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ELR؉-CXC 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 family members CXCL2 and CXCL7, along with their preferred receptor CXCR2, were expressed at the earliest stages of metaneph- ric 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 (-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 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

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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 pro- genitor 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 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 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 an adaptive change with culturing. The and tubule formation, as previously demonstrated,1 CXCL7 expression of CXCL7 in MM was confirmed by Western blot- treatment failed to induce expression of markers (Figure 3) or ting (Figure 1C), and in situ hybridization demonstrated che- tubular morphogenesis in MM even after 12 d in culture, sug-

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Figure 1. Expression of CXCR1 and CXCR2 receptors and their ligands in rat metanephric progenitors, metanephroi, and renal cell lines. (A) Affymetrix data for GeneChip probes D11445 (rat gene Gro-␣/CXCL1), U45965 (MIP-2/Gro-␤/CXCL2), U90448 (LIX/CXCL5), rc_AI169104 (PF4/CXCL4), and U17035 (IP10/mob-1/CXCL10). RNA was purified from the RIMM-18 or the RUB1 cells and processed as described in the Concise Methods section. (B and D) Semiquantitative reverse transcriptase–PCR (RT-PCR). (C) Western blot analysis of protein extracts from 16-d postcoitus (dpc), 19-dpc, newborn, adult kidney, and 13-dpc metanephric mesenchyme (MM) with CXCR1 (sc-988, Santa Cruz), CXCR2 (sc-683, Santa Cruz), or CXCL7 antibody (AF1116; R&D Systems, Minneapolis, MN). (E) In situ hybridization of 16-dpc rat kidney probed with a CXCL7 antisense riboprobe. (F) Immunohistochemical staining of 16-dpc rat kidney for CXCR2. Bar ϭ 100 ␮m. gesting that ELRϩ-CXC chemokines do not function in tubule for 9 d with a wide range of chemokine concentrations (50 to development. 2000 ng/ml; data not shown). Inhibition of CXCR2 activity using the selective antagonistic compound SB225002 caused .ELR؉-CXC Chemokines Function in Cell Survival massive cell death of both primary MM and RIMM-18 cells ELRϩ-CXC chemokines can enhance cell survival and prolif- Administration of SB225002 to 13-dpc metanephroi impaired eration of some cultured cell types (e.g., human umbilical vein renal morphogenesis in a concentration-dependent manner. endothelial cells, tumor cell lines). Using a 3-(4,5-dimethyl- Both UB branching and MM tubulogenesis were greatly af- thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell fected (Figure 4), but administration of SB225002 to embry- viability test, we assessed the growth effects of CXCL2 or onic kidney at later stages of development (14- or 16-dpc kid- CXCL7 on RIMM-18 cells or primary MM explants. However, neys) was significantly less inhibitory as shown by staining with we observed no growth advantage for cells that were incubated TO-PRO-1 reagent (Figure 5). This staining for nonviable cells

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Figure 4. Blocking of CXCR2 with a selective antagonist SB225002 impairs kidney development. SB225002 (Calbiochem) was applied at various concentrations to 13-dpc embryonic kid- neys 1 d after explantation to collagen-coated filters as described in the Concise Methods section. After 24 h in SB225002, the Figure 2. Cultivation of primary MM changes chemokine expres- tissues were fixed in methanol and immunostained with WT1 sion profiles. Semiquantitative RT-PCR of RNA purified from un- antibody (green) and Dolichos biflorus agglutinin (DBA; red). cultivated MM (none) and from MM incubated for 24 h on colla- Magnification, ϫ100. gen-coated filters in the presence of 30 ng/ml basic fibroblast growth factor (bFGF) and 20 ng/ml TGF-␣ (FTa) or the same factors plus conditioned medium from the RUB1 cells (CFTa). and mesangial cell]), migration of endothelial cells, and tube formation and fusion with other blood vessels. revealed that undifferentiated MM in the cortical nephrogenic To evaluate directly the response of MM to chemokine sig- zone is more sensitive to SB225002 treatment. For 13-dpc kid- naling and determine genomic profiles after stimulation, we ney, it involved almost the entire metanephros except the cen- screened Affymetrix GeneChips for expression of that tral area where the UB is located (Figure 5B). At 16 dpc, only were upregulated in explanted MM by treatment with CXCL7. the cortical nephrogenic zone of kidney was affected (Figure Such analyses revealed genes that were associated with survival 5F). At higher magnification, nephron formation was observed and with angiogenesis, including endothelial and mesangial despite SB225002 treatment, although there are many fewer markers as well as metalloprotease Mmp9 (Table 1, Figure 7A), condensates (as visualized by WT1 staining) in the periphery of which functions in invasive growth, kidney organogenesis, and the metanephros (Figure 6, A and B). UB branching was also vasculogenesis.18,19 The upregulated genes included markers of inhibited in the periphery (Figure 6, D and F versus C and E), early vascular development: Vegf, Fgfr1, Fgfr2, Tgf␤1 and its but staining with TO-PRO-1 does not indicate that cells in the receptor Tgf␤RII, and urokinase-type . UB are dying in greater numbers than in untreated cultures Results were confirmed by semiquantitative RT-PCR as were (Figures 5, E and F, and 6, G and H), suggesting that it is a other markers of mesangial and endothelial cells, platelet-en- secondary effect as a result of loss of the MM. dothelial cell adhesion molecule (Pecam-1), Thy-1, and Megsin (Figure 7, B and C). Cultivation of MM for1dinthepresence ELR؉-CXC Chemokines Function in Angiogenesis of FGF2 and TGF-␣ resulted in upregulation of these markers Another established property of ELRϩ-CXC chemokines is in comparison with uncultivated MM, but addition of CXCL7 stimulation of angiogenesis. This process implies proteolytic induced much higher levels of expression. In addition, eleva- degradation of the basement membrane, proliferation of the tion of PECAM-1 in CXCL7-treated MM explants was con- two major cell types that populate the microvasculature (en- firmed by Western analysis (Figure 7D). dothelial and vascular smooth muscle cells [i.e., the pericyte For assessment of a possible role of chemokine signaling in

