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Originally published In Press as doi:10.1074/jbc.M001964200 on April 14, 2000

J. Biol. Chem., Vol. 275, Issue 28, 21233-21240, July 14, 2000
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Distinctive Cyclic AMP-dependent Protein Kinase Subunit Localization Is Associated with Cyst Formation and Loss of Tubulogenic Capacity in Madin-Darby Canine Kidney Cell Clones*

Stephanie A. OrellanaDagger § and Carmela Marfella-Scivittaro

From the Departments of Pediatrics and Dagger  Physiology and Biophysics, Case Western Reserve University School of Medicine and The Rainbow Center for Childhood PKD at Rainbow Babies and Children's Hospital of the University Hospitals of Cleveland, Cleveland, Ohio 44106

Received for publication, March 8, 2000, and in revised form, April 6, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Polycystic kidney disease is characterized by abnormal morphological development. Mechanisms that regulate cyst development may involve multiple signaling pathways. Cyst formation by Madin-Darby canine kidney (MDCK) cells in three-dimensional culture is assumed to be cyclic AMP-dependent and due to cyclic AMP-dependent protein kinase (cAPK) activation based on pharmacological responsiveness. To determine if different cyclic AMP (cAMP) pathways are associated with morphological development, the role of cAMP in regulating morphological change was examined in MDCK clones that form tumor-like or tubular structures under basal conditions. Pharmacological cAMP pathway activators induce cyst formation and diminish formation of other structures in three clones, whereas one clone is unaffected. Tyrosine kinase-mediated morphogens have little effect. Although all clones have intact cAMP signaling pathways, each has a unique subcellular distribution of cAPK regulatory subunits. This may reflect distinct mechanisms for cAPK anchoring, allowing cAPK subtype regulation of the unique phenotypic character of each clone through preferential access to substrates. These observations suggest a molecular basis for differential cAMP responsiveness in cells that develop distinct morphological phenotypes. This evidence establishes these MDCK clones as models for understanding the mechanism and functional significance of cAPK subunit localization and may have broader implications for cystogenesis in polycystic kidney disease.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Formation of three-dimensional structures in developing kidney is crucially important for normal renal functioning. Although specific genes and developmental sequences have been defined (reviewed in Refs. 1 and 2), there are still gaps in basic knowledge about intracellular steps required to induce and extend normal tubular nephron structure and to develop its functional capacity. This basic information is important to the study of renal developmental disorders like polycystic kidney disease, where defective nephrogenesis can lead to abnormal structure and result in loss of renal function.

Madin-Darby canine kidney cells (3) are a renal epithelial cell line whose collecting tubule-like cells have been used to characterize renal transport physiology, to study polarized sorting of epithelial cell membrane proteins, and to study morphogenesis. The range of phenotypic variety observed in MDCK1 cells is now accepted as the result of both cellular heterogeneity and some cellular multipotence (Refs. 4-7 and comments associated with Ref. 8). In three-dimensional collagen gel culture, MDCK cells can form spherical cysts by clonal growth (9), can be induced to enlarge after stimulation with cAMP analogues (10-12), and can form branching tubules in response to HGF (13, 14). Many more reports have implicated tyrosine kinases, phosphoinositide metabolism, and beta -catenins in tubule formation and branching morphogenesis (14-19). Although cellular mechanisms for tubule formation have been addressed in detail, reports addressing MDCK cyst formation have used pharmacological responsiveness to activators and inhibitors of cAMP pathway components to demonstrate cAMP-dependence (Refs. 6, 10-12, 14, and 20 and reviewed in Ref. 7). Several reports have addressed the role of the cAMP pathway in MDCK cell proliferation and chloride and fluid transport (10, 11, 21-25). None of these studies have fully addressed links between cAMP, the specific effector of cAMP action, and cystogenesis.

The cAMP-dependent protein kinase is the primary cellular mediator of cAMP action and is a holoenzyme composed of four subunits: two regulatory (R) and two catalytic (C). The binding of two cAMP molecules to each R subunit promotes the release of C subunits, which then phosphorylate substrate proteins on serine residues. Both R and C subunits exist as multiple isoforms that probably contribute to the range of cellular functions attributed to cAMP, including modulation of enzyme activity, membrane channel activity, and gene expression (reviewed in Refs. 26 and 27). In addition to diversity generated by subunit isoforms, cAPK can be localized to specific intracellular locations by A-kinase anchoring proteins (AKAPs) (reviewed in Ref. 28), thus allowing cAPK action to be directed to discrete subcellular sites and targets (29, 30). Nuclear C subunits translocated from the cytoplasm to regulate gene expression are inhibited and shuttled back out by the endogenous cAPK inhibitor, PKI (protein kinase inhibitor) (31-37). Kinase activity is turned off in the cytoplasm as cellular cAMP levels decline and cAPK holoenzyme is reformed.

