despite different cytosolic domains, sheddingof KIM-1a is regulated in a similar manner to KIM-1.
Figure 4. Constitutive and accelerated ectodomain shedding of KIM-1a, a splice variant of KIM-1. (A) Schematic representation of the cytosolic domain of human KIM-1 (359 amino acids) and the splice variant KIM-1a (334 amino acids). (B) Subconfluent LLC-PK1 cells were infected for 48 h with a recombinant adenoviral vector expressing KIM-1a (AdKIM-1a) in culture medium supplemented with 2% FBS. AdLacZ served as a control for infection. The multiplicity of infection (MOI) is indicated. At the end of infection, the conditioned media were collected for analysis. (C) After infection with AdKIM-1a or AdLacZ for 48 h, cells were incubated in serum-free medium with 50 µM pervanadate for 30 min, and the conditioned media were assessed for shed KIM-1 as in Figure 1.
MAPK Signaling Pathway Regulates Ectodomain Shedding of KIM-1
The rapid increase in KIM-1 ectodomain shedding after pervanadatetreatment suggests that this effect is not dependent on geneexpression. Indeed, we observed that pretreatment of 769-P cellswith cycloheximide, a protein synthesis inhibitor, had no effecton pervanadate-induced KIM-1 shedding (data not shown). Becausethe MAPK family regulates shedding in other systems, we investigatedwhether such signaling pathways regulate pervanadate-inducedKIM-1 ectodomain shedding. Extracellular signal–regulatedkinase (ERK) and p38 MAPK activation were assessed using phospho-specificantibodies that recognize only activated ERK or p38. Pervanadatetreatment induced dosage-dependent (Figure 5A) and rapid p38phosphorylation with activation by 5 min and peaking by 15 min(Figure 5B). This is well before the peak of activation of KIM-1ectodomain cleavage at this dosage (Figure 1B). ERK activationwas also observed upon pervanadate treatment (50 µM),occurring within 15 min and reaching a peak at 30 min (Figure 5C).The rapid activation of P38 and ERK suggested that these kinasesmight be involved in pervanadate-induced KIM-1 shedding.
Figure 5. Pervanadate activates p38 and ERK MAPK. (A) 769-P cells were incubated in serum-free medium for 30 min with various concentrations of pervanadate, and activation of p38 in cell lysates was determined by Western blot analysis using a p38 phospho-specific antibody (top lane). The same blot was stripped and reprobed with an antibody against total p38 (bottom lane) to control for protein loading. (B) For determination of the time course for p38 activation, cells were incubated in serum-free medium in the absence (Con) or presence of 50 µM pervanadate for various times, and both activated p38 and total p38 were determined as in A. (C) Cells were incubated in serum-free medium for 30 min with various concentrations of pervanadate (top) or in the presence of 50 µM pervanadate for various times (bottom), and the activation of ERK in cell lysates was determined by Western blot analysis using a phospho-specific antibody (top lane). The same blot was stripped and reprobed with an antibody against total ERK (bottom lane).
