Protein Kinase A Is a Negative Regulator of Renal Branching Morphogenesis and Modulates Inhibitory and Stimulatory Bone Morphogenetic Proteins*

Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961–F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.

Branching morphogenesis, defined as growth and branching of epithelial tubules during embryonic development, arises in the kidney through reciprocal mesenchymal-epithelial tissue interactions between the mesenchymal metanephric blastema and the epithelial ureteric bud (and its derivative collecting ducts) (1). These interactions are mediated by secreted peptide growth factors. Members of the bone morphogenetic protein (BMP) 1 family, a subset of the TGF-␤ superfamily, are expressed in a temporal and spatial pattern consistent with their role as regulators of tubular growth and branching (2,3). BMP-2 inhibits renal branching morphogenesis, whereas BMP-7 is stimulatory at low doses and inhibitory at higher doses (3). Although a growing body of evidence suggests that signals derived from inhibitory and stimulatory BMPs, as well as other growth factors, are integrated by the ureteric bud/ collecting duct cells to determine the number and phenotype of tubules (4), the intracellular events that act in concert with these signals are largely undefined.
Protein kinase A (PKA), an intracellular kinase, has been implicated in the control of morphogenesis in diverse nonrenal tissues including the central nervous system (5,6), the eye (7), and the limb (8,9). Within these tissues, PKA interacts with growth factor signaling pathways to regulate morphogenetic responses to BMPs (7)(8)(9). Within the embryonic limb, BMP-2mediated stimulation of chondrogenesis is dependent on PKA (9). Furthermore, the mechanism underlying the interdependence between PKA and BMP-2 signaling within the limb may be controlled by BMP-2 because treatment with BMP-2 induces PKA activity (9).
The function of PKA during kidney morphogenesis is undefined. However, evidence in cell culture models suggests a role for PKA in growth factor signaling in the kidney. In the Madin-Darby canine kidney cell model of tubulogenesis, PKA modulates responses to hepatocyte growth factor (HGF), a known regulator of renal branching morphogenesis (10). Additionally, in renal mesangial cells, TGF-␤ stimulates PKA by a cAMP independent mechanism (11), which provides further evidence that PKA can act downstream of TGF-␤ superfamily members.
We demonstrate here that PKA inhibits renal branching morphogenesis and modulates the effects of both inhibitory and stimulatory BMPs. Using embryonic renal explants and an in vitro cell culture model of tubulogenesis, we demonstrate that inhibition of PKA rescues ureteric buds or collecting ducts from the inhibitory effects of BMPs by decreasing tubular cell apoptosis. Consistent with these effects, activation of PKA abrogates the stimulatory effects of low dose BMP-7. To determine whether the effect of PKA is mediated by known signaling events in the BMP-dependent Smad1 signaling pathway, we showed that PKA has no effect on endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. To determine whether the dependence of inhibitory BMPs on PKA is mediated by regulation of the PKA signaling pathway by BMPs, we demonstrated that BMP-2 increases endogenous PKA activity and induces phosphorylation of its downstream effector, cAMP-response element-binding protein (CREB). Taken together, our results show that PKA acts via a pathway parallel to ligand-induced Smad1 to inhibit renal branching morphogenesis and that PKA activity is positively regulated by BMP-2.

Embryonic Kidney Organ Culture and Treatment with Recombinant
Proteins-Murine embryos were surgically resected from embryonic (E) day 12 pregnant CD1 mice and the kidneys were cultured as described previously (3). Explants were grown in Dulbecco's modified Eagle's medium-Ham's F-12 nutrient mixture (DMEM-F12) with Richter's modification (Life Technologies, Inc.) containing 50 g/ml transferrin (Sigma) and either no ligand, 5 nM BMP-2 (kindly provided by Genetics Institute), 0.25 nM BMP-7 (originally named OP-1) (kindly provided by Creative Biomolecules), 30 nM BMP-7 alone, or combinations of each of these growth factors with 5-10 M H-89 (Calbiochem) for 48 -60 h. Explants were fixed in 4% fomaldehyde for 10 min, washed four times with PBS, and then embedded in paraffin. 5-m tissue sections were stained with hematoxylin and eosin and imaged by light microscopy.
