Stat3 調控NHE3 基因之分子機制與上皮細胞分化之關聯(1/3)

行政院國家科學委員會專題研究計畫 期中進度報告

Stat3 調控 NHE3 基因之分子機制與上皮細胞分化之關聯

(1/3)

中 華 民 國 95 年 7 月 7 日

Cell Confluence-Induced Activation of Stat3 Triggers Epithelial Dome Formation

via Augmentation of NHE3 Expression

Hsiao-Wen Su1, Hsuan-Heng Yeh1, Shainn-Wei Wang2, Meng-Ru Shen3, Pawel R. Kiela5, Fayez K. Ghishan5 and Ming-Jer Tang4*

1_{Institute of Basic Medical Sciences,} 2_{Institute of Molecular Medicine,} 3_Department

of Pharmacology and 4Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. 5Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA

*Corresponding Author:

Ming-Jer Tang, MD, PhD. Department of Physiology, College of Medicine, National Cheng Kung University, 1 Da-Hsueh Road, Tainan 701, Taiwan. Tel.: 886-6-235-3535 ext 5425; Fax: 886-6-236-2780; E-mail: [email protected]

Running Title: Stat3 regulates dome formation via NHE3 expression

Subject Categories: Signal Transduction

ABSTRACT

Cell confluence induces the activation of signal transducer and activator of transcription-3 (Stat3) in various cancer and epithelial cells, yet the biological implications and the associated regulatory mechanisms remain unclear. Since polarized epithelia in confluence results in dome formation and sodium influx that mimic the onset of epithelial differentiation, we sought to elucidate the role of Stat3 in this biological consequence in association with the regulation of epithelial transporters. This study established the correlation of JAK-dependent Stat3 activation with cell confluence-induced dome formation in Madin-Darby canine kidney cells (MDCK) by chasing Stat3 activation events in dome forming cells. Epi-flurorescent and confocal microscopy provided evidence showing specific localization of phosphorylated Stat3 Tyr705 at nuclei of dome forming cells at initial stages. The relationship was further elucidated by the establishment of tetracycline-inducible MDCK cells expressing constitutive Stat3 mutant (Stat3-C) and stable cell lines (MDCK and NMuMG) expressing dominant negative Stat3 (Stat3-D). Dome formation was promoted by the expression of Stat3-C but inhibited by Stat3-D. Two trans-epithelial transporters, NHE3 and ENaC α-subunit, were found to be increased during cell confluence. Interestingly, NHE3 expression could be specifically up-regulated by Stat3-C but inhibited by Stat3-D through promoter regulation, whereas NHE1 and ENaC

α-subunit were not affected by Stat3 expression. Application of NHE3 shRNA, NHE3 inhibitors (EIPA and S3226), or JAK inhibitor (AG490) suppressed confluence-induced dome formation in MDCK or NMuMG cells. These results demonstrated a cell confluence-induced JAK/Stat3 signaling pathway in epithelial cells in triggering dome formation through NHE3 augmentation.

INTRODUCTION

Domes are multicellular hemicyst structures unique to polarized epithelia in culture (44) and show functional equivalence to epithelium with trans-epithelial transport of absorbed solutes (8, 29, 41). They occur sporadically in small areas during cell confluence that mark the initial differentiation process of a functional epithelial monolayer. While turning into an impermeable substratum with preceded expression of tight junction proteins (41), the dome structure sets off with diminishing cellular adherence ability as a result of liquid accumulation between the cell layer and the underlying support (8, 29, 41). This feature is conceived by the coordinated development of trans-epithelium transport systems provided by morphologically polarized cells, which in kidney, intestine, or other epithelia mostly involves the differential localization and activation of sodium channels (e.g., ENaC) or other sodium transporters (e.g., Na+_-H+ _{Exchanger: NHEs) on the apical membrane and}

Na+_,K+_{-ATPase ubiquitously expressed on the basolateral sites to maintain Na}+ _and

fluid homeostasis (8, 14, 29, 33, 34, 47). Most importantly, the net unidirectional transport of Na+ _{coincided with intracellular alkalosis has been implicated in dome}

formation (8, 10, 36).

The epithelial cell line MDCK (Madin-Darby canine kidney) (12) is a model system widely used to study dome formation associated renal trans-epithelial transport

during cell confluence (29). At least three possible mechanisms exist to coordinate the vectorial sodium transport across MDCK monolayer from apical to basolateral sites. One is the ouabain-sensitive Na+_-K+ _{exchange mechanism, the second is the}

furosemide-sensitive Na+_-Cl-_/K+_-Cl- _{co-transport mechanism, and the third is the}

amiloride-sensitive ENaC (18, 21, 28). Because MDCK cells used to be considered cell line of distal nephron, whether NHE3, a marker for proximal tubule cells, plays a role in dome formation in MDCK cells has not been implicated before. Since abundant studies indicated the involvement of Na+_-H+ _{exchangers (NHEs) and}

epithelial sodium channel (ENaC) during renal development or epithelial cell differentiation (2, 5, 17, 35, 50), we hypothesized the involvement of these sodium transporters to coordinate with Na+_,K+_{-ATPase in the initiation of dome formation in}

MDCK cells. The physiological roles of NHE is to maintain intracellular and systemic pH, trans-cellular absorption of NaCl and NaHCO3, and intracellular volume and

body fluid balance (1, 37, 38). There are nine isoforms of NHEs (33). Among these NHEs, NHE1 is ubiquitously expressed and localized at the basolateral membrane of polarized epithelial cells, whereas NHE2 and NHE3 is localized at the apical membrane of small intestine, colon and renal tubular cells (33). In mammalian kidney, NHE3 is the key factors acting to reabsorb sodium and water to across renal proximal tubule epithelial cells (37).

Recently, a ligand-independent activation of Stat3 (signal transducer and activator of transcription-3) by cell confluence has been demonstrated in multiple cancer and normal epithelial cell lines (43, 45). These studies revealed cdk2- (cyclin dependent kinase 2) regulated activation of Stat3 by cell growth arrest and envisioned a novel role of JAK- (Janus kinases) dependent Stat3 signaling in modulating the survival related physiological functions during cell confluence. This raises the possibility that signaling of Stat3 (or their cognate family members involving in the growth control, such as Stat1 and 5 (3)), may result from cell confluence to modulate trans-epithelial sodium transport for dome formation. STAT proteins, which include 7 latent cytoplasmic transcription factors, are normally activated through phosphorylation of their tyrosine residues by several tyrosine kinases. The key tyrosine residue phosphorylated in Stat1 is Tyr701, in Stat3 is Tyr705, and in Stat5 is Tyr694 (6). Phosphorylated STATs are dislodged from the receptor or non-receptor associated kinase complex and may crosstalk between each other to form immediate hetero- (e.g. Stat1-Stat3) or homo-dimmers (e.g. Stat1-Stat1 or Stat3-Stat3)(6). They are then translocated into nucleus to work coordinately with different co-activators and induce transcription of distinct genes based on their DNA binding affinities for promoter responsive elements (9, 26). In general, STAT signaling events are triggered by membrane receptors in response to wide varieties of cytokines and growth factors

to regulate physiological responses (3, 6, 26). Receptors for cytokines are devoid of intrinsic kinase activity and require the mediation of JAKs for STATs phosphorylation (3, 6).

