BASIC RESEARCH www.jasn.org NFkB-Induced Periostin Activates Integrin-b3 Signaling to Promote Renal Injury in GN † †‡ Niki Prakoura,* Panagiotis Kavvadas,* Raphaёl Kormann,* Jean-Claude Dussaule,* † † Christos E. Chadjichristos,* and Christos Chatziantoniou* *Institut National de la Santé Et de la Recherche Médicale, Unité Mixte de Recherche Scientifique 1155, Tenon Hospital, Paris, France; †Sorbonne Université, Université Pierre-et-Marie-Curie Paris 6, Paris, France; and ‡Department of Physiology, Saint-Antoine Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France ABSTRACT De novo expression in the kidney of periostin, a protein involved in odontogenesis and osteogenesis, has been suggested as a biomarker of renal disease. In this study, we investigated the mechanism(s) of induction and the role of periostin in renal disease. Using a combination of bioinformatics, reporter assay, and chromatin immunoprecipitation analyses, we found that NFkB and other proinflammatory transcription factors induce periostin expression in vitro and that binding of these factors on the periostin promoter is enriched in glo- meruli during experimental GN. Mice lacking expression of periostin displayed preserved renal function and structure during GN. Furthermore, delayed administration of periostin antisense oligonucleotides in wild-type animals with GN reversed already established proteinuria, diminished tissue inflammation, and improved renal structure. Lack of periostin expression also blunted the de novo renal expression of integrin-b3and phosphorylation of focal adhesion kinase and AKT, known mediators of integrin-b3 signaling that affect cell motility and survival, observed during GN in wild-type animals. In vitro, recombinant periostin increased the expression of integrin-b3 and the concomitant phosphorylation of focal adhesion kinase and AKT in podo- cytes. Notably, periostin and integrin-b3 were highly colocalized in biopsy specimens from patients with inflammatory GN. These results demonstrate that interplay between periostin and renal inflammation orches- trates inflammatory and fibrotic responses, driving podocyte damage through downstream activation of integrin-b3 signaling. Targeting periostin may be a novel therapeutic strategy for treating CKD. J Am Soc Nephrol 28: ccc–ccc, 2016. doi: 10.1681/ASN.2016070709 CKD is a major burdenworldwide with currently no highly induced in vitro by TGF-b1,1,5,8 and it was also available early prognostic marker or efficient ther- found to be upregulated by angiotensin II in cardiac apeutic treatment. Although various stimuli can fibroblasts.8 IL-4 and IL-13 increased periostin in lung lead to CKD, there are common mechanisms char- fibroblasts and bronchial epithelial cells, associating acterized by chronic inflammation and accumulation its induction with asthma.9 of extracellular matrix within the kidney, resulting in Periostin has been found to be upregulated in decline of renal function and finally, ESRD. renal disease models and kidney biopsy specimens. Periostin is a matricellular protein abundant in periosteum, periodontal ligament, bone, skin, and de- veloping heart.1,2 Periostin interacts with extracellular Received July 1, 2016. Accepted October 31, 2016. matrix components, including collagen I, tenascin-C, Published online ahead of print. Publication date available at and fibronectin, promoting collagen fibrillogenesis www.jasn.org. and increased strength performance of the extracellu- Correspondence: Dr. Christos Chatziantoniou, Institut National 3–5 lar matrix. Interaction of periostin with cell-surface de la Santé et de la Recherche Médicale, Unité Mixte de Re- integrins avb3andavb5 has been associated with in- cherche Scientifique 1155, Tenon Hospital, 4 rue de la Chine, creased adhesion and migration of cancer cells and 75020 Paris, France. Email: [email protected] vascular smooth muscle cells (SMCs).6,7 Periostin is Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2016 ISSN : 1046-6673/2805-ccc 1 BASIC RESEARCH www.jasn.org It was found highly overexpressed in experimental polycystic transfection with the transcription factor plasmids, confirm- kidney disease promoting cyst growth and fibrosis.10 Periostin ing the results of the luciferase assay (Figure 1E). tissue expression and urinary levels were inversely correlated We performed chromatin immunoprecipitation (ChIP) ex- with renal function in patients with lupus nephritis and type 2 periments to verify the in vivo binding of these transcription diabetes, respectively.11,12 Periostin mRNA expression was factors on periostin promoter in a chronic renal disease highly upregulated in both glomeruli and tubulointerstitium model, the NTS-induced GN. Indeed, p65 was highly enriched in patients with different