histone h3 serine 10 phosphorylation facilitates ...€¦ · ized human podocytes (provided by m....

14
Histone H3 Serine 10 Phosphorylation Facilitates Endothelial Activation in Diabetic Kidney Disease Tamadher A. Alghamdi, 1 Sri N. Batchu, 1 Mitchell J. Hadden, 1 Veera Ganesh Yerra, 1 Youan Liu, 1 Bridgit B. Bowskill, 1 Suzanne L. Advani, 1 Laurette Geldenhuys, 2 Ferhan S. Siddiqi, 3 Syamantak Majumder, 1 and Andrew Advani 1 Diabetes 2018;67:26682681 | https://doi.org/10.2337/db18-0124 The posttranslational histone modications that epige- netically affect gene transcription extend beyond con- ventionally studied methylation and acetylation patterns. By examining the means by which podocytes inuence the glomerular endothelial phenotype, we identied a role for phosphorylation of histone H3 on serine residue 10 (phospho-histone H3Ser10) in mediating endothelial activation in diabetes. Culture media conditioned by podocytes exposed to high glucose caused glomeru- lar endothelial vascular cell adhesion protein 1 (VCAM- 1) upregulation and was enriched for the chemokine CCL2. A neutralizing anti-CCL2 antibody prevented VCAM-1 upregulation in cultured glomerular endothelial cells, and knockout of the CCL2 receptor CCR2 dimin- ished glomerular VCAM-1 upregulation in diabetic mice. CCL2/CCR2 signaling induced glomerular endothelial VCAM-1 upregulation through a pathway regulated by p38 mitogen-activated protein kinase, mitogen- and stress-activated protein kinases 1/2 (MSK1/2), and phosphorylation of H3Ser10, whereas MSK1/2 inhibition decreased H3Ser10 phosphorylation at the VCAM1 pro- moter. Finally, increased phospho-histone H3Ser10 lev- els were observed in the kidneys of diabetic endothelial nitric oxide synthase knockout mice and in the glomeruli of humans with diabetic kidney disease. These ndings demonstrate the inuence that histone protein phos- phorylation may have on gene activation in diabetic kidney disease. Histone protein phosphorylation should be borne in mind when considering epigenetic targets amenable to therapeutic manipulation in diabetes. Posttranslational histone modications have emerged as pivotal mediators of diabetes complications by either per- mitting or preventing cellular injury. Most of the evidence that associates histone modications with the development and progression of diabetes complications comes from the study of histone (de)methylation or histone (de)acetylation (1). However, several other modications also can affect histone proteins, including phosphorylation, ubiquitination, O-GlcNAcylation, ADP-ribosylation, and sumoylation (1). Although these alternative modications have important biological functions, their potential contribution to the development of diabetes complications has largely been overlooked. Diabetes is an inammatory disease. Release of inam- matory cytokines by resident cells within the diabetic kid- ney, for instance, facilitates the recruitment of leukocytes that in turn contribute to the progressive brosis that characterizes later-stage nephropathy. Diabetes is also an endothelial disease, and the enhanced expression of endo- thelial adhesion molecules that facilitate leukocyte recruit- ment has long been linked to the development of diabetes complications (2). The endothelial expression of cell surface adhesion molecules that facilitate leukocyte recruitment is termed endothelial activation (3). One of the prototypical endothelial adhesion molecules indicative of endothelial activation is vascular cell adhesion protein 1 (also called vascular cell adhesion molecule 1 [VCAM-1]) (3). VCAM-1 primarily functions as the ligand for the b1-integrin sub- family member very late antigen-4 (VLA-4, a4b1) present on the leukocyte plasma membrane, and its upregulation 1 Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michaels Hospital, Toronto, Ontario, Canada 2 Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada 3 Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Corresponding author: Andrew Advani, [email protected]. Received 26 January 2018 and accepted 30 August 2018. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0124/-/DC1. S.M. is currently afliated with the Department of Biological Sciences, Birla Institute of Technology and Sciences, Pilani, Rajasthan, India. © 2018 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for prot, and the work is not altered. More information is available at http://www.diabetesjournals .org/content/license. 2668 Diabetes Volume 67, December 2018 COMPLICATIONS

Upload: others

Post on 08-Aug-2020

1 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

Histone H3 Serine 10 Phosphorylation FacilitatesEndothelial Activation in Diabetic Kidney DiseaseTamadher A. Alghamdi,1 Sri N. Batchu,1 Mitchell J. Hadden,1 Veera Ganesh Yerra,1 Youan Liu,1

Bridgit B. Bowskill,1 Suzanne L. Advani,1 Laurette Geldenhuys,2 Ferhan S. Siddiqi,3 Syamantak Majumder,1

and Andrew Advani1

Diabetes 2018;67:2668–2681 | https://doi.org/10.2337/db18-0124

The posttranslational histone modifications that epige-netically affect gene transcription extend beyond con-ventionally studied methylation and acetylation patterns.By examining the means by which podocytes influencetheglomerular endothelial phenotype,we identifieda rolefor phosphorylation of histone H3 on serine residue10 (phospho-histone H3Ser10) in mediating endothelialactivation in diabetes. Culture media conditioned bypodocytes exposed to high glucose caused glomeru-lar endothelial vascular cell adhesion protein 1 (VCAM-1) upregulation and was enriched for the chemokineCCL2. A neutralizing anti-CCL2 antibody preventedVCAM-1 upregulation in cultured glomerular endothelialcells, and knockout of the CCL2 receptor CCR2 dimin-ished glomerular VCAM-1 upregulation in diabetic mice.CCL2/CCR2 signaling induced glomerular endothelialVCAM-1 upregulation through a pathway regulatedby p38 mitogen-activated protein kinase, mitogen- andstress-activated protein kinases 1/2 (MSK1/2), andphosphorylation of H3Ser10, whereas MSK1/2 inhibitiondecreased H3Ser10 phosphorylation at the VCAM1 pro-moter. Finally, increased phospho-histone H3Ser10 lev-els were observed in the kidneys of diabetic endothelialnitric oxide synthase knockout mice and in the glomeruliof humans with diabetic kidney disease. These findingsdemonstrate the influence that histone protein phos-phorylation may have on gene activation in diabetickidney disease. Histone protein phosphorylation shouldbe borne in mind when considering epigenetic targetsamenable to therapeutic manipulation in diabetes.

Posttranslational histone modifications have emerged aspivotal mediators of diabetes complications by either per-mitting or preventing cellular injury. Most of the evidencethat associates histone modifications with the developmentand progression of diabetes complications comes from thestudy of histone (de)methylation or histone (de)acetylation(1). However, several other modifications also can affecthistone proteins, including phosphorylation, ubiquitination,O-GlcNAcylation, ADP-ribosylation, and sumoylation (1).Although these alternative modifications have importantbiological functions, their potential contribution to thedevelopment of diabetes complications has largely beenoverlooked.

Diabetes is an inflammatory disease. Release of inflam-matory cytokines by resident cells within the diabetic kid-ney, for instance, facilitates the recruitment of leukocytesthat in turn contribute to the progressive fibrosis thatcharacterizes later-stage nephropathy. Diabetes is also anendothelial disease, and the enhanced expression of endo-thelial adhesion molecules that facilitate leukocyte recruit-ment has long been linked to the development of diabetescomplications (2). The endothelial expression of cell surfaceadhesion molecules that facilitate leukocyte recruitment istermed endothelial activation (3). One of the prototypicalendothelial adhesion molecules indicative of endothelialactivation is vascular cell adhesion protein 1 (also calledvascular cell adhesion molecule 1 [VCAM-1]) (3). VCAM-1primarily functions as the ligand for the b1-integrin sub-family member very late antigen-4 (VLA-4, a4b1) presenton the leukocyte plasma membrane, and its upregulation

1Keenan Research Centre for Biomedical Science and Li Ka Shing KnowledgeInstitute of St. Michael’s Hospital, Toronto, Ontario, Canada2Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada3Department of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada

Corresponding author: Andrew Advani, [email protected].

Received 26 January 2018 and accepted 30 August 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0124/-/DC1.

S.M. is currently affiliated with the Department of Biological Sciences, BirlaInstitute of Technology and Sciences, Pilani, Rajasthan, India.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

2668 Diabetes Volume 67, December 2018

COMPLIC

ATIO

NS

Page 2: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

has been reported to occur in the kidneys of both diabeticmice (4) and humans with diabetes (5).