We report here that cell confluence-induced activation of Stat3 regulates dome formation in MDCK and NMuMG (normal mouse mammary gland) cells by augmentation of NHE3 transporter in a JAK dependent manner. By coupling dome formation as a biological trait to chase Stat3 signaling events in MDCK or NMuMG cells during cell confluence, a causal effect of Stat3 resulting from cell-cell contact on dome formation was revealed. The morphologic characteristics of dome formation in terms of numbers and sizes resulting from the transcriptional regulation of Stat3 in constitutive or dominant negative forms were assessed and found to correlate with the effect of Stat3 on the expression of NHE3 sodium transporter. Lastly, by functional inhibition of JAK or NHE3 activities, we demonstrated a novel JAK/Stat3-mediated NHE3 augmentation signaling pathway in regulating the differentiation of a functional epithelial monolayer during cell-cell contact.

MATERIALS AND METHODS

Cell culture and stable transfections. MDCK cells and NMuMG cells were maintained in Dulbecco’s modified minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) under 5% CO2 at 37 °C. The

active-form Stat3 (Stat3-C) plasmid was kindly provided by Dr. James Darnell Jr (4). To generate the tetracycline-inducible Stat3-C system, we sub-cloned Stat3-C gene from pRc/CMV plasmid to rtTA-responsive pTRE2-hyg plasmid (CLONTECH Laboratories, Inc.). Stable transfections of inducible FLAG epitope-taggedStat3-C (4) to MDCK cells were conducted by using 20 μl of LipofectAMINE to establish clonal lines constitutively expressing rtTA encoded by the pTet-ON regulator plasmid. The pTet-ON plasmid contains a neomycin resistance gene to permit G418 selection (0.5 mg/ml; CLONTECH Laboratories, Inc.). One of these rtTA clones was selected for secondary stable transfection with rtTA-responsive pTRE2-hyg hygromycin plasmid containing Stat3-C, and colonies were selected by 0.2 mg/ml hygromycin B (Invitrogen Co.). Stable clones were screened after 24 h induction with 10 μg/ml doxycycline (Sigma, Co.) for Stat3-C expression by Western blot using anti-FLAG antibody. For dome formation assay, MDCK (2.5x105_{) cells or NMuMG (3x10}5_{) cells}

were plated on 6-cm culture dish in medium containing 10% FCS and the medium was changed every 2 days till dome formation.To generate cells stably expressing

Stat3-F, subconfluent MDCK cells grown on 6-cm dish were co-transfected with 1 μg of pRc/CMV neomycin resistance empty vector and 4 μg of pMS1 plasmid encoding Stat3-F (20) using 20 μl of LipofectAMINE (Life Technologies,Inc.). One day after transfection, cells were re-seeded on 10-cm dish at an appropriate density in the medium containing0.5 mg/ml G418. Neomycin-resistantcell clones were selected and screened forexogenous Stat3-F expression by immunoblotting with anti-Stat3 and anti-Stat3 Tyr705-P polyclonal antibodies. Two positive clones (clones #11 and #12) expressing Stat3-F were used for dome formation assay. To generate cells stably expressing HA epitope-taggedStat3-D, which was kindly provided by Dr. T Hirano (30), subconfluent MDCK (and NMuMG) cells grown on 6-cm dish were transfected with 4 μg of pCAGGSneo plasmid encoding Stat3-D using 20 μl of LipofectAMINE. Neomycin-resistantcell clones were selected following aforementioned procedures and were screened forexogenous Stat3-D expression by immunoblotting with anti-HA monoclonal antibody. Multiple positive clones expressing Stat3-D were obtained for dome formation assay. The numbers or diameters of domes were quantified by counting and measuring domes from at least 20 independent fields at high power magnification (100 x). AG490 (JAK inhibitor), EIPA (NHEs inhibitor) and S3226 (NHE3 inhibitor) were used for inhibition of dome formation. Confluent cells were incubated with each inhibitor at different concentrations till dome appeared in control

cells.

Preparation of Nuclear Extracts. Nuclear extracts were collected according to the method of Wang et al. (46) with minor modifications. Cells grown to different confluence on 6-cm plastic dishes were washed 3 times with phosphate-buffered saline (PBS) (137 mM sodium chloride, 2.7 mM potassium chloride, 10mM dibasic sodium phosphate, and 2 mM monobasic potassium phosphate) and scraped off the plate after incubation with 400 μl of buffer A (10 mM HEPES, pH 7.9,1.5 mM MgCl2, and 10 mM KCl) on ice for 10 min. Cells were precipitated by centrifugation

at 7,500 x g for 20–30 s and the pellet was resuspended in 100 μl of buffer C (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 420 mM NaCl). The resulting

suspension was centrifuged at 7,500 x g for 2 min. The supernatants were collected and stored at -70 °C. Buffer A and buffer C contained 0.5 mM dithiothreitol, 2 μg/ml leupeptin, 1 mM orthovanadate, 2 μg/ml pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF).

DNA affinity precipitation assay (DAPA). This assay was performedaccording to the method of Wang et al. (46) with minor modifications.The binding assay was performed by mixing 200 μg of nuclearextract proteins, 2 μg of biotinylated m67 oligonucleotides specific to Stat3 binding (14), and 20 μg streptavidin-agarosebeads in TE buffer (pH 7.9). The mixture was incubated at roomtemperature for 1 h with

rotation followed by precipitation at low speed microcentrifuge and washedwith cold PBS for 3 times. The bound proteins were separated by SDS- polyacrylamide gel electrophoresis (PAGE), followed by Westernblot analysis probed with Stat1 or Stat3 antibody(Cell Signaling).

Reverse Transcription-PCR. Total cellular RNA was extracted by RNeasy Mini kit (Qiagen). For RT-PCR, first-strand cDNAwas synthesized from 0.2–1 μg of total RNA with an oligo-dT primer and the Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega). The following primers fromCynomys ludovicia used

for NHE3 (NCBI accession# U75970) are: forward (5'-GCTGGTCTTCATCTCTG TGT-3') and reverse (5'-GAGGTTCTTCTCCTTGACCT-3'), and the resulting PCR product was 185 bp. Stat3 primers are: forward (5'-CTAAAGTCAGGTTGCTG GTC-3') and reverse (5'-AAGGAGTGGGTCTCTAGGTC-3'), and the resulting PCR product was 337 bp. GAPDH primers are: forward (5'-ACGGCACAG TCAAGGCTGAG-3') and reverse (5'-GGAGGCCATGTAGACCATGAGG-3'), and the resulting PCR productwas 558 bp. The PCR protocol performed with the NHE3 and GAPDH primers was: 94 °Cfor 30 s, 55 °C for 30 s, and 72 °C for 45 s (30 cycles), followed by 72 °C for 7 min. The PCR protocol performed with the Stat3 primers was: 94 °C for 30 s,60 °C for 45 s, and 72 °C for 1 min (28 cycles), followed by 72 °C for 10 min.