Observations from studies of four novel MDCK cell clones isolated from original American Type Culture Collection MDCK CCL-34 (6) support the suggestion that induction of intracellular cAMP formation negatively regulates tubular structure formation in certain clones and implies that cystogenesis may represent a "default pathway" for impaired tubulogenesis. It is possible that cross-talk between signaling pathways, specific target substrates, and/or variable expression of pathway component isoforms may regulate the appearance of morphological phenotypes in different cell types. Therefore, the focus of this study is the role of cAPK in mediating both cyst formation and the inhibition of tubule formation. The present study uses the four previously described MDCK clones (6) as tools to understand the molecular mechanism for cAMP regulation of MDCK morphology. Pharmacological, biochemical, microscopic, and molecular genetic techniques were used to examine cAPK in these clones. Differences in cAPK components were found that may be important for in vitro cyst formation and for inhibition of tubule formation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Stocks of MDCK cell clones (clone OR93.22.D6 is designated 22 in this report, OR55.25.II20 is designated 25, OR55.28.V2 is designated 28, and OR55.29 is designated 29) were cultured as described previously (6). Cells in three-dimensional Vitrogen-100® (Cohesion, Palo Alto, CA) gels were cultured in defined medium (6) with experimental changes noted in the figure legends. Quantitation of structure formation after 5-9 days of gel culture was performed as described previously (6). Briefly, for each sample, the incidence of specific cellular structures (process-forming, cystic, and tumor-like) was scored relative to the total number of structures observed in the sample. Process formation is a predictor of tubule formation, cystic structures have defined cyst walls and open interior lumens, and dense cell spheres or cysts with wall thicknesses greater than the diameter of a lumen are designated "tumor-like." MDCK cells used for intracellular cAMP determinations, cAMP-dependent protein kinase assays, reporter gene assays, Western analysis of protein expression, and immunofluorescence studies were seeded in 0.4-µm pore Transwell-Clear filter inserts (Corning Costar Corp., Cambridge, MA) at approximately 1800 cells/cm2. Under these conditions, cells formed confluent monolayers by 1 week in culture. Although time frames for experimental determinations in filter culture were not the same as for three-dimensional structure formation in gel culture, intact epithelial layers had been established before assay or treatment in all cases, as demonstrated by transepithelial resistance measurements (6). All filter-grown cultures were maintained in serum-containing Dulbecco's modified Eagle's medium/F-12 (1:1, Life Technologies Inc.) until used for experiments, when growth medium was replaced with defined medium lacking PGE1 (6). All growth and experimental incubations of live cell cultures took place in a tissue culture incubator with a humidified 37 °C atmosphere of 5% CO2, 95% air.

Intracellular cAMP Determinations-- Cells were seeded onto 12-mm diameter Transwell-Clear inserts (Corning Costar Corp.) and maintained in culture as described above. Growth medium was removed, and confluent cell monolayers were washed 3× with PBS (10 mM Na3PO4, 150 mM NaCl, pH 6.9). Defined medium with additions indicated in the figure legends was added, and cultures were again placed in the tissue culture incubator for 1 h. Monolayers were then placed on ice, washed 3× with PBS, prepared for intracellular cAMP determinations using the cell lysis and assay reagents from the BIOTRAK cAMP enzyme immunoassay kit (RPN225, Amersham Life Science), and assayed according to the manufacturer's protocol. Values were normalized for protein content.

Cyclic AMP-dependent Protein Kinase Assays-- Cells were seeded onto 24-mm diameter Transwell-Clear inserts (Corning Costar Corp.) and maintained in culture as described above. Samples were prepared for assay as described (38). Cyclic AMP-dependent protein kinase activity was measured using the Colorimetric PKA SpinZymeTM Format assay kit (29500, Pierce) in the presence or absence of cAMP. The difference between the two values reflects cAMP-dependent activity. In the present study, activity in the absence of cAMP was negligible. The assay, conducted according to the manufacturer's protocol, detects the incorporation of phosphate into a specific dye-labeled cAPK substrate, Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Purified C subunit from bovine heart (Pierce) was used to generate a standard activity curve. All sample values were normalized for protein content.

Reporter Gene Assay-- To measure the ability of each clone to induce cAMP-mediated gene expression, all clones were transiently co-transfected with expression vectors encoding luciferase and beta -galactosidase cDNAs, as described previously (39). Luciferase cDNA is under the control of a promoter that confers sensitivity to activation of cAPK (40), whereas the beta -galactosidase cDNA is under the control of a constitutive promoter. Confluent Transwell-Clear filter cultures of MDCK clones were transfected with cDNA and LipofectAMINETM (Life Technologies, Inc.). Mixtures of LipofectAMINETM (2.7 µg/12 mm insert), alpha -168-luciferase cDNA (91 µg/12-mm insert), and Rous sarcoma virus-beta -galactosidase cDNA (91 µg/12-mm insert) were prepared according to the manufacturer's protocol in Dulbecco's modified Eagle's medium/F-12 (1:1) medium. Lipid/cDNA mixtures were left on the cells for 24 h. Medium was removed and replaced with defined medium plus additions indicated in Fig. 4 for 6 h. Cell lysates were harvested as described previously (39) and assayed using Dual-LightTM reagents (Tropix, Inc., Bedford, MA). Luciferase activity (in arbitrary units) was determined by dividing luciferase values by beta -galactosidase values for each sample replicate to correct for any variability in transfection efficiency between samples.