Immunofluorescence-To study nuclear translocation of Smad1, mIMCD-3 cells were grown in monolayer on glass coverslips in DMEM-F12 culture medium supplemented with 0.2% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 units/ml) in 5% CO 2 at 37°C for 12 h. Cells were treated for 1 h with either 5 nM BMP-2, 5 M H-89, or 5 M 8-Br-cAMP or combinations of BMP-2/H-89 or BMP-2/8-Br-cAMP and then fixed in 4% formaldehyde. After three washes in PBS, the cells were permeabilized with methanol for 15 min at room temperature and then blocked with 10% goat serum and 3% bovine serum albumin in PBS for 1 h at room temperature. Cells were incubated with anti-Smad 1 antibody (12) (1:50 dilution) overnight at 4°C, washed with PBS, and then incubated with fluorescein isothiocyanateconjugated goat anti-rabbit antibody (Jackson Immunologicals; 1:200 dilution) for 60 min at room temperature. Coverslips were mounted and imaged using a Zeiss Axioskop confocal laser microscope.
In Vitro Kinase Assay for PKA Activity-mIMCD-3 cells were grown in DMEM-F12 with 0.2% FBS and treated with 5 nM BMP-2, 5 M 8-Br-cAMP, 5 M H-89, 8-Br-cAMP/H-89, or BMP-2/H-89. Cell lysates were prepared by suspending the cells in cold extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ␤-mercaptoethanol, 1 g/ml leupeptin, 1 g/ml aprotinin) and then homogenized using a Dounce homogenizer. Cell lysates were centrifuged, and then the supernatant was used in a nonradioactive assay for cAMP-dependent protein kinase (PKA assay system, Promega). The amount of protein added to the kinase assay was standardized by measuring absorption by cell lysates at 280 nm using a Hewlett-Packard UV spectrophotometer. Phosphorylated and unphosphorylated forms of the PKA peptide substrate were separated by agarose gel electrophoresis and imaged by UV light.
TUNEL Assay-In situ TUNEL assays were performed in embryonic kidney explants and collagen gels using commercially available reagents (Apop Tag kit, Oncor). E12 embryonic kidneys were cultured for 2 days in Richter's modified DMEM-F12 in the presence of 10 M H-89, 5 nM BMP-2, or both. Deparaffinized tissue sections were treated with proteinase K followed by 3% hydrogen peroxide to quench endogenous peroxidase activity. Sections were preincubated with 100 l of terminal deoxynucleotidyl transferase equilibration buffer for 15 min at 37°C and then incubated with 100 l of terminal deoxynucleotidyl transferase mixed in reaction buffer according to the manufacturer's instructions. The reaction was stopped by immersing slides in 300 mM NaCl, 30 mM sodium citrate buffer, pH 8.0 (stop/wash buffer), for 30 min at 37°C. Sections were blocked in blocking buffer for 1 h and subsequently treated with 100 l of sheep monoclonal anti-digoxigenin F(ab) fragments antibody (Roche Molecular Biochemicals) diluted 1:20 in blocking buffer for 1 h at room temperature. After washing sections in PBS, 100 l of DAB substrate diluted 1:10 in 1ϫ DAB buffer was added for Collagen gels containing mIMCD-3 cells were individually transferred from wells of a 96-well culture plate to 1.5-ml microcentrifuge tubes and treated with 0.2% trypsin at 37°C for 2 h. Gels were washed four times in PBS, preincubated in terminal deoxynucleotidyl transferase equilibration buffer for 1 h, and subsequently treated with terminal deoxynucleotidyl transferase in reaction buffer for 5 h at 37°C. The reaction was terminated in stop/wash buffer for 30 min at 37°C. Cells were left overnight in blocking buffer at 4°C and then incubated with 100 l of anti-digoxigenin antibody diluted 1:20 in blocking buffer for 4 h at 37°C. After four 5-min washes in PBS, cells were stained with 3-amino-9-ethylcarbazole (Zymed Laboratories Inc.) for 10 -20 min and rinsed with PBS. Cells were counterstained with 1 g/ml bisbenzamide (Hoechst No. 33258; Sigma) for 30 min at 4°C, followed by two washes in PBS. Cells were directly imaged by DIC and fluorescent microscopy.