Assays for NHE 3 promoter activity and dual-luciferase assay. NHE3 promoter regulation by Stat3 was conducted in Stat3-C inducible (St3C-3) and non-inducible (mock) MDCK cells according to a promoter-based dual luciferase reporter assay. Cells (2x105 _{cells) were plated in 6-cm dish and transiently transfected with a Renilla}

luciferase reporter gene containing plasmid alone (either phRG-b vector, pNHE3 -450/+58, or pRL-TK) or in combination with a pCMV-Luc vector (firefly luciferase). Transient transfection was carried out by the Arrest-InTM _{reagent (Openbiosystens)}

according to the manufacturer’s instructions. The phRG-b vector was a promoterless vector harboring the Renilla luciferase gene (Promega, Co.). The pNHE3 -450/+58 was the phRG-b vector fused with a rat NHE3 promoter (-450 to +58 region) to drive

Renilla luciferase expression (22), as canine NHE3 promoter sequence was currently

unidentified. The pRL-TK contained the Renilla luciferase gene driven by thymidine kinase (TK) promoter (Promega). The pCMV-Luc contained the firefly luciferase gene driven by Cytomegalovirus CMV promoter (a gift kindly provided by Dr. Wen-Tsan Chang, College of Medicine, National Cheng-Kung University). After 12 h of transfection,the transfectants were reseeded in six-well plates and cultured in the medium with or without doxycycline (10 μg/ml) for 20 h. The cells were washed 3 times in PBS and the lysates were prepared by scraping the cells from platesin the presence of 1x passive lysis buffer (Promega). The dual luciferaseassays for Renilla

luciferase and firefly luciferase activities were then performed on cell lysates by using Dual-Luciferase Assay System (Promega) and a Sirius luminometer (Berthold Detection System,Pforzheim, Germany).

RNA interference. To knockdown NHE3 expression in NMuMG epithelial cells, 19-mer short hairpin RNA (shRNA) against mouse NHE3 was synthesized by Open Biosystems. Sequences for shNHE3 are: 5’-GCGTCTGTCTCATATTTCT-3’ and 5’-AGAAATATGAGACAGACGC-3’. For stable transfection, shNHE3 was cloned in pSM2 expression vector (Open Biosystems) and then transfected into NMuMG cells using Arrest-In TM _{transfection reagent (Open Biosystems) according to}

manufacturer's instruction (Open Biosystems). After 24 h transfection, cells were subjected to puromycin (1 μg/ml) selection for at least 1 week. Subsequently, puromycin-resistantcell clones were selected and screened forNHE3 expression by immunoblotting with NHE3 polyclonal antibody. Multiple positive clones (clones #4 and #10) with minimal NHE3 expression were obtained for dome formation assay as described above.

Immunofluorescence and confocal study. MDCK cells were culturedat different time intervals followed by washing 3 times with PBS and fixed with 4% para-formaldehyde prepared in PBS for 20min at room temperature. After cells were washed 3 timesin PBS and permeabilized with 0.5% Triton X-100 in PBS for 10min

at room temperature, they were incubated with Stat3 Tyr 705-P or Stat3 (Cell Signaling) polyclonal antibody, or with ezrin (Transduction Lab.) monoclonal antibody for 1 h. Cells were then washed and incubated with Alexa Fluor 488-conjugated goat anti-rabbit or anti-mouse antibody (Molecular Probes) and Hoechst 33258 for 1 h. The immunofluorescentimages were taken by epi-fluorescent microscopy (Olympus BX-51) or confocal microscopy (Olympus, FV-1000).

Western blotting. Cells were grown to different confluences and proteins were extracted using lysis buffer (20 mM HEPES, pH 7.9, 0.5% NP-40, 7.5% Glycerol, 300 mM NaCl, 1 mM EDTA) containing protease inhibitors (0.5 mM PMSF, 0.2 units/ml aprotinin, 1 mM DTT, 10 mM NaF, 1 mM Na3VO4 and 20 μg/ml leupeptin).

For each blotting analysis, 50 μg of clarified cell extract was resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Hybound-ECL). The membranes were blocked with 5% nonfat milk for at least 1 h followed by an overnight incubation in primary antibody. Immunodetection was performed using antibodies against the Stat3 Tyr705-P (Cell Signalling), total Stat3 (Cell Signalling), Stat3 Ser727-P (Cell Signalling), Stat1 Tyr701-P (Cell Signalling), total Stat1 (Cell Signalling), Stat5 Tyr694-P (Cell Signalling), FLAG (Sigma), HA (Boehringer Mannheim), Ki-67 (Santa Cruz), PCNA (Zymed), Lamin A/C (Santa Cruz), β-actin (Sigma), NHE1 (Chemicom), NHE3 (Chemicom), ENaC α-subunit (Santa Cruz), Na+_,K+_{-ATPase α1}

(Santa Cruz), or Na+,K+-ATPas β1 (Upstate) followed by HRP-conjugated goat secondary antibodies (Biosource). The bands were visualized using enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (Perkin- Elmer Life Sciences).

Statistics. All data were expressed as the means ± standard error (SE). Differences between groups were determined by Student’s t-test. Multi-group comparisons were determined by one-way ANOVA. Differences in the comparison were considered to be significant when p values were less than 0.05.

RESULTS

Cell confluence induces Stat3 Tyr705 phosphorylation and DNA binding, which

is correlated with the dome formation in MDCK cells. MDCK cells were monitored daily in culture to establish the sequential events from cell confluence to dome formation (Fig. 1A). They formed initial cell islets and proliferated to reach confluence with few highly cell-condensed clusters occasionally present at day 4. These cells organized into small blister-like structures which represent for dome formation at day 5 and phased into a relatively larger dimension at day 6. These domes continued to bulge with decreased adherence to plate and may keep developing for an additional week in association with floating cells and apoptotic bodies (data not shown). Cell growth reached saturation at day 4, and cell number was sustained as 100% confluence at day 5~6 based on a standard growth curve (data not shown). The result indicated that the initial stages of dome formation with retarded cell proliferation were captured within the time frame.

Possible phosphorylation events of Stat1, Stat3, or Stat5 during cell culture at different stages were investigated (Fig. 1B). The level of Stat3 Tyr705-P sharply increased at day 3 and continuously augmented till day 5, which correlated with the microscopic observations of large patches of cells in confluence at day 3, highly condensed cell-clusters at day 4, and early dome formation at day 5. A significant

drop of Stat3 Tyr705-P was observed at day 6. The Stat3 Ser727-P maintained at low levels and increased slightly from day 5 to day 6. Since Stat3 were constitutively maintained (Stat1 and Stat5 not shown) within the time frame, the timing of Stat3 Tyr705-P induction rather than that of Stat3 Ser727-P showed a better correlation to confluence-induced dome formation. No apparent correlation of Stat1 Tyr701-P or Stat5 Tyr694-P to these sequential events was observed except that Stat1 Tyr701-P was increased prior to cell reaching confluence.

