Novel MAPK-dependent and -independent tubulogenes identified via microarray analysis of 3D-cultured Madin-Darby canine kidney cells.
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.
['Chacon-Heszele|Maria F|MF|', 'Zuo|Xiaofeng|X|', 'Hellman|Nathan E|NE|', 'McKenna|Sarah|S|', 'Choi|Soo Young|SY|', 'Huang|Liwei|L|', 'Tobias|John W|JW|', 'Park|Kwon Moo|KM|', 'Lipschutz|Joshua H|JH|']
D000818:Animals D016764:Cell Polarity D004285:Dogs D020869:Gene Expression Profiling D018507:Gene Expression Regulation, Developmental D015971:Gene Expression Regulation, Enzymologic D055785:Gene Knockdown Techniques D053263:Gene Regulatory Networks D017228:Hepatocyte Growth Factor D007684:Kidney Tubules D020935:MAP Kinase Signaling System D061985:Madin Darby Canine Kidney Cells D020781:Matrix Metalloproteinase 1 D020928:Mitogen-Activated Protein Kinases D020411:Oligonucleotide Array Sequence Analysis D038081:Organogenesis D034622:RNA Interference D012333:RNA, Messenger D060888:Real-Time Polymerase Chain Reaction D015203:Reproducibility of Results D046509:Tissue Culture Techniques
kidney tubulogenesis matrix metalloproteinase development mitogen-activated protein kinase