In the current study, we explored the mechanisms thatcontrol endothelial activation within the kidney glomerulusin diabetes. We started from the premise that the ordinaryfunctioning of the kidney glomerulus depends on the or-chestrated interaction of its cellular constituents. In partic-ular, paracrine communication between podocytes lining theurinary space and endothelial cells lining the glomerularcapillary walls maintains the permselective integrity of theglomerular filtration barrier (6). We hypothesized thatfactors secreted by glomerular podocytes under conditionsof high glucose may promote glomerular endothelial cell(GEC) activation characterized by VCAM-1 upregulation.While testing this hypothesis, we discovered a pivotal rolefor the phosphorylation of histone protein H3 on serineresidue 10 (phospho-histone H3Ser10) in facilitating glo-merular endothelial activation in diabetic kidney disease.

RESEARCH DESIGN AND METHODS

Cell CultureExperiments were conducted in conditionally immortal-ized human podocytes (provided by M. Saleem, Universityof Bristol, Bristol, U.K.) (7) and in primary cultured humanrenal GECs (hGECs) (ScienCell Research Laboratories,Carlsbad, CA) (8,9). hGECs were cultured under controlconditions (5.6 mmol/L glucose) or with the addition of19.4 mmol/L glucose (final concentration 25 mmol/L, highglucose) or 19.4 mmol/L mannitol for 16 h. To generatehuman podocyte–conditioned medium, differentiated hu-man podocytes were incubated under control conditions(5.6 mmol/L glucose [hpod_CM]) or high-glucose condi-tions (25 mmol/L [hpod_HGCM]) for 48 h. The HumanCytokine 41-Plex Discovery Assay was performed by EveTechnologies (Calgary, Alberta, Canada). Neutralizing an-tibody experiments were performed by incubating hGECsfor 16 h in high glucose or hpod_HGCM that had beenpreincubated with an anti–C-C motif ligand 2 (CCL2)neutralizing antibody (#16-7096-81; Thermo Fisher Sci-entific, Rockford, IL) at a concentration of 20 mg/mL (10)for 1 h. Recombinant angiopoietin 1, angiopoietin 2, orendothelin 1 were applied to hGECs for 16 h at the fol-lowing concentrations: angiopoietin 1 100 ng/mL (11)(#923-AN; R&D Systems, Minneapolis, MN), angiopoietin2 100 ng/mL (#623-AN; R&D Systems), and endothelin1 10 nmol/L (12) (#1160; Tocris Bioscience, Bristol, U.K.).Recombinant human CCL2 (#RPA087Hu01; Cloud-CloneCorp., Katy, TX) was applied to hGECs at a concentrationof 0.5 ng/mL (13,14) for 16 h. For CCR2 antagonism,hGECs were incubated with RS504393 (IC50 ,100 nmol/L[15]; Tocris Bioscience) at a concentration of 10 mmol/L(16). For inhibition of p38 mitogen-activated proteinkinase (MAPK), hGECs were incubated with SB203580(IC50 0.6 mmol/L [17]; Tocris Bioscience) at a concentrationof 10 mmol/L (18). For inhibition of mitogen- and stress-activated protein kinase 1/2 (MSK1/2), hGECswere incubated

with SB-747651A (IC50 11 nmol/L [19]; Tocris Bioscience) ata concentration of 5 mmol/L (19).

ImmunoblottingImmunoblotting was performed with antibodies in thefollowing concentrations: VCAM-1 1:1,000 (#sc-8304;Santa Cruz Biotechnology, Dallas, TX), b-actin 1:10,000(#A1978; Sigma-Aldrich, Oakville, Ontario, Canada), CCL21:1,000 (#NBP1-07035; Novus Biologicals, Littleton, CO),CCR2 1:1,000 (#NBP2-35334; Novus Biologicals), intra-cellular adhesion molecule 1 1:1,000 (#4915S; Cell Signal-ing Technology, Danvers,MA), E-selectin 1:1,000 (#ab18981;Abcam, Cambridge, MA), P-selectin 1:1,000 (#ab59738;Abcam), phospho-p38 MAPK threonine 180/tyrosine 182(Thr180/Tyr182) 1:1,000 (#9216; Cell Signaling Technology),total p38 MAPK 1:1,000 (#9212; Cell Signaling Technology),phospho-histone H3Ser10 1:1,000 (#ab5176; Abcam), andtotal histone H3 1:1,000 (#9715; Cell Signaling Technology).

Animal StudiesIn study 1, male C57BL/6 mice (wild type [WT]) (TheJackson Laboratory, Bar Harbor, ME) and Ccr2 knockoutmice (CCR22/2) (The Jackson Laboratory) age 8 weekswere made diabetic by administration of a daily intraper-itoneal injection of streptozotocin (STZ) (55 mg/kg) in 0.1mol/L citrate buffer (pH 4.5) (or citrate buffer control)after a 4-h fast for 5 consecutive days. Mice were followedfor 14 weeks from the first intraperitoneal injection of STZ(WT, n = 9; CCR22/2, n = 6; STZ-WT, n = 8; STZ-CCR22/2,n = 7). Blood glucose was determined using OneTouchUltraMini (LifeScan Canada, Burnaby, British Columbia,Canada). Glomerular VCAM-1 was determined in frozenkidney sections after immunostaining with a VCAM-1antibody 1:50 dilution (#550547; BD Biosciences, SanJose, CA) and horseradish peroxidase–conjugated donkeyanti-rat IgG (H&L) 1:100 dilution (CLAS10-1115; Cedar-lane, Burlington, Ontario, Canada). Glomerular VCAM-1immunostaining was quantified using ImageScope 11.1software (Leica Microsystems, Concord, Ontario, Canada)in an average of seven glomerular profiles per mouse kidneyand is represented as fold change relative to WT. In study 2,diabetes was induced in male WT and endothelial nitricoxide synthase knockout (eNOS2/2) mice (The JacksonLaboratory) age 8 weeks by five daily intraperitoneal injec-tions of STZ 55 mg/kg (or citrate buffer) as previouslydescribed (8,20). Mice were followed for 3 weeks fromthe first injection of STZ (WT, n = 12; STZ-WT, n = 14;eNOS2/2, n = 11; STZ-eNOS2/2, n = 9). Urine CCL2excretion was determined by ELISA (#MJE00; R&D Sys-tems) after 24-h metabolic caging. All experimental proce-dures adhered to the guidelines of the Canadian Council onAnimal Care and were approved by the St. Michael’s Hos-pital Animal Care Committee.

Chromatin ImmunoprecipitationFor chromatin immunoprecipitation (ChIP), hGECs wereincubated in the presence or absence of 5 mmol/LSB-747651A for 1 h. ChIP was performed using a Magna

diabetes.diabetesjournals.org Alghamdi and Associates 2669

Page 3: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

ChIP kit (EMDMillipore, Etobicoke, Ontario, Canada) withan antibody against phospho-histone H3Ser10 (1:50 di-lution) (#ab5176; Abcam) or an equal concentration ofnormal rabbit IgG (Santa Cruz Biotechnology) as previ-ously described (21,22). Quantitative real-time PCR wasperformed using primers specific for the human VCAM1promoter (forward 59-GAGCTTCAGCAGTGAGAGCA-39,reverse 59-CCTTCAAGGGGAAACCCAGG-39) in hGECs orfor the mouse Vcam1 promoter (59-ATCTCTGTCTTTGCT-GTCAC-39, reverse 59-CTCTCCTGAAAAGATGATTG-39) inthe kidneys of male WT and CCR22/2 mice age 22 weeks(n = 4/group).