Immunological Reagents-- Primary antibodies used were: anti-cAPK-catalytic subunit alpha  (sc-903), a rabbit IgG against human Calpha (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-cAPK-catalytic subunit (P73420), a mouse IgG against human C (Transduction Laboratories, Lexington, KY); anti-cAPK-regulatory subunit type I (P19920), a mouse IgG against mouse RI (Transduction Laboratories); anti-cAPK-regulatory subunit type Ialpha (P53620), a mouse IgG against human RIalpha (Transduction Laboratories); anti-cAPK-regulatory subunit type IIalpha (sc-909), a rabbit IgG against mouse RIIalpha (Santa Cruz Biotechnology, Inc.); and anti-cAPK-regulatory subunit type IIalpha (P55120), a mouse IgG against human RIIalpha (Transduction Laboratories). The fluorescent-tagged secondary antibody used was Oregon Green 488 goat anti-mouse IgG conjugate (O-6380, Molecular Probes, Eugene, OR). Peroxidase-linked secondary antibodies used were: goat IgG against rabbit IgG (A0545, Sigma); rabbit IgG against mouse IgG (A9044, Sigma); goat IgG against rabbit IgG (sc-2004, Santa Cruz Biotechnology, Inc.); and goat IgG against mouse IgG (sc-2005, Santa Cruz Biotechnology, Inc.).

Western Analysis of Protein Expression-- To characterize protein expression in cultured cells, samples grown on filter inserts were harvested, subjected to SDS-polyacrylamide gel electrophoresis, immunoblotting, and signal detection with ECLTM (Amersham Pharmacia Biotech) essentially as described previously (41). Protein loaded in each lane was equal for all clones (10 µg). Primary antibodies used (listed above) were: anti-Calpha sc-903 at 1:1000; anti-RI P19920 at 1:100-250 and P53620 at 1:100; and anti-RIIalpha sc-909 at 1:250-500 and P55120 at 1:250. Peroxidase-linked secondary antibodies were used at 1:1000-10,000 dilutions. Specific primary and secondary antibody combinations are indicated in the figure legends. Densities of the resulting specific bands shown in Fig. 5 were measured with a Sci Scan 5000 densitometer using the OS-Scan Image Analysis System 0306-371-10001 Release 4.2 (Oberlin Scientific Corp., Oberlin, OH) of the Molecular Biology Core Laboratory at Case Western Reserve University. Sample values are expressed as arbitrary units relative to the value resulting from the least dense band (set to 1 unit) in each subunit group.

Localization Studies-- Confocal laser scanning microscopy was used to localize specific proteins identified by immunofluorescence. Cells were seeded onto 12-mm-diameter Transwell-Clear inserts and maintained in culture as described above. Primary antibodies (listed above) used were anti-Calpha P73420, anti-RI P19920, and anti-RIIalpha P55120. Nonspecific binding sites were blocked, as described below, with normal serum from the host animal used to raise the secondary antibody, in this case, GS (Sigma). The secondary antibody O-6380, with a fluorescent tag, used is listed above. The specificity of antibody immunoreactivity was demonstrated using controls lacking primary antibodies, which in all cases resulted in no detectable fluorescent signal (data not shown).

All steps were performed at room temperature unless noted and are essentially as described previously (42). Similar methods have been used to visualize cytoskeletal and tight junction proteins in confluent MDCK filter cultures (43, 44). Insert cultures were fixed with 3.7% paraformaldehyde in PBS for 10 min. Surfaces were washed 3× with PBS for 5 min each. Monolayers were permeabilized from the apical surface with 0.1% Triton X-100 in PBS for 5 min. Both surfaces were then washed 2× with FBS/PBS (10% FBS in PBS) and then with GS/PBS (10% GS in PBS). The apical GS/PBS wash was removed and replaced with primary antibody diluted 1:100 in GS/PBS. After a 1-h incubation in the 37 °C humidified incubator, samples were washed 3× with FBS/PBS. The last FBS/PBS wash was removed from the apical surface and replaced with the appropriate secondary antibody diluted 1:250 in GS/PBS. After a 30-min incubation at 37 °C, surfaces were washed 2× in FBS/PBS and then in PBS. The Transwell-Clear membrane was cut out and mounted on a standard glass microscope slide with SlowFadeTM Antifade kit component A (Molecular Probes).

All samples were examined, as described previously (42), with a Zeiss LSM 410 scanning laser confocal microscope using the 488/568-nm wavelength lines of an argon-krypton laser. Using a Zeiss 100× Neofluor objective and Zeiss LSM software, the cell monolayer was optically sectioned every 0.5 µm. Image resolution was 512 × 512 pixels.