Figure 3. CXCL7 does not induce epithelial markers in culture in explanted MM. (A) Semiquantitative RT-PCR of RNA purified from MM induced by conditioned medium (CM; 20 ␮l/ml) or CXCL7 (200 ng/ml) for different time points. (B) RT-PCR analysis of expression of nephron epithelial marker sFrp2 in MM induced with CM or CXCL7. Values are relative units determined by the LabWork software (mean Ϯ SD) based on triplicate samples.

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Figure 5. The toxic effect of SB225002 depends on the stage of kidney development. Metanephroi at ages 13 (A and B), 14 (C and D), or 16 dpc (E and F) were cultured for 20 h and then treated (B, D, and F) or not (A, C, and E) with 1.1 ␮M SB225002. After 24 h of incubation, explants were stained with 2% TO-PRO-1 reagent (Molecular Probes) for 30 min at 37°C. Magnification, ϫ40.

Figure 6. Inhibition of CXCR2 signaling with SB225002 primarily cell migration/invasion, isolated MM and RIMM-18 cells were affects the cortical nephrogenic zone of cultured metanephroi. (B, treated with CXCL7 and evaluated using Matrigel invasion D, F, and H) One day of treatment with 1.1 ␮M SB225002. (A, C, chambers. In these experiments, addition of CXCL7 signifi- E, and G) Untreated explants. (A and B) Immunostaining with WT1 cantly increased the invasiveness of both MM and RIMM-18 antibody (arrows show WT1-positive structures: condensed mes- cells through a layer of Matrigel (Figure 8). Typically, we ob- enchyme and nephron epithelia). (C and D) DBA staining of served a 15 to 35% increase in invasion of MM (Figure 8B). To ureteric bud (UB). (E and F) Skeletonized images of DBA-stained confirm that this activity was dependent on signaling through UB. (G and H) TO-PRO-1 staining of nonviable cells (brackets CXCR2, we treated cultures with SB225002. In these studies, denote peripheral blastemal area). Magnification, ϫ100. this CXCR2-specific inhibitor dramatically decreased MM and RIMM-18 cell invasiveness through a Matrigel layer without nephric development, we speculated that they would ex- affecting cell migration through a control membrane, suggest- press CXCR and respond to the CXCR2 inhibitor SB225002. ing that the inhibitor was applied at nontoxic levels. These To evaluate the potential cell-selective toxicity of SB225002, studies demonstrate that CXCL7 can elicit an angiogenic re- we compared its effect on RUB1 and RIMM-18 cells as well sponse in embryonic kidney by upregulation of Mmp9 and as on two Wilms’ tumor cell lines, SK-NEP-1 and WiT49, endothelial and mesangial markers and further suggest that and three renal cell carcinoma lines, CRL, Caki-1, and renal progenitors may use CXCR2 signaling for tissue invasion. Caki-2. All analyzed cell lines showed expression of the CXCR2 receptor, but SB225002 induced apoptosis only in Chemokine Signaling in Renal Tumors blastemal/mesenchymal cell lines (RIMM-18, SK-NEP-1, Chemokine signaling has been reported to play a role in the and WiT49 lines) and not RUB1 cells, as demonstrated by pathogenesis of a variety of tumors, and CXCR1 or 2 specif- caspase-3 activity or poly(ADP-ribose) polymerase (PARP) ically has been implicated in the growth or metastasis of cleavage (Figure 9). Administration of a high (2 ␮g/ml) con- melanomas and lung and colon tumor cells.20–22 Because centration of CXCL7 to SB225002-treated cells to activate Wilms’ tumors originate from MM and caricature meta- CXCR1 did not rescue the cells from apoptosis (data not