PKA Controls Renal Branching Morphogenesis and Modulates the Effects of BMPs in Embryonic Kidney
Explants-We investigated the role of PKA and its interaction with BMPs that control renal morphogenesis because PKA has been impli-cated in the control of cellular responses to BMPs (9). To determine whether PKA regulates renal development, we manipulated its activity in embryonic kidney explants and identified alterations in collecting duct morphogenesis. H-89 is a direct reversible antagonist of the activated catalytic subunit of PKA (14,15). E12 murine metanephric explants were grown for 48 h in the presence or absence of 10 M H-89. As shown in BMP-7. We were not able to demonstrate enhancement of tubule formation by H-89 in the presence of low doses of BMP-7 because BMP-7, alone, exerted a potent stimulatory effect (3) (data not shown). In summary, these results suggest that antagonism of PKA promotes branching morphogenesis in embryonic explants and attenuates the inhibitory effects of BMPs.
Direct Effects of PKA during Collecting Duct Morphogenesis in Vitro-The cellular complexity of the organ culture explant precludes analysis of PKA effects on specific cells within the collecting duct system. We have used mIMCD-3 cells, which are derived from the terminal inner medullary collecting duct of the SV40 transgenic mouse and form branched tubules in three dimensional matrices (3,16), to investigate the direct effects of PKA on branching morphogenesis. mIMCD-3 cells were seeded in type I collagen, cultured for 48 h in the presence of H-89 or the specific activator of PKA, 8-Br-cAMP (17), and then imaged by DIC microscopy. Tubules treated with H-89 were longer and more highly branched than tubules grown under control conditions consistent with our observations in renal explants ( Fig.  2A). Tubules treated with 8-Br-cAMP were similar in phenotype to control tubules (data not shown). Because we also observed an effect of H-89 and 8-Br-cAMP on tubule number, we quantitated the number of tubules formed in the presence of these agents. 10 M H-89 increased tubule number by 883% compared with control (p Ͻ 0.001) (Fig. 2B). At 50 M H-89, no tubules were formed secondary to toxic effects on the cell, as indicated by morphologic evidence of cell death. In contrast, 8-Br-cAMP exerted a dose-dependent inhibitory effect with 61% inhibition at 1 nM (p Ͻ 0.005) (Fig. 2C). Taken together, the opposing effects of H-89 and 8-Br-cAMP on mIMCD-3 tubulogenesis suggest that PKA directly controls renal branching morphogenesis at the level of tubule number.