Other Information-- Statistical determinations are indicated in the figure legends. Protein was quantitated using either the Bio-Rad or BCA (Pierce) protein assay reagents, according to the manufacturer's protocol. Reagents not specifically listed above were either cell culture or molecular biology grade and were obtained from Sigma, Calbiochem-Novachem Corp., Life Technologies, Inc., or R&D Systems Inc. (Minneapolis, MN)

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Clonal Differences Were Observed in Morphological Responsiveness to cAMP Pathway Agonists-- In our previous report (6), Vitrogen-100® gel cultures of MDCK clones 22, 28, and 29 formed tumor-like structures in the absence of cAMP agonists, and clone 25 formed tubular structures. All 4 clones had similar transepithelial resistances, but only clone 29 formed domes in monolayer culture (6). To examine the role of cAMP-signaling system components in regulating clonal cyst and tubule formation, gel cultures of each clone were overlaid with defined medium lacking PGE1. Cultures were then incubated with pharmacological agents targeted to cell surface receptors (PGE1, arginine-8-vasopressin, isoproterenol), heterotrimeric GTP-binding protein Galpha s (cholera toxin), adenylyl cyclase (forskolin), cAMP phosphodiesterase (IBMX), or cAPK (cell-permeable cAMP analog 8BrcAMP), and the formation of specific structures was scored as described under "Experimental Procedures." The phosphodiesterase inhibitor IBMX had little effect on structure formation at 100 µM and was included in all other conditions to block the breakdown of intracellular cAMP generated by specific agonists (Fig. 1) (11). As described previously (6) and shown here as a positive control (Fig. 1), increasing concentrations of PGE1 resulted in cyst formation in all clones except clone 28, which formed spontaneous tumor-like structures and was previously shown to be incapable of PGE1-mediated cyst formation. Neither vasopressin nor isoproterenol had any effect on cyst formation in these clones (data not shown), unlike previously described MDCK clonal cell lines (reviewed in Ref. 7). Cholera toxin, forskolin, and cyclic 8-bromo-adenosine 3',5'-monophosphate 8BrcAMP effects were similar to those of PGE1 (Fig. 1). These agents also decreased the formation of processes (clone 25) and tumor-like structures (clones 22 and 29) in responsive clones that expressed these phenotypes but had no effect on clone 28. Doses required for stimulation of cyst formation and loss of original phenotype were nearly identical within each responsive clone and were similar between clones (Fig. 1). Also, the order of effectiveness was the same for all responsive clones. Therefore, activation of the cAMP pathway at several levels resulted in cyst formation and loss of the original phenotype of the clone in all clones except clone 28. 


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  		Fig. 1.   All clones except 28 respond to activators of the cAMP signaling system with decreases in process or tumor-like structure formation and increases in cyst formation. Experiments were performed as described under "Experimental Procedures" using the indicated concentrations. The cyclic 8-bromo-adenosine 3',5'-monophosphate (8BrcAMP) experiments were scored at 7 days, whereas all others were scored at 5 days. The IBMX time 0 condition was incubated in defined medium only; all others were in the presence of 100 µM IBMX. Clone names (22, 25, 28, or 29) are indicated in each panel. Values are the means ± S.D. of n = 3-7 experiments performed in duplicate.

Clonal Differences in cAMP Responsiveness Were Not Due to Lack of cAMP Pathway Components or Ability to Activate cAMP-mediated Gene Expression-- To determine if the clone 28 lack of responsiveness to cyst-promoting agonists was the result of missing or defective cAMP pathway components, intracellular cAMP accumulation in response to agonists was measured in clones grown on filter insert cultures. As shown in Fig. 2, all clones had similar basal levels of cAMP. All clones showed increases in intracellular cAMP levels in response to PGE1 and forskolin, although the magnitude of the responses differed. Clones 22, 25, and 28 responded to PGE1 or forskolin with cAMP levels between 2.5-7.4-fold greater than with IBMX alone. In general, clone 29 was less responsive; cAMP accumulation in response to forskolin was1.8-fold greater than IBMX alone and in response to PGE1 was 1.5-fold greater than IBMX alone. Clone 25 displayed the greatest difference in cAMP levels produced in response to the two stimuli. Nevertheless, these results support the conclusion that coupling of receptor, GTP-binding proteins, and adenylyl cyclase in each clone was intact and that the inability of clone 28 to form cysts was not the result of an inability to elevate intracellular cAMP levels.


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  		Fig. 2.   Intracellular cAMP levels in all clones increase in response to extracellular agonists. Experiments were performed as described under "Experimental Procedures," with either defined medium plus 100 µM IBMX, 70 nM PGE1 plus 100 µM IBMX (PGE1), or 10 µM forskolin plus 100 µM IBMX (Forskolin). Clone names (22, 25, 28, or 29) are indicated in each panel. Values are mean ± S.D. of n = 3-4 experiments performed in duplicate.

To determine if the lack of responsiveness of clone 28 to cyst-promoting agonists was the result of missing or defective cAPK, total cAPK activity was measured in clones grown on filter insert cultures. While the activity assay does not distinguish between Type I and Type II subtypes, as shown in Fig. 3 all clones had measurable cAMP-dependent cAPK activity. No cAMP-independent, basal activity was detected in any clone. Therefore, the inability of clone 28 to form cysts was not the result of a lack of cAPK activity, although clonal differences were observed in overall activity levels.


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  		Fig. 3.   All clones have cAPK activity. Experiments were performed as described under "Experimental Procedures." Values are mean ± S.D., n = 3-5 experiments performed in duplicate.