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Table 1. Selected genes that showed up- or downregulation of expression after1dof stimulation of MM with CXCL7 compared with cultivated MMa Fold GeneChip Number Gene Name Difference Upregulated genes U77697_at PECAM 1.4 X06801_cds_i_ Vascular ␣-actin (VAA) 2.0 Z14118cds_g_at Platelet-derived growth factor receptor ␣ (PDGFR␣) 6.4 U24441_at Matrix metalloprotease 9 (MMP9) 3.7 X63434_at Urokinase-type plasminogen activator 1.8 M32167_g_at VEGF-A 4.2 rc_AI231472_s_at Collagen type 1 ␣ 1 (Col1A1) 2.4 X52498cds_at TGF-␤1 1.7 L09653_s_at TGF-␤R2 3.5 D12498_s_at FGFR1 2.7 L19112_at FGFR2 4.7 AF051895_at Annexin V 1.9 U49930_at Caspase 3 12.1 Downregulated genes rc_AI070295_g_at Growth arrest and DNA-damage-inducible 45 ␣ (GADD45) 0.4 rc_AA944 M60921 B-cell translocation gene (Btg2) 0.7 rc_AA891591_at Programmed cell death 8 (Pdcd8) 0.6 U89653_at Breast cancer susceptibility gene (Brca2) 0.7 aFGFR, fibroblast growth factor receptor; MM, metanephric mesenchyme; PECAM, platelet-endothelial cell adhesion molecule. shown). We also evaluated primary tumors for CXCR2 and This report describes the first evidence of possible functions found expression in all Wilms’ tumors but not in renal cell for ELRϩ-CXC chemokines in metanephric development. We carcinomas (Figure 10). These data support a role for found by RT-PCR and Western blot analysis that in rat em- CXCR2 in maintaining survival of normal mesenchymal bryos, both CXCR2 and CXCR1 are expressed in MM and the progenitors and Wilms’ tumors but not epithelial progeni- UB. Western blot analysis of both receptors showed one major tors or renal carcinomas. band for each, which corresponds in size to reported forms.32–34 Receptor expression was slightly downregulated at birth (more obvious for CXCR1) and subsequently lost from DISCUSSION adult kidneys. These findings are consistent with CXCR1 ex- Chemokines have been described as chemotactic agents and pression patterns obtained by screening rat kidney at different pre- and postnatal stages using microarray.35 activators of leukocytes during physiologic and inflammatory ϩ processes23; however, more recent investigations have revealed All ELR -CXC chemokines bind CXCR2 with high affinity. a nonhematopoietic role for these factors, particularly for the Whereas CXCL8 exhibits a high affinity for CXCR1 as well, ELRϩ-CXC chemotactic , which promote mitosis, CXCL1, CXCL6, and CXCL7 show approximately a 100-fold modulate apoptosis, enhance cell survival, and stimulate an- lower affinity for this receptor.7,36 In agreement with this, two giogenesis. Accordingly, their receptors, CXCR1 and CXCR2, concentration optima were observed in the effects of CXCL1 originally shown to be expressed in leukocytes,24 now have and CXCL7 on neutrophil chemotaxis.6 Because rodents lack a been reported on nonhematopoietic cells (e.g., keratinocytes, CXCL8 equivalent, signaling through CXCR1 depends on neurons, neuroendocrine, and oligodendroglial cells).25–29 other CXC chemokines. In these studies, we show that CXCL2 More recently, receptors have been described in a variety of and CXCL7 are expressed in rat metanephric tissues, including embryonic populations. CXCR2 is widely distributed in hu- 13-dpc UB and MM. Adult human renal cells can express man tissues including brain (undifferentiated neurons), heart CXCL8, CXCL1, or CXCL7 with proinflammatory stimuli.23 (myocardiocytes), lung (bronchial epithelial cells), liver (hepa- Here, we demonstrate that, under conditions of normal ϩ tocytes), and kidney (early glomeruli and collecting duct).30 In growth and development, ELR -CXC chemokines are ex- mouse embryos, CXCR2 expression was detected in brain, car- pressed and presumably function in organogenesis. Because diovascular system, and condensing cartilage.31 The temporal MM is avascular up to 14 dpc, CXC chemokines that are syn- pattern of receptor expression and the wide embryonic tissue thesized directly by renal progenitors may be the only renal distribution argue for a role in organogenesis and the kidney in source of CXCR ligands. Of course, we cannot rule out the particular, yet the role(s) of ELRϩ-CXC chemokines and their possibility that some noted effects resulted from changes in the receptors in development remains unclear. microenvironment as a result of culturing. For example,

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from more than one tissue source, there is sufficient evidence to indicate that they originate at least in part from the endoge- nous blastemal population.37 A comparison of profiles of uninduced MM that were treated with CXCL7 revealed a group of genes related to angiogenesis. We detected elevated levels of endothelial and mesangial markers (Pecam-1 and Megsin) and other angiogenesis-related genes (Mmp9 and Vegf). We did not apply hy- poxic conditions for MM cultivation, which are normal for embryonic development and a stimulus to angiogenesis. In cultured MM, the hypoxia-induced angiogenic stimulator Vegf38 was significantly downregulated rela- tive to primary tissue isolates (data not shown), so it is possible that exposure of ex- plants to hypoxia could further enhance the effect of CXCL7 on these markers, including Vegf. In these studies, the induction of some markers (e.g., Pecam-1, Vaa, Pdgfr␣) was greatest using very high concentrations of CXCL7 (2.0 ␮g/ml). This may reflect a con- tribution of CXCR1, which has a lower af- finity for CXCL7 and, unlike CXCR2, is rap- idly re-expressed upon internalization.39 Lower concentrations of CXCL7 still in- duced these markers but may be less effec- Figure 7. CXCL7 upregulates mesangial and endothelial markers and matrix metal- tive as a result of the upregulation of Mmp9, loprotease 9 (MMP9) expression. (A) Affymetrix data for GeneChip probes U77697_at which inactivates the chemokine.40 Con- (platelet-endothelial cell adhesion molecule 1 [PECAM-1]), X06801cds_i (VAA), versely, MMP9 may contribute to angiogen- Z14118cds_g_at (PDGFR␣), and U24441_at (MMP9). (B) RT-PCR of RNA purified from esis by degrading extracellular matrix and MM incubated for 24 h with 30 ng/ml bFGF and 20 ng/ml TGF-␣, with or without facilitating the sprouting of growing blood CXCL7. (C) Densitometry of PCR gels. Values are relative units determined by the vessels.19 The findings on metalloprotease LabWork software. Data shown are the typical results of one of the four independent upregulation obtained by Affymetrix and experiments. In each set of experiments, four or five MM plated on a filter were used RT-PCR assays were supported by migra- as one experimental point for in vitro stimulation and consequent RNA preparation, RT, and PCR amplifications. As uninduced material, 15 to 45 freshly prepared MM tion/invasion studies. We detected a mea- were used. (D and E) PECAM-1 expression in MM induced with 0.2 ␮g/ml CXCL7 for surable basal rate of invasion by cells from 48 h. Immunostaining of MM plated on collagen-coated filters (two typical MM of 48 the MM, presumably as a result of the pres- total analyzed in two independent experiments are shown; D). (E) Western blotting. ence of FGF2 and TGF-␣, which are neces- sary to sustain these cells and can induce CXCL1 expression increased in cultured rudiments, whereas metalloprotease activity.41,42 Cultured MM had a six-fold CXCL7 expression was downregulated. The differences, greater expression level for Mmp9 than freshly isolated MM. In though, were quantitative and not qualitative in nature and addition, Matrigel itself contains multiple growth factors, therefore probably reflect either normal compensatory re- which may also facilitate invasiveness. Despite this high basal sponses or possible feedback regulatory relationships among level, we observed a significant increase in invasion of MM family members. The significance of such changes, however, is upon stimulation with CXCL7. The CXCR2 antagonist unclear, because ligand substitutions may not have a signifi- SB225002 inhibited both basal and CXCL7-stimulated inva- cant impact on CXCR signaling. sion of MM at nontoxic concentrations, supporting the role of The angiogenic properties of ELRϩ-CXC chemokines are the receptor in cell motility/invasion. RIMM-18 cells gave sim- well documented, so it is reasonable to hypothesize such a role ilar results, except cells were less dependent on added growth in the metanephros. Although the origin of endothelial ele- factors, presumably as a result of E1A immortalization. ments in the metanephros remains controversial and may arise Genomic profiling of CXCL7-treated MM also revealed a