Whether the elevation of Stat3 Tyr705-P during dome formation contributed to the active fraction of nuclear Stat3 with DNA binding was characterized. The fractions of nuclear Stat3 in different cell stages precipitated by the Stat3-specific binding oligomer m67 were profiled and found to be in homogeneic form as they were recognized by Stat3 antibody but not by Stat1 antibody (Fig. 1C, upper panel). The specificity and validity of the DNA affinity precipitation assay (DAPA) were reflected by the detection of Stat1 and Stat3 in a heterologous positive control. Moreover, the profile of Stat3 with DNA binding in comparison with that of nuclear Stat3 Tyr705-P showed similar transactivation pattern (Fig. 1C, lower panel), corresponding with decreased cell proliferation markers, such as Ki-67 and PCNA, and the initiation of dome formation at days 4~5 (Fig. 1C, lower panel). This result suggests that Stat3 Tyr705-P contributed to the active form of Stat3 for possible

downstream signaling and reflected a distinctive differentiation nature of sustained Stat3 Tyr705-P signaling during cell confluence and early stages of dome formation. Cell density triggers Stat3 Tyr705 phosphorylation and DNA binding in MDCK

cells. Whether cell density is a factor independent of cell proliferation and capable of activating Stat3 for DNA binding was investigated by seeding cells at low cell density (LCD) or high cell density (HCD). After being cultured for 1 day, cells at LCD formed islets, while cells at HCD reached confluence (Fig. 2A). It was also found that Stat3 Tyr705-P, but not Stat3 Ser727-P and Stat3, was substantially higher in HCD lysates (Fig. 2B), which indicated a density effect on Stat3 Tyr705-P phosphorylation and correlated with the higher nuclear Stat3 DNA binding in HCD (Fig. 2C, upper panel). Since Ki-67 and PCNA were found to sustain in both HCD and LCD cultures, the elevation of nuclear Stat3 Tyr705-P was independent of cell proliferation (Fig. 2C, lower panel). These results strongly suggested that cell density triggered the phosphorylation of Stat3 Tyr705, which corresponded with the nuclear translocalization of Stat3.

Stat3 Tyr705-P is localized in the nuclei of initial stages of dome forming MDCK

cells. We examined the physical localization of Stat3 Tyr705-P in cultures of confluent and post-confluent cells by epi-fluorescent (Fig. 3A and 3B) and confocal microscopy (Fig. 3C). Stat3 Tyr705-P was found specifically localized at highly

condensed areas of confluent cell monolayer at day 4 (Fig. 3A, upper panel). Once the dome was formed at day 5, Stat3 Tyr705-P was specifically localized at central area of the dome, and not detected from the surrounding adhesion cells (Fig. 3A, middle panel). Interestingly, Stat3 Tyr705-P at day 6 disappeared from the dome and replaced by the appearance of heavily Hoechst-stained apoptotic bodies (Fig. 3 A, bottom panel). The percentage of domes stained positive for Stat3 Tyr705-P exhibited in dome areas at day 5 and day 6 was quantified (Fig. 3B) and the results reflected a positive correlation of Stat3 Tyr705-P induction with the initial stages of dome formation.

Confocal microscopy detected the sub-cellular localization of Stat3 Tyr705-P and Stat3 in the dome areas. As shown in Fig. 3C (top panel), most of the Stat3 Tyr705-P co-localized with Hoechst-staining in the nuclei of dome forming cells at day 5. Similar result of the colocalization of Stat3 with Hoechst-staining was seen in the nuclei except that a small portion of Stat3 could be detected at cytoplasm of dome forming cells at day 5 (Fig 3C, second panel). At day 6, most of the Stat3 proteins were predominantly localized at cytoplasm, not overlapped with Hoechst-staining in the nuclei (Fig 3C, third panel). This observation confirmed that the reduction of Stat3 Tyr705-P at day 6 correlated with the loss of signaling for Stat3 nuclear translocalization.

Constitutive expression of dominant active Stat3 (Stat3-C) promotes the initial

number and size of domes. To examine whether Stat3 dominated the regulation of dome formation in MDCK cells, selected cell lines expressing FLAG epitope-tagged Stat3-C, a constitutively active form of Stat3 (4), and tetracycline (tet)-responsive transcriptional activator (rtTA) were established based on a tetracycline inducible (Tet-On) system. Upon Stat3-C induction by a tetracycline analogue doxycycline after 5 days, no apparent changes with respect to the morphology or growth characteristics were found in any of the selected clones (data not shown). Stat3-C expression from the stable transfectants (St3C-3 and St3C-4) was dose-dependent on doxycycline induction in contrast to the control of mock transfectant expressing rtTA alone (Fig. 4A). Furthermore, the extent of dome formation in terms of number and size corresponding to the exogenous Stat3-C induction in the 5th _{day culture of St3C-3 and}

St3C-4 cells was far more advanced than that observed in mock cells (Fig. 4B). Quantification of the result confirmed a substantial increase in averaged number and diameter of domes in St3C-3 and St3C-4 in responding to increasing amounts of doxycycline (Fig. 4C and 4D).

In addition, the extent of dome formation in St3C-4 was lower than that in St3C-3 cells at day 5, corresponding to the lower Stat3-C expression in St3C-4 cells. This indicates that the number and size of domes are very sensitive to the level of

Stat3-C (Fig. 4A, 4C, and 4D). This sensitivity to doxycycline stimulation, however, did not cause accelerated dome formation earlier than day 5. Therefore, it is unlikely that Stat3-C promoted dome formation through promoting local cell proliferation. Independently, equivalent numbers of live cells were counted during cell confluence and dome formation among these experimental groups (data not shown). This observation reinforced that the Stat3-C augmentation for dome formation is cell confluence-dependent and reflected a distinct Stat3 signaling pathway in promoting the extent of dome formation by both number and size right at the initiation process. Constitutive expression of dominant negative Stat3 mutants suppresses the

number and size of domes. Whether expression of dominant negative Stat3 can offset the dome formation in MDCK cells was examined. Individual colonies of MDCK cells stably transfected with Stat3-F (a tyrosine 705 mutant) or HA epitope-tagged Stat3-D (a DNA-binding domain mutant) were selected and characterized for Stat3 expression and Tyr705 phosphorylation (Fig. 4E). Compared to control cell line, the 5th _{day cultures of stable Stat3-F transfectants (clones #11 and}

#12) exhibited elevated cellular Stat3 expression but reduced Stat3 Tyr705-P, (Fig. 4E, left panel), while the stable Stat3-D transfectants (clones #2, #3, #5, and #10), exhibited elevated levels of Stat3 and Stat3 Tyr705-P (Fig. 4, right pannel). These data suggest that over-expression of Stat3-F partially reduced the overall Stat3 Tyr705

phosphorylation, whereas overexpression of Stat3-D did not. Quantitative estimation of dome formation for the corresponding 5th _{day cell cultures reflected the dominant}

negative effects of Stat3-F and Stat3-D expression on the number of domes (Fig. 4F). Both St3F-11 and St3F-12 transfectants exhibited less number of domes (~18/field and ~11/field respectively) than the Neo control (~28/field), whereas all of the Stat3-D stable transfectants dramatically suppressed dome formation in comparison with the control MDCK cells (Fig. 4F). As the size of domes was extremely small at day 5 under the influence of dominant negative Stat3 (data not shown), the dome size was measured on the 6th _{day culture. The average diameter of domes in St3F-11 at day}

6 was the same as the Neo control (~120 μm), while that of the St3F-12 showed a significant reduction by 33% (~80 μm) (Fig. 4G). In contrast, the dominant negative effect of Stat3-D (both St3D-2 and St3D-5) had an apparent potential of reducing up to 50% of the average diameter of domes comparing to control (MDCK) cells (Fig. 4H). While the reason for the less effectiveness of Stat3-F on dome formation is not immediately clear, it is possible that over expression of Stat3-D, via inhibition of the DNA binding, may compete more extensively than Stat3-F in masking the effects from the endogenous Stat3. Regardless, these results showed that both Stat3-F and Stat3-D had dominant negative effect on dome formation, implicating the pivotal role of activated Stat3 in tuning the initiation of dome formation.

