An established and productive investigator in the field of endothelin signaling plans to continue investigating intracellular signaling mechanisms through which endothelin-1 stimulates mesangial cell proliferation. This work will, however, remain focused on mesangial cells in vitro.
Aim 1 seeks to determine whether c-Src mediates the mitogenic response to ET-1, and whether Raf-1 is in this pathway. Previous work has shown that c-Src is required for c-fos promoter activation. A transient transfection assay in which the fraction of transfected cells (lac-Z marker) taking up BrdU is used to quantify DNA synthesis; quantification of clones emerging from transient transfection will be used to determine cell proliferation. The effect of transiently tranfected dominant neg. Src (or a kinase competent Src) on ET-1 stimulated DNA/cell proliferation will be examined. If constitutively active Raf-1 rescues ET-1 mitogenesis in Src kinase deficient cells, Raf-1 will be judged to be downstream of Src. Transfection with Csk (to block Src) and with v-Src or constitutively active Raf-1 to overcome the block, will also be used as additional evidence for the role of c-Src. Another approach to investigate whether c-Src is required for ET-1 mesangial cell proliferation is to use mesangial cells cultured from Src -/-mice. They will be shown to be responsive to ET-1 by determining receptor expression and ET-1 Ca mobilization.
Aim 2 will determine whether FAK and/or Pyk2 may be upstream activators of c-Src in the ET-1 ras-MAPkinase-mitogenic signal. 1. Preliminary data show that Pyk2 is present in mesangial cells but not activated by ET-1, while it is activated by Ca ionophore and phorbol ester. Full time- course and dose-responses of ET-1 P-tyr of FAK and Pyk2 will be determined, it will be determined whether FAK and/or Pyk2 complex with Src. Whether overexpression of FAK or Pyk2 leads to activation of Src in response to ET-1 will be determined by co-expressing HA-tagged Src, immunoprecipitation and immunoblotting with an antibody that recognizes only active Src. The effect of dominant negative mutants of FAK and Pyk2 on ET-1 stimulated c-Src activation will also be determined. The involvement of PI-3 kinase in activation of cSrc and MAP kinase cascade will be sought by determining ET-1 stimulated Src activation, and c-fos induction in the presence of Wortmannin, and alternatively by overexpression of active-, or a dominant negative mutant of PI-3kinase.
Aim 3 will determine the role of Src independent pathways in ET-1 stimulated cell cycle progression. Preliminary data show that Rho A dominant negative mutant inhibits the c-fos induction by ET-1, and that the effect is independent of Src. Transient transfection with constitutively active mutants of Rho A, rac-1, and dominant negative Rho-A and rac-1, and their effect on c-fos induction are planned. To further test for the involvement of rho, the botulinum C3 exoenzyme (which ADP-ribosylates rho and inactivates it), will be used. Finally, whether rho is in the Src pathway or not, will be examined by cotransfection with constitutively active v-Src/dominant negative RhoA and vice versa and evaluation of c-fos induction/ DNA synthesis.
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