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Figure 8. SB225002 (0.13 ␮M) inhibits CXCL7- stimulated invasion through a Matrigel layer. (A) The migration-invasion data for the RIMM-18 cells represent the number of cells per field of view (mean Ϯ SD, n ϭ 4). (B) The migration-invasion data for the MM cells represent the mean gray values (mean Ϯ SD, n ϭ 3). Images of Giemsa-stained membranes were converted to gray mode, then color inverted and evaluated using an ImageJ program. (C) Giemsa-stained membranes with primary MM. group of apoptosis- and growth-related genes. CXCL7-medi- genes in MM results in renal agenesis,43 so their expression is ated downregulation of antiproliferative (Gadd45, Btg2), ap- essential to MM survival. However, proapoptotic genes (An- optosis-inducing (Pdcd8), and tumor suppressor (Brca2) nexin V and Caspase 3) were also upregulated with CXCL7 genes and upregulation of Fgfr1 and Fgfr2 genes argue for a administration. This may explain why an MTT test showed no prosurvival role of CXCL7. In fact, targeted loss of both Fgfr growth advantage for CXCL7-treated MM. More probable, any proapoptotic effects may be offset by the observed upregu- lation of CXCL1 and CXCL2 in control MM cultures, which may be sufficient to maintain the survival of explants indepen- dent of CXCL7 in culture medium, but this will require further investigation. It seems, however, that CXCR2 is critical for survival of undifferentiated MM, because SB225002 caused its selective death. MM cells from metanephroi or established mesenchy- mal lines, including two Wilms’ tumors, were sensitive to SB225002-induced apoptosis, unlike renal epithelial (UB) cells, suggesting that blastemal cells require signaling through CXCR2. It is known that phosphatidylinositol-3 kinase/Akt and extracellular signal–regulated kinase prosurvival signaling cascades can be triggered via CXCR2.44,45 Furthermore, neu-

Figure 9. SB225002 induces apoptosis selectively in mesenchy- mal/blastemal/Wilms’ tumor cells but not in epithelial/carcinoma cells. (A) Image of RUB1 and RIMM-18 cells cultivated in serum- free conditions with or without 1.1 ␮M SB225002 for 24 h. (B) Caspase-3 activity is upregulated after 24 h of treatment with 1.1 ␮M SB225002 in the RIMM-18 but not in the RUB1 cells. (C) Western blot analysis of cell lines of renal progenitors (RUB1 and RIMM-18), Figure 10. CXCR2 receptor is expressed in Wilms’ tumors but not renal cell carcinoma (CRL, Caki-1, and Caki-2), and Wilms’ tumors in renal cell carcinomas (RCC) or most normal counterparts. Kid- (SK-NEP-1 and WiT49) with CXCR2 rabbit polyclonal, a poly(ADP- ney tumor samples and normal adjacent tissue were obtained ribose) polymerase (PARP) rabbit polyclonal ( Technol- from the Children’s Oncology Group Renal Tumor Biologic Sam- ogy, Beverly, MA), or a glyceraldehyde-3-phosphate dehydroge- ples Bank via the Cooperative Human Tissue Network. Eight of 16 nase (GAPDH) mouse mAb (Ambion, Austin, TX). analyzed Wilms’ tumor samples are shown.