Since one of the results of cAMP pathway activation is regulation of cAMP-inducible genes, differences in the capacity of the four clones to induce cAMP-mediated gene expression were evaluated. Confluent clones grown on filter inserts were transiently transfected with a cAMP-responsive luciferase reporter gene as described under "Experimental Procedures." After a 6-h incubation with PGE1 or forskolin, agonists that induce intracellular elevations of cAMP and presumably activate cAPK in all clones (Figs. 2 and 3), induction of reporter activity was measured. All clones showed some amount of increased gene expression in response to the cAMP phosphodiesterase inhibitor, IBMX (Fig. 4). Interestingly, clone 28 had severalfold higher levels of reporter activity than the 3 clones that responded to agonists with cyst formation. Clone 25 showed the largest PGE1-mediated induction of gene expression over IBMX alone, compared with other clones that showed minimal increases. Consistent with the difference observed in cAMP accumulation, forskolin did not produce a gene expression response in clone 25. Clone 29, the clone able to form the largest cysts, displayed little PGE1 or forskolin-stimulated gene expression greater than with IBMX alone. However, all clones were capable of some level of reporter gene expression.


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  		Fig. 4.   Expression of a cAMP-dependent luciferase reporter gene is induced in all clones. Experiments were performed in duplicate as described under "Experimental Procedures," with either defined medium alone (Control), 100 µM IBMX, 70 nM PGE1 plus 100 µM IBMX (PGE1), and 10 µM forskolin plus 100 µM IBMX (Forskolin). Clone names (22, 25, 28, or 29) are indicated in each panel. Values are the mean ± S.D., n = 3-4, except for clone 25 IBMX and PGE1, clone 28 forskolin, and clone 29 control and IBMX, which are means of 2 experiments.

Clonal Differences in cAPK Subunit Localization Were Observed-- The results of experiments to characterize intracellular cAMP signal transduction and the expression of specific clonal phenotypes in gel culture suggested differences could occur at the level of the cAPK or beyond. Therefore, expression and localization of the most widely expressed cAPK subunit isoforms RIalpha , RIIalpha , and Calpha were determined (reviewed in Ref. 27). All clones expressed cAPK subunit proteins RIalpha , RIIalpha , and Calpha , as shown by the results of Western analyses of confluent Transwell cultures (Fig. 5). Relative expression levels for RIIalpha were 1.9, 2.2, 1.0, and 2.0 arbitrary units for clones 22, 25, 28, and 29, respectively. Relative expression levels for RIalpha were 1.4, 1.7, 1.0, and 2.3 units. Relatively greater expression of Calpha was detected in samples from clones 22 (4.1 units) and 25 (3.2 units) compared with clones 28 (1.5 units) and 29 (1.0 unit), but this greater relative mass was not correlated with greater cAPK activity (Fig. 3). However, all clones had the potential to express both Type I and Type II cAPK activities.


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  		Fig. 5.   All MDCK clones expressed cAPK subunits RIIalpha , RIalpha , and Calpha . Western analysis of clonal cAPK subunit expression in unstimulated, confluent cultures was performed as described under "Experimental Procedures," and data shown are representative of 2-3 experiments. Antibody combinations for Western analyses shown were: anti-RIIalpha sc-909 (1:250), anti-RI P19920 (1:250), or anti-Calpha sc-903 (1:1000), each with the appropriate peroxidase-linked secondary antibody sc-2004 or sc-2005 (each at 1:2000). Each pair of cAPK subunit antibodies tested ("Experimental Procedures") resulted in immunoreactive bands at the apparent molecular weights indicated in a total of 4-5 experiments performed on samples grown either in defined or serum-containing media. Clone names (22, 25, 28, or 29) are indicated above the appropriate lanes.

Confocal laser scanning microscopy of immunostained filter-grown cultures demonstrated that Calpha subunit was expressed similarly in all clones and was primarily distributed throughout the cytoplasm (Fig. 6). However, each clone had a unique cellular distribution of cAPK regulatory subunits RIalpha and RIIalpha (Fig. 6). In clones 22 and 25, RIalpha was mainly cytoplasmic. In clone 28, RIalpha was primarily near the apical surface with some punctate staining. Clone 29 RIalpha immunostaining was characterized by clear punctate staining near the apical surface with less cytoplasmic staining. In clone 22, RIIalpha appeared outside of the nucleus but showed more intense staining on the apical perinuclear side of the cell layer. Clone 25 had a unique RIIalpha staining pattern, with RIIalpha clearly localized next to, and outside of, the nucleus. In clone 28, RIIalpha immunostaining was again outside the nucleus, with more intense staining in cytoplasmic regions of the cells. Clone 29 RIIalpha was cytoplasmic with some cells having more intense regions of staining, both perinuclear and cytoplasmic, than others. Therefore, although all clones had similar distributions of C subunit, there were clear clonal differences in the subcellular location of R subunits.


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  		Fig. 6.   Although all MDCK clones exhibit similarities in cellular expression, there are differences in localization of cAPK subunits Calpha , RIalpha , and RIIalpha . Confocal immunolocalization of cAPK subunits in unstimulated, confluent cultures was performed as described under "Experimental Procedures," and data shown are representative of three experiments. Antibodies used for confocal laser scanning microscopy were anti-Calpha P73420, anti-RI P19920, or anti-RIIalpha P15520, each with the secondary Oregon Green conjugate O-6380. Square images are single xy scans, and rectangular images are cross-sections reconstructed from a series of z scans. Bar in 22, Calpha represents 25 µm. Clone names (22, 25, 28, or 29) are indicated in each panel.

