Full PapersModulation of HGF-Induced Tubulogenesis and Branching by Multiple Phosphorylation Mechanisms
Abstract
MDCK cells cultured in Type I collagen gels can be induced to develop branching tubular structures with demonstrable lumens in the presence of hepatocyte growth factor (HGF). As we have now shown by immunofluorescent localization of specific marker proteins, these tubules retain apical-basolateral polarity. However, the secondary signaling events which lead to these characteristic morphogenetic changes induced by HGF remain largely unelucidated. In order to examine these signaling events, particularly the role of protein phosphorylation in the formation of branching tubular structures, Madin-Darby canine kidney (MDCK) cells in collagen gels were treated with HGF plus well-characterized agents that affect protein phosphorylation. We quantified the formation of branching processes, an early step in the development of tubular structures in this model. Protein kinase C (PKC) inhibition resulted in more complex branching processes in the presence of HGF, when compared with HGF alone. In contrast, treatment with activators of protein kinase A (PKA), as well as calmod ulin antagonists, caused a marked decline in process formation. Consistent with an important role for protein phosphorylation in HGF-induced morphogenesis, protein phosphatase inhibition by okadaic acid or calyculin A was found to markedly inhibit process formation. Tyrosine kinase (TK) inhibition also decreased the percentage of processes. This is consistent with data indicating that one of the HGF receptors is identical to the c-met protooncogene product, which is known to possess TK activity. Our results suggest that the HGF-mediated induction of branching processes in MDCK cells, an early step in the development of branching tubular structures, can be modulated by multiple phosphorylation mechanisms including those mediated by PKC, PKA, and Ca2+/calmodulin-dependent kinase(s). We discuss how these phosphorylation events may play crucial roles in determining the degree of tubule formation and their length, as well as the extent of their arborization during the early development of epithelial tissues.
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Molecular and Cellular Mechanisms of Kidney Development
2013, Seldin and Geibisch's The KidneyMolecular and Cellular Mechanisms of Kidney Development
2012, Seldin and Giebisch's The Kidney: Physiology and PathophysiologyHyperglycemia induced down-regulation of renal P-glycoprotein expression
2012, European Journal of PharmacologyThe purpose of this study is to investigate the regulation of P-glycoprotein expression in the kidney under diabetic condition. Renal P-glycoprotein expression was examined in inbred mice with type 1 or type 2 diabetes by Western blotting. The underlying mechanisms of P-glycoprotein regulation were examined in Madin–Darby canine kidney type II (MDCK-II) cells by Western blotting or qRT-PCR. 3H-digoxin uptake was measured for P-glycoprotein activity in cells under various treatments. The results showed that P-glycoprotein expression was lower in kidneys of diabetic mice than in controls. In MDCK-II cells, treatments with insulin or IL-6 did not cause any change in P-glycoprotein expression, whereas TNF-α tended to increase P-glycoprotein expression at a concentration of 1 ng/ml. On the other hand, P-glycoprotein expression was reduced under high glucose conditions (450 mg/dl), while superoxide production was increased, and the reduction in P-glycoprotein expression was abolished by N-acetylcysteine (an antioxidant) and staurosporine (a nonselective PKC inhibitor). Treatment with oxidizing agents (H2O2, BSO) or PMA (a PKC activator) reduced P-glycoprotein expression. Antioxidant (N-acetylcysteine or glutathione) co-treatment abolished the H2O2-induced and BSO-induced reduction in P-glycoprotein expression, whereas it did not prevent the effect of PMA. The PMA-induced P-glycoprotein down-regulation was prevented by co-treatment of LY333531 (a PKC-β inhibitor). 3H-digoxin levels were higher in MDCK-II cells with high glucose, PMA or H2O2 treatments. In conclusion, P-glycoprotein expression is lower in kidneys of diabetic mice and in MDCK-II cells under high glucose conditions. Hyperglycemia induced reactive oxygen species and activated PKC in MDCK-II cells, leading to the decrease in P-glycoprotein expression.
A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development
2012, Developmental BiologyGenetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.