2366 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2359–2370, 2007 www.jasn.org BASIC RESEARCH tralization of CXCR2 signaling was shown to modulate anti- Herman Yeger (Hospital for Sick Children, Toronto, Canada). Other and proapoptotic proteins in endothelial cells and negatively tumor lines were obtained from the ATCC (Rockville, MD). affect their survival.46 However, identification of the specific mechanism(s) responsible for caspase 3–dependent, Isolation and Cultivation of Rat Embryonic Kidneys SB225002-initiated apoptosis in MM cells is to be the subject of and MM future study. Impairment of UB branching after SB225002 ad- Embryonic kidneys were excised from F344 rat embryos. MM were ministration seems to be a secondary event, because UB cells enzymatically separated from embryonic day 13.5 UB. Metanephroi (both primary and from the RUB1 cell line) did not show signs or MM were cultured on polycarbonate filters (Whatman-Nuclepore, of apoptosis or death. In all probability, the loss of branching Florham Park, NJ) coated with type IV collagen (BD Bioscience, Bed- may be due to the loss of MM-secreted branch-inducing fac- ford, MA) as described previously.1 tors, such as glial cell line–derived neutrophic factor (GDNF).47 Participation of CXCR2 in the survival of blastemal Affymetrix GeneChip Analysis cells in kidney may be critical, because levels of CXCL7 that RNA was extracted from cells using TRIzol reagent (Invitrogen, Carls- could activate the CXCR1 receptor did not rescue the cells bad, CA). Total RNA (5 ␮g) from duplicate samples was converted from SB225002-induced apoptosis. In this regard, it is possible into cDNA and purified by phenol/chloroform extraction. cDNA la- that the induction of angiogenic markers by CXCL7 is indirect beling, hybridization to U34A GeneChips, and scanning were per- and due instead to enhanced survival of angiogenic progeni- formed according to Affymetrix instructions, and data were analyzed tors as a part of blastemal cell population. For nonblastemal by GeneChip software. cells, other signaling pathways may contribute to survivability, even though they express CXCR2. ϩ Semiquantitative RT-PCR Analysis Although ELR -CXC chemokines have been implicated in RT reactions and PCR amplifications were performed using total the control of cell differentiation,48,49 we did not detect mor- RNA (0.2 or 1 ␮g) as described previously.54 Primer sequences, an- phologic changes that are characteristic of mesenchymal-to- nealing temperatures, and numbers of cycles are shown in Table 2. No epithelial conversion of MM or even CXCL7-induced expres- RT controls were included to eliminate the possibility of DNA con- sion of specific epithelial markers. Although this may simply tamination. All PCR products were sequenced to confirm identities. reflect the inadequacies of cell culture conditions, it may also be attributable to the cooperative nature of inductive signaling Western Blot Analysis as we have demonstrated.3 Therefore, CXCL7 may function in tubulogenesis in combination with other factors. Protein lysates were obtained from rat kidneys at various stages of The results obtained for CXCR2-deficient mice confirm the development (16 dpc, 19 dpc, newborn, or adult) or from isolated angiogenic role of the receptor. Decreased vascular density as 13-dpc MM (20 to 30) and analyzed by Western blotting as described well as marked reduction of tumor growth and its metastatic previously.54 potential were shown in these animals.50,51 CXCR2 knockout mice also have retarded wound healing, another process that Immunocytostaining depends on microvascularization.52 However, there is no re- MM or kidney explants cultivated on polycarbonate filters were ported evidence of kidney involvement in CXCR2 null mice. fixed in methanol, washed, preblocked with 10% sheep serum This may be explained by redundancy in receptor expression (Sigma-Aldrich, St. Louis, MO), and stained for WT1 (rabbit poly- (i.e., CXCR1), which was recently discovered in mice53 and clonal, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA) or PE- which may substitute for some CXCR2 functions. CAM-1 (mouse monoclonal, 1:50; Chemicon Int., Temecula, CA), These data provide evidence of a prosurvival and proangio- using Alexa488-conjugated secondary antibodies (1:100; Molecu- genic role for ELRϩ-CXC chemokines and their receptor lar Probes, Eugene, OR) in 1% sheep serum overnight at 4°C. CXCR2 during metanephric development. Expression of the Filters were washed, stained if needed with 20 ␮g/ml Dolichos CXCR2 receptor in blastemal tumors of the kidney and apop- biflorus agglutinin (Sigma), and mounted using a ProLong Anti- tosis-promoting properties of its selective antagonist may pro- fade Kit (Molecular Probes). vide a novel approach for chemotherapeutic intervention in Wilms’ tumors. In Situ Hybridization and Immunohistochemistry Frozen sections (20 ␮m) of 4% paraformaldehyde-fixed, 30% sucrose permeabilized, and OCT-embedded 16-dpc rat kidneys were probed CONCISE METHODS with a 200-bp digoxigenin-labeled riboprobe according to Tuttle et al.55 using a chromogenic alkaline phosphatase substrate BM Purple Cell Cultures (Roche, Palo Alto, CA). For immunohistochemistry, similarly pre- RUB1 and RIMM-18 cell lines were established from rat UB and un- pared frozen sections were probed with a rabbit polyclonal antibody differentiated MM, respectively, and were characterized previously for CXCR2 (sc-683, Santa Cruz Biotechnology) or nonimmune rabbit and grown as described.4,54 Wilms’ tumor line WiT49 was a gift of Dr. IgG at a 1:100 dilution. Staining was visualized using a Vectastain ABC