Cell confluence induces augmentation of NHE3 through Stat3 activation in

MDCK cells. One of the requirements for successful dome formation in epithelium is the activation of sodium transport from apical to basolateral site (8, 25, 29, 34). Our results point to the sodium transporters, NHE3 and ENaC α-subunit, induced by cell confluence, as the most likely candidates involved in dome formation. As shown in Fig. 5, the protein expression levels of NHE3 and ENaC α-subunit increased from day 1 to day 5 following cells reaching confluence and then decreased after day 6, which bear similarity to the Stat3 Tyr705 phosphorylation pattern, as shown in Fig. 1B. Of other sodium transporters, Na+,K+-ATPase α1 was slightly increased from day 0 to day 2 but decreased afterwards, while Na+,K+-ATPase β1 and NHE1 were stably maintained during the 6-day period (Fig. 5A).

Although the above data suggest that NHE3 and ENaC α-subunit are the candidates augmented following Stat3 activation, only NHE3 expression was specifically promoted by Stat3 (Fig. 5B). Induction of Stat3-C in St3C-3 cells at day 2 by optimal dose of doxycycline enhanced the expression of NHE3, but not ENaC α-subunit, in a time-dependent fashion from 36 h to 96 h after induction (Fig. 5B). This approximated the time-dependent increase of Stat3-C detected by FLAG antibody from 24 h to 96 h. Furthermore, the expression of NHE3, in contrast to that of NHE1 and ENaC α-subunit, was augmented by doxycycline in a dose dependent

manner in St3C-3 cells but not mock control cells (Fig. 5C). Comparison of the NHE3 expression levels between Stat3-D transfectants (clones #2, #3, #5 and #10) and the control MDCK cells by Western blot showed substantial dominant negative effect of Stat3-D in reducing the level of NHE3 but not NHE1 (Fig. 5D).

In order to distinguish whether Stat3 augmented NHE3 through up-regulation of NHE3 mRNA level, RT-PCR was performed to detect the mRNA level of NHE3 in St3C-3 cells (Fig. 5E) and Stat3-D transfectans (Fig. 5F). Induction of Stat3-C mRNA by doxycycline was associated with increased NHE3 mRNA level in St3C-3 cells but not in the control mock cells (Fig. 5E); whereas the expression of exogenous Stat3-D mRNA was associated with reduced NHE3 mRNA level in St3D-10 cells in contrast to what was observed in MDCK control cells (Fig. 5F). These results taken together suggest the effect of Stat3 in promoting NHE3 production through transcription regulation.

Stat3 regulates NHE3 promoter activity in MDCK cells. A dual luciferase assay system was performed to delineate whether NHE3 promoter activity could be regulated by Stat3. Stat3-C inducible (St3C-3) and non-inducible mock cells were transiently co-transfected with a firefly luciferase reporter plasmid (pCMV-Luc) in combination with Renilla luciferase plasmids (either pNHE3 -450/+58 or phRG-b vector) for 12 h and treated with doxycycline for another 20 h. Dual luciferase assay

was performed. As shown in Fig. 6A, the results reflected a positive correlation of Stat3-C induced by doxycycline with Renilla luciferase activity driven by NHE3 promoter. After normalization with firefly luciferase activity, the Renilla luciferase activity in St3C-3 cells harboring only phRG-b vector was minimal regardless of doxycycline induction. Cells harboring only pNHE3 -450/+58 had about 7~9 fold induction of Renilla luciferase activity without doxycycline induction, which reflected the basal NHE3 promoter activity in response to the regular level of endogenous Stat3 activity. Upon induction by doxycycline, the Renilla luciferase activity in St3C-3 cells significantly increased by 26 fold comparing to the basal level in control St3C-3 cells and about 3.7 fold comparing to that in St3C-3 cells with pNHE3 -450/+58 but without doxycycline treatment.

In addition, St3D-10 cells harboring pNHE3 -450/+58 down-regulated the NHE3 promoter and had 6 fold reduction of luciferase activity comparing to the MDCK cells harboring the same promoter plasmid only (Fig. 6B). To exclude that Stat3 coordinated NHE3 expression through translational regulation, a pRL-TK constitutively expressing Renilla luciferase was used to replace the pNHE3 -450/+58 in a dual luciferase assay system to investigate whether Stat3-C augmentation can indirectly up-regulate Renilla luciferase activity from the constitutive thymidine kinase (TK) promoter. The Renilla luciferase activity was decreased in St3C-3 cells in

proportion to that in the control mock cells upon doxycycline treatment (Fig. 6C). Thus, Stat3-C did not regulate the luciferase activity through translational regulation. These results indicate that Stat3-C up-regulate the NHE3 through transcriptional regulation.

Cell confluence-induced Stat3 activation regulates NHE3 expression and dome

formation in NMuMG cells. We examined whether cell confluence induced Stat3 Tyr705 phosphorylation and NHE3 expression could be applied to other dome forming epithelial cells and employed NMuMG cells. As shown in Fig. 7A, cell confluence-induced activation of Stat3 and augmented expression of NHE3 in NMuMG cells were similar to those observed in MDCK cells except that the time frame from seeding to dome formation extended up to 8 days. On the other hand, cell confluence induced an early onset of Stat5 Tyr694-P and late onset of Stat1 Tyr701-P. There was not further increase in Stat5 Tyr694-P levels at day 6. Thus, the correlation of Stat1 with NHE3 expression is excluded. We also observed that Stat3 Tyr705-P levels were decreased at the 8th _{day. These results suggest similar Stat3 Tyr705-P}

activation and NHE3 augmentation during dome formation in NMuMG cells. Furthermore, cells seeded at HCD displayed higher level of Stat3 Tyr705-P than cells seeded at LCD (Fig. 7B). In addition, Stat3 regulated NHE3 expression during dome formation in NMuMG cells, as selected dominant negative Stat3-D transfectants of

NMuMG cells (clones #2, #7 and #18) expressed lower levels of NHE3 than those in the non-transfectant control (Fig. 7C). Moreover, dome formation was significantly reduced in all Stat3-D transfectants compared to control NMuMG cells (Fig. 7D). These results again support the notion that cell density-dependent Stat3 Tyr705-P and NHE3 expression are the key factors driving dome formation.

JAK is involved in cell confluence-regulated dome formation. Since Janus kinase (JAK) is a common tyrosine kinase to phosphorylate Stat3 Tyr705 (23), studies were conducted to examine the involvement of JAK in cell confluence-induced dome formation through Stat3 signaling pathway. Application of JAK inhibitor, AG490, to confluent MDCK and NMuMG cells reduced the level of Stat3 Tyr705-P substantially within 4 h and resulted in reduced NHE3 expression in a dose-dependent manner after 24 h as compared to control cells exposed to the solvent, DMSO (Fig. 8A). AG490 also significantly inhibited dome formation in both MDCK and NMuMG cells in a dose-dependent fashion (Fig. 8B). It was not unexpected that the number of domes increased after DMSO treatment, since DMSO is known to be a differentiation inducer, which stimulates dome formation (52). These results indicate the involvement of JAK in the confluence-induced Stat3 activation signaling pathway for dome formation.