Quantitative RT-PCRRNA was isolated from snap-frozen kidney tissue or celllysates using TRIzol reagent (Thermo Fisher Scientific),and cDNA was reverse transcribed from 1 mg RNA usingSuperScript III Reverse Transcriptase (Thermo Fisher Sci-entific). Primer sequences (Integrated DNA Technologies,Coralville, IA) were as follows: human VCAM1 forward 59-ATTTCACTCCGCGGTATCTG-39, reverse 59-CCAAGGATCA-CGACCATCTT-39; human RPL13a forward 59-AGCTCATG-AGGCTACGGAAA-39, reverse 59-CTTGCTCCCAGCTTCCT-ATG-39; mouse Vcam1 forward 59-CCCAAGGATCCAGAG-ATTCA-39, reverse 59-TAAGGTGAGGGTGGCATTTC-39;and mouse b-actin forward 59-AGAGGGAAATCGTGCGT-GAC-39, reverse 59-CAATAGTGATGACCTGGCCGT-39. Fordetermination of micro RNA (miR)-93 levels, RNA iso-lation was performed using TRIzol reagent; poly(A) tailingwas performed using Poly(A) Polymerase, Yeast (#E017;Applied Biological Materials, Richmond, British Columbia,Canada); and cDNA was synthesized using an miRNA cDNAsynthesis kit (Applied Biological Materials). Primers forhsa-miR-93 and U6 small nuclear RNA were from AppliedBiological Materials (#MPH02022 and #MPH00001). SYBRgreen–based quantitative RT-PCRwas conducted using a ViiA7 real-time PCR system (Thermo Fisher Scientific), and dataanalysis was performed using the Applied Biosystems Com-parative CT method.

Human Tissue StudyArchived kidney tissue was examined from eight individ-uals without diabetes (control subjects) and nine individ-uals with diabetic kidney disease (22). Tissue had beenobtained at the time of nephrectomy for conventionalrenal carcinoma and was examined from regions of thekidney unaffected by tumor. Immunohistochemistry wasperformed with an antibody against phospho-histoneH3Ser10 (1:200 dilution) (#ab5176; Abcam), and the ratioof positively immunostaining glomerular nuclei to totalglomerular nuclei was calculated in 10 glomeruli per kidneysection. All histological analyses were performed by aninvestigator masked to the study groups. The study wasapproved by the Nova Scotia Health Authority ResearchEthics Board (Halifax, Nova Scotia, Canada) and the Re-search Ethics Board of St. Michael’s Hospital and wasconducted in accordance with the Declaration of Helsinki.A waiver of consent was provided by the Nova Scotia

Health Authority Research Ethics Board on the basis ofimpracticability criteria.

In Situ HybridizationIn situ hybridization for Vcam1 was performed with RNA-Scope (Advanced Cell Diagnostics, Hayward, CA) accordingto the manufacturer’s instructions and using custom soft-ware as previously described (23). Briefly, sections offormalin-fixed paraffin-embedded mouse or human kidneytissue were baked for 1 h at 60°C before deparaffinization,dehydration, and air drying. Slides were treated witha peroxidase blocker before boiling in target retrieval solu-tion for 15 min. Protease plus was applied for 30 min at40°C, and target probes were hybridized for 2 h at 40°Cbefore signal amplification and washing. Hybridization sig-nals were detected using Fast Red, and RNA staining wasidentified as red puncta on light microscopy. For immuno-fluorescence microscopy, in situ hybridization was followedby immunostaining for nephrin, CD31, or phospho-histoneH3Ser10 using the following antibodies: mouse nephrin1:200 (#AF3159; R&D Systems), secondary antibody AlexaFluor 647 donkey anti-goat 1:100 (#A21447; Thermo FisherScientific); mouse CD31 1:100 (#ab124432; Abcam), sec-ondary antibody Alexa Fluor 488 donkey anti-rabbit 1:100(#A21206; Thermo Fisher Scientific); human nephrin 1:100(#ab136894; Abcam), secondary antibody Alexa Fluor488 donkey anti-rabbit 1:100 (#A21206; Thermo FisherScientific); human CD31 1:100 (#3528; Cell Signaling Tech-nology), secondary antibody Alexa Fluor 647 donkey anti-mouse 1:100 (#A31571; Thermo Fisher Scientific); andhuman phospho-histone H3Ser10 1:200 (#ab5176; Abcam),secondary antibody Alexa Fluor 488 donkey anti-rabbit1:100 (#A21206; Thermo Fisher Scientific). DAPI wasfrom Cell Signaling Technology and was used at a concen-tration of 1:10,000. Slides were viewed using a Zeiss LSM700 confocal microscope (Carl Zeiss Canada, Toronto,Ontario, Canada) with a 363 optic.

Statistical AnalysisData are expressed as mean 6 SD. Statistical significancewas determined by one-way ANOVA followed by Fisherleast significant difference post hoc test for more than twogroups and two-tailed Student t test for two-groupcomparisons. Statistical analyses were performed usingGraphPad Prism for Mac OS X (GraphPad Software, LaJolla, CA).

RESULTS

Podocyte-Derived CCL2 Promotes VCAM-1Upregulation in hGECs, and CCR2–/– DecreasesGlomerular VCAM-1 Upregulation in Diabetic MiceIn our first series of experiments, we set out to determinewhich, if any, podocyte-derived cytokines or chemokinespromote the expression of VCAM-1 by GECs under high-glucose conditions. By immunoblotting, we observed thatVCAM-1 is expressed by cultured hGECs and that themagnitude of VCAM-1 expression is unaffected by exposure

2670 H3Ser10 Phosphorylation in Diabetic Nephropathy Diabetes Volume 67, December 2018

Page 4: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

of hGECs to high (25 mmol/L) glucose or mannitol (os-motic control) alone (Fig. 1A). Likewise, when we exposedhGECs to hpod_CM, we similarly observed no change inhGEC VCAM-1 expression (Fig. 1B). In contrast, VCAM-1expression was significantly increased when hGECs wereincubated in hpod_HGCM (Fig. 1B). To determine whichcytokines or chemokines are secreted by human podocytesin culture and which of these are altered by high glucose, weperformed a multiplex array (Table 1). The most abundantcytokines/chemokines present in the culture media of podo-cytes under normal conditions were C-X-C motif ligand1 (CXCL1)/CXCL2/CXCL3 (pan-GRO), interleukin-6 (IL-6),IL-8, CCL2, and vascular endothelial growth factor A. Of all41 cytokines/chemokines surveyed, CCL2 was the only oneto be significantly upregulated with the addition of highglucose to the culture medium (Table 1). Incubating hGECsin hpod_HGCM together with an anti-CCL2 neutralizingantibody resulted in a significant lowering of VCAM-1mRNA (Fig. 1C) and protein (Fig. 1D) levels, confirmingthat CCL2 contributes to VCAM-1 upregulation.

We recognized that podocytes secrete other factors thatwere not included in our cytokine/chemokine multiplexarray (e.g., angiopoietin 1, angiopoietin 2, endothelin 1)and that some of these factors have been implicated inpodocyte-endothelial communication (6,24). However, whenwe exposed hGECs to recombinant angiopoietin 1, angio-poietin 2, or endothelin 1, we observed that each recombi-nant protein actually downregulated VCAM-1 expression(Supplementary Fig. 1). By immunoblotting, we observedthat hGECs express the principal receptor for CCL2 CCR2 (aswell as CCL2 itself) and that neither CCR2 nor CCL2 werealtered in their expression by high glucose in hGECs (Fig. 1Eand F). To determine whether CCR2 regulates glomerularVCAM-1 expression in vivo, we examined the kidneys ofnondiabetic and diabetic WT and CCR22/2 mice 14 weeksafter the initial induction of diabetes with STZ. Althoughelevated blood glucose levels were unaffected by CCR2knockout (Fig. 1G), glomerular VCAM-1 upregulation wassignificantly attenuated in diabetic CCR22/2 mice (Fig. 1H).By immunofluorescence microscopy we observed VCAM-1transcript to be present in CD31+ GECs in the kidneys ofboth mice and humans (Supplementary Fig. 2). However,GECs were not the only cells to express the adhesionmolecule; VCAM-1 mRNA also was detectable in nephrin-positive podocytes (Supplementary Fig. 2).

CCL2/CCR2 Signaling Controls GEC VCAM-1Expression Through p38 MAPK- and MSK1/2-Dependent PathwaysHaving discovered that CCL2 regulates VCAM-1 expres-sion in cultured hGECs and that knockout of the CCL2receptor CCR2 diminishes glomerular VCAM-1 upregula-tion in diabetic mice, we next set out to determine thepathways through which CCL2/CCR2 signaling controlsVCAM-1. We observed that exposure of hGECs to recombi-nant CCL2 more than doubled VCAM-1 protein levels andthat this increase was negated by antagonism of CCR2 (Fig.