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Table 2. PCR primers for amplification reactionsa Accession Product Melting No. of Primer Sequence No. Size Temperature Cycles CXCL1 D11445 U 5Ј-CCA GCC ACA CTC CAA CAG 388 57.0 33 D5Ј-CCC TCA ATA GAA ATC GTA AAA TG CXCL2 XM_346458 U 5Ј-ATC AGG GTA CAG GGG TTG TTG 238 57.0 33 D5Ј-GGT CAG TTA GCC TTG CCT TTG CXCL5 U90488 U 5Ј-CGT CAT TCA CCC TGC TGG CAT 316 57.2 35 D5Ј-GCA AGT GCA TTC CGC TTT GTT TTC CXCL7 AF349115 U 5Ј-ATG GGC TTC AGA CTC AGA CCT AC 209 57.0 33 D5Ј-AAC ACA TTC ACA CGG GAG ATA GAG CXCR1 U71089 U 5Ј-TTG GAA ATA TCA CCC GAA TGC TG 480 59.5 32 D5Ј-AAG ATG GCA AAA GGC AGA GAC CXCR2 D63584 U 5Ј-TTC TGA CCC GCC CTT TAC TCT GT 630 59.8 34 D5Ј-CGC AGT GTG AAC CCA TAG CAG Megsin AF105329 U 5Ј-TGA ATG TGT TTC TCC CCC AGT TC 193 55.4 31 D5Ј-GCC TCG GTG CCT TCT TCT GAG MMP9 U24441 U 5Ј-CCA CCG CCA ACT ATG ACC AG 877 59.8 35 AGC CCC AAC TTA TCC AGA CTC CT PECAM-1 U77697 U 5Ј-AGG CAT CGG CAA AGT GGT CAA GAG 693 59.4 28 GCT GCA ACT ATC AAG GCG GCA ATG PDGFR␣ Z14118 U 5Ј-TCA AAC TCC CGT CCA TCA AAC TG 747 58.8 27 D5Ј-CTC TGT TCC CAA TGC CAA GGT C sFRP-2 U88567 U 5Ј-GCC TCG CTG CTG CTG CTA GTC 538 55.2 34 D5Ј-TGT CGT TGT CGT CCT CAT TCT TG Thy-1 X03150 U 5Ј-ATA ACA CCA ACT TGC CCA TCC AG 371 58.2 30 D5Ј-CCC AAC CAG TCA CAG AGA AAT GAA VAA X06801 U 5Ј-ATG CTC CCA GGG CTG TTT TC 159 57.6 24 D5Ј-TGG TGA TGA TGC CGT GTT CTA TC GAPDH AB017801 U 5Ј-CCA TGC CAT CAC TGC CAC TCA GAA G 372 60.7 22 D5Ј-GCA ATG CCA GCC CCA GCA TCA AAG aGAPDH, glyceraldehyde-3-phosphate dehydrogenase.

kit (Vector Laboratories, Burlingame, CA), and sections were lightly invasion chambers and the culture wells. After 20 h of incubation, non- counterstained with hematoxylin. invading cells were removed with cotton swabs, and membranes were fixed in 100% methanol and stained with Giemsa solution. Measurement of Caspase-3 Activity Using a Fluorometric Substrate ␮ Caspase-3 activity in cells was measured using 20 M caspase-3 flu- ACKNOWLEDGMENTS orometric substrate Ac-DEVD-amc (Upstate Biotech, Lake Placid, ␮ NY) and 1 M caspase-3 inhibitor IV Ac-VEID-CHO (for negative This research was supported in part by the Intramural Research Pro- control; Calbiochem, San Diego, CA), according to manufacturers’ gram of the National Institutes of Health, National Cancer Institute, instructions. Fluorescence was measured at excitation 380 nm and Center for Cancer Research. emission 460 nm using a Luminescence Spectrometer (Perkin-Elmer LS50B, Waltham, MA). Protein contents were quantified with a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL), and fluores- DISCLOSURES cence was normalized to protein content. None. Invasion Assay Invasiveness of RIMM-18 or MM cells was determined using BD Biocoat Matrigel Invasion Chambers (BD Biosciences) according to the manu- REFERENCES facturer’s recommendations. RIMM-18 cells (3 ϫ 104 cells/well) or MM (1 MM/well) were added to the invasion chambers in 500 ␮l of medium 1. Karavanova ID, Dove LF, Resau JH, Perantoni AO: Conditioned me- dium from a rat ureteric bud cell line in combination with bFGF with FGF2 (10 ng/ml); 700 ␮l of the same medium was added to the induces complete differentiation of isolated metanephric mesen- culture wells. CXCL7 (200 ng/ml; Peprotech, Rocky Hill, NJ) was added chyme. Development 122: 4159–4167, 1996 to the culture wells; inhibitors, when applied, were added to both the 2. Barasch J, Yang J, Ware CB, Taga T, Yoshida K, Erdjument-Bromage

