The authors have declared that no competing interests exist.
Conceived and designed the experiments: RJH RSB MHW. Performed the experiments: RJH LC RSB. Analyzed the data: RJH RAE. Contributed reagents/materials/analysis tools: MHW. Wrote the paper: RJH RAE MHW.
Since inflammatory bowel diseases (IBD) represent significant morbidity and mortality in the US, the need for defining novel drug targets and inflammatory mechanisms would be of considerable benefit. Although protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been primarily studied in an oncogenic context, it was noted that PTK6 null mice exhibited significantly enhanced colonic epithelial barrier function. Considering that the inflammatory functions of PTK6 have not yet been explored, we hypothesized that cytokines responsible for mediating IBD, such as TNFα/IFNγ, may solicit the action of PTK6 to alter barrier function. After first assessing critical mediators of TNFα/IFNγ driven epithelial barrier dysfunction, we further explored the possibility of PTK6 in this inflammatory context. In this report, we showed that PTK6 siRNA and PTK6 null young adult mouse colonic epithelial cells (YAMC) exhibited significant attenuation of TNFα/IFNγ induced barrier dysfunction as measured by electric cell-substrate impedance sensing (ECIS) assay and permeability assays. In addition, PTK6 null cells transfected with PTK6 cDNA displayed restored barrier dysfunction in response to TNFα/IFNγ, while the cells transfected with vector alone showed similar attenuation of barrier dysfunction. Furthermore, using subcellular fractionation and immunocytochemistry experiments, we found that PTK6 plays a role in FoxO1 nuclear accumulation leading to down-regulation of claudin-3, a tight junction protein. Moreover, we searched for relevant miRNA candidates putative for targeting PTK6 in order to identify and assess the impact of microRNA that target PTK6 with respect to TNFα/IFNγ induced barrier dysfunction. Subsequently, we assayed likely targets and determined their effectiveness in attenuating PTK6 expression as well as cytokine induced barrier dysfunction. Results showed that miR-93 reduced PTK6 expression and attenuated TNFα/IFNγ imposed decrease in transepithelial electrical resistance (TER), as well as excluded FoxO1 from the nucleus. Our results indicate that PTK6 may act as a novel mediator of intestinal epithelial permeability during inflammatory injury, and miR-93 may protect intestinal epithelial barrier function, at least in part, by targeting PTK6.
Abnormal intestinal epithelial barrier function is often observed in patients with inflammatory bowel diseases characterized by inflammation driven relapsing diarrhea [
The distantly Src-related protein kinase termed protein tyrosine kinase 6 (PTK6) has been identified as playing a role in intestinal epithelial barrier function in that PTK6 knockout led to several fold increase in basal intestinal epithelial barrier function as evidenced by resistance measurements of cultured monolayers [
Barrier function is a dynamic process that demands regulation at the epigenetic level. As shown by Ghatak et al, interruption of the machinery required to process microRNA resulted in high trans-epidermal water loss in mice [
The conditionally immortalized cell line, young adult mouse colonic epithelial cells (PTK6+/+ = “wild type” or WT YAMC; and PTK6-/- YAMC) were a gift from Dr. Whitehead at Vanderbilt University [
YAMC barrier function was determined as previously described [
YAMC treated (cytokine cocktail: (TNFα [100ng/ml]/ IFNγ [500U/ml]) or vehicle control (VC) 0.1% BSA in PBS) 16 hours were lysed in RNAzol (Molecular Research Center, Inc) followed by RNA extraction as described by manufacturer. The mRNA fraction was quantified and equal masses of RNA were reverse transcribed to cDNA using random hexamers prior to qPCR. Samples without reverse transcriptase (No RT) were run as negative control. Equal volumes of cDNA were used to quantify PTK6 message using specific primers (Bio-Rad) and SYBR master mix (Bio-Rad) according to manufacturer’s instructions. Samples were assayed in triplicate and No RT controls were used to ensure absence of genomic DNA (data not shown). Relative mRNA expression levels were determined by using GAPDH as the housekeeping gene via CFX96 Touch Real-Time PCR detection system and software (Bio-Rad). Data are representative of 4 experiments *p<0.05.
Sodium fluorescein-flux assays were conducted as previously described [
WT or PTK6-/- YAMC monolayers were grown in 2-chamber slides (Nunc) for immunocytochemistry analysis. Fixation and immunolabeling were performed using standard protocols. Briefly, cells were washed twice [PBS with 2mM CaCl2, 2mM MgCl2] followed by incubation in 4% paraformaldehyde for 10 minutes. Slides were rinsed twice in PBS, permeabilized [PBS, 0.1% Triton X-100], then blocked [PBS, 3% BSA, 0.01% Tween-20] for 2 hours at room temperature. Cells were incubated with primary antibodies (ZO-1, Cell Signaling/ FoxO1, Millipore) 16 hours at 4°C diluted 1:50 [PBS, 0.01% Tween-20, 3% BSA], washed 3 times for 10 minutes each [PBS, 0.1% Tween-20], incubated with appropriate secondary antibody diluted 1:200 [PBS, 0.01% Tween-20, 2% BSA] (AlexaFluor-conjugated; Life Technologies). Slides were mounted with anti-fade media containing DAPI (Life Technologies). Immunofluorescence confocal microscopy was performed with Olympus FV1000 MPE multiphoton laser scanning microscope (Olympus). Images were performed from 3 independent experiments and representative confocal micrograph images are presented. Images were analyzed using Image J software as previously described [
Treated YAMC (TNFα [100ng/ml]/ IFNγ [500U/ml]) or vehicle control (VC) 0.1% BSA in PBS 16 hours) were subjected to nuclear fractionation in order to assess changes in FoxO1 nuclear accumulation relative to treatment and PTK6 expression status. Cells were lysed in fractionation buffer (250 mM sucrose, 20 mM HEPES pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1X phosphatase inhibitor cocktail (Pierce)) followed by several passages through 25 G needle. Nuclear pellets were obtained by centrifugation (3,000 RPM, 4°C, 5 minutes) and washing 4 X in PBS. Pellets were then sonicated and resuspended in nuclear buffer. The nuclear fractions were subjected to BCA protein assay (Pierce) prior to Western blot analysis. Equal masses of protein were loaded to 4–20% polyacrylamide gels (Bio-Rad) for SDS-PAGE followed by Western blotting using FoxO1 antibody (Cell Signaling), or lamin A/C antibody (Cell Signaling) to ensure equal protein loading of nuclear proteins. Results are representative of 3 individual experiments.