2A). In support of a relative specificity for the regulation inthe expression of VCAM-1 by CCL2, we found that theexpression of other adhesion molecules (i.e., intracellularadhesion molecule 1, E-selectin, P-selectin) was unaffectedby treatment of hGECs with recombinant CCL2 (Supple-mentary Fig. 3). We recognized the importance of p38MAPK as a downstream regulator of CCR2 signaling (25)and found that recombinant CCL2 increases hGEC p38MAPK Thr180/Tyr182 phosphorylation (Fig. 2B), indica-tive of p38 MAPK activation. As expected, pretreatment ofhGECs with the CCR2 antagonist RS504393 negated theincrease in p38 MAPK phosphorylation induced by CCL2(Fig. 2C). We observed that the p38 MAPK inhibitorSB203580 (17) prevented hGEC VCAM-1 upregulationinduced by CCL2 (Fig. 2D), confirming that p38 MAPKactivation is required for hGEC VCAM-1 expression. Next,we considered how p38 MAPK induces VCAM-1 upregula-tion. Two downstream kinases that are activated by p38MAPK are MSK1 andMSK2. We preincubated cells with theMSK1/2 inhibitor SB-747651A (19) and observed that likep38 MAPK inhibition, MSK1/2 inhibition prevented theupregulation in hGEC VCAM-1 induced by CCL2 (Fig. 2E).

CCL2 Induces H3Ser10 Phosphorylation, Which IsEnriched at the VCAM1 Promoter in hGECs and theVcam1 Promoter in Mouse KidneysMSK1/2 is known to regulate gene expression by directlyphosphorylating histone protein H3, including phospho-histone H3Ser10, which is a mark of active gene transcrip-tion. Accordingly, we next probed to see whether phospho-histone H3Ser10 levels are altered by CCL2 in hGECs.Aligned with this hypothesis, CCL2 induced an increase inH3Ser10 phosphorylation levels in hGECs, and this effectwas negated by antagonism of CCR2 (Fig. 3A) or inhibi-tion of either p38 MAPK (Fig. 3B) or MSK1/2 (Fig. 3C).Furthermore, by using ChIP, we observed enrichment ofH3Ser10 phosphorylation at the promoter region ofVCAM1 in hGECs, whereas this enrichment was dimin-ished (although not negated) by MSK1/2 inhibition (Fig.3D). To determine whether H3Ser10 phosphorylation isalso enriched at the Vcam1 promoter in vivo and whetherthis is affected by upstream CCR2-regulated signaling, weperformed ChIP experiments in the kidneys of WT andCCR22/2 mice. Although H3Ser10 phosphorylation wasenriched at the Vcam1 promoter in WT mouse kidneys,enrichment was approximately two-thirds lower in thekidneys of CCR22/2 mice (Fig. 3E). miR-93 recently hasbeen implicated in regulating podocyte MSK-mediatedH3Ser10 phosphorylation in diabetic kidney disease (26),but we saw no change in miR-93 levels in hGECs afterCCL2 treatment (Supplementary Fig. 4).

Phospho-Histone H3Ser10 Is Increased in Murine andHuman Diabetic Kidney DiseaseHaving identified a role for H3Ser10 phosphorylation infacilitating CCL2/CCR2-mediated VCAM-1 upregulation,we set out to determine whether H3Ser10 phosphoryla-tion levels are altered in diabetic kidney disease. For

diabetes.diabetesjournals.org Alghamdi and Associates 2671

Page 5: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

Figure 1—Anti-CCL2 neutralizing antibody diminishes VCAM-1 upregulation induced by exposure of hGECs to culturemedia conditioned byhigh-glucose (HG)–exposed podocytes, and knockout of the CCL2 receptor CCR2 decreases glomerular VCAM-1 upregulation in diabeticmice. A: Immunoblotting for VCAM-1 in hGECs incubated for 16 h under control conditions (5.6 mmol/L glucose) or in the presence of HG(25 mmol/L) or mannitol (osmotic control) (n = 3/condition). B: Immunoblotting for VCAM-1 in hGECs incubated for 16 h under control or HGconditions or in the presence of hpod_CM or hpod_HGCM (control, n = 5; HG, n = 5; hpod_CM, n = 6; hpod_HGCM, n = 7). C: Quantitative

2672 H3Ser10 Phosphorylation in Diabetic Nephropathy Diabetes Volume 67, December 2018

Page 6: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

these experiments, we chose to study both a mouse modelof diabetic kidney disease that is characterized by endo-thelial dysfunction and podocytopathy (STZ-diabeticeNOS2/2 mice) (9) and the glomeruli of humans withdiabetic kidney disease (22). Compared with nondiabeticmice, 3 weeks after the first intraperitoneal STZ injection,STZ-diabetic eNOS2/2 mice exhibited renal enlargementand heavy albuminuria (Supplementary Table 1) that wereaccompanied by increased urinary excretion of CCL2 (Fig.4A), increased renal H3Ser10 phosphorylation (Fig. 4B),and increased renal VCAM-1 mRNA (Fig. 4C and D) andprotein (Fig. 4E) levels.

To explore the relationship between H3Ser10 phos-phorylation and VCAM-1 expression in human diabetickidney disease, we studied kidney tissue from individualswith histopathologically confirmed diabetic glomeruloscle-rosis and compared it with kidney tissue from individualswithout diabetes. The clinical characteristics of the indi-viduals from whom kidney tissue was obtained have beenreported before (22). In brief, we examined kidney tissuefrom eight control subjects (five male, three female; age,69 6 11 years; serum creatinine, 85 6 11 mmol/L; esti-mated glomerular filtration rate, 746 11 mL/min/1.73 m2)and nine individuals with diabetic kidney disease (five male,four female; age, 67 6 10 years; serum creatinine, 107 639 mmol/L; estimated glomerular filtration rate, 61 625 mL/min/1.73 m2). Five of the individuals with diabetickidney disease had stage 3 chronic kidney disease or worse.We observed that in the kidney sections from humans withdiabetic glomerulosclerosis, there was an approximatelythreefold increase in the proportion of glomerular nucleipositively immunostaining for phospho-histone H3Ser10(Fig. 5A), including H3Ser10 phosphorylation in VCAM1-expressing glomerular cells (Fig. 5B).

DISCUSSION

Every cell that lies within the kidney glomerulus is affectedby diabetes, and every cell that lies within the kidneyglomerulus is affected by the actions of its neighbors. Inthe current study, we unearthed a signaling cascade thatregulates expression of the adhesion molecule VCAM-1 byGECs. Specifically, ligand binding by the receptor CCR2expressed by GECs induces VCAM-1 upregulation througha pathway that is regulated by the MSK1/2-dependentphosphorylation of H3Ser10. Heightened phospho-histone H3Ser10 levels in experimental and human dia-betic kidney disease and recent improvements in MSK1/2

inhibitor specificity (19) should galvanize efforts to ex-plore the modulation of histone phosphorylation asa means of attenuating kidney disease in diabetes.

As a marker of endothelial activation, we focused on theregulation of expression of VCAM-1, an immunoglobulinsuperfamily member that is expressed on the surface ofendothelial cells in response to proinflammatory cyto-kines. VCAM-1 promotes the firm adhesion and spreadingof leukocytes on the endothelium, enabling their trans-migration across the endothelial barrier. Several studieshave linked circulating VCAM-1 levels to diabetic kidneydisease or mortality risk (27–29), and likewise, a number ofreports have described an association between renal ex-pression or urinary excretion of the CCR2 ligand CCL2 andthe extent of diabetic kidney disease (30–32). However,even though CCL2, CCR2, and VCAM-1 often are consid-ered together in the same context of inflammation, thisdescription that CCL2/CCR2 binding can directly triggerglomerular endothelial VCAM-1 upregulation is the firstto our knowledge.