Clonal Phenotype Regulation by Tubular Morphogens Was Minimal-- The previous experiments showed that cysts were formed and tubular or tumor-like structures were inhibited as the cAMP pathway was activated in responsive clones. However, what mechanisms promote formation of tubular or tumor-like structures in the absence of cAMP activation? Since branching morphogenesis was studied extensively in other MDCK cells (14-18), roles for "tubular" growth factors were investigated in the four clones. Qualitative data in a previous report (6) suggested that rhHGF could modulate process formation in clones 22 and 28 but had little effect on the spontaneous process-forming clone 25 or on clone 29. To determine if rhHGF sensitivities were responsible for differences in clonal tubule and cyst formation, gel cultures were overlaid with defined medium with a range of rhHGF concentrations (1-100 ng/ml). A modest increase in incidence of process formation (15-20%) was observed only in rhHGF-responsive clones 22 and 28 with 30-100 ng/ml rhHGF in the presence of FBS (Fig. 7). For most clones, in the absence of FBS or in the presence of PGE1, the range of rhHGF doses tested failed to induce process formation and had little effect on cyst incidence or tumor-like structure incidences (Fig. 7). In clone 29, 10 ng/ml rhHGF was able to inhibit cyst formation induced by PGE1 by 30-40% (Fig. 7). Therefore, rhHGF may modulate cyst formation in clone 29, but it seems unlikely that clonal differences in rhHGF sensitivity account for a lack of rhHGF-inducible process formation in clones 25 and 29. No change in cAMP levels was observed in response to 30 ng/ml rhHGF (data not shown). No effect of rhHGF on cAMP-mediated gene expression was observed, since luciferase activity in response to 30 ng/ml rhHGF plus IBMX (0.454 ± 0.097, 0.243 ± 0.169, 2.623 ± 0.903, 0.052 ± 0.062 for clones 22, 25, 28, and 29, respectively; n = 3) was similar to activity in response to IBMX alone (Fig. 4). Therefore, there were no apparent clonal differences in HGF stimulation of process formation in the four clones. Since HGF-mediated tubule formation was demonstrated in Type I collagen and Vitrogen (95-98% Type I, remainder Type III) gel cultures (10, 14, 18), it is unlikely that a larger response might have been observed in the present study had a culture matrix other than Vitrogen-100® been used. Also, although the heterodimeric HGF available from the same source (R&D Systems Inc.) might have been more effective than the predominantly uncleaved single chain form used, this possibility was not evaluated since the effects of serum, which should have provided the necessary protease activity, were examined.


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  		Fig. 7.   Effects of rhHGF on clonal morphogenesis in gel culture are modest. Experiments were performed as described under "Experimental Procedures," with concentrations of rhHGF indicated in defined medium alone (Control), defined medium with 100 µM IBMX (IBMX), defined medium with 70 nM PGE1 alone (pge1), defined medium with 70 nM PGE1 plus 100 µM IBMX (PGE1), and standard growth medium plus 5% FBS (FBS). Clone 25 was scored at 5 days; all others were scored at 7 days. Clone names (22, 25, 28, or 29) are indicated in each panel. Values are mean ± S.D., n = 3-7.

Epidermal growth factor acted as a cyst initiator in human kidney cells (20) and a tubular morphogen in mIMCD-3 (murine intramedullary collecting duct-3) cells (18) but not in the MDCK cells used in those reports. To determine if EGF, an activator of intracellular tyrosine kinase activity like HGF, could play a role in modulating structure in the four MDCK clones of the present study, clonal responsiveness to EGF was evaluated. In clone 22, increasing EGF concentrations prevented the formation of PGE1-induced cysts and maintained tumor-like structures in the presence of PGE1 (Fig. 8). In clones 25, 28, and 29, in the presence of 100 µM IBMX or 70 nM PGE1 plus IBMX, EGF (1-30 ng/ml) had no effect on the incidence of each type of structure formed in gel culture (data not shown). Therefore EGF and HGF may modulate three-dimensional phenotypes in some clones, but overall the effects of tyrosine kinase-mediated morphogens on the types of structures formed by these four MDCK clones were relatively modest.


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  		Fig. 8.   EGF inhibits PGE1-induced cyst formation in clone 22. Experiments were performed as described under "Experimental Procedures," with concentrations of EGF indicated in defined medium with 100 µM IBMX (IBMX) or defined medium with 70 nM PGE1 plus 100 µM IBMX (PGE1). Structure formation was scored at 7 days. Clone name (22) is indicated in each panel. Values are mean ± S.D., n = 3-7.

