title: Protein kinase A regulates GDNF/RET-dependent but not GDNF/Ret-independent ureteric bud outgrowth from the Wolffian duct.
abstract: Embryonic kidney development begins with the outgrowth of the ureteric bud (UB) from the Wolffian duct (WD) into the adjacent metanephric mesenchyme (MM). Both a GDNF-dependent and GDNF-independent (Maeshima et al., 2007) pathway have been identified. In vivo and in vitro, the GDNF-dependent pathway is inhibited by BMPs, one of the factors invoked to explain the limitation of UB formation in the unbudded regions of the WD surrounding the UB. However, the exact mechanism remains unknown. Here a previously described in vitro system that models UB budding from the WD was utilized to study this process. Because Protein kinase A (PKA) activation has been shown to prevent migration, morphogenesis and tubulogenesis of epithelial cells (Santos et al., 1993), its activity in budded and non-budded portions of the GDNF-induced WD was analyzed. The level of PKA activity was 15-fold higher in the unbudded portions of the WD compared to budded portions, suggesting that PKA activity plays a key role in controlling the site of UB emergence. Using well-characterized PKA agonists and antagonists, we demonstrated that at various levels of the PKA-signaling hierarchy, PKA regulates UB outgrowth from the WD by suppressing budding events. This process appeared to be PKA-2 isoform specific, and mediated by changes in the duct rather than the surrounding mesenchyme. In addition, it was not due to changes in either the sorting of junctional proteins, cell death, or cell proliferation. Furthermore, the suppressive effect of cAMP on budding did not appear to be mediated by spread to adjacent cells via gap junctions. Conversely, antagonism of PKA activity stimulated UB outgrowth from the WD and resulted in both an increase in the number of buds per unit length of WD as well as a larger surface area per bud. Using microarrays, analysis of gene expression in GDNF-treated WDs in which the PKA pathway had been activated revealed a nearly 14-fold decrease in Ret, a receptor for GDNF. A smaller decrease in GFRα1. a co-receptor for GDNF, was also observed. Using Ret-null WDs, we were able to demonstrate that PKA regulated GDNF-dependent budding but not GDNF-independent pathway for WD budding. We also found that BMP2 was higher in unbudded regions of the GDNF-stimulated WD. Treatment of isolated WDs with BMP2 suppressed budding and resulted in a 3-fold increase in PKA activity. The data suggests that the suppression of budding by BMPs and possibly other factors in non-budded zones of the WD may be regulated in part by increased PKA activity, probably partially through downregulation of Ret/GFRα1 coreceptor expression.
authors.affiliation: Department of Pediatrics, University of Calgary and Alberta Children's Hospital, Alberta, Canada.
authors.forename: James B
authors.initials: JB
authors.lastname: Tee
authors.forename: Yohan
authors.initials: Y
authors.lastname: Choi
authors.forename: Mita M
authors.initials: MM
authors.lastname: Shah
authors.forename: Ankur
authors.initials: A
authors.lastname: Dnyanmote
authors.forename: Derina E
authors.initials: DE
authors.lastname: Sweeney
authors.forename: Tom F
authors.initials: TF
authors.lastname: Gallegos
authors.forename: Kohei
authors.initials: K
authors.lastname: Johkura
authors.forename: Chiharu
authors.initials: C
authors.lastname: Ito
authors.forename: Kevin T
authors.initials: KT
authors.lastname: Bush
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Nigam
mesh_terms: D000818:Animals; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism / Q000494:pharmacology; D049109:Cell Proliferation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D047108:Embryonic Development / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism* / Q000494:pharmacology; D007668:Kidney / Q000196:embryology*; D008297:Male; D008648:Mesoderm / Q000196:embryology; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D011247:Pregnancy; D051096:Proto-Oncogene Proteins c-ret / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D014513:Ureter / Q000196:embryology*; D014928:Wolffian Ducts / Q000196:embryology*
chemical_list: C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D017931:DNA Primers; D051100:Glial Cell Line-Derived Neurotrophic Factor; D011994:Recombinant Proteins; D051096:Proto-Oncogene Proteins c-ret; C494999:Ret protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases
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