SB202190, a potent and specific inhibitor of p38 MAPK, efficientlyblocked pervanadate-induced ectodomain shedding of KIM-1 (Figure 6A).The inhibitory effect was observed at dosages of 10 µMand greater. The dosage-response relationship of p38 MAPK phosphorylationparalleled that of inhibition of KIM-1 ectodomain shedding withSB202190 upon treatment with pervanadate (Figure 6B). A similardosage of this inhibitor was used in other studies to blockpervanadate-mediated tyrosine phosphorylation of keratins 8and 19 via a p38 MAPK pathway.23 By contrast, the MEK inhibitorU0126 at various concentrations showed only minimal inhibitionof pervanadate-induced KIM-1 shedding (Figure 6C). These experimentssuggest that the pervanadate-induced ectodomain shedding ofKIM-1 is mediated by the p38 MAPK pathway. Although sheddingof certain transmembrane proteins is sensitive to the receptortyrosine kinase inhibitor genistein and the Src kinase inhibitorPP2, neither of these agents had any effect on pervanadate-inducedectodomain shedding of KIM-1 (Figure 6, D and E).24,25
Figure 6. p38 MAPK signaling pathway regulates pervanadate-induced ectodomain shedding of KIM-1. (A) Confluent 769-P cells were preincubated in serum-free medium with various concentrations of SB202190, a p38 MAPK inhibitor, for 1 h before the addition of pervanadate (50 µM for 30 min). The conditioned media were analyzed for shed KIM-1 as in Figure 1. (B) SB202190 was added to cells in serum-free medium for 1 h and then exposed to pervanadate (50 µM) for an additional 30 min. The activation of p38 in cell lysates was determined using a phospho-specific antibody. (C through E) Cells were preincubated in serum-free medium with various concentrations of U0126 (C), genistein (D), the Src kinase inhibitor PP2 or its control PP3 (E) for 1 h before the addition of pervanadate (50 µM for 30 min), and KIM-1 shedding was assessed.
769-P cells exhibit constitutive shedding in the absence ofpervanadate that is detectable with longer supernatant collectionperiods. Unlike its ability to block pervanadate-induced KIM-1shedding, basal KIM-1 shedding was unaffected by p38 inhibition(Figure 7A). Like pervanadate-induced shedding, the constitutiveshedding pathway is inhibited by the metalloproteinase inhibitorGM6001 (Figure 7B). In contrast to its lack of an effect onpervanadate-induced shedding of KIM-1 ectodomain, U0126, a specificinhibitor of MEK, significantly blocked the constitutive sheddingof KIM-1 ectodomain (Figure 7B), suggesting the involvementof the ERK MAPK pathway in this process. Genistein was alsowithout effect (Figure 7B). By contrast, the Src kinase inhibitorPP2 enhanced constitutive shedding of KIM-1 ectodomain (Figure 7B).These experiments indicate that the constitutive and acceleratedshedding pathways are differentially regulated.
Figure 7. ERK MAPK signaling pathway regulates the constitutive shedding of KIM-1 ectodomain. (A and B) Confluent 769-P cells were maintained in medium supplemented with 2% FBS in the presence of SB202190 (A), at various concentrations, or other inhibitors (B), including GM6001 (25 µM), U0126 (20 µM), genistein (100 µM), and PP2 (100 µM). The conditioned media were collected for 24 h and then subjected to Western blot analysis for assessment of shed KIM-1. (C) For determination of whether constitutive activation of ERK MAPK would stimulate shedding, subconfluent 769-P cells were infected for 48 h with a recombinant adenoviral vector expressing MEK1-DD (AdMEK-DD; the constitutively activated MEK1 mutant) in culture medium supplemented with 2% FBS. AdLacZ served as a control for infection. The MOI is indicated. After infection, cells were maintained in serum-free medium for 24 h, and the conditioned media were assessed for shed KIM-1 (top). Activated ERK (p-ERK, top lane of bottom) and total ERK (t-ERK; bottom lane of bottom) were determined as described in Figure 5C. (D) After infection with AdMEK-DD or Ad LacZ for 48 h, medium was changed to serum-free medium for an additional 24 h in the presence or absence of U0126 (20 µM). The conditioned media were analyzed for shed KIM-1 (top). p-ERK (top lane of bottom) and t-ERK (bottom lane of bottom) were determined as described in Figure 5C.
For further examination of the role of the ERK MAPK pathwayon the regulation of constitutive shedding of KIM-1 ectodomain,cells were infected with adenovirus encoding MEK1-DD (AdMEK-DD),a constitutively active upstream kinase of ERK. 769-P cellsinfected with AdMEK-DD had increased levels of constitutiveshedding of KIM-1 compared with AdLacZ-infected control cells(Figure 7C). The activation of ERK MAPK after viral infectionwas confirmed using anti–phospho-ERK antibody (Figure 7C).Enhanced constitutive shedding of KIM-1 with MEK1-DD is abolishedin the presence of U0126 (Figure 7D, top), suggesting that theeffect of MEK1-DD on constitutive shedding of KIM-1 was indeeddue to activation of ERK MAPK pathway. The detection of KIM-1shedding in conditions without ERK activation indicates thatother signaling pathways are also involved in the constitutiveshedding of KIM-1 ectodomain (Figure 7D).