Identifying microRNAs for study was performed by analyzing the PTK6 3’UTR at several levels (
The confirmation of miR-93 targeting the 3’UTR of PTK6 was determined by using the LightSwitch Luciferase assay system (SwitchGear Genomics). YAMC cells were plated at 5 X 105 cells/ml in a white 96 well culture plate the day before initiating the assay. On the following day, 30ng/μl of the PTK6 3’UTR, mutated PTK6 3’UTR (mutated at putatitive miR-93 binding sites), or reporter vector only were co-transfected with 100nM miR-93, mutated miR-93, scrambled sequence, or miR-518 for 24 hours. The luciferase assay was performed the following day according to the manufacturer’s instructions. Knockdown was calculated by determining the luciferase signal ratio for each specific combination and comparing that to signal intensity for the reporter vector and scrambled sequence. Experiments were performed in triplicate and repeated three times for statistical significance.
One-way analysis of variance was used to determine significantly different changes among sets of 3 or more groups. Significance (p<0.05) was noted between treated and vehicle control groups using Students
Since colonic epithelial cells from PTK6-/- mice showed enhanced barrier function under basal conditions, we wanted to determine if the expression of PTK6 was regulated by inflammatory stimulation. Therefore, WT YAMC were grown to confluence then treated with TNFα/IFNγ cocktail or vehicle control (VC) 16 hours followed Western blotting and qPCR. In order to examine the precise role that PTK6 may play in YAMC under these conditions, we probed for a number of signaling molecules known to mediate pathways that characterize epithelial inflammation. Results showed that TNFα/IFNγ (100ng/ml, 500U/ml) induced PTK6 expression at the level of mRNA and phosphorylation at Y342 (
A) Mortalized, 2-day post-confluent YAMC monolayers were treated with vehicle control (0.1% BSA in PBS) or TNFα (100ng/ml) and IFNγ (500U/ml) at the indicated time points then assayed for changes in expression of the indicated proteins (please refer to
Next, we sought to determine whether decreasing the expression of PTK6 was beneficial to barrier function in YAMC treated with TNFα/IFNγ cocktail. We first determined the impact of TNFα/IFNγ at various concentrations to identify the best concentration for the assay. Results showed that TNFα/IFNγ at 100ng/ml and 500U/ml yielded the strongest response (
A) YAMC were electroporated with PTK6 siRNA or vehicle control (TE) then plated on ECIS arrays and allowed to express for 48 hours. Monolayers were then treated with vehicle control or indicated cytokines then resistance measurements were recorded every 90 seconds B) Either WT or PTK6-/- YAMCs were grown on gold-plated ECIS arrays, stimulated with either vehicle control (VC) or TNFα (100ng/ml) and IFNγ (500U/ml), then resistance measurements were recorded every 90 seconds. Values were normalized to timepoint zero and shaded area represents standard error (n = 4). C) PTK6-/- cells were transfected with empty vector (EV) or PTK6 cDNA then treated with either vehicle control or TNFα (100ng/ml) and IFNγ (500U/ml). Inlay shows successful overexpression of PTK6 cDNA vs. EV transfected cells. D) Either WT or PTK6-/- YAMCs were grown on transwell inserts at 2 X 105 cells/ml until reaching 2-days post-confluence then treated with vehicle control (VC) or TNFα (100ng/ml) and IFNγ (500U/ml) for 24 hours. Permeability coefficients were calculated as described in methods. Error bars represent standard error (*p<0.05 compared to WT, n = 3). E) Westerns showing relative expression of PTK6 in siRNA treated and epithelial cells from PTK6-/- knockout mice (Please see
In order to exclude the possibility that barrier changes observed in response to TNFα/IFNγ between PTK6 -/- and WT YAMC was erroneous, we transfected PTK6-/- cells with PTK6 cDNA or empty vector (EV) then compared TER values in response to treatment. Results showed that PTK6 expression in PTK6-/- cells sensitized monolayers to cytokine treatment and dropped resistance by approximately 10%, whereas PTK6-/- cells transfected with empty vector showed slightly enhanced resistance despite treatment (
Next, to determine the differences in paracellular permeability between PTK6-/- and WT YAMC under inflammatory conditions, we utilized permeability assays for analysis of sodium fluorescein flux [
Next, considering that TNFα promotes FoxO1 nuclear accumulation [
Post-confluent, mortalized WT or PTK6-/- YAMC monolayers were treated with vehicle control (0.1% BSA in PBS) or TNFα (100ng/