nephropathies.13 We previously re- on its respective binding sites on periostin promoter after NTS ported that periostin expression is highly correlated with dis- administration (Figure 1F). p-c-Jun and p-Stat1 were also en- ease progression in a model of hypertensive nephropathy, and riched on several of their predicted binding sites on periostin we subsequently showed that mice lacking periostin are pro- promoter in the NTS group (Figure 1, G and H). On the con- tected against the structural alterations induced by unilateral trary, p-Smad3 was not enriched on periostin promoter after ureteral obstruction.14,15 NTS, in agreement with its low efficiency to induce periostin Despite these findings, the mechanisms of induction and promoter activity or mRNA expression (Figure 1I). function of periostin in renal disease are largely unknown. In this study we investigated these mechanisms in an established Genetic Deletion of Periostin Preserves Renal chronic renal disease model induced by nephrotoxic serum Structure and Function in the NTS Model (NTS) administration. We found that periostin is induced We used periostin null mice in the C57BL/6 background and by key proinflammatory transcription factors, with the major confirmed whether periostin expressionwas induced after NTS factor being NFkB, and showed that periostin colocalizes with administration. Transcriptional and protein analysis showed integrin-b3 at sites of renal damage driving activation of increased renal expression of periostin in wild-type NTS mice p-FAK– and p-AKT–dependent pathways. Finally, using a compared with control littermates treated with PBS, whereas pharmacogenetic approach of in vivo antisense administration knockout (KO) mice did not display any periostin expression after severe proteinuria and the disease onset, we showed that (Figure 2, A and B). Next, we analyzed functional and struc- inhibition of periostin can be used as a therapeutic strategy to tural alterations indicative of disease progression. The increase alleviate renal disease progression. To our knowledge, we de- in weight caused by edema and the severe increase of protein- scribe for the first time how periostin is induced to mediate the uria were markedly attenuated in KO mice, showing that lack progression of renal disease and underline periostin targeting of periostin resulted in preservation of kidney function (Fig- as therapeutic treatment against CKD. ure 2, C and D). Histologic examination of kidney sections stained with Masson trichrome showed that mice lacking per- iostin developed less fibrotic lesions and had highly preserved RESULTS kidney structure together with decreased number of injured glomeruli (Figure 2, E and F). Periostin Is Induced by Proinflammatory Transcription We next examined the expression of known proinflamma- Factors in the Model of NTS-Induced GN tory mediators and fibrotic molecules, as well as the inflam- To find transcription factors that may induce periostin expres- matory cell infiltration profile of the diseased kidneys. Both sion, we performed bioinformatics analysis of a 2-kb region of collagens I and III and profibrotic TGF-b1 were upregulated in periostin promoter using the Genomatix software. We focused wild-type NTS mice, but their expression was highly attenu- on known proinflammatory and profibrotic transcription fac- ated in KO mice (Figure 2, G–I). In parallel, the induction of tors and mapped their predicted binding sites on the periostin proinflammatory mediators, such as MCP-1, Rantes, and promoter sequence (Figure 1A). VCAM-1, in wild-type mice after NTS was completely blunted To examine the efficiency of these transcription factors to in mice lacking periostin (Figure 2, J–L). Infiltration of mac- affect periostin expression, we performed luciferase assays rophages and lymphocytes was markedly reduced in KO NTS using 2 and 1 kb constructs of human or mouse periostin mice (Figure 2, M and N), establishing a close connection promoter, together with expression plasmids of these tran- between periostin and inflammation probably leading to de- scription factors. The large subunit of NFkB, p65, highly velopment of fibrosis and renal damage. induced the activity of both human periostin promoter con- structs and, even more impressively, that of mouse promoter Delayed Inhibition of Periostin Prevents the constructs (Figure 1B). Other proinflammatory transcription Development of Renal Disease in the NTS Model factors (c-Jun, Stat1a, and Stat6) induced a two-fold increase To assess whether inhibition of periostin can be used as ther- in the activity of both human and mouse constructs (Figure apeutic approach in renal disease, we inhibited periostin 1C). In contrast, from the group of profibrotic transcription expression via constant