CXC Chemokines and Their Receptors in Early Metanephric Development

BASIC RESEARCH www.jasn.org ELR؉-CXC Chemokines and Their Receptors in Early Metanephric Development Zoia B. Levashova,* Nirmala Sharma,* Olga A. Timofeeva,* Jeffrey S. Dome,† and Alan O. Perantoni* *Laboratory of Comparative Carcinogenesis, National Cancer Institute, National Institutes of Health, Frederick, Maryland; and †Division of Oncology, Children’s National Medical Center, Washington, DC ABSTRACT Although originally identified as mediators of inflammation, it is now apparent that chemokines play a ϩ fundamental role in tissue development. In this study, ELR -CXC chemokine family members CXCL2 and CXCL7, along with their preferred receptor CXCR2, were expressed at the earliest stages of metanephric development in the rat, and signaling through this receptor was required for the survival and maintenance of the undifferentiated metanephric mesenchyme (MM). A specific antagonist of the CXCR2 receptor SB225002 induced apoptosis in this population but did not affect more mature structures or cells in the ureteric bud. CXCL7 treatment of isolated MM elicited an angiogenic response by upregulation of matrix metalloprotease 9 and endothelial and mesangial markers (platelet-endothelial cell adhesion molecule, Megsin, Thy-1, PDGF receptor ␣, and vascular ␣-actin) and induced SB225002- sensitive cell invasion through a matrix. Because Wilms’ tumor cells may similarly depend on CXCR2 signaling for survival, primary tumor samples were analyzed, and 15 of 16 Wilms’ tumors were found to be CXCR2 positive, whereas grossly normal kidney tissues from tumor patients or renal cell carcinomas were CXCR2 negative. Furthermore, cell lines derived from Wilms’ tumors but not those from renal cell carcinomas were sensitive to SB225002-induced apoptosis. These data provide evidence for a prosur- ϩ vival and proangiogenic role of ELR -CXC chemokines and their receptor CXCR2 during metanephric development and suggest a novel mechanism for chemotherapeutic intervention in Wilms’ tumor. J Am Soc Nephrol 18: 2359–2370, 2007. doi: 10.1681/ASN.2006040380 Metanephric development requires mutual interac- presence of an N-terminal tripeptide motif gluta- tions between the ureteric bud (UB) and the meta- mate-leucine-arginine (ELR) adjacent to the CXC nephric mesenchyme (MM). MM induces growth motif. All ELRϩ-CXC chemokines act through and branching of the UB, whereas survival and dif- CXC chemokine receptor type 1 or type 2 (CXCR1 ferentiation of the MM into nephronic epithelia de- and CXCR2), which are rhodopsin-like seven- pends on factors secreted by the UB.1 Nephron-in- transmembrane G-protein–coupled receptors. ducing UB-secreted factors have been identified CXCR1 exhibits a high affinity for CXCL8 (IL-8) and include leukemia inhibitory factor and TGF- but a 10- to 100-fold lower affinity for CXCL6 ␤2.2,3 In a search for new UB-secreted inductive (GCP-2), CXCL7 (NAP-2), or CXCL1 (GRO-␣/ molecules, we applied microarray technology to a MGSA-␣).6,7 Rat species possess two homologous rat UB-derived cell line4 and implicated chemo- receptors, CXCR1 and CXCR2, and four ELRϩ- kines as novel participants in kidney development. Chemokines belong to one of four families of Received April 20, 2006. Accepted May 9, 2007. secreted polypeptides, initially identified for their Published online ahead of print. Publication date available at ability to induce migration of leukocytes.5 The CXC www.jasn.org. family is defined by four conserved cysteine resi- Correspondence: Dr. Alan O. Perantoni, NCI-Frederick, Building dues; the first two are separated by one noncon- 538, Room 205E, Frederick, MD 21702-1201. Phone: 301-846- served residue (hence the CXC designation). This 6529; Fax: 301-846-5946; E-mail: [email protected] group can be further subdivided on the basis of the Copyright © 2007 by the American Society of Nephrology J Am Soc Nephrol 18: 2359–2370, 2007 ISSN : 1046-6673/1808-2359 2359 BASIC RESEARCH www.jasn.org CXC chemokines (CXCL1, CXCL2, CXCL5, and CXCL7). mokine expression in both the UB and cortical MM (Figure 1E, However, they lack an equivalent for CXCL8, the key human purple staining). These studies demonstrate that ELRϩ-CXC ϩ ELR -CXC chemokine. family members are expressed at the earliest stages of meta- Besides their “classical” function of providing migrational nephric development in both inductor UB and nephron pro- ϩ signals for leukocytes in adults, ELR -CXC chemokines progenitor MM, and they persist throughout renal development. mote angiogenesis, cell proliferation, and survival during de- To assess renal cell competence to respond to ELRϩ chemo- velopment.8–10 CXCL8 stimulated cell migration, prolifera- kines, we evaluated kidney tissues at various stages of develop- tion, and differentiation in the developing