PKA Modulates the Effects of BMPs in Vitro on Renal
Branching Morphogenesis-Having determined that PKA potently modulates renal branching morphogenesis, we determined its interactions with the BMP signaling pathway. The results of our experiments in embryonic kidney explants ( Fig.  1) suggested that BMP-mediated inhibition of ureteric bud/ collecting duct growth and branching is dependent on PKA activity because antagonism of PKA rescued the inhibitory effects of BMPs. To test this more directly, we determined the interactions between BMPs and PKA in the mIMCD-3 culture model. Addition of H-89 to cultures treated with 5 nM BMP-2 induced a rescue of BMP-2-mediated inhibition (Fig. 3A). Quantitation of these effects showed that 5 M H-89 enhanced tubule formation in the presence of BMP-2 by 328% (p Ͻ 0.05) (Fig. 3B). To obtain independent evidence that H-89 was acting to inhibit PKA, we determined the effects of the PKI amide 14 -22 in our in vitro model of tubulogenesis. PKI rescued tubule formation in BMP-2-treated cultures in a dose-dependent manner (maximum effect: 1 nM, 190%, p Ͻ 0.05) (Fig. 3C). In contrast to H-89 and PKI, when 5 nM BMP-2 was combined with 1 M 8-Br-cAMP, tubulogenesis was inhibited to the same degree as observed with BMP-2 treatment alone (Fig. 2B). Because BMP-2 exerts a strong inhibitory effect on tubule formation, we were not able to determine whether 8-Br-cAMP acted synergistically with BMP-2 to inhibit. Similarly, addition of H-89 to mIMCD-3 cultures treated with high doses (30 nM) of BMP-7 induced a rescue of BMP-7-mediated inhibition by 70% (p Ͻ 0.05) (Fig. 3D). However, when 30 nM BMP-7 was combined with 1 M 8-Br-cAMP, the inhibitory effect of BMP-7 was unchanged. In summary, these data indicate that inhibitory BMPs, including BMP-2 and high dose BMP-7, are dependent on PKA to exert their inhibitory effects on renal branching morphogenesis. BMP-7, at low doses (Ͻ0.25 nM), acts to stimulate renal branching morphogenesis (3). To determine whether PKA might also play a role in modulating this stimulatory pathway, we tested the interactions between H-89, 8-Br-cAMP and low doses of BMP-7. H-89 enhanced BMP-7 stimulated tubulogenesis by 52% compared with BMP-7 alone (p Ͻ 0.05) (Fig. 4A). In contrast, 8-Br-cAMP decreased the stimulatory effect of BMP-7 by 37% (p Ͻ 0.05). These results indicate that PKA negatively modulates stimulation by BMP-7 and are consistent with the dependence of inhibitory BMPs on PKA. To determine whether PKA modulates other stimulatory pathways, including those downstream of receptor tyrosine kinases, we tested the interaction of H-89 or 8-Br-cAMP with HGF. As shown in Fig. 4B, H-89 enhanced the stimulatory effect of HGF on tubulogenesis by 13% (p Ͻ 0.005). In contrast, 8-Br-cAMP exerted a dose-dependent inhibitory effect in the presence of HGF. Taken together, these data suggest that PKA modulates stimulatory pathways mediated by both receptor tyrosine (HGF) and receptor serine/threonine (BMPs) kinases.
Inhibition of PKA Activity Blocks BMP-2-mediated Collecting Duct Apoptosis-Our studies in embryonic kidney explants and in the mIMCD-3 model of branching morphogenesis indicate that PKA acts at a morphogenetic level to alter the phenotype of renal collecting tubules. The morphogenetic pathway for any particular tissue or structure is derived from a composite of cellular events, such as cell proliferation, apoptosis, cell shape change and cell migration (18). Because antagonism of PKA rescues BMP-mediated inhibition of tubulogenesis, we determined whether this was due to a change in collecting duct cell number. Specifically, we determined the effects of BMP-2, H-89, and both together on apoptosis in the developing collect- ing system. E12 embryonic kidney explants were cultured for 48 h in the presence of 5 nM BMP-2, 10 M H-89, or both. Histologic sections were then prepared from fixed tissue, and ureteric bud/collecting ducts were identified by staining with Dolichos biflorus agglutinin, and apoptotic cells were assessed using an in situ TUNEL assay (Fig. 5A). BMP-2 markedly enhanced the number of apoptotic cells in collecting tubules and decreased tubule formation. In contrast, H-89 induced the formation of large, elongated, and highly branched collecting tubules in which apoptotic cells were rarely observed. Treatment with H-89 in the presence of BMP-2 partially rescued the BMP-2 phenotype and ameliorated the extent of apoptosis. The effect of these treatments on ureteric bud/collecting duct apoptosis was quantitated by determining the ratio of apoptotic cells to the total number of ureteric bud/collecting duct cells in each tissue section. Because there was no statistically significant difference between the number of ureteric bud or collecting duct cells in control compared with BMP-2-treated explants (control, 774 Ϯ 60 versus BMP-2, 604 Ϯ 110; p ϭ 0.21), and in BMP-2 compared with BMP-2/H89-treated explants (BMP-2, 604 Ϯ 110 versus BMP-2/H-89, 503 Ϯ 212; p ϭ 0.53), the ratio of apoptotic to the total number of cells reflects only the effect of these treatments on apoptosis. As shown in Fig. 5B, BMP-2 increased apoptosis by 1.4-fold compared with control (no li-gand), whereas antagonism of PKA attenuated collecting duct apoptosis in embryonic kidney explants by 0.4-fold compared with control and by 2.0-fold compared with BMP-2-treated explants (p Ͻ 0.05). Treatment with H-89 in the presence of BMP-2 attenuated apoptosis to an extent similar to that observed with H-89 alone (2.0-fold) (p Ͻ 0.05).
