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 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 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, through downregulation of Ret/GFRα1 coreceptor expression.

Parasympathetic Innervation Regulates Tubulogenesis in the Developing Salivary Gland

“…Pharmacological manipulations of PKA have been shown to affect epithelial cell polarity and morphogenesis in vitro (Nedvetsky et al, 2012; Tee et al, 2010). However, we identify an endogenous upstream signal engaging PKA in the regulation of epithelial tubulogenesis.…”

BMP regulates regional gene expression in the dorsal otocyst through canonical and non-canonical intracellular pathways

“…A possible molecular pathway mediating PKA activation by BMP non-canonical signaling Several studies have reported that BMP signaling can upregulate PKA activity (Gangopahyay et al, 2011;Lee and Chuong, 1997;Liu et al, 2005;Tee et al, 2010). However, the molecular pathway by which this occurs remains unclear.…”

Multimodal Eph/Ephrin signaling controls several phases of urogenital development

“…Bar: ERK, AKT, phosphatidylinositol-3 0 -kinase, and protein kinase A are also known to play a role in wolffian duct budding. [20][21][22] To investigate these pathways, we measured multiple phosphorylated proteins simultaneously by the enzyme-linked immunosorbent assay microarray technique 23 in microdissected urogenital systems from embryonic day (ED) 11.5 dKO and control embryos. In 2 of 3 comparisons, ERK2 phosphorylation was decreased (Figure 6a and e).…”

“…CND regression critically depends on retinoic acid and Ret signaling. 7-9,31 ERK is a downstream target of Ret and Eph/Ephrin signaling, [18][19][20] and inhibition of ERK led to failure in UB formation and CND aplasia. 31 Our analysis at ED 11.5 localized high ERK1/2 activation in the UGS and the caudal CND with a gradual decrease toward the UB.…”