intestine and central ment by immunoblotting for CXCR1 and 2. By RT-PCR and nervous system11,12 and enhanced endothelial cell survival and immunoblotting, both receptors were expressed in rat kidney proliferation and the production of matrix metalloproteinases from 13 dpc through birth but not in adult kidney (Figure 1, C for matrix reconstruction and angiogenesis.10 and D). The data indicate that both UB and MM progenitor Because they are expressed by UB cells, we hypothesized ϩ populations express Cxcr1 and 2, and this is supported by the that ELR -CXC chemokines may also function in metaneph- observation that CXCR2 is detectable by immunoblotting and ric differentiation, cell proliferation, survival, angiogenesis, or RT-PCR (data not shown) in the RUB1 and RIMM-18 cell migration. In this study, we demonstrate that these chemo- lines. Immunohistochemistry with anti-CXCR2 antibody re- kines are expressed in the metanephros and that they promote vealed a prominent staining in UB, cortical mesenchyme, and survival, angiogenesis, and cell migration. Furthermore, we re- newly formed epithelia such as S-shaped bodies (Figure 1F). port that Wilms’ tumors express CXCR2, suggesting that tu- These findings indicate that this receptor is widely expressed in morigenesis may depend in part on these factors. metanephric progenitors during development and suggest that both UB and MM progenitors are capable of responding to ELRϩ chemokines. RESULTS CXC Chemokines Do Not Induce Differentiation in MM Expression of CXC Chemokines and Their Receptors in Because CXC chemokines have been implicated in a number of Rat Embryonic Kidney biologic processes relevant to embryogenesis (progenitor cell Because metanephric inductive factors have heretofore been differentiation, cell survival and proliferation, cell migration/ identified primarily through tedious protein purification invasion, and angiogenesis), we assessed their effects on these methods, we sought to elucidate the majority of factors various processes. For this, we used an explant culture system through genomic analysis. We applied Affymetrix Microarray of isolated uninduced MM from 13-dpc rat kidneys or intact Gene Chip technology (Affymetrix, Santa Clara, CA) to our metanephroi of the same age. Culture conditions require the cell lines RUB1 and, for comparative purposes, RIMM-18. addition of fibroblast growth factor 2 (FGF2) and TGF-␣, From these studies, we found that besides known UB cell known survival factors for MM13,14 and endothelial cells.15,16 markers (e.g., Claudin 3, Claudin 9, Bmp3, Bmp7, c-Met, Cyto- keratins 18 and 19 [data not shown]), RUB1 cells expressed To minimize their inductive and angiogenic effects, we sought members of the ELRϩ group of chemokines, namely, Cxcl1, to limit levels of these factors in the culture medium. We found ␣ Cxcl2, and Cxcl5. Expression of these chemokines in unin- that 30 ng/ml FGF2 and 20 ng/ml TGF- maintained the sur- duced RIMM-18 cells was negligible in comparison with RUB1 vival of explanted MM but did not induce morphologic cells, and both lines failed to express ELRϪ chemokines Cxcl4 changes in the explants. However, such concentrations slightly and Cxcl10 (Figure 1A). induced expression of Cxcl1 and Cxcl2 (Figure 2, second col- Because immortalized RUB1 cell expression profiles may umn versus first), so tissues were unavoidably exposed to these differ from those of primary tissues, we also evaluated expres- chemokines as a result of endogenous production when culti- sion in freshly isolated 13-d postcoitus (dpc) UB or MM, 16- or vated ex vivo. Conversely, Cxcl7 was downregulated in cultured 19-dpc metanephroi, and adult kidneys from rats by reverse MM under these conditions. In efforts to control for endoge- transcriptase–PCR (RT-PCR) for all members of the ELRϩ- nous exposure, experiments included both uncultured and ex- CXC subfamily and their common receptors Cxcr1 and Cxcr2. plant cultured MM and the specific CXCR2 inhibitor RNA from RUB1 or RIMM-18 cells was included to confirm SB22500217 in some studies. ϩ microarray findings. At 13 dpc, both MM and UB expressed For testing whether ELR -CXC chemokines function in tu- Cxcl2, Cxcl5, and Cxcl7 (Figure 1B), although levels by semi- bular development, explanted MM was treated with CXCL7 quantitative RT-PCR were higher for all of these in the MM. and examined on subsequent days for markers of tubular dif- Whereas the RIMM-18 cell line expressed Cxcl7 like its MM ferentiation, sFrp2, Lim1, and E-cadherin. Whereas treatment progenitor, the RUB1 cell line differed significantly from the of control cultures of MM with conditioned medium from UB progenitor cells in that Cxcl1 (and not Cxcl7) was highly RUB1 cells induced expression of these markers (Figure 3A) expressed, suggesting

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