CCL2 (also called MCP-1) is a member of the CCchemokine family. Although CCL2 is best known for itsfunction as the ligand for the receptor CCR2, which isexpressed on the surface of monocytes and macrophages,the actions of CCL2 and CCR2 are not limited to inflam-matory cells, and the relationship between CCL2 and CCR2is not monogamous. For instance, podocytes themselvesare known to express both CCL2 and CCR2 (33,34), and weobserved that hGECs also express both ligand and re-ceptor. In terms of ligand-receptor specificity, CCL2 alsobinds to CCR4 (35), and CCR2 also may be bound by CCL7,CCL8, CCL12 (mouse only), CCL13, and CCL16 (humanonly) (36). In a similar nonreductionist context, althoughwe focused on VCAM-1 upregulation as a marker ofendothelial activation (3), it is noteworthy that otherglomerular cells, including both podocytes (37) and mesan-gial cells (38), also are capable of expressing VCAM-1. Inthe current study, we observed that 1) VCAM-1 levels wereincreased in hGECs incubated in culture medium that hadbeen conditioned by podocytes exposed to high glucose, 2)secretion of CCL2 by podocytes into the culture mediumwas upregulated by high glucose, 3) an anti-CCL2–neutralizing antibody diminished hGEC VCAM-1 expres-sion, and 4) recombinant CCL2 induced hGEC VCAM-1upregulation in a CCR2-dependent manner. Thus, whereasCCL2/CCR2 signaling to hGECs could be paracrine inorigin, autocrine in origin, or both and the relationship

RT-PCR for VCAM1 in hGECs incubated for 16 h with HG or hpod_HGCM that had been preincubated with an anti-CCL2 neutralizingantibody (20 mg/mL) for 1 h (control, n = 5; HG, n = 6; hpod_HGCM, n = 6; hpod_HGCM+ anti-CCL2 antibody [Ab], n = 6). D: Immunoblottingfor VCAM-1 in hGECs under control conditions or incubated for 16 h with HG or hpod_HGCM preincubated with an anti-CCL2 neutralizingantibody (20 mg/mL) for 1 h (n = 5/condition). E and F: Immunoblotting hGECs for CCR2 (E) or CCL2 (F ) under control conditions or afterincubation with HG or mannitol (osmotic control) for 48 h (n = 4/condition).G andH: WT andCCR22/2mice 14weeks after diabetes inductionwith STZ. Blood glucose (G) and immunohistochemistry (H) for VCAM-1 and quantification of glomerular VCAM-1 immunostaining (WT, n = 9;CCR22/2, n = 6; STZ-WT, n = 8; STZ-CCR22/2, n = 7). Original magnification3400. AU, arbitrary unit. Data are mean6 SD. *P, 0.05, **P,0.01, ***P , 0.001, ****P , 0.0001 by one-way ANOVA followed by Fisher least significant difference post hoc test.

diabetes.diabetesjournals.org Alghamdi and Associates 2673

Page 7: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

Table 1—Chemokine and cytokine content of hpod_CM and hpod_HGCM

Cytokine/chemokine Control High glucose Mannitol

Basic FGF 252 6 13 219 6 18a 205 6 24b

CCL2 3,236 6 517 4,223 6 1,034c 3,299 6 717

CCL3 1,016 6 362 678 6 195 547 6 262c

CCL4 78 6 23 52 6 12c 40 6 20a

CCL5 30 6 9 31 6 7 23 6 4

CCL7 64 6 14 44 6 9a 33 6 8b

CCL11 145 6 49 109 6 15 149 6 45

CCL22 50 6 27 61 6 12 48 6 6

C-X-C motif chemokine 10 73 6 31 76 6 21 69 6 26

CX3CL1 111 6 37 97 6 19 109 6 24

EGF 3.9 6 0.8 4.8 6 1.3 4.2 6 1.1

FMS-like tyrosine kinase 3 ligand 9.0 6 2.1 9.5 6 1.0 7.3 6 2.2

Granulocyte CSF 1,116 6 360 762 6 229c 433 6 248b

Granulocyte-macrophage CSF 1,904 6 492 1,576 6 197 1,295 6 477c

IFN-a2 41 6 18 45 6 3 45 6 10

IFN-g 4.9 6 2.4 4.9 6 1.8 5.2 6 2.7

IL-1a 161 6 35 126 6 19 108 6 31a

IL-1b 3.1 6 0.7 3.9 6 0.7 3.3 6 0.9

IL-1 receptor antagonist 41 6 12 46 6 6 41 6 6

IL-2 1.8 6 0.7 1.7 6 0.7 2.0 6 0.7

IL-3 ,0.64 ,0.64 ,0.64

IL-4 4.1 6 0.9 3.9 6 1.4 4.8 6 1.1

IL-5 0.5 6 0.1 0.4 6 0.1 0.4 6 0.1c

IL-6 12,463 6 9,486 8,915 6 2,522 6,994 6 2,124

IL-7 3.5 6 0.3 3.3 6 0.7 3.5 6 0.5

IL-8 .10,000 .10,000 .10,000

IL-9 0.7 6 0.2 0.6 6 0.2 0.7 6 0.4

IL-10 3.3 6 1.2 2.7 6 0.6 2.2 6 0.4c

IL-12 subunit p40 16 6 6 12 6 7 13 6 6

IL-12 subunit p70 1.6 6 0.5 1.6 6 0.9 2.0 6 0.7

IL-13 1.3 6 0.5 1.2 6 0.5 1.2 6 0.6

IL-15 10.0 6 2.9 10.0 6 1.7 8.5 6 2.6

IL-17A 4.6 6 1.4 2.2 6 0.4a 1.2 6 1.0b

Pan-GRO 8,972 6 1,683 8,200 6 2,202 14,559 6 10,435

PDGF-AA 473 6 158 629 6 233 433 6 147

PDGF-BB 161 6 25 144 6 16 153 6 37

Soluble CD40 ligand 6.1 6 2.3 6.9 6 2.5 6.7 6 1.6

TNF-a 22 6 7 24 6 4 18 6 7

TNF-b 1.8 6 1.0 1.5 6 1.1 1.8 6 0.6

TGF-a 40 6 14 30 6 6 30 6 10

VEGF-A 2,370 6 784 2,153 6 435 2,375 6 1,233

Data are mean 6 SD and expressed in pg/mL. Boldface type highlights CCL2 levels. CSF, colony-stimulating factor; EGF, epidermalgrowth factor; FGF, fibroblast growth factor; IFN, interferon; Pan-GRO, CXCL1/CXCL2/CXCL3; PDGF, platelet-derived growth factor;TGF, transforming growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor. aP, 0.01 vs. control; bP, 0.001vs. control; cP , 0.05 vs. control by one-way ANOVA followed by Fisher least significant difference post hoc test.

2674 H3Ser10 Phosphorylation in Diabetic Nephropathy Diabetes Volume 67, December 2018

Page 8: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

between CCL2 and CCR2 is not exclusive, the evidenceherein demonstrates that CCR2 signaling regulates glo-merular VCAM-1 expression, including CCL2-inducedVCAM-1 upregulation by hGECs.

In unraveling the cascade by which signaling throughCCR2 induces VCAM-1 upregulation in hGECs, we discov-ered important roles for p38 MAPK and MSK1/2 and anassociated enrichment of the phospho-histone H3Ser10mark at the VCAM1 promoter. Chromatin modifications,such as phospho-histone H3Ser10 rarely control geneactivation or repression in isolation. Rather, an interplayexists whereby histone marks function alongside other

epigenetic regulators, other histone marks, and canonicaltranscription factors to coordinate gene expression in anintegrated manner (39). For instance, H3Ser10 phosphor-ylation (like histone acetylation) can facilitate gene acti-vation by affecting the electrostatic charge relationshipbetween histone proteins and DNA, associating with openchromatin during interphase, and allowing access to DNAby the transcriptional machinery. Separately, H3Ser10phosphorylation also may promote gene transcriptionby virtue of its proximity to other histone marks. Forinstance, the histone acetyltransferase Gcn5 can acetylatelysine residue 14 (K14) on histone H3 more effectively