To determine the direct effects of H-89 on BMP-2-treated collecting duct cells, we measured mIMCD-3 cell apoptosis in three-dimensional cultures. mIMCD-3 cells were cultured for 48 h in the presence of BMP-2, H-89, or both. Apoptotic cells were identified by the TUNEL assay (Fig. 5C). Because the numbers of cells at the time cultures were initiated and after 48 h of culture did not differ, the ratio of the number of TUNEL positive cells to the total number of cells (that is, the number of bisbenzamide-staining cells) reflects only an effect on apoptosis. As shown in Fig. 5D, treatment with BMP-2 induced a 3-fold increase in apoptosis compared with control (BMP-2, 11.0 Ϯ 2.1% versus control, 3.7 Ϯ 0.7%, p ϭ 0.002). This BMP-2 induced increase was similar in degree to that observed after culture of mIMCD-3 cells in serum-free medium (12.3 Ϯ 1.8%). In contrast, treatment with H-89 had no significant effect on apoptosis and totally abrogated the pro-apoptotic effect of BMP-2. Taken together, our results in embryonic kidney explants and in the mIMCD-3 model of collecting duct morpho- genesis demonstrate that inhibition of PKA, alone and in the presence of BMP-2, protects the collecting duct cell from undergoing apoptosis.
Interaction of PKA with Intracellular Signaling Events Downstream of BMP-2-We have demonstrated that PKA modulates morphogenetic and cellular responses to BMPs. Our results provide a basis for determining the interactions between PKA and BMPs at a molecular level. Several events downstream of BMP/cell surface receptor molecular complexes have been described (19). Specifically, activated BMP receptor phosphorylates the cytoplasmic protein Smad1. Phosphorylated Smad1 complexes to Smad4 and then Smad1 translocates to the nucleus, where it is postulated to act as a transcription factor. Because BMP-mediated inhibition of branching morphogenesis is dependent on PKA, we hypothesized that PKA could induce one or more events downstream of the BMP receptor. Likewise, we predicted that inactivation of PKA would be disruptive to BMP signaling.
First, we investigated the effects of PKA on BMP-2-induced Smad1 phosphorylation in mIMCD-3 cells. mIMCD-3 cells were labeled with [ 32 P]phosphate and then treated with either 5 nM BMP-2, 5 M 8-Br-cAMP, 5 M H-89, BMP-2/H-89, or BMP-2/8-Br-cAMP for 1 h. Cellular proteins were immunoprecipitated with anti-Smad1 antibody, and the immunoprecipitated proteins were then analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 6A, BMP-2 induced a marked increase in Smad1 phosphorylation as indicated by both an increase in radioactive signal and a decrease in the electrophoretic migration of Smad1. This mobility shift is a direct result of phosphorylation of residues in the MH2 domain, inducing a conformational change in the protein and causing it to migrate more slowly than unphosphorylated Smad1 (19). In contrast, under basal conditions, phosphorylation of Smad1 occurs at residues located outside of the MH 2 domain, does not result in a conformational change, and does not result in formation of Smad1-Smad4 complexes and nuclear translocation (12). Treatment with 8-Br-cAMP or H-89 did not induce a shift in Smad1 mobility. Moreover, treatment either with 8-Br-cAMP or with H-89 in addition to BMP-2 did not modify the increase in Smad1 phosphorylation or the shift in Smad1 mobility seen in cultures treated with BMP-2 alone. The small degree of variation in the intensity of the Smad1 protein bands observed reflects fluctuations in the yield of Smad1 isolated in the immunoprecipitation procedure. Taken together, these data indicate that the BMP-2 and PKA pathways do not converge at the level of Smad1 phosphorylation.