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H, Tempst P, Parravicini E, Malach S, Aranoff T, Oliver JA: Mesenchy- lated oncogene (gro) in human colon carcinoma cells with different mal to epithelial conversion in rat metanephros is induced by LIF. Cell metastatic potential and its role in regulating their metastatic pheno- 99: 377–386, 1999 type. Clin Exp Metastasis 21: 571–579, 2004 3. Plisov SY, Yoshino K, Dove LF, Higinbotham KG, Rubin JS, Perantoni 22. Varney ML, Johansson SL, Singh RK: Distinct expression of CXCL8 and AO: TGF beta 2, LIF and FGF2 cooperate to induce nephrogenesis. its receptors CXCR1 and CXCR2 and their association with vessel Development 128: 1045–1057, 2001 density and aggressiveness in malignant melanoma. Am J Clin Pathol 4. Perantoni A, Kan FW, Dove LF, Reed CD: Selective growth in culture 125: 209–216, 2006 of fetal rat renal collecting duct anlagen. Morphologic and biochem- 23. Segerer S, Nelson PJ, Schlondorff D: Chemokines, chemokine recep- ical characterization. Lab Invest 53: 589–596, 1985 tors, and renal disease: From basic science to pathophysiologic and 5. Baggiolini M: Novel aspects of inflammation: -8 and related therapeutic studies. J Am Soc Nephrol 11: 152–176, 2000 chemotactic cytokines. Clin Investig 71: 812–814, 1993 24. Chuntharapai A, Lee J, Hebert CA, Kim KJ: Monoclonal antibodies 6. Ludwig A, Petersen F, Zahn S, Gotze O, Schroder JM, Flad HD, Brandt detect different distribution patterns of IL-8 receptor A and IL-8 re- E: The CXC-chemokine neutrophil-activating peptide-2 induces two ceptor B on human peripheral blood leukocytes. J Immunol 153: distinct optima of neutrophil chemotaxis by differential interaction 5682–5688, 1994 with interleukin-8 receptors CXCR-1 and CXCR-2. Blood 90: 4588– 25. Schulz BS, Michel G, Wagner S, Suss R, Beetz A, Peter RU, Kemeny L, 4597, 1997 Ruzicka T: Increased expression of epidermal IL-8 receptor in psoria- 7. Wuyts A, Proost P, Lenaerts JP, Ben-Baruch A, Van Damme J, Wang sis. Down-regulation by FK-506 in vitro. J Immunol 151: 4399–4406, JM: Differential usage of the CXC chemokine receptors 1 and 2 by 1993 interleukin-8, granulocyte chemotactic protein-2 and epithelial-cell- 26. Kondo S, Yoneta A, Yazawa H, Kamada A, Jimbow K: Downregulation derived neutrophil attractant-78. Eur J Biochem 255: 67–73, 1998 of CXCR-2 but not CXCR-1 expression by human keratinocytes by 8. Belperio JA, Keane MP, Arenberg DA, Addison CL, Ehlert JE, Burdick UVB. J Cell Physiol 182: 366–370, 2000 MD, Strieter RM: CXC chemokines in angiogenesis. J Leukoc Biol 68: 27. Horuk R, Martin AW, Wang Z, Schweitzer L, Gerassimides A, Guo H, Lu 1–8, 2000 Z, Hesselgesser J, Perez HD, Kim J, Parker J, Hadley TJ, Peiper SC: 9. Mockenhaupt M, Peters F, Schwenk-Davoine I, Herouy Y, Schraufstat- Expression of chemokine receptors by subsets of neurons in the ter I, Elsner P, Norgauer J: Evidence of involvement of CXC-chemo- central nervous system. J Immunol 158: 2882–2890, 1997 kines in proliferation of cultivated human melanocytes. Int J Mol Med 28. Tecimer T, Dlott J, Chuntharapai A, Martin AW, Peiper SC: Expression 12: 597–601, 2003 of the chemokine receptor CXCR2 in normal and neoplastic neuroen- 10. Li A, Dubey S, Varney ML, Dave BJ, Singh RK: IL-8 directly enhanced docrine cells. Arch Pathol Lab Med 124: 520–525, 2000 endothelial cell survival, proliferation, and matrix metalloproteinases 29. Nguyen D, Stangel M: Expression of the chemokine receptors CXCR1 production and regulated angiogenesis. J Immunol 170: 3369–3376, and CXCR2 in rat oligodendroglial cells. Brain Res Dev Brain Res 128: 2003 77–81, 2001 11. Maheshwari A, Lu W, Lacson A, Barleycorn AA, Nolan S, Christensen 30. Dame JB, Juul SE: The distribution of receptors for the pro-inflamma- RD, Calhoun DA: Effects of interleukin-8 on the developing human tory cytokines interleukin (IL)-6 and IL-8 in the developing human intestine. 20: 256–267, 2002 fetus. Early Hum Dev 58: 25–39, 2000 12. Ragozzino D: CXC chemokine receptors in the central nervous system: 31. Luan J, Furuta Y, Du J, Richmond A: Developmental expression of two Role in cerebellar neuromodulation and development. J Neurovirol 8: CXC chemokines, MIP-2 and KC, and their receptors. Cytokine 14: 559–572, 2002 253–263, 2001 13. Perantoni AO, Dove L, Karavanova I: Basic fibroblast growth factor can 32. Samanta AK, Oppenheim JJ, Matsushima K: Identification and char- mediate the early inductive events in renal development. Proc Natl acterization of specific receptors for monocyte-derived neutrophil Acad Sci U S A 92: 4696–4700, 1995 chemotactic factor (MDNCF) on human neutrophils. J Exp Med 169: 14. Rogers SA, Ryan G, Hammerman MR: Metanephric transforming 1185–1189, 1989 growth factor-alpha is required for renal organogenesis in vitro. Am J 33. Tikhonov II, Fomin IK, Doroshenko TM, Chalyi IuV, Voitenok NN: Physiol 262: F533–F539, 1992 Expression of cDNA for human interleukin-8 type one receptor in 15. Montesano R, Vassalli JD, Baird A, Guillemin R, Orci L: Basic fibroblast BALB 3T3 fibroblasts and characteristics of products of expression [in growth factor induces angiogenesis in vitro. Proc Natl Acad Sci U S A Russian]. Mol Biol (Mosk) 30: 1014–1021, 1996 83: 7297–7301, 1986 34. Ludwig A, Ehlert JE, Flad HD, Brandt E: Identification of distinct 16. Vinals F, Pouyssegur J: Transforming growth factor beta1 (TGF-beta1) surface-expressed and intracellular CXC-chemokine receptor 2 glyco- promotes endothelial cell survival during in vitro angiogenesis via an forms in neutrophils: N-glycosylation is essential for maintenance of autocrine mechanism implicating TGF-alpha signaling. Mol Cell Biol receptor surface expression. J Immunol 165: 1044–1052, 2000 21: 7218–7230, 2001 35. Stuart RO, Bush KT, Nigam SK: Changes in gene expression patterns 17. White JR, Lee JM, Young PR, Hertzberg RP, Jurewicz AJ, Chaikin MA, in the ureteric bud and metanephric mesenchyme in models of kidney Widdowson K, Foley JJ, Martin LD, Griswold DE, Sarau HM: Identifi- development. Kidney Int 64: 1997–2008, 2003 cation of a potent, selective non-peptide CXCR2 antagonist that 36. Schumacher C, Clark-Lewis I, Baggiolini M, Moser B: High- and low- inhibits interleukin-8-induced neutrophil migration. J Biol Chem 273: affinity binding of GRO alpha and neutrophil-activating peptide 2 to 10095–10098, 1998 receptors on human neutrophils. Proc Natl Acad Sci 18. Lelongt B, Trugnan G, Murphy G, Ronco PM: Matrix metalloprotein- USA89: 10542–10546, 1992 ases MMP2 and MMP9 are produced in early stages of kidney mor- 37. Hyink DP, Tucker DC, St John PL, Leardkamolkarn V, Accavitti MA, phogenesis but only MMP9 is required for renal organogenesis in Abrass CK, Abrahamson DR: Endogenous origin of glomerular endo- vitro. J Cell Biol 136: 1363–1373, 1997 thelial and mesangial cells in grafts of embryonic kidneys. Am J Physiol 19. Lambert V, Munaut C, Jost M, Noel A, Werb Z, Foidart JM, Rakic JM: 270: F886–F899, 1996 Matrix metalloproteinase-9 contributes to choroidal neovasculariza- 38. Shweiki D, Itin A, Soffer D, Keshet E: Vascular endothelial growth tion. Am J Pathol 161: 1247–1253, 2002 factor induced by hypoxia may mediate hypoxia-initiated angiogene- 20. Zhu YM, Webster SJ, Flower D, Woll PJ: Interleukin-8/CXCL8 is a sis. Nature 359: 843–845, 1992 growth factor for human lung cancer cells. Br J Cancer 91: 1970–1976, 39. A Chuntharapai A, Kim KJ: Regulation of the expression of IL-8 recep- 2004 tor A/B by IL-8: Possible functions of each receptor. J Immunol 155: 21. Li A, Varney ML, Singh RK: Constitutive expression of growth regu- 2587–2594, 1995