Protein Secondary Structure in the Juxtamembrane Region Is Important for the Cleavage of KIM-1 Ectodomain
One mAb to KIM-1 (Figure 8A), ABE3, blocks constitutive cleavage,indicating that the cleavage site may overlap the ABE3 bindingsite.10 We investigated whether ABE3 also blocked pervanadate-inducedKIM-1 shedding, and indeed ABE3 did inhibit the acceleratedshedding pathway dosage dependently (Figure 8B). A differentKIM-1 mAb, ACA12, whose epitope is farther from the KIM-1 transmembranedomain than ABE3, did not inhibit KIM-1 shedding (Figure 8B).
Figure 8. Antibody directed to the juxtamembrane region of KIM-1 blocks the cleavage of KIM-1 ectodomain. (A) Schematic representation of the 359–amino acid human KIM-1 protein structural domains and the relative site of KIM-1 cleavage. The approximate sites of antibody (AKG7, ACA12, ABE3, and 1400) recognition are indicated. (B) Effect of mAb against various regions of KIM-1 ectodomain on pervanadate-induced ectodomain of KIM-1. Confluent 769-P cells were preincubated in serum-free medium with various dilutions of ABE3 (top) or ACA12 (bottom) for 30 min and then exposed to pervanadate (50 µM, final concentration) for an additional 30 min. The conditioned media were subjected to Western blot analysis for assessment of shed KIM-1 using AKG7Ab. S, signal peptide; TM, transmembrane domain.
For further characterization of the structural requirementsfor KIM-1 shedding, KIM-1 protein secondary structure was predictedwith the NNPREDIT program (http://www.cmpharm.ucsf.edu/nomi/nnpredict.html).The model predicts a helical polypeptide structure in the juxtamembraneregion, partially overlapping with the ABE3 binding site (Figure 9A).To distinguish whether the primary amino acid sequence and/orthe secondary structure at the cleavage site is important forthe cleavage of KIM-1 ectodomain, we constructed several deletionmutants as depicted in Figure 9B. These mutants, along withthe wild-type KIM-1 construct, were transiently transfectedinto COS-7 cells that do not express endogenous KIM-1.1 Mutant246 to 273, in which a region between the end of the ACA12 bindingsite and part of the ABE3 binding site is deleted, was robustlyshed in response to pervanadate, generating an appropriatelysmaller shed fragment compared with the wild-type protein (Figure 9C,left). Another mutant, 264 to 281, in which the entire ABE3binding site is deleted, had a similar shedding response topervanadate (Figure 9C, left). Of note, the juxtamembrane helicalstructure is predicted to remain intact for both mutants (Figure 9B).By contrast, a mutant in which the helical structure is predictedto be disrupted by deletion of six amino acids just outsidethe ABE3 binding site (278 to 283) exhibited dramatically reducedshedding in response to pervanadate with shed KIM-1 virtuallyabsent in the conditioned medium (Figure 9C, left). The expressionof KIM-1 mutants in transfected cells was confirmed in celllysates by Western blot analysis (Figure 9C, right). The highermolecular mass mature KIM-1 band for 246 to 273 is reduced incomparison with the wild-type KIM-1 (Figure 9C, right), despiterobust shedding of the 246 to 273 ectodomain (Figure 9C, left).This suggests that either the 246 to 273 mutant is shed moreefficiently than the wild-type KIM-1 or the 246 to 273 matureprotein is unstable. Taken together, we conclude that the secondarystructure in the juxtamembrane region is critical for cleavage.Because the mutant with the least pervanadate-induced sheddingalso was predicted to disrupt the juxtamembrane helical structurethe most, we conclude that KIM-1 secondary structure determinesthe accessibility of the protease to its substrate. This conclusionis supported by the lack of effect of 264 to 281 on sheddingeven though the ABE3 antibody, which recognizes an epitope inthis region, did inhibit shedding.
