title: Novel MAPK-dependent and -independent tubulogenes identified via microarray analysis of 3D-cultured Madin-Darby canine kidney cells.
abstract: Cystogenesis and tubulogenesis are basic building blocks for many epithelial organs, including the kidney. Most researchers have used two-dimensional (2D) cell culture to investigate signaling pathways downstream of hepatocyte growth factor (HGF). We hypothesize that three-dimensional (3D) collagen-grown Madin-Darby canine kidney (MDCK) cells, which form cysts and then tubulate in response to HGF, are a much more in vivo-like system for the identification of novel tubulogenes. With the use of a canine microarray containing over 20,000 genes, 2,417 genes were identified as potential tubulogenes that were differentially regulated, exclusively in 3D-grown MDCK cells. Among these, 840 were dependent on MAPK signaling. Importantly, this work shows that many putative tubulogenes, previously identified via microarray analysis of 2D cultures, including by us, do not change in 3D culture and vice versa. The use of a 3D-culture system allowed for the identification of novel MAPK-dependent and -independent genes that regulate early renal tubulogenesis in vitro, e.g., matrix metalloproteinase 1 (MMP1). Knockdown of MMP1 led to defects in cystogenesis and tubulogenesis in 3D-grown MDCK cells, most likely due to problems establishing normal polarity. We suggest that data obtained from 2D cultures, even those using MDCK cells treated with HGF, should not be automatically extrapolated to factors important for cystogenesis and tubulogenesis. Instead, 3D culture, which more closely replicates the biological environment and is therefore a more accurate model for identifying tubulogenes, is preferred. Results from the present analysis will be used to build a more accurate model of the signaling pathways that control cystogenesis and tubulogenesis.
authors.affiliation: Renal, Electrolyte and Hypertension Division, Rm. 405C, Clinical Research Bldg., Univ. of Pennsylvania, Philadelphia, PA 19104. jhlipsch@mail.med.upenn.edu.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Chacon-Heszele
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zuo
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Hellman
authors.forename: Sarah
authors.initials: S
authors.lastname: McKenna
authors.forename: Soo Young
authors.initials: SY
authors.lastname: Choi
authors.forename: Liwei
authors.initials: L
authors.lastname: Huang
authors.forename: John W
authors.initials: JW
authors.lastname: Tobias
authors.forename: Kwon Moo
authors.initials: KM
authors.lastname: Park
authors.forename: Joshua H
authors.initials: JH
authors.lastname: Lipschutz
mesh_terms: D000818:Animals; D016764:Cell Polarity; D004285:Dogs; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D053263:Gene Regulatory Networks; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D007684:Kidney Tubules / Q000201:enzymology* / Q000254:growth & development / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000235:genetics*; D061985:Madin Darby Canine Kidney Cells; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D038081:Organogenesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D046509:Tissue Culture Techniques
chemical_list: D012333:RNA, Messenger; D017228:Hepatocyte Growth Factor; D020928:Mitogen-Activated Protein Kinases; D020781:Matrix Metalloproteinase 1
keywords: development; kidney; matrix metalloproteinase; mitogen-activated protein kinase; tubulogenesis