J Am Soc Nephrol 18: 2359–2370, 2007 Chemokines in Metanephric Development 2369 BASIC RESEARCH www.jasn.org

40. Van den Steen PE, Proost P, Wuyts A, Van Damme J, Opdenakker G: the ureteric epithelium during kidney development is coordinated by Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminot- the opposing functions of GDNF and Sprouty1. Dev Biol 299: 466– erminal processing, whereas it degrades CTAP-III, PF-4, and GRO- 477, 2006 alpha and leaves RANTES and MCP-2 intact. Blood 96: 2673–2681, 48. Merz D, Liu R, Johnson K, Terkeltaub R: IL-8/CXCL8 and growth- 2000 related oncogene alpha/CXCL1 induce chondrocyte hypertrophic dif- 41. Liu JF, Crepin M, Liu JM, Barritault D, Ledoux D: FGF-2 and TPA ferentiation. J Immunol 171: 4406–4415, 2003 induce matrix metalloproteinase-9 secretion in MCF-7 cells through 49. Emadi S, Clay D, Desterke C, Guerton B, Maquarre E, Charpentier A, PKC activation of the Ras/ERK pathway. Biochem Biophys Res Com- Jasmin C, Le Bousse-Kerdiles MC; French INSERM Research Network mun 293: 1174–1182, 2002 on MMM: IL-8 and its CXCR1 and CXCR2 receptors participate in the 42. Ganser GL, Stricklin GP, Matrisian LM: EGF and TGF alpha influence in control of megakaryocytic proliferation, differentiation, and ploidy in vitro lung development by the induction of matrix-degrading metal- myeloid metaplasia with myelofibrosis. Blood 105: 464–473, 2005 loproteinases. Int J Dev Biol 35: 453–461, 1991 50. Keane MP, Belperio JA, Xue YY, Burdick MD, Strieter RM: Depletion of 43. Poladia DP, Kish K, Kutay B, Hains D, Kegg H, Zhao H, Bates CM: Role CXCR2 inhibits tumor growth and angiogenesis in a murine model of of fibroblast growth factor receptors 1 and 2 in the metanephric lung cancer. J Immunol 172: 2853–2860, 2004 mesenchyme. Dev Biol 291: 325–339, 2006 51. Mestas J, Burdick MD, Reckamp K, Pantuck A, Figlin RA, Strieter RM: 44. Knall C, Young S, Nick JA, Buhl AM, Worthen GS, Johnson GL: The role of CXCR2/CXCR2 ligand biological axis in renal cell carci- Interleukin-8 regulation of the Ras/Raf/mitogen-activated protein ki- noma. J Immunol 175: 5351–5357, 2005 nase pathway in human neutrophils. J Biol Chem 271: 2832–2838, 52. Devalaraja RM, Nanney LB, Du J, Qian Q, Yu Y, Devalaraja MN, 1996 Richmond A: Delayed wound healing in CXCR2 knockout mice. J In- 45. Limatola C, Ciotti MT, Mercanti D, Santoni A, Eusebi F: Signaling vest Dermatol 115: 234–244, 2000 pathways activated by chemokine receptor CXCR2 and AMPA-type 53. Moepps B, Nuesseler E, Braun M, Gierschik P: A homolog of the glutamate receptors and involvement in granule cells survival. J Neu- human chemokine receptor CXCR1 is expressed in the mouse. Mol roimmunol 123: 9–17, 2002 Immunol 43: 897–914, 2006 46. Li A, Varney ML, Valasek J, Godfrey M, Dave BJ, Singh RK: Autocrine 54. Levashova ZB, Plisov SY, Perantoni AO: Conditionally immortalized role of interleukin-8 in induction of endothelial cell proliferation, sur- cell line of inducible metanephric mesenchyme. Kidney Int 63: 2075– vival, migration and MMP-2 production and angiogenesis. Angiogen- 2087, 2003 esis 8: 63–71, 2005 55. Tuttle R, Nakagawa Y, Johnson JE, O’Leary DDM: Defects in thalamo- 47. Basson MA, Watson-Johnson J, Shakya R, Akbulut S, Hyink D, Costan- cortical axon pathfinding correlate with altered cell domains in Mash- tini FD, Wilson PD, Mason IJ, Licht JD: Branching morphogenesis of 1-deficient mice. Development 126: 1903–1916, 1999

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