Figure 9. Protein secondary structure in the juxtamembrane region is critical for the cleavage of KIM-1 ectodomain. (A) Schematic representation of the juxtamembrane region of KIM-1 ectodomain. The sequences at the approximate sites of antibody recognition are underlined. Protein secondary structure was predicted using the NNPREDICT program and predicted helix and strand regions marked. (B) The predicted protein secondary structure in the juxtamembrane region of wild-type KIM-1 and its deletion mutants. The deleted amino acids are boxed. (C) Effect of pervanadate on ectodomain shedding of KIM-1 in KIM-1 deletion mutants. Subconfluent COS-7 cells were transfected with cDNA of either wild-type KIM-1 (WT) or its deletion mutants (246 to 273, 264 to 281, and 278 to 283) for 48 h and then incubated with pervanadate (50 µM) in serum-free medium for 30 min. The conditioned media were analyzed for shed KIM-1 as in Figure 1 (left). Total KIM-1 expression was evaluated by Western blot analysis in cell lysates using AKG7 (right). H, helix; E, strand.
A 14-kDa Tyrosine-Phosphorylated and Cell Membrane–Associated Fragment of KIM-1 Is Generated after Ectodomain Cleavage of KIM-1
As shown in Figure 10A, pervanadate treatment of 769-P cellsresults in an increased amount of a 14-kDa fragment of KIM-1,representing the remnant transmembrane and cytosolic domainthat remain after ectodomain cleavage. This fragment is recognizedby polyclonal sera directed against the cytosolic domain ofKIM-1 (1400) but not by the mAb recognizing the ectodomain ofKIM-1 (data not shown). The 14-kDa fragment is associated primarilywith the insoluble fraction of the cells (Figure 10B), suggestingthat it remains membrane bound.
Figure 10. A tyrosine-phosphorylated and cell membrane–associated fragment of KIM-1 is generated after pervanadate treatment. (A) A 14-kDa fragment of KIM-1 is generated after pervanadate treatment. Confluent 769-P cells were incubated in serum-free medium in the presence or absence of pervanadate (50 µM) for 30 min, and total KIM-1 in cell lysates was determined by Western blot analysis using an antibody against the cytosolic domain of KIM-1 (1400). (B) The 14-kDa fragment of KIM-1 is associated with cell membranes. After incubation with or without pervanadate, confluent 769-P cells were mechanically disrupted in isotonic buffer solution without detergents and separated into soluble (cytosolic) and insoluble (membrane) fractions. Aliquots of each fraction were examined for KIM-1 protein by Western blot analysis using 1400. (C) The 14-kDa fragment of KIM-1 is tyrosine phosphorylated. Confluent 769-P cells were incubated in serum-free medium in the presence or absence of pervanadate (50 µM of final concentration) for various times. The cell lysates were immunoprecipitated using 1400 followed by Western blot analysis with an antibody against phosphotyrosine (top). The same blot was stripped and reprobed with the 1400 antibody (bottom). Control cells were kept for 30 min in serum-free medium in the absence of pervanadate.
For examination of whether the 14-kDa truncated fragment istyrosine-phosphorylated, cell lysates were immunoprecipitatedwith antibody 1400, followed by Western blotting with anti-phosphotyrosineantibodies. A tyrosine-phosphorylated band at 14 kDa was apparentafter pervanadate treatment, and it was confirmed to be the14-kDa fragment of KIM-1 by stripping and reprobing the sameblot with 1400 (Figure 10C).