Figure 2—CCL2 increases hGEC VCAM-1 levels through CCR2-, p38 MAPK-, MSK1/2-regulated mechanisms. A: Immunoblotting forVCAM-1 in hGECs incubated with or without the CCR2 antagonist RS504393 (10 mmol/L) for 1 h before exposure to recombinant CCL2(0.5 ng/mL) for 16 h (control, n = 5; RS504393, n = 5; CCL2, n = 3; CCL2+ RS504393, n = 3). B: Immunoblotting for p38 MAPK Thr180/Tyr182phosphorylation (phospho-p38 MAPK) in hGECs incubated in the presence or absence of CCL2 (0.5 ng/mL) for 16 h (n = 7/condition). C:Immunoblotting for phospho-p38 MAPK in hGECs incubated in the presence or absence of the CCR2 antagonist RS504393 (10 mmol/L) for1 h before exposure to recombinant CCL2 (0.5 ng/mL) for 16 h (n = 4/condition). D: Immunoblotting for VCAM-1 in hGECs incubated with orwithout the p38 MAPK inhibitor SB203580 (10 mmol/L) for 1 h before exposure to recombinant CCL2 (0.5 ng/mL) for 16 h (control, n = 7;SB203580, n = 6; CCL2, n = 7; CCL2+ SB203580, n = 6). E: Immunoblotting for VCAM-1 in hGECs incubated with or without the MSK1/2inhibitor SB-747651A (5 mmol/L) for 1 h before exposure to recombinant CCL2 (0.5 ng/mL) for 16 h (control, n = 5; SB-747651A, n = 4; CCL2,n = 5; CCL2+ SB-747651A, n = 5). AU, arbitrary unit. Data are mean6 SD. *P, 0.05, **P, 0.01, ****P, 0.0001 by one-way ANOVA followedby Fisher least significant difference post hoc test (A and C–E ) and two-tailed Student t test (B).

diabetes.diabetesjournals.org Alghamdi and Associates 2675

Page 9: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

when H3Ser10 is phosphorylated, with H3K14ac beingfound at actively transcribed promoters (40). A numberof kinases have been reported to phosphorylate histone H3on serine residue 10, but the best characterized of theseis MSK1/2, which is a substrate for p38 MAPK (41), itselfactivated by CCR2 (25). The observation that MSK1/2inhibition reduces but does not negate H3Ser10 phosphor-ylation at the VCAM1 promoter may suggest an additionalrole for other kinases (e.g., calcium/calmodulin-dependentprotein kinase II [42,43]). Similarly, H3Ser10 is not theonly substrate of MSK1/2, with the transcription factorCREB also being phosphorylated by the kinase (44). In-deed, VCAM-1 expression induced by tumor necrosisfactor-a in endothelial cells has been reported to in-volve p38 MAPK-mediated CREB phosphorylation (45).

Moreover, transcription factor binding at specific sites onthe genome itself depends on histone modifications and isboth histone modification and protein family specific (46).Thus, aligned with the current perspective of coordinatedinterplay between epigenetic modifications and canonicaltranscription factors, CCR2-regulated VCAM-1 expressionby the glomerular endothelium likely involves both effectsthat are mediated through histone protein posttrans-lational modifications and effects that are regulated byassociated transcription factor responses. Nonetheless,a role for H3Ser10 phosphorylation in regulating endo-thelial activation in diabetes is supported by increasedH3Ser10 phosphorylation at the VCAM1 promoter anda reduction with MSK1/2 inhibition that coincides witha decrease in VCAM-1 protein levels in hGECs. H3Ser10

Figure 3—CCL2 increases hGEC phospho-histone H3Ser10, and phospho-histone H3Ser10 is enriched at the VCAM-1 promoter in hGECsand mouse kidneys. A: Immunoblotting hGECs for phospho-histone H3Ser10 incubated with or without the CCR2 antagonist RS504393(10 mmol/L) for 1 h before exposure to recombinant CCL2 (0.5 ng/mL) for 16 h (n = 5/condition). B: Immunoblotting hGECs for phospho-histone H3Ser10 incubated with or without the p38 MAPK inhibitor SB203580 (10 mmol/L) for 1 h before exposure to recombinant CCL2(0.5 ng/mL) for 16 h (n = 5/condition). C: Immunoblotting hGECs for phospho-histone H3Ser10 incubated with or without the MSK1/2 in-hibitor SB-747651A (5mmol/L) for 1 h before exposure to recombinant CCL2 (0.5 ng/mL) for 16 h (control, n = 6; SB-747651A, n = 5; CCL2, n =6; CCL2+ SB-747651A, n = 5). D: ChIP for the presence of phospho-histone H3Ser10 at the VCAM1 promoter in hGECs in the presence orabsence of SB-747651A (5 mmol/L) for 1 h (n = 7/condition). E: ChIP for phospho-histone H3Ser10 at the Vcam1 promoter in WT andCCR22/2 mouse kidneys (n = 4/group). ChIP data were determined by quantitative real-time PCR. AU, arbitrary unit. Data are mean 6 SD.*P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001 by one-way ANOVA followed by Fisher least significant difference post hoc test.

2676 H3Ser10 Phosphorylation in Diabetic Nephropathy Diabetes Volume 67, December 2018

Page 10: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

phosphorylation could regulate VCAM-1 expression bydirectly affecting CCR2-mediated signaling, or it couldhave parallel effects, facilitating canonical transcriptionfactor–mediated gene transcription (Fig. 6).

Consistent with its contributory role to the develop-ment of diabetic kidney disease, we observed increasedlevels of H3Ser10 phosphorylation both in the kidneys ofSTZ-eNOS2/2 mice (a model considered to mimic humandisease more closely [47]) and in the glomeruli of humanswith diabetic kidney disease. We studied STZ-eNOS2/2

mice soon after the induction of diabetes because wepreviously found that the heavy albuminuria that these

mice develop coincides with the onset of hyperglycemia(9). Even at this early time point, we observed increasedurine CCL2 excretion in STZ-eNOS2/2mice that coincidedwith increases in both renal H3Ser10 phosphorylation andVCAM-1 expression. Of note, however, is that distinctfrom its role in transcriptional activation, H3Ser10 phos-phorylation also marks highly condensed chromatin dur-ing mitosis. Thus, whether the increased kidney cellH3Ser10 phosphorylation in diabetes is indicative of mi-totic cell division, a generalized shift in the epigenomiclandscape that supports transcriptional activation, ora combination of the two is unclear. Also of note, the

Figure 4—Urine CCL2 excretion and renal phospho-histone H3Ser10 and VCAM-1 expression are increased in STZ-diabetic eNOS2/2mice.Shown areWT and eNOS2/2mice 3weeks after the initiation of diabetes inductionwith STZ.A: Urine CCL2 excretion (WT, n = 6; STZ-WT, n =7; eNOS2/2, n = 5; STZ-eNOS2/2, n = 5). B: Renal phospho-histone H3Ser10 (WT, n = 7; STZ-WT, n = 7; eNOS2/2, n = 6; STZ-eNOS2/2, n =6). C: Renal Vcam1 mRNA levels (WT, n = 10; STZ-WT, n = 11; eNOS2/2, n = 9; STZ-eNOS2/2, n = 9). D: In situ hybridization for Vcam1.Original magnification 3400. E: Immunoblotting of kidney homogenates for VCAM-1 (n = 4/group). AU, arbitrary unit. Data are mean 6 SD.*P , 0.05, **P , 0.01 by one-way ANOVA followed by Fisher least significant difference post hoc test.

diabetes.diabetesjournals.org Alghamdi and Associates 2677

Page 11: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

findings are aligned with a recent study that reportedincreased global H3Ser10 phosphorylation levels in podo-cytes exposed to high glucose and in glomerular cells oftype 2 diabetic db/db mice (26).

As already highlighted, our study has limitations. Ofnote, paracrine podocyte-derived CCL2 may not be the onlyactivator of hGEC CCR2 signaling, H3Ser10 phosphoryla-tion by MSK1/2 may not be the sole means through whichCCR2 signaling regulates VCAM-1 expression, and increases

in kidney cell H3Ser10 phosphorylation in experimental andhuman diabetic kidney disease will not solely reflect thechanges occurring at the VCAM-1 promoter. On the basisof our initial experiments, despite containing appreciablelevels of CCL2, media of podocytes grown under normalglucose conditions did not induce hGEC VCAM-1 upregu-lation, suggesting the presence of other nonquantifiedfactors in the culture media. For instance, we observeda reduction in VCAM-1 expression by exposure of hGECs

Figure 5—Phospho-histone H3Ser10 is increased in human diabetic kidney disease. A: Immunohistochemistry for phospho-histoneH3Ser10 in kidney sections from control subjects (h_Control) (n = 8) and individuals with diabetic kidney disease (h_Diabetes) (n = 9) andquantification of the proportion of phospho-histone H3Ser10–positive glomerular nuclei. Original magnification3400. B: In situ hybridizationfor VCAM1 and immunostaining for phospho-histone H3Ser10 in kidney sections from a control subject and an individual with diabeticglomerulosclerosis. Original images takenwith an363 optic. The zoomed images are enlargements of the boxed areas. Data aremean6SD.****P , 0.0001 by two-tailed Student t test.