Second, we determined the effect of PKA on BMP-2 induced Smad1-Smad4 complex formation in mIMCD-3 cells (Fig. 6B). For this, we subjected lysates from mIMCD-3 cells to anti-Smad1 immunoprecipitation followed by immunoblotting with an anti-Smad 4 antibody as described previously (12). Under basal conditions, whereas cell lysates contained Smad4 protein (Fig. 6B, Basal), Smad 4 was not detected in association with Smad1, indicating that Smad1-Smad4 complexes do not exist under basal conditions. Similar to these controls, cells treated with 8-Br-cAMP or H-89 did not contain Smad1-Smad4 complexes. In contrast, treatment with 5 nM BMP-2 for 60 min resulted in the formation of Smad1-Smad4 complexes, and the efficiency of association was unaffected by 8-Br-cAMP or H-89. Taken together, these data indicate that the BMP-2 and PKA pathways do not converge at the level of Smad1-Smad4 complex formation.
Smad1-Smad4 complexes translocate to the nucleus, where they function to activate BMP-responsive genes. Thus, we determined whether PKA altered BMP-2 induced Smad1 cytoplasmic to nuclear translocation in collecting duct cells using immunofluorescence and confocal laser microscopy (60 cells analyzed/treatment group) (Fig. 6C). Under basal conditions, Smad1 protein was detected in a punctate distribution throughout the cytoplasm of mIMCD-3 cells similar to previous , or preimmune sera (basal and BMP-2) and then analyzed by immunoblotting using antibody to Smad4 (arrow). Lower panel, immunoblot analysis with anti-Smad1 antibody of cellular proteins immunoprecipitated with anti-Smad1 antibody. A band corresponding to the nonphosphorylated form of Smad 1 is seen in all lanes. A slower migrating protein, marked by an asterisk, is seen in the BMP-2, BMP-2/8-Br-cAMP, and BMP-2/H-89 treatment groups. This band corresponds to the phosphorylated form of Smad1. C, effect of PKA on BMP-2-dependent Smad 1 nuclear translocation in mIMCD-3 cells. Immunostaining was performed using an antibody to Smad1, followed by a fluorescein-conjugated secondary antibody. Images were obtained by confocal laser microscopy (ϫ 480 magnification).
reports for transiently expressed Smad5 (20) and Smad2/3 (21,22). However, after a 60-min treatment with 5 nM BMP-2, translocation of Smad1 protein was identified by a shift in fluorescent signal from cytoplasm to nucleus in 78% cells analyzed. No signal was detected using preimmune antisera and fluorescein-conjugated secondary antibody (data not shown). Treatment with either 5 M 8-Br-cAMP or with 5 M H-89 alone did not induce Smad1 nuclear translocation and did not interfere with BMP-2 induced Smad1 nuclear translocation. Taken together, our experiments indicate that activation of PKA alone does not induce any of the known steps in the BMP-2 pathway. Furthermore, although antagonism of PKA is dominant over the inhibitory effect of BMP-2 on renal branching morphogenesis, it does not disrupt the known biochemical events that are activated in response to BMP-2. These findings suggest that PKA controls the actions of BMP-2 via a parallel signal transduction pathway.