Generation of the tyrosine-phosphorylated 14-kDa fragment wasstimulated by APMA, and GM6001 completely blocked formationof the tyrosine-phosphorylated 14-kDa fragment (data not shown).Taken together, these experiments indicate that generation ofthe 14-kDa, tyrosine-phosphorylated C-terminal fragment is theconsequence of metalloproteinase-mediated KIM-1 ectodomain cleavage.Tyrosine phosphorylation does not seem to be required for cleavage,however, because the KIM-1a splice variant, which lacks Y350and Y356, still underwent pervanadate-induced ectodomain cleavage(Figure 4).
Although KIM-1 shedding is not stimulated by the typical sheddaseactivator PMA, we have demonstrated that pervanadate, a potentinhibitor of protein tyrosine phosphatases, induces a strikingincrease in the shedding of KIM-1 ectodomain. We speculate thatphysiologic stimuli, such as growth factors, may also regulateshedding in vivo and that pervanadate mimics this process; however,we have not identified a more physiologic stimulus to date.Pervanadate activates the shedding of a variety of cell surfaceproteins. Some of them, such as HER4,26 MUC1,27 and L-adhesionmolecule,24 are also shed in response to PMA. By contrast, theshedding of HER2,28 betaglycan,29 and TNF-related activation-inducedcytokine30 can be stimulated by pervanadate but not by PMA,although protein kinase C–activated shedding of otherproteins was found in the cell lines tested.28 In our previousstudy,22 we reported that hydroxamate metalloproteinase inhibitors,including BB-94 and GM6001, blocked the constitutive sheddingof KIM-1 ectodomain.10 This study demonstrates that pervanadate-inducedectodomain shedding of KIM-1 is inhibited by GM6001 and activatedby APMA, a general activator of metalloproteinases. Thus, bothaccelerated and constitutive KIM-1 shedding is mediated by ametalloproteinase.
Protein tyrosine phosphorylation is controlled by coordinateactions of protein tyrosine kinases and protein tyrosine phosphatases.31Growth factors, hormones, and cytokines shift the balance byrapidly stimulating protein tyrosine kinase activity, therebyinducing tyrosine phosphorylation and initiating signal transduction.However, inhibition of protein tyrosine phosphatases mimicscertain aspects of signal transduction that are normally triggeredby tyrosine kinase activation. Cross-talk between protein tyrosinekinase and MAPKs has been reported.32 Pervanadate, which isa potent inhibitor of protein tyrosine phosphatases and thereforeis able to sustain protein tyrosine phosphorylation-dependentevents,33 also leads to activation of MAPKs.34,35
MAPKs are integral to many signal transduction pathways, andpervanadate activates both ERK and p38 MAPK in 769-P cells.Because pervanadate activates p38 before KIM-1 shedding is detectedand because inhibition of p38 also strongly attenuates pervanadate-inducedshedding, we conclude that the p38 pathway mediates pervanadate-induced,accelerated KIM-1 shedding. Although ERK is also activated bypervanadate, the lack of effect seen with the MEK inhibitorsuggests that this pathway is less important for regulationof accelerated shedding. We did find evidence that the ERK (butnot p38) pathway regulates constitutive shedding of KIM-1 ectodomain.The differential regulation of constitutive KIM-1 shedding bythe ERK pathway and accelerated shedding by the P38 pathwaysuggest that KIM-1 shedding is a physiologically important processunder tight regulation. Our data contrast with TGF- in whichthe basal and growth factor–accelerated shedding of TGF-are regulated by p38 and ERK pathways, respectively,36 suggestingthat no simple rule can account for the regulation of constitutiveand accelerated shedding activities of unrelated membrane proteins.
The mechanism linking MAPK pathways to the regulation of ectodomainshedding of membrane proteins has not been elucidated. TNF-–convertingenzyme, a prototype sheddase, is phosphorylated at threonine735 by the ERK pathway,37 and MAPKs have also been shown tomediate the expression and activation of a variety of metalloproteinases,including matrix metalloproteinases 1, 2, 3, 9, 10, and 13;membrane type 1 (MT1)-matrix metalloproteinase; and a disintegrin-likemetalloproteinase domain with thrombospondin type 1 motifs.38–43Thus, it is possible that MAPK pathways may lead to direct activationof the metalloproteinases that mediate KIM-1 ectodomain shedding.