Effects of tyrosine kinases on MDCK cell growth rather than structure formation were suggested by a recent report that HGF induced MDCK cell proliferation in culture on plastic or filter inserts but only after the culture reached confluence (45). To determine if tyrosine kinase signaling could affect MDCK cell growth, the nonspecific tyrosine kinase inhibitor, genistein, was included in gel cultures for 7 days. Genistein inhibited overall clonal growth under all conditions tested (defined medium by itself, with 70 nM PGE1 or 30 ng/ml rhHGF, or serum-containing growth medium); however, although resulting structures were ~5% of the sizes seen without the inhibitor, all 3 types were present (data not shown). The concentration of genistein used was well below 350 µM, the IC50 value at which genistein might have affected protein kinase C or cAPK (46). Therefore, the inhibitory effect of 20 µM genistein universally affected clonal expansion, and its effect was exerted primarily on tyrosine kinase-mediated growth, not structure formation.

Only clone 25 consistently produced tubular structures in gel culture, and these appeared spontaneously (6). In fact, when clone 25 was grown on filter inserts well past the point of confluence (7-9 days), tubular structures developed on the monolayer (Fig. 9). A similar phenomenon was previously reported after long term culture of classic Type II MDCK cells on plastic (44). In contrast, clone 29 produced spherical structures that were partially attached to the monolayer, and clones 22 and 28 remained simple monolayers (Fig. 9). The results suggest that constitutive expression of a cellular component associated with tubule or lumen formation accounted for the apparent spontaneity of the phenotype of clone 25. 


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  		Fig. 9.   Only clone 25 filter cultures form tubular structures. Filter insert cultures were seeded and maintained as described under "Experimental Procedures." Confluence was reached by 7 d, and cells were photographed on day 15 in bright field. Magnification is noted, and boxes indicate areas shown at 100×. Clone names (22, 25, 28, or 29) are indicated in each panel. Data shown are representative of more than 10 samples.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epithelial layers of all MDCK clones used in the present study had intact intracellular cAMP pathways despite differential morphological responses to cAMP system activators in gel culture. This result demonstrated that clonal differences in morphological phenotypes were not due to diminished sensitivity to cAMP pathway agonists or a defect in cAMP signaling. Effects of tubular morphogens were modest, showing that tyrosine kinase-mediated mechanisms for morphological change were unlikely to be involved in regulating clonal phenotype. However, the cellular balance and location of Type I and Type II cAPK subtypes in each clone are predicted to differ as a result of clonal differences observed in RIalpha and RIIalpha location and in relative levels of Calpha expression (Fig. 10). Initiation of cAMP signaling could preferentially activate cAPK subtypes whose proximity to differentially expressed cellular substrates would lead to negative regulation of tubulogenesis or tumor-like structure formation and promotion of cystogenesis, depending upon the clone. These results support a mechanistic role for cAPK subtype regulation of the unique phenotypic character of each clone.


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  		Fig. 10.   Each MDCK cell clone has a unique predicted pattern of cAPK subtype expression associated with a distinct morphological phenotype. This figure summarizes data shown in previous figures. The likely location of cAPK subtype I is indicated by I and of cAPK subtype II is indicated by II. The nucleus is represented by n.

The speculation that cAPK subtypes regulate MDCK morphogenesis by providing a cAMP signal access to specific substrates at discrete subcellular locations is supported by several reports describing compartmentalization of cellular responsiveness to cAMP. Even in the present study, differences in the responsiveness of clone 25 (cAMP accumulation and gene expression) to PGE1 and forskolin suggested cellular compartmentalization of cAMP pathway components associated with specific PGE1 receptor-mediated signaling. Reports using clonal MDCK lines selected for PGE1-independence and dibutyryl cAMP-resistance (22, 23, 25) suggested that PGE1 affected growth and differentiated function by different cAMP pathway mechanisms, including modulation of cAPK subtypes and cAMP phosphodiesterases. However, these studies did not address morphogenesis. Later reports that used either the parent MDCK line from the American Type Culture Collection or a single clonal line showed that cAMP stimulated cell proliferation, fluid secretion, and cyst formation, but that cAMP-mediated proliferation by itself was insufficient for cyst formation (10, 11). A report that investigated the role of protein kinase-mediated phosphorylation in modulation of MDCK cell tubulogenesis in gel cultures using pharmacological activators and inhibitors showed that cAPK activation promoted cyst formation and inhibited process formation in the clone used (14). Subtypes of cAPK were not examined in these reports, but instances of cAPK subtype regulation of cellular functions have been reported more recently. For example, Type I cAPK activity was found to be higher in cancer cells than in normal or non-proliferating cells, and selective inhibition of Type I cAPK in breast cancer cells blocked mitogenesis (reviewed in Ref. 47). A clear role for a cAPK subtype was demonstrated by the report that a unique AKAP (A-kinase anchoring protein) was responsible for presenting Type II cAPK and protein phosphatase-1 to a specific substrate, the N-methyl-D-aspartate receptor, whose ion channel activity was regulated by phosphorylation (48). Therefore, compartmentalization of cAMP responsiveness resulting in a range of functional outcomes has been demonstrated and may result from cAPK subtype expression.