2678 H3Ser10 Phosphorylation in Diabetic Nephropathy Diabetes Volume 67, December 2018

Page 12: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

to recombinant angiopoietin 1, which is constitutivelyexpressed by podocytes and downregulated in diabetes(48). Thus, overall effects of podocyte-conditioned me-dium likely reflect the overall balance of its constituents,which are not limited to proteins but may also involvebioactive lipids, nucleic acids, and microparticles (6).

Despite their limitations, the current experiments pro-vide important new insights. First, they demonstrate howproinflammatory cytokines/chemokines can have directeffects on the glomerular endothelium, which could haveimplications for the interpretation of the mechanism ofaction of anti-inflammatory therapies recently trialed in thetreatment of diabetic kidney disease (49,50). Second, theyhighlight the emerging role for H3Ser10 phosphorylation

and its regulatory kinases MSK1/2 in facilitating the acti-vation of genes important to the development of kidneydisease in diabetes, specifically the expression of VCAM-1 byGECs. Moreover, the elucidation of these actions in culturedcells of human origin and the observation of heightenedglomerular cell H3Ser10 phosphorylation in human diabetickidney disease lend weight to the significance of the findingsin an era when the value of rodent models is under scrutiny.

In summary, ligand binding by CCR2 initiates an in-tracellular signaling cascade in GECs that involves the p38MAPK, MSK1/2-regulated phosphorylation of H3Ser10,facilitating the expression of the inducible proinflammatoryadhesion molecule VCAM-1, a marker of endothelial acti-vation. Histone protein phosphorylation should be placed

Figure 6—Schematic illustration of the role phospho-histone H3Ser10 plays in regulating glomerular endothelial VCAM-1 expression andendothelial activation in diabetes. High glucose causes increased secretion of the chemokine CCL2 by podocytes. CCL2 may function ina paracrine fashion (e.g., arising from podocytes) or an autocrine fashion (arising from the glomerular endothelium) and binds to its cognatereceptor CCR2 on GECs. CCR2 can induce signaling that leads to canonical transcription factor (TF) effects, and it can induce signaling thatresults in epigenetic effects, each of which may promote VCAM-1 expression. CCR2 signaling can induce epigenetic effects througha pathway that involves the sequential activation of p38 MAPK, the nuclear kinases MSK1/2, and phospho-histone H3Ser10. AntagonizingCCR2 or inhibiting p38 MAPK or MSK1/2 (numbered circles) limits both H3Ser10 phosphorylation and VCAM-1 expression by GECs.H3Ser10 also may be phosphorylated by other kinases, and MSK1/2 also can phosphorylate other TFs.

diabetes.diabetesjournals.org Alghamdi and Associates 2679

Page 13: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

alongside previously better-studied histone modificationswhen considering potentially druggable candidates suitablefor targeted intervention in diabetic kidney disease.

Acknowledgments. The authors thank Dr. Hana Klassen Vakili (currentlyaffiliated with the Department of Pathology, University of Texas SouthwesternMedical Center, TX) for technical assistance.Funding. T.A.A. is supported by a King Abdullah Foreign Scholarship. S.N.B.was supported by the Kidney Foundation of Canada through a Keenan FamilyFoundation Kidney Research Scientist Core Education and National TrainingProgram Post-Doctoral fellowship, a Heart and Stroke/Richard Lewar Centerof Excellence fellowship, and a Banting & Best Diabetes Centre, Universityof Toronto, postdoctoral fellowship. M.J.H. is a recipient of a St. Michael’sHospital scholarship from the Research Training Centre and a Banting &Best Diabetes Centre-Novo Nordisk studentship. S.M. and A.A. weresupported by a Diabetes Canada postdoctoral fellowship and diabetesinvestigator award, respectively. These studies were supported by a Cana-dian Institutes of Health Research operating grant (MOP-133631) and in partby a Heart and Stoke Foundation of Canada grant-in-aid (G-17-0018231)to A.A.Duality of Interest. A.A. has received research support through hisinstitution from AstraZeneca and Boehringer Ingelheim and is listed as an inventoron an unrelated patent application by Boehringer Ingelheim. No other potentialconflicts of interest relevant to this article were reported.Author Contributions. T.A.A. designed and performed the experiments,analyzed the data, and wrote the manuscript. S.N.B. and S.M. designed andperformed the experiments and analyzed the data. M.J.H., V.G.Y., Y.L., B.B.B., andS.L.A. performed the experiments. L.G. and F.S.S. contributed to the human data.A.A. designed the experiments, supervised the study, and wrote the manuscript.A.A. is the guarantor of this work and, as such, had full access to all the data in thestudy and takes responsibility for the integrity of the data and the accuracy of thedata analysis.Prior Presentation. Parts of this work were presented at the WorldDiabetes Congress, Vancouver, British Columbia, Canada, 30 November–4December 2015, and the 78th Scientific Sessions of the American DiabetesAssociation, Orlando, FL, 7–11 June 2018.

References1. Keating ST, van Diepen JA, Riksen NP, El-Osta A. Epigenetics in diabeticnephropathy, immunity and metabolism. Diabetologia 2018;61:6–202. Navarro-González JF, Mora-Fernández C, Muros de Fuentes M, García-PérezJ. Inflammatory molecules and pathways in the pathogenesis of diabetic ne-phropathy. Nat Rev Nephrol 2011;7:327–3403. Liao JK. Linking endothelial dysfunction with endothelial cell activation. J ClinInvest 2013;123:540–5414. Ina K, Kitamura H, Okeda T, et al. Vascular cell adhesion molecule-1 ex-pression in the renal interstitium of diabetic KKAy mice. Diabetes Res Clin Pract1999;44:1–85. Seron D, Cameron JS, Haskard DO. Expression of VCAM-1 in the normal anddiseased kidney. Nephrol Dial Transplant 1991;6:917–9226. Siddiqi FS, Advani A. Endothelial-podocyte crosstalk: the missing link be-tween endothelial dysfunction and albuminuria in diabetes. Diabetes 2013;62:3647–36557. Saleem MA, O’Hare MJ, Reiser J, et al. A conditionally immortalized humanpodocyte cell line demonstrating nephrin and podocin expression. J Am SocNephrol 2002;13:630–6388. Batchu SN, Majumder S, Bowskill BB, et al. Prostaglandin I2 receptor ag-onism preserves b-Cell function and attenuates albuminuria through nephrin-dependent mechanisms. Diabetes 2016;65:1398–14099. Yuen DA, Stead BE, Zhang Y, et al. eNOS deficiency predisposes podocytesto injury in diabetes. J Am Soc Nephrol 2012;23:1810–1823