Growth factor-dependent branching of the ureteric bud is modulated by selective 6-O sulfation of heparan sulfate
2011, Developmental BiologyCitation Excerpt :Multiple growth factors have been identified as modulators of UB branching and architecture. Of these, some of the most prominent members have belonged to the FGF and TGFβ families (Bush et al., 2004; Qiao et al., 1999b, 2001; Sakurai et al., 1997; Santos et al., 1993). FGFs have been shown to affect the formation of UB tips and stalks, and signaling through a variety of FGF receptors has been shown to play a role in UB branching and stromal mesenchymal patterning (Poladia et al., 2006; Qiao et al., 2001; Zhao et al., 2004).
Heparan sulfate proteoglycans (HSPGs) are found in the basement membrane and at the cell-surface where they modulate the binding and activity of a variety of growth factors and other molecules. Most of the functions of HSPGs are mediated by the variable sulfated glycosaminoglycan (GAG) chains attached to a core protein. Sulfation of the GAG chain is key as evidenced by the renal agenesis phenotype in mice deficient in the HS biosynthetic enzyme, heparan sulfate 2-O sulfotransferase (Hs2st; an enzyme which catalyzes the 2-O-sulfation of uronic acids in heparan sulfate). We have recently demonstrated that this phenotype is likely due to a defect in induction of the metanephric mesenchyme (MM), which along with the ureteric bud (UB), is responsible for the mutually inductive interactions in the developing kidney (Shah et al., 2010). Here, we sought to elucidate the role of variable HS sulfation in UB branching morphogenesis, particularly the role of 6-O sulfation. Endogenous HS was localized along the length of the UB suggesting a role in limiting growth factors and other molecules to specific regions of the UB. Treatment of cultures of whole embryonic kidney with variably desulfated heparin compounds indicated a requirement of 6O-sulfation in the growth and branching of the UB. In support of this notion, branching morphogenesis of the isolated UB was found to be more sensitive to the HS 6-O sulfation modification when compared to the 2-O sulfation modification. In addition, a variety of known UB branching morphogens (i.e., pleiotrophin, heregulin, FGF1 and GDNF) were found to have a higher affinity for 6-O sulfated heparin providing additional support for the notion that this HS modification is important for robust UB branching morphogenesis. Taken together with earlier studies, these findings suggest a general mechanism for spatio-temporal HS regulation of growth factor activity along the branching UB and in the developing MM and support the view that specific growth factor-HSPG interactions establish morphogen gradients and function as developmental switches during the stages of epithelial organogenesis (Shah et al., 2004).
Stage-dependent regulation of mammary ductal branching by heparan sulfate and HGF-cMet signaling
2011, Developmental BiologySpecific interactions of growth factors with heparan sulfate may function as "switches" to regulate stages of branching morphogenesis in developing mammalian organs, such as breast, lung, salivary gland and kidney, but the evidence derives mostly from studies of explanted tissues or cell culture (Shah et al., 2004). We recently provided in vivo evidence that inactivation of Ndst1, the predominant N-deacetylase/N-sulfotransferase gene essential for the formation of mature heparan sulfate, results in a highly specific defect in murine lobuloalveolar development (Crawford et al., 2010). Here, we demonstrate a highly penetrant dramatic defect in primary branching by mammary epithelial-specific inactivation of Ext1, a subunit of the copolymerase complex that catalyzes the formation of the heparan sulfate chain. In contrast to Ext1 deletion, inactivation of Hs2st (which encodes an enzyme required for 2-O-sulfation of uronic acids in heparan sulfate) did not inhibit ductal formation but displayed markedly decreased secondary and ductal side-branches as well as fewer bifurcated terminal end buds. Targeted conditional deletion of c-Met, the receptor for HGF, in mammary epithelial cells showed similar defects in secondary and ductal side-branching, but did not result in any apparent defect in bifurcation of terminal end buds. Although there is published evidence indicating a role for 2-O sulfation in HGF binding, primary epithelial cells isolated from Hs2st conditional deletions were able to activate Erk in the presence of HGF and there appeared to be only a slight reduction in HGF-mediated c-Met phosphorylation in these cells compared to control. Thus, both c-Met and Hs2st play important, but partly independent, roles in secondary and ductal side-branching. When considered together with previous studies of Ndst1-deficient glands, the data presented here raise the possibility of partially-independent regulation by heparan sulfate-dependent pathways of primary ductal branching, terminal end bud bifurcation, secondary branching, ductal side-branching and lobuloalveolar formation.