Regulation of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) expression in podocytes.
BACKGROUND
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) is expressed constitutively in the adult glomerular podocytes at high levels; however, the regulation of its production is unclear. Recent data from podocyte-specific knockout mice suggest that VPF/VEGF-A is critical for the proper maintenance of glomerular filtration barrier and the glomerular endothelial fenestrae. We hypothesized that the glomerular basement membrane (GBM) matrix-podocyte interaction may play a role in the constitutive expression of VPF/VEGF-A in the adult glomerulus.


METHODS
VPF/VEGF-A mRNA levels in a human podocyte cell line grown in the presence of various extracellular matrices were quantitated by real-time polymerase chain reaction (PCR) experiments. VPF/VEGF-A protein levels in the culture supernatant from the same conditions were measured by enzyme-linked immunosorbent assay (ELISA). Promoter activity of VPF/VEGF-A gene in these cells was performed by transfecting full length (2.6 kb) VPF/VEGF-A promoter, which is fused with luciferase reporter gene. Immunoprecipitation and Western blot experiments were carried out in order to detect the association of hypoxia-inducible factor-alpha (HIF-alpha) and p300 in podocyte cells.


RESULTS
In this study, we provide preliminary evidence that signaling through the extracellular matrix proteins and, in particular, laminin and its receptor alpha(3)beta(1) integrin may regulate VPF/VEGF-A production in cultured podocytes in vitro. We also present data that increased activity of the transcription factor HIF-alphas in podocyte is not related to hypoxia and may lead to up-regulation of VPF/VEGF-A transcription. The classical type protein kinase C (PKC) may be a potential intermediate signaling molecule in this event.


CONCLUSIONS
These data suggest a novel nonhypoxic regulation of VPF/VEGF-A production in the glomerulus of the kidney during physiologic states. These observations may form the basis of more elaborate studies that will finally provide the detailed signaling pathway for VPF/VEGF-A synthesis in podocytes and will help our understanding of the pathogenesis of various VPF/VEGF-A-related diseases in the glomerulus of the kidney.
Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA. Datta.Kaustubh@mayo.edu
Kaustubh
K
Datta
Jinping
J
Li
S Ananth
SA
Karumanchi
Enfeng
E
Wang
Eric
E
Rondeau
Debabrata
D
Mukhopadhyay
D051792:Basic Helix-Loop-Helix Proteins; D002461:Cell Line, Transformed; D005109:Extracellular Matrix / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D039201:Integrin alpha3beta1 / Q000378:metabolism; D007678:Kidney Glomerulus / Q000166:cytology / Q000502:physiology*; D007797:Laminin / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D039201:Integrin alpha3beta1; D007797:Laminin; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human