10. Shah R, Hinkle CC, Ferguson JF, et al. Fractalkine is a novel human adi-pochemokine associated with type 2 diabetes. Diabetes 2011;60:1512–151811. Satchell SC, Anderson KL, Mathieson PW. Angiopoietin 1 and vascularendothelial growth factor modulate human glomerular endothelial cell barrierproperties. J Am Soc Nephrol 2004;15:566–57412. Collino F, Bussolati B, Gerbaudo E, et al. Preeclamptic sera induce nephrinshedding from podocytes through endothelin-1 release by endothelial glomerularcells. Am J Physiol Renal Physiol 2008;294:F1185–F119413. Gibson DA, Greaves E, Critchley HO, Saunders PT. Estrogen-dependent

regulation of human uterine natural killer cells promotes vascular remodelling viasecretion of CCL2. Hum Reprod 2015;30:1290–130114. Salcedo R, Ponce ML, Young HA, et al. Human endothelial cells express CCR2and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor pro-gression. Blood 2000;96:34–4015. Mirzadegan T, Diehl F, Ebi B, et al. Identification of the binding site for a novelclass of CCR2b chemokine receptor antagonists: binding to a common chemokinereceptor motif within the helical bundle. J Biol Chem 2000;275:25562–2557116. Simonson MS, Ismail-Beigi F. Endothelin-1 increases collagen accumulationin renal mesangial cells by stimulating a chemokine and cytokine autocrine

signaling loop. J Biol Chem 2011;286:11003–1100817. Cuenda A, Rouse J, Doza YN, et al. SB 203580 is a specific inhibitor ofa MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995;364:229–23318. Clerk A, Michael A, Sugden PH. Stimulation of the p38 mitogen-activatedprotein kinase pathway in neonatal rat ventricular myocytes by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine: a role in cardiacmyocyte hypertrophy? J Cell Biol 1998;142:523–53519. Naqvi S, Macdonald A, McCoy CE, Darragh J, Reith AD, Arthur JS. Char-

acterization of the cellular action of the MSK inhibitor SB-747651A. BiochemJ 2012;441:347–35720. Thieme K, Majumder S, Brijmohan AS, et al. EP4 inhibition attenuates thedevelopment of diabetic and non-diabetic experimental kidney disease. Sci Rep2017;7:344221. Siddiqi FS, Majumder S, Thai K, et al. The histone methyltransferase enzymeenhancer of zeste homolog 2 protects against podocyte oxidative stress and renalinjury in diabetes. J Am Soc Nephrol 2016;27:2021–203422. Majumder S, Thieme K, Batchu SN, et al. Shifts in podocyte histone

H3K27me3 regulate mouse and human glomerular disease. J Clin Invest 2018;128:483–49923. Wang F, Flanagan J, Su N, et al. RNAScope: a novel in situ RNA analysisplatform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 2012;14:22–2924. Daehn I, Casalena G, Zhang T, et al. Endothelial mitochondrial oxidative

stress determines podocyte depletion in segmental glomerulosclerosis. J ClinInvest 2014;124:1608–162125. WerleM, Schmal U, Hanna K, Kreuzer J. MCP-1 induces activation of MAP-kinasesERK, JNK and p38 MAPK in human endothelial cells. Cardiovasc Res 2002;56:284–29226. Badal SS, Wang Y, Long J, et al. miR-93 regulates Msk2-mediated chromatinremodelling in diabetic nephropathy. Nat Commun 2016;7:1207627. Liu JJ, Yeoh LY, Sum CF, et al.; SMART2D Study. Vascular cell adhesionmolecule-1, but not intercellular adhesionmolecule-1, is associated with diabetic kidneydisease in Asians with type 2 diabetes. J Diabetes Complications 2015;29:707–71228. Rubio-Guerra AF, Vargas-Robles H, Lozano Nuevo JJ, Escalante-AcostaBA. Correlation between circulating adhesion molecule levels and albuminuria intype-2 diabetic hypertensive patients. Kidney Blood Press Res 2009;32:106–10929. Stehouwer CD, Gall MA, Twisk JW, Knudsen E, Emeis JJ, Parving HH. In-

creased urinary albumin excretion, endothelial dysfunction, and chronic low-gradeinflammation in type 2 diabetes: progressive, interrelated, and independentlyassociated with risk of death. Diabetes 2002;51:1157–116530. Har R, Scholey JW, Daneman D, et al. The effect of renal hyperfiltration onurinary inflammatory cytokines/chemokines in patients with uncomplicated

type 1 diabetes mellitus. Diabetologia 2013;56:1166–1173

2680 H3Ser10 Phosphorylation in Diabetic Nephropathy Diabetes Volume 67, December 2018

Page 14: Histone H3 Serine 10 Phosphorylation Facilitates ...€¦ · ized human podocytes (provided by M. Saleem, University ofBristol,Bristol,U.K.)(7)andinprimaryculturedhuman renal GECs

31. Wada T, Furuichi K, Sakai N, et al. Up-regulation of monocyte chemo-

attractant protein-1 in tubulointerstitial lesions of human diabetic nephropathy.

Kidney Int 2000;58:1492–149932. Tashiro K, Koyanagi I, Saitoh A, et al. Urinary levels of monocyte chemo-

attractant protein-1 (MCP-1) and interleukin-8 (IL-8), and renal injuries in patients

with type 2 diabetic nephropathy. J Clin Lab Anal 2002;16:1–433. Tarabra E, Giunti S, Barutta F, et al. Effect of the monocyte chemoattractant

protein-1/CC chemokine receptor 2 system on nephrin expression in streptozo-

tocin-treated mice and human cultured podocytes. Diabetes 2009;58:2109–211834. Lee EY, Chung CH, Khoury CC, et al. The monocyte chemoattractant protein-

1/CCR2 loop, inducible by TGF-beta, increases podocyte motility and albumin

permeability. Am J Physiol Renal Physiol 2009;297:F85–F9435. Power CA, Meyer A, Nemeth K, et al. Molecular cloning and functional

expression of a novel CC chemokine receptor cDNA from a human basophilic cell

line. J Biol Chem 1995;270:19495–1950036. Chu HX, Arumugam TV, Gelderblom M, Magnus T, Drummond GR, Sobey CG.

Role of CCR2 in inflammatory conditions of the central nervous system. J Cereb

Blood Flow Metab 2014;34:1425–142937. Visweswaran GR, Gholizadeh S, Ruiters MH, Molema G, Kok RJ, Kamps JA.

Targeting rapamycin to podocytes using a Vascular Cell Adhesion Molecule-1

(VCAM-1)-harnessed SAINT-based lipid carrier system. PLoS One 2015;10:

e013887038. Ishibashi Y, Matsui T, Yamagishi S. Olmesartan blocks advanced glycation

end products-induced vcam-1 gene expression in mesangial cells by restoring

Angiotensin-converting enzyme 2 level. Horm Metab Res 2014;46:379–38339. Yerra VG, Advani A. Histones and heart failure in diabetes. Cell Mol Life Sci.

22 June 2018 [Epub ahead of print]. DOI: 10.1007/s00018-018-2857-140. Lo WS, Trievel RC, Rojas JR, et al. Phosphorylation of serine 10 in histone H3

is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14.

Mol Cell 2000;5:917–926

41. Thomson S, Clayton AL, Hazzalin CA, Rose S, Barratt MJ, Mahadevan LC. Thenucleosomal response associated with immediate-early gene induction is me-diated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase. EMBO J 1999;18:4779–479342. Awad S, Kunhi M, Little GH, et al. Nuclear CaMKII enhances histone H3phosphorylation and remodels chromatin during cardiac hypertrophy. NucleicAcids Res 2013;41:7656–767243. Smedlund K, Tano JY, Vazquez G. The constitutive function of native TRPC3channels modulates vascular cell adhesion molecule-1 expression in coronary en-dothelial cells through nuclear factor kappaB signaling. Circ Res 2010;106:1479–148844. Arthur JS, Cohen P. MSK1 is required for CREB phosphorylation in responseto mitogens in mouse embryonic stem cells. FEBS Lett 2000;482:44–4845. Ono H, Ichiki T, Ohtsubo H, et al. CAMP-response element-binding proteinmediates tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1expression in endothelial cells. Hypertens Res 2006;29:39–4746. Xin B, Rohs R. Relationship between histone modifications and transcriptionfactor binding is protein family specific. Genome Res. 11 January 2018 [Epubahead of print]. DOI: 10.1101/gr.220079.11647. Fu J, Wei C, Zhang W, et al. Gene expression profiles of glomerular en-dothelial cells support their role in the glomerulopathy of diabetic mice. Kidney Int2018;94:326–34548. Gnudi L. Angiopoietins and diabetic nephropathy. Diabetologia 2016;59:1616–162049. de Zeeuw D, Bekker P, Henkel E, et al.; CCX140-B Diabetic NephropathyStudy Group. The effect of CCR2 inhibitor CCX140-B on residual albuminuria inpatients with type 2 diabetes and nephropathy: a randomised trial. Lancet DiabetesEndocrinol 2015;3:687–69650. Tuttle KR, Brosius FC III, Adler SG, et al. JAK1/JAK2 inhibition by baricitinibin diabetic kidney disease: results from a Phase 2 randomized controlled clinicaltrial. Nephrol Dial Transplant. 22 February 2018 [Epub ahead of print]. DOI:10.1093/ndt/gfx377

diabetes.diabetesjournals.org Alghamdi and Associates 2681