A mAb directed to the juxtamembrane region of KIM-1 (ABE3) effectivelyblocked the ectodomain shedding of KIM-1 that either occursconstitutively or is induced by pervanadate. Deleting the ABE3epitope had no effect on shedding, however, suggesting thata mechanism other than peptide sequence might regulate metalloproteinaserecognition of KIM-1. Our mutagenesis studies, based on predictionof KIM-1 secondary structure, indicate that the protein structurein the juxtamembrane region is the most important factor determiningthe cleavage of KIM-1. Specifically, maintenance of the helicalstructure in the juxtamembrane region of KIM-1 may be the criticalvariable for cleavage to occur, probably by regulating accessibilityof the protease to its substrate. Our findings are consistentwith other reports in which no apparent primary sequence similarityat the cleavage site of various transmembrane proteins or minimalconsensus shedding sequence were identified.14,15
The functional significance of KIM-1 ectodomain shedding isunknown. There are three tyrosine residues in the cytosolicdomain of human KIM-1, and two of them are conserved in mouseand rat. In this study, a 14-kDa truncated membrane-bound fragmentof KIM-1, in which the tyrosine residues are phosphorylated,was generated after pervanadate treatment. The generation ofthis tyrosine-phosphorylated 14-kDa fragment also occurs afterAPMA treatment, which has no independent effect on tyrosinephosphorylation. One interpretation is that phosphorylationof tyrosine residues in the 14-kDa fragment of KIM-1 is a consequenceof KIM-1 ectodomain cleavage by metalloproteinases. Previousstudies have shown that ectodomain cleavage by metalloproteinasesgenerates tyrosine-phosphorylated membrane-associated fragmentsthat retain tyrosine kinase activities in the Heregulin receptorErbB-2, ErbB-4, and TrkA neurotrophin receptors.20,44,45 Phosphorylationis an important means of propagating intracellular signals.Although KIM-1 itself does not have a kinase domain that couldinitiate a phosphorylation cascade, it does have a predictedtyrosine kinase phosphorylation motif, QAEDNIY, in its cytosolicdomain. Tyrosine phosphorylation of KIM-1 may provide dockingsites for downstream transducers and effectors to regulate cellularfunctions. The regulated shedding of KIM-1 may represent a mechanismto limit cell exposure to a KIM-1 ligand, and soluble extracellularKIM-1 could bind and neutralize KIM-1 ligand. Testing this hypothesisrequires identification of a KIM-1 ligand in kidney.
The shedding of KIM-1 into the urine of patients with acutekidney injury is clinically significant, and elevated urinaryKIM-1 levels are associated with adverse outcomes in this population.18In both preclinical and clinical studies using several mechanisticallydifferent models of kidney injury, urinary KIM-1 serves as anearlier and more specific diagnostic indicator of kidney injurywhen compared with any of the conventional biomarkers (plasmacreatinine, blood urea nitrogen, glycosuria, proteinuria, urinaryN-acetyl--d-glucosaminidase, -glutamyltransferase, or alkalinephosphatase).17,46 Recently, a consortium of pharmaceuticalcompanies the Preventive Safety Testing Consortium (PSTC), hasbeen working on identifying and qualifying protein biomarkersfor predictive nephrotoxicity in preclinical drug development.The Consortium has concluded that KIM-1 is an excellent urinarymarker of nephrotoxicity when correlated with histopathologyin the rodent.47 Understanding the pathways that regulate theappearance of KIM-1 ectodomain in human urine will be informativeas we further qualify KIM-1 as a useful biomarker of nephrotoxicityin humans.