BMP-2 Induces Activation of PKA and the PKA Effector, CREB, in Collecting Duct Cells-Because the inhibitory effect of BMP-2 on renal branching morphogenesis requires PKA activation, and PKA cannot induce BMP-2-mediated signaling events, we hypothesized that BMP-2 positively regulates PKA, thereby regulating its own activity. We investigated this possibility by measuring PKA kinase activity in collecting duct cells. Under basal conditions, PKA activity, as measured by the phosphorylated state of a PKA-specific target peptide, was low (Fig. 7A). However, after 10 min of treatment of mIMCD-3 cells with BMP-2, we observed a modest increase in the phosphorylated fraction of the PKA substrate peptide, suggesting increased PKA kinase activity (data not shown). Because we observed the effects of BMP-2 on collecting duct morphogenesis 48 h after cell cultures were established (Figs. 1 and 3A), we determined the effect of BMP-2 on PKA activity at this time point. At 48 h, BMP-2 markedly increased PKA activity, which was inhibited by either a short (2 h) or long (48 h) co-incubation with H-89. Induction of PKA activity by BMP-2 was qualitatively similar to that observed after treatment for 48 h with 5 M 8-Br-cAMP alone (Fig. 7A). To determine whether BMP-2 also positively regulates molecules known to function as transcription factors downstream of PKA, we measured phosphorylation of CREB, a PKA substrate (23). Treatment with BMP-2 induced phosphorylation of CREB within 1 h of treatment (Fig. 7B). This induction was completely abrogated by treatment with H-89, indicating that PKA is required for BMP-2-mediated phosphorylation of CREB. Taken together, these results demonstrate that PKA and CREB are positively regulated by BMP-2.

DISCUSSION
Several types of evidence strongly suggest that members of the BMP family regulate renal branching morphogenesis. The spatial and temporal expression pattern of BMP-2 and BMP-7 is consistent with their role in modulating the inductive tissue interactions that control branching morphogenesis (2). Mutational inactivation of Bmp-7 causes kidney maldevelopment, including abnormal branching morphogenesis (24,25). Recombinant BMP-2 and BMP-7 protein control branching morphogenesis in embryonic kidney explants and in the mIMCD-3 model of collecting duct morphogenesis. Interestingly, these BMPs exert specific and dose-dependent effects. BMP-2 is inhibitory, whereas BMP-7 stimulates at low doses (Ͻ0.5 nM) and inhibits at higher doses (3). The stimulatory effects of BMP-7 taken together with the phenotype of the Bmp-7 Ϫ/Ϫ mouse suggest that low dose BMP-7 acts via a stimulatory signaling pathway. In contrast, high doses of BMP-7 and BMP-2 act via the inhibitory Smad 1 signaling pathway. 2 The ureteric bud or collecting duct cells exist within a complex environment of growth factors, including BMPs, and must integrate these diverse signals to form a tubular network. PKA is an intracellular kinase that interacts with both receptor tyrosine kinase (10) and serine/threonine kinase pathways in several tissue types, including the central nervous system (5,6), the eye (7), and the limb (8). In this work, we show that PKA directly controls renal branching morphogenesis and, within this context, modifies the morphogenetic effects of stimulatory and inhibitory BMPs, and HGF. Our results in an in vitro culture model of tubulogenesis and in embryonic renal explants demonstrate that PKA directly controls renal branching morphogenesis. When PKA is activated, tubulogenesis is inhibited, whereas when PKA is antagonized, tubulogenesis is markedly enhanced. Our work also shows that PKA modifies specific growth factor-induced signaling events in the collecting duct cell. PKA inhibits the stimulatory effect of low dose BMP-7 and HGF. Consistent with these effects, antagonism of PKA in the presence of either stimulatory factor further enhances tubulogenesis. In addition, antagonism of PKA rescues the inhibitory effects of BMP-2 and high dose BMP-7. These observations suggest that PKA functions to modulate the cellular response to stimulatory and inhibitory signals during renal branching morphogenesis.