NMuMG cells. To further examine whether NHE3 augmentation was indeed required for dome formation, RNA interference technique was employed to knockdown NHE3 expression. Individual colonies of NMuMG cells stably transfected with shRNA of NHE3 (shNHE3) were selected and characterized for NHE3 expression. Comparing to control cell lines (NMuMG harboring with or without pSM2 vector), the 6th _day

cultures of stable shNHE3 transfectants (clones #4 and #10) exhibited lower level of NHE3 (Fig. 9A) and reduced dome formation (Fig. 9B). Quantitative estimation of dome formation for the corresponding 6th _{day cell cultures revealed the knockdown}

effects of shNHE3 in reducing the number of domes observed per microscopic filed (100x), showing ~5/field and ~1/field for shNHE3-4 and shNHE3-10, respectively in comparison with the control NMuMG (~48/field) and pSM2 control (~46/field) cells (Fig. 9C). These results suggest the role of NHE3 in regulating the initiation stage of dome formation.

NHE3 inhibitors block epithelial dome formation. The role of NHE3 in dome formation was further examined by using NHE3 inhibitors, EIPA (specific NHE family inhibitor) and S3226 (specific NHE3 inhibitor). These inhibitors were applied to confluent MDCK or NMuMG cells and then dome formation was assessed. As shown in Fig. 10, after 24 h treatment with different doses of EIPA or S3226, the number of domes in both MDCK (upper panel) and NMuMG (lower panel) cells was

decreased substantially in a dose-dependent manner. In addition, there was a tendency that EIPA was more effective in reducing the number of domes formed than S3226. This may indicate the involvement of other members from the NHE family in the process of dome formation.

DISCUSSION

Activation of Stat3 during cell confluence has been shown to play important physiological roles in cell growth arrest and survival (15, 43, 45). This has led us to speculate that a distinct Stat3 signaling event in cell differentiation may be functionally and biologically relevant to dome formation in epithelial cells. This study provides evidence for a distinct JAK/Stat3/NHE3 signaling pathway during cell confluence to coordinate MDCK epithelial dome formation. The evidence includes the followings: (1) Cell confluence induces Stat3 signaling through Stat3 Tyr705-P translocalization in a homogeneic form. (2) The translocalization of Stat3 Tyr705-P to nucleus is associated with DNA binding and regulation of NHE3 promoter activities during the initial but not the later stage of dome formation. (3) The extent of dome formation during the initiation stage is governed by Stat3 downstream effectors which most likely involve NHE3. (4) Inhibition of NHE3 diminishes the confluence-induced dome formation. (5) JAK inhibitor blocks Stat3 activation, NHE3 expression and dome formation.

The study reported here demonstrates a novel role of Stat3 in transcriptional regulation of NHE3. To our knowledge, there is virtually no information regarding to Stat3 regulation of NHE3. Although our result does not directly demonstrate the binding of Stat3 to NHE3 promoter per se, potential STAT responsive sequences,

TTGGCCAA (-359 to -367), in the rat NHE3 promoter could be found by using TFSearch (16). In addition, Stat3 is known to synergize with other transcription regulators acting on a different site in the promoter. Whether certain transcriptional factors, such as AP-1, AP-2, C/EBP, NF-1, Oct-1, Sp1, glucocorticoid receptor and thyroid receptor (7, 24, 32), known to bind to the rat NHE3 promoter (19), involve in the synergistic regulation of Stat3 to modulate NHE3 promoter for dome formation require further exploration.

Our result demonstrates a distinctive role of Stat3 activation in promotion of epithelial differentiation. The specific translocalization of Stat3 Tyr705-P in early dome structure (Fig. 3) and the effect of exogenous Stat3-C and Stat3-D in modulating the process of dome formation provide solid evidence for such a unique biological consequence. Although Stat3 in most instances promotes cell proliferation or transformation (4, 13), it does not affect cell proliferation in MDCK cells (data not shown), ruling out the possibility that Stat3 regulated dome formation through promoting cell proliferation. This is further supported by the decreased cell proliferation markers following cell confluence and by the observation of cell proliferation-independent elevation of nuclear Stat3 Tyr705-P in HCD culture during confluence. In addition, nuclear staining of the 5th _{day confluent monolayer with}

area (unpublished data). It has been well established that cell proliferation of normal epithelial cells is inhibited by cell contact and cell differentiation, such as dome formation, is triggered by cell confluence. This study strongly indicates that local accumulation of Stat3 Tyr705-P in confluent epithelial monolayer mediates the cell density effect to drive dome formation.

Corresponding to the cell density-induced Stat3 activation, dome formation in MDCK (or NMuMG) epithelial cells was correlated with the augmentation of NHE3 through Stat3 mediated transcriptional regulation. Early induction of NHE3 by Stat3 did not result in dome formation before cell confluence. Instead, the expression of NHE3 reaches the highest level during the appearance of dome structures and declined with decreased Stat3 activity soon after dome formation. It has been demonstrated that localization of NHE3 to the apical site in morphologically polarized epithelial cells is critical for the execution of its function (1, 38, 48). Inhibition of NHE3 expression and activity respectively by iRNA and functional inhibitors (EIPA and S3226) further strengthen the essential role of NHE3 activity in dome formation. In addition, augmentation of different sodium transporters, Na+_,K+_{-ATPase and ENaC,}

has been reported in responding to several differentiation inducers, such as DMSO, 8Br-cAMP, or steroid hormones (e.g. estrogen, aldosterone, glucocoticoid, etc.), to drive dome formation in epithelial cells (5, 21, 31, 52). We observed concurrent

expression of ENaC following Stat3 activation, yet the elevation of ENaC was not regulated by Stat3. In addition, the expression level of Na+,K+-ATPase β1 or NHE1 was not regulated by Stat3. These data indicate importance of NHE3 in regulation of dome formation.

In marking the onset of differentiation, Stat3 activation, however, appears to coordinate with other Stat3 related activities to modulate dome formation in MDCK monolayer. Elevated Stat3 activity has been observed in cells entering the growth arrest phase which precedes the differentiation of keratinocytes (15). Similar growth arrest phenomenon during cell confluence has also been observed in cancer cells (43). In that study, a possible mechanism of negative regulation of Stat3 by cyclin-dependent kinase 2 (cdk2), which is depressed upon cell confluence, has been reported (43). Furthermore, disruption of Stat3 Tyr705-P by specific peptide results in apoptosis in confluent NIH3T3 fibroblast cells, suggesting that cell confluence-induced Stat3 activity is critical for cell survival (43, 45). We have found that MDCK cells exhibit markedly enhanced apoptosis at post-confluent stage at day 6. In this study, we show that Stat3 phosphorylation and activity is down-regulated at the same time, suggesting the role of Stat3 activation in cell survival. On the other hand, despite that dominant negative Stat3-D reduces dome formation, it neither affects cell proliferation, nor enhances apoptosis. This may simply suggest that Stat3

activation-induced cell survival only occur to dome forming cells.