We report that ectodomain shedding of KIM-1 is stimulated bypervanadate, a potent inhibitor of protein tyrosine phosphatases.The constitutive and pervanadate-induced shedding of KIM-1 ismediated by metalloproteinases and regulated by ERK and p38MAPK, respectively. We provide evidence that the protein secondarystructure in the juxtamembrane region of KIM-1 is importantfor its cleavage. We also demonstrate that ectodomain cleavageof KIM-1 results in generation of a truncated 14-kDa cell membrane–associatedand tyrosine-phosphorylated KIM-1 fragment. Because shed KIM-1is a sensitive urinary biomarker for kidney injury, understandingthe regulation of KIM-1 shedding at the cellular level willultimately inform our interpretation of elevated urinary KIM-1levels in human disease.
Cells and Reagents
769-P cells (human renal cell adenocarcinoma, CRL-1933) andCOS-7 cells were grown in RPMI supplemented with 10% FBS andmaintained at 37°C in 5% CO2. LLC-PK1 cells (porcine renaltubular epithelial cell line, CRL-1390) were grown in DMEM supplementedwith 10% FBS and maintained at 37°C in 5% CO2. MAb againstphosphotyrosine was purchased from Upstate Biotechnology (Upstate,NY). Murine mAb against the extracellular domain of KIM-1 (AKG7,ACA12, and ABE3) and rabbit polyclonal antibody against thecytosolic domain of KIM-1 (1400) have been described.10 GM6001(Ilomastat) was from Chemicon Int. (Temecula, CA), and APMAwas from Sigma Chemical Co. (St. Louis, MO).
Western Blot Analysis
Conditioned media were collected, and cell extracts from confluentcell monolayers were prepared on ice in lysis buffer (1% TritonX-100, 20 mM HEPES [pH 7.4], 2 mM EGTA, 1 mM DTT, 50 mM -glycerophosphate,10% glycerol, 1 mM NaVO4, 2 µM leupeptin, and 400 µMPMSF). Samples were separated by SDS-PAGE, transferred to polyvinylidenedifluoride membranes, and subjected to Western analysis usingstandard procedures. Each experiment was repeated independentlyat least twice with similar results.
Construction of Recombinant Adenoviral Vectors
A recombinant adenoviral vector carrying a constitutive activemutant of MEK-1 (MEK1-DD) cDNA (AdMEK-DD) was constructed asdescribed previously,48 and the activation of ERK was confirmedin LLC-PK1 cells infected with AdMEK-DD in our previous study.49The adenovirus carrying the Escherichia coli LacZ gene (AdLacZ)was provided by Dr. Roger Hajjar (Cardiovascular Research Center,Massachusetts General Hospital, Boston, MA).
For creation of an adenovirus for expression of human KIM-1a,a KIM-1a cDNA was subcloned into the shuttle vector pAdTrack-CMV.This was linearized with PmeI and transformed together withsupercoiled adenoviral backbone vector (pAdEasy-1) into E. colistrain BJ5183. The recombinant adenoviral construct was amplifiedin DH 5 cells and plasmid purified by CsCl banding. Virus wascreated by transfecting PacI linearized adenoviral constructinto 293 cells using Lipofectamine and OptiMEM (Life Technologies,Gaithersburg, MD).
Immunoprecipitation and Phosphotyrosine Detection
Cell lysates were prepared as described previously, immunoprecipitatedwith antibody against the cytosolic domain of KIM-1 (1400) for2 h on ice, separated with protein A–agarose beads (BoehringerMannheim, Mannheim, Germany), and incubated for 1 h at 4°Cwith end-over-end rotation. Beads were washed with lysis buffer,resuspended in 2x SDS-PAGE sample buffer, and boiled for 5 min.Aliquots of the boiled samples were fractionated on 10% SDS-PAGEgels, transferred to polyvinylidene difluoride membranes (Millipore,MA), and subjected to Western analysis using standard techniques.