We have demonstrated the effect of PKA on collecting duct formation at a morphogenetic level. Tubule formation requires a unique combination of cellular events including cell prolifer- ation, apoptosis, cell shape change, and cell movement. Our morphogenetic data suggest that PKA exerts an effect on tubular cell number, as indicated by increased tubule formation. Indeed, our results in embryonic kidney explants demonstrate that antagonism of PKA significantly decreases collecting duct apoptosis and abrogates the apoptotic effect of BMP-2. Thus, the protubulogenic effect of H-89 and PKI may arise secondary to an increase in the population of collecting duct cells as a direct result of decreased cellular apoptosis. Further studies will be required to identify other cellular events that may be implicated in the control of tubulogenesis by PKA.
We have examined the function of PKA at a molecular level within the BMP pathway. Our morphogenetic data suggest that BMP-2 and high dose BMP-7 are dependent on PKA. Events downstream of BMP cell surface receptors have been well characterized (19). The cytoplasmic protein Smad1 plays a central role in these events. Smad1 is phosphorylated as a result of BMP/receptor complex formation. This, in turn, permits its translocation to the nucleus. We determined that activation of PKA does not induce Smad1 signaling. Likewise, antagonism of PKA in the presence of BMP-2-induced signaling did not disrupt Smad1 activity. These results suggest that PKA interacts, directly or indirectly, with BMP signaling at the level of the cell nucleus. Such an interaction could be mediated either by PKA itself or one of its downstream targets. The best characterized downstream nuclear target of PKA is CREB (reviewed in Ref. 26). Activation of the PKA catalytic subunit leads to phosphorylation of CREB at serine 133, which allows recruitment of the coactivator CREB-binding protein and binding of this complex to cAMP-response element DNA sequences (23). The possibility that PKA controls BMP-mediated gene transcription is supported by the observation that the BMP-2 homologue dpp stimulates transcription of the ultrabithorax gene in Drosophila by inducing CREB binding to a cAMPresponse element-responsive element (27). Indeed, the ability of TGF-␤-inducible Smads (Smad2, Smad3, and Smad4) to interact with CREB-binding protein has recently been demonstrated (28 -31). These observations suggest that the Smad and PKA signaling pathways converge to coordinate gene expression during renal branching morphogenesis. Further studies are required to identify genes that are targeted by CREBbinding protein-BMP-inducible Smad complexes.
Our results that indicate that PKA kinase activity is low in mIMCD-3 cells under basal conditions and that BMP-2 positively regulates PKA and CREB provides insight into the mechanisms that underlie tubulogenesis during renal development. These findings suggest that PKA activity is low in ureteric bud or collecting duct cells that are not exposed to BMP-2 (or inhibitory doses of BMP-7), thus promoting tubulogenesis. However, in areas of tissue where collecting ducts are exposed to inhibitory BMPs, the activity of the PKA signaling pathway is increased, thus promoting inhibition of tubulogenesis.
The mechanism by which BMP-2 activates PKA remains to be determined. PKA activation was originally described as being dependent on cAMP induction (reviewed in Ref. 26). However, recent evidence demonstrates that cAMP-independent activation of PKA can occur via the association of the catalytic subunit of PKA with IkB-a or IkB-b in a NF-kB-IkB-PKAc complex (32). Signals that cause the degradation of IkB release the catalytic subunit of PKA, thereby activating it. Interestingly, TGF-␤ has recently been reported to induce PKA kinase activity by a cAMP-independent mechanism that is not yet fully characterized (11). Future studies aimed at determining the interactions between BMP signaling intermediates, PKA, and other intracellular proteins will provide insight into the mechanisms that underlie the induction of PKA by BMP-2 in collecting duct cells.
The spatial and temporal patterning of the renal collecting system is precisely controlled (1). Our previous work, as well as that of others, suggests that this development is controlled simultaneously by stimulatory and inhibitory peptide growth factors. Our current work provides insight into how these signals are integrated within the collecting duct cell and provide a basis for further investigating the genetic control of tubulogenesis.