MDCK diversity in the intracellular cAMP signals available to activate cAPK subtypes was demonstrated by clonal differences in cAMP accumulation in response to extracellular agonists. This could reflect the action of subsets of phosphodiesterase activities in each clone, since IBMX will not inhibit all subtypes, and forskolin was expected to activate a larger proportion of the available adenylyl cyclase of the cell than PGE1 would acting through a receptor-specific mechanism. Clonal diversity in substrates available to mediate cAPK action can be speculated upon, although at this point, the evidence that each clone exhibited a unique pattern of cAPK subunit localization suggests that subcellular co-localization of a cAPK subtype with a universally expressed substrate is equally likely. However, specific cAPK substrates or genes affected as cysts form and other structures are lost in the four clones are presently unknown. In clone 28, cAMP and cAPK were unlikely to have stimulated the fluid secretion required for cyst expansion, since structures were characteristic of those in which cAMP-mediated fluid secretion was blocked and uncoupled from cell proliferation (10). However, the magnitude of clone 28 cAPK-mediated gene expression response suggested that components of this pathway were very sensitive to agonists or were localized near the site of cAMP synthesis, compared with those in other clones. In clone 29, cyst formation was unrelated to cAPK-mediated gene expression and probably involved more direct actions on cellular enzyme activities or channels that lead to fluid transport. The existence of these clonal differences supports the hypothesis that mechanisms at (subtypes) and/or downstream of (specific gene targets, cellular enzyme activities, or ion channels and fluid transport) cAPK were responsible for phenotypic differences observed. Intracellular variability in the cellular balance between the two cAPK subtypes along with the clear clonal differences in subcellular distribution could be the basis for preferential modulation of substrates.

For example, the cellular component responsible for the spontaneous tubule formation of clone 25 under a variety of conditions must be subject to cAMP-mediated regulation since activation of the cAMP pathway downregulated the incidence of tubular processes as the incidence of cystic structures rose. Several cellular proteins, growth factors, and receptors have been reported to be associated with tubulogenesis (reviewed in Refs. 2, 49, and 50). A previous report demonstrated that specifically localized beta -catenins were involved in the development of tubular processes from an initial cystic structure of MDCK cells (19). Perhaps the specific location of cAPK subtypes in clone 25 allowed a cellular modulator access to a protein complex involved in control of the developing tubular process.

The developmental renal disease family of inherited PKDs is characterized by progressive enlargement of nephrons to form many fluid-filled cysts. Researchers have hypothesized that protein products of the three known PKD genes (polycystin-1, polycystin-2, and the Tg737 gene product are components of a membrane-associated signaling complex involved in nephrogenesis and that mutations contribute to the pathogenesis of PKD by altering the function of the complex (reviewed in Refs. 51 and 52). Polycystin-1 was found to be a cAPK substrate (53, 54). Genetic studies showed that in addition to the three known genes, several other independent chromosomal loci were linked to the phenotypic expression of PKD (reviewed in Ref. 2). However, a consistent phenotype of all affected PKD-collecting tubular epithelial cells is the altered expression of the EGF receptor, and EGF receptor tyrosine kinase-dependent cyst formation (reviewed in Refs. 2, 55, and 56). Preliminary studies demonstrated no apparent EGF receptor cDNA sequence defect in human autosomal recessive PKD,2 cAPK subunit localization differences in cells derived from cystic kidneys of murine models of PKD, and abnormal cell proliferation possibly due to cAMP/EGF receptor interactions that did not occur in non-cystic cells.3 If a PKD protein complex is involved in regulating normal nephrogenesis, then mutations that altered its interaction with, or regulation by, cAPK subtypes could contribute to cyst formation. In summary, intracellular diversity in the cellular complement of Type I and Type II cAPK activities, their subcellular locations, and their preferred targets is likely to account for the ability of cAPK to regulate the complicated process of MDCK morphogenesis. These results are potentially important with respect to the functional significance of mechanisms that regulate subcellular location of cAPK subunits and may be relevant to renal development and disease.

    FOOTNOTES

* This work was supported by the National Institutes of Health Grant DK50707, the Polycystic Kidney Research Foundation, and the Department of Pediatrics of Rainbow Babies and Children's Hospital of the University Hospitals of Cleveland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Mail Stop 6003, 11100 Euclid Ave., Cleveland, OH 44106-6003. Tel.: 216-844-7360; Fax: 216-844-7642; E-mail: sao3@po.cwru.edu.

Published, JBC Papers in Press, April 14, 2000, DOI 10.1074/jbc.M001964200

2 S. A. Orellana, M. Hobert, and C. R. Carlin, unpublished observation.

3 C. Marfella-Scivittaro, A. Quiñones, and S. A. Orellana, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: MDCK, Madin-Darby canine kidney; C, cyclic AMP-dependent protein kinase catalytic subunit; R, cyclic AMP-dependent protein kinase regulatory subunit; cAPK, cyclic AMP-dependent protein kinase; EGF, epidermal growth factor; FBS, fetal bovine serum; GS, goat serum; HGF, hepatocyte growth factor; rhHGF, recombinant human HGF; IBMX, isobutylmethylxanthine; IgG, immunoglobulin G; PBS, phosphate-buffered saline; PGE1, prostaglandin E1; PKD, polycystic kidney disease.

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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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C. Marfella-Scivittaro, A. Quinones, and S. A. Orellana
cAMP-dependent protein kinase and proliferation differ in normal and polycystic kidney epithelia
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