Fractionation of Soluble and Insoluble KIM-1 Proteins
Confluent cell monolayers were washed once with ice-cold PBS;scraped; and sonicated briefly in isotonic buffer solution withoutdetergents containing 130 mM KCL, 20 mM NaCl, 2 mM EDTA, 1 mMEGTA, 50 mM Tris-HCl (pH 7.4), 1 mM PMSF, and 10 µg/mlleupeptin on ice. The homogenates were centrifuged at 100,000x g for 10 min at 4°C to recover the soluble and insolublefractions in the supernatant and pellet, respectively. Equivalentamounts of supernatant and pellet were subjected to Westernblot analysis using antibody directed against the cytosolicdomain of KIM-1 (1400).
Construction of KIM-1 cDNA and Mutagenesis
The coding region for human KIM-1 (amino acids 1 to 359) wasgenerated by PCR using phKIM1.2 as template and then subclonedinto the BamH1 and XhoI sites of eukaryotic expression vectorpcDNA3 (Invitrogen, Carlsbad, CA). Deletions in the juxtamembraneregion of the KIM-1 ectodomain were produced using the QuickChangesite-directed mutagenesis kit (Stratagene, La Jolla, CA). Thesequences of all constructs were confirmed by DNA sequencing.
Mammalian Cell Transfection
COS-7 cells were seeded in 6-cm tissue culture dishes at a densityof 4 x 105 cells per dish and cultured for 24 h in completemedium. The expression plasmids containing the cDNA encodingfor wild-type KIM-1 or its mutants (5 µg each) were transfectedinto COS-7 cells using Superfect reagent (Qiagen, Valencia,CA). Empty vector (pcDNA3) served as a control for transfection.Experiments were performed 48 h after transfection.
Therapeutic use of KIM-1 has been very recently licensed toBiogen Idec Corp. JVB holds patents on KIM-1.
Acknowledgments
This work was supported by the Joseph E. Murray Award from theNational Kidney Foundation of Massachusetts, Rhode Island, NewHampshire & Vermont; and National Institutes of Health IndividualNational Research Service Award 1F32DK10126 to Z.Z., and NationalInstitutes of Health grants DK 73628 to B.D.H. and DK 39773and DK 72381 to J.V.B.
We thank Dr. W. Wu for technical assistance and Amy Parker foreditorial assistance. Our gratitude goes also to Drs. RohanSamarakoon and Vishal S. Vaidya for helpful discussions.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Z.Z.'s current affiliation is Renal Division, St. Louis Universityand VA Medical Center, St. Louis, Missouri.
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Top
Abstract
Introduction
nomi/nnpredict.html). The model predicts a helical polypeptide structure in the juxtamembrane region, partially overlapping with the ABE3 binding site (Figure 9A). To distinguish whether the primary amino acid sequence and/or the secondary structure at the cleavage site is important for the cleavage of KIM-1 ectodomain, we constructed several deletion mutants as depicted in Figure 9B. These mutants, along with the wild-type KIM-1 construct, were transiently transfected into COS-7 cells that do not express endogenous KIM-1.1 Mutant 
246 to 273, in which a region between the end of the ACA12 binding site and part of the ABE3 binding site is deleted, was robustly shed in response to pervanadate, generating an appropriately smaller shed fragment compared with the wild-type protein (Figure 9C, left). Another mutant, 
in which the basal and growth factor–accelerated shedding of TGF-
-d-glucosaminidase, 
-glutamyltransferase, or alkaline phosphatase).17,46 Recently, a consortium of pharmaceutical companies the Preventive Safety Testing Consortium (PSTC), has been working on identifying and qualifying protein biomarkers for predictive nephrotoxicity in preclinical drug development. The Consortium has concluded that KIM-1 is an excellent urinary marker of nephrotoxicity when correlated with histopathology in the rodent.47 Understanding the pathways that regulate the appearance of KIM-1 ectodomain in human urine will be informative as we further qualify KIM-1 as a useful biomarker of nephrotoxicity in humans. 