That Stat3 activation modulates cell differentiation process has also been described in a ligand-dependent manner by accepting signals from specific cytokines or growth factors (6, 30, 42). For instance, Stat3 responds to EGF to induce keratin 13 expression for squamous epithelium differentiation (49) and to G-CSF-induced expression of cdk inhibitor p27kip1 _{in myeloid cells differentiation of non-epithelial}

cell origins (40). However, Stat3 activation observed in our system and others (45) are distinct from those ligand-dependent cell differentiation systems, as disruption of cell-to-cell contacts in confluent cells by calcium chelater (e.g. EDTA) reduced Stat3 activity levels and serum starvation in MDCK cells could not depress cell density-induced Stat3 Tyr705 phosphorylation (data not shown). These results corroborate with two previous reports showing that neither serum starvation nor disruption of the potential growth controllers, such as EGF, IGF-1 or Ras-mediated signaling, influences cell density-induced Stat3 activation in fibroblast, normal breast epithelial, or breast carcinoma cell lines (43, 45). It is possible that the signaling pathway of the confluence-mediated Stat3 activation may prevalently exist in various cell types with certain biological roles of many kinds for confluence-associated survival (45).

characteristics of epithelial cell differentiation and its underlining mechanisms. Given that cell-cell adhesion is the primary step in association with subsequent formation of tight junction, cell polarity, and microvilli for epithelial cell differentiation (39, 41), it is appreciable that our result unveils the molecular mechanism whereby activation of Stat3 triggered by cell confluence participates in transepithelial transport through activation of NHE3 during the epithelial differentiation process. A novel JAK/Stat3/NHE3 signaling pathway induced by cell confluence, which is distinct to ligand-dependent stimulation, is accountable for modulating such an event. Many important questions remain to be investigated, including (1) the up-stream membrane molecules mediating cell density-induced Stat3 activation, (2) the detailed regulatory mechanism of NHE3 promoter by Stat3 and (3) the functional relevance of NHE3 in coordinating the modulation of dome formation. As dome formation mimics the status terminal differentiation of renal epithelium during epithelial cell remodeling in vivo (11, 27, 51), our findings open the possibility to further unravel the role of JAK/Stat3/NHE3 signaling pathways in epithelial cell differentiation.

ACKNOWLEDGMENTS

We are grateful to Dr. Norimoto Yanagawa for his thorough review and discussion of our manuscript, and to Ms. Tsu-Ling Chen for her technical assistance. This work was supported by National Science Council grant NSC 94-2320-B006-060 and the Ministry of Education Program for Promoting Academic Excellence in Universities grant 91-B-FA09-1-4 to Ming-Jer Tang.

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FIGURE LEGENDS

FIG. 1. Cell confluence triggers Stat3 Tyr705 phosphorylation and DNA binding

which precedes dome formation in MDCK cells. (A) Cell confluence was monitored by phase-contrast microscopy (100x) from day 1 to day 6. Dome formation (pointed with arrows) was initiated at day 5 and enhanced at day 6. Cultured MDCK cells reached confluence at day 4 and the confluence was coined as 100%. The percentage of cell confluence, as shown below each picture, was assessed by dividing the cell number with confluent cell number (day 4). (B) Whole cell lysates collected at indicated days were subjected to Western blot analysis with indicated antibodies to detect phosphorylation events of Stat1, Stat3, and Stat5. Phosphorylation of Stat1 and Stat5 showed no correlations with cell confluence although they were expressed in most epithelial cells. Jurkat T cells treated with IL-6 were used as a positive control (PC) for Stat5 Tyr694 phosphorylation and β-actin was used as an internal control. (C) Nuclear extracts of MDCK cells collected at indicated days were subjected to DNA affinity precipitation assay (DAPA) using Stat1 and Stat3 specific oligomer m67 as a probe (14). Western blot analysis was then employed for the determination of Stat1 and Stat3 levels (upper panel). The nuclear extract of NIH 3T3 cells stimulated with IL-6 subjected to DAPA was used as the positive control (PC). In addition, the nuclear extracts were analyzed by Western blot in a parallel experiment to correlate Stat3

Tyr705-P with cell confluence and the markers of cell proliferation, such as Ki-67 and PCNA (lower panel). A nuclear matrix protein Lamin A was used as the internal control. IB indicates immunoblotting.

FIG. 2. High cell density triggers Stat3 Tyr705 phosphorylation and DNA binding

in MDCK cells. (A) MDCK cells seeded at low cell density (LCD, 1x106 cells/10-cm dish) and high cell density (HCD, 6x106 _{cells/10-cm dish) were monitored by}

phase-contrast microscopy after 1 day of culture. (B) Similar amounts of whole cell lysates from LCD or HCD cultures were subjected to Western blot analysis with indicated antibodies to correlate Stat3 Tyr705 phosphorylation with cell density. (C) Similar amounts of nuclear extracts from LCD or HCD cultures were subjected to DAPA (upper panel) and Western blot analysis using indicated antibodies (lower panel) to correlate cell density with Stat3-DNA binding and the level of Tyr705 phosphorylated Stat3 in the nucleus, respectively. The nuclear extract of NIH 3T3 cells stimulated with IL-6 was used as the positive control (PC) for DAPA. “Bead only” was a negative control without adding m67 probe in nuclear extracts. The Ki-67 and PCNA were cell proliferation markers. A nuclear matrix protein Lamin A was used as the nuclear internal control.

FIG. 3. Nuclear localization of Stat3 Tyr705-P during dome formation. (A) Cultured MDCK cells at indicated days were double-stained with anti-Stat3 Tyr705-P

antibody (green) and nuclear indicator Hoechst 33258 (blue). Note that domes composed of monolayer of cells were flattened by fixation. Photographs were taken by epi-fluorescent microscopy. At day 4, cells at a condensed region prior to dome formation were stained positive for Stat3 Tyr705-P, whereas at day 5, Stat3 Tyr705-P was observed predominantly within domes. At day 6, little staining of Stat3 Tyr705-P in the dome was observed. (B) The percentage of domes stained positive for Stat3 Tyr705-P during formation was quantified. At day 5, 45 out of 59 (76.3%) domes stained positively of Stat3 Tyr705-P, while at day 6, 10 out of 51 (19.6%) domes were positive for Stat3 Tyr705-P. (C) Confocal microscopic examination of sub-cellular localizations of Stat3 Tyr705-P and Stat3 at the central area of the dome during and after dome formation. MDCK cells at indicated days were double-stained with Hoechst 33258 (blue) and either Stat3 Tyr705-P (green) or Stat3 (green) antibodies. Negative control (NC) indicates day 5 cells receiving staining by secondary anti-rabbit-alex488 (green) antibody alone. Dome area stained with an ezrin (green) antibody was used as a positive control for the specificity of membrane localization. FIG. 4. Stat3 activation regulates dome formation in MDCK cells. (A) Doxycycline induced dose-dependent Stat3-C expression in two tetracycline inducible MDCK clones (St3C-3 and St3C-4 cells). MDCK transfectants expressing either rtTA alone (mock) or the rtTA-controlled FLAG epitope-tagged Stat3-C (St3C-3 and

St3C-4) were treated with doxycycline (0, 1, 10 μg/ml) for 5 days. The cell lysates were subjected to Western blot analysis with antibodies against FLAG, Stat3 or β-actin. (B) Dome formation in mock, St3C-3, and St3C-4 cells in response to doxycycline treatment for 5 days was visualized by phase-contrast microscopy. The number (C) and the diameter (D) of domes at day 5 were quantified by averaging from at least 20 independent fields (100x) in 3 independent experiments. (E) Selected MDCK transfectants expressing dominant negative Stat3 mutants (Stat3-F: Stat3-Y705F mutant; Stat3-D: DNA-binding domain mutant) were cultured for 5 days and their levels of Stat3 or Stat3 Tyr705-P were analyzed by Western blot with indicated antibodies. A corresponding dome formation assay for Stat3-F and Stat3-D cells at day 5 was also conducted to estimate the number (F) and the size (G and H) of domes according to the method mentioned above. Values were plotted as means ± SE from at least 3 independent experioments. *, p <0.05; **, p <0.01; ***, p <0.001;

t-test.

FIG. 5. Stat3 regulates NHE3 expression in MDCK cells. (A) Whole cell lysates harvested daily from day 0 to day 6 were subjected to Western blot analysis with antibodies against NHE3, NHE1, ENaC α-subunit, Na,K-ATPase α1 or β1 subunit, or β-actin. (B) St3C-3 cells seeded for 2-day and then treated with (+) or without (-) doxycycline (10 μg/ml) for 24 h to 96 h. The cell lysates were subjected to Western

blot analysis for the detection of NHE3 and ENaC α-subunit. (C) St3C-3 cells treated with different doses of doxycycline (0, 1, 10 μg/ml) for 4 days. The cell lysates were subjected to Western blot analysis for the detection of NHE3, NHE1 and ENaC α-subunit. Expression of NHE3 in St3C-3 cells was up-regulated by doxycyline treatments in a dose dependent manner. (D) Stat3-D stable transfectants (St3D clones #2, #3, #5, and #10) cultured for 4 days were subjected to Western blot analysis for detection of NHE3 and NHE1 levels. The result indicated that dominant negative Stat3 inhibited NHE3 expression. (E) and (F) show NHE3 and Stat3 mRNA levels in cell lines corresponding to (C) and (D), respectively, as assessed by RT-PCR analysis using primers specific to canine NHE3, Stat3 or GAPDH. The results indicated that active Stat3 augmented and dominant negative Stat3 inhibited NHE3 mRNA levels. FIG. 6. Stat3 regulates NHE3 promoter activity in MDCK cells. (A) Induction of Stat3-C in St3C-3 cells by doxycycline up-regulated the rat NHE3 promoter activity (-450 to +58 region) in a dual luciferase reporter assay. St3C-3 and mock cells were co-transfected with a pCMV-Luc vector (firefly luciferase) in combination with a

Renilla luciferase reporter gene containing plasmid (either NHE3 promoter-driven

pNHE3 -450/+58 or promoterless phRG-b vector). The activity of the dual luciferase reporter assay system was analyzed after 20 h of doxycycline (10 μg/ml) treatment. The levels of Renilla luciferase activity were normalized with firefly luciferase

activity as the internal control for transfection efficiency. The results were presented as the normalized fold-induction of Renilla luciferase activity average from 3 independent experiments, as compared to the basal level luciferase activity from St3C-3 cells transfected with phRG-b in the absence of doxycycline in triplicate. (B) Stable cell line (St3D-10) harboring dominant negative Stat3 (Stat3-D) and wild type MDCK cells were used to evaluate the NHE3 promoter activity in a dual luciferase reporter system as described in (A). (C) To verify whether Stat3-C augmented luciferase activity were affected at translational levels, a thymidine kinase promoter driven plasmid (pRL-TK) expressing Renilla luciferase was used in a dual luciferase reporter assay system as described in (A) except that the pNHE3 -450/+58 was replaced by pRL-TK. The relative luciferase activity average from mock cells transfected with pRL-TK without doxycycline (10 μg/ml) induction was regarded as 100%. Values were plotted as means ± SE from at least three independent experiments. *, p <0.05; ***, p <0.001; t-test.

FIG. 7. Cell confluence-induced Stat3 activation regulates NHE3 expression and

dome formation in NMuMG cells. (A) Whole cell lysates of NMuMG collected every 2 days were subjected to Western blot analysis with indicated antibodies. β-actin was used as an internal control. (B) Cell lysates collected from low cell density (LCD, seeding at 1x106 _{cells/10-cm dish) or high cell density (HCD, seeding}

at 6x106 _{cells/10-cm dish) of NMuMG cells cultured after 24 h were subjected to}

Western blot with indicated antibodies. (C) Cell lysates collected from the day 6 NMuMG cells expressing Stat3-D with HA tag were subjected to Western blot with indicated antibodies. (D) NMuMG cells and Stat3-D transfectants of NMuMG cells corresponding to (C) were subjected to dome formation assay and quantitatively estimated by counting domes under at least 20 un-overlapping (100x) fields in 3 independent experiments. Values were plotted as means ± SE from at least 3 independent studies. ***, p <0.001; t-test.

FIG. 8. JAK is involved in cell confluence-regulated dome formation. Confluent MDCK (upper panel) or NMuMG (lower panel) cells at day 4 or day 6, respectively, were treated with JAK inhibitor AG490 (20 or 40 μM) for 24 h. (A) Whole cell lysates collected at indicated times were subjected to Western blot analysis with indicated antibodies. DMSO (1:1000 dilution) was used as a solvent control for AG490. The results showed that JAK inhibitor AG490 effectively blocked Stat3 Tyr705 phosphorylation as well as NHE3 expression. (B) Dome formation assay was performed by counting domes under at least 20 un-overlapping (100x) fields in each experiment. Values were plotted as means ± SE from at least 3 independent experiments. p <0.001, ANOVA, n=60.

retards dome formation. Control NMuMG cells, pSM2 or shNHE3 transfectants (#4 and #10) were cultured for 6 days. (A) Cell lysates were subjected to Western blot with indicated antibodies. NHE3 shRNA significantly reduced NHE3 levels in both transfectants. (B) Dome formation in the above cell lines was visualized by phase-contrast microscopy. (C) Dome formation per field was quantitatively estimated by counting the number of domes under at least 20 un-overlapping (100x) fields. NHE3 shRNA significantly blocked dome formation in NMuMG cells in both transfectants. Values were plotted as means ± SE from 3 independent experiments. ***, p <0.001; t-test.

FIG. 10. NHE3 inhibitors block dome formation in MDCK and NMuMG cells. Confluent MDCK (upper panel) or NMuMG (lower panel) cells were treated with different doses of indicated inhibitors, EIPA (1, 10, 102 _{nM in DMSO, specific for}

NHE family) and S3226 (10, 102_{, 10}3 _{nM in DMSO, specific for NHE3). Cells treated}

with (+) DMSO was used as solvent controls. After additional 24 h of incubation, dome formation was quantitatively estimated by counting domes under at least 20 un-overlapping (100x) fields. Values were plotted as means ± SE from at least 3 independent experiments. p <0.01, ANOVA, n=60.

B

IB: whole cell lysate

Stat1 Tyr701-P Stat5 Tyr694-P PC

C

Stat3 Tyr705-P Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

A

5.25% 17.7% 66% 100% 100%+ 1 d 100%+ 2 d

Stat3 Tyr705-P Stat3 β-actin Stat3 Ser727-P IB: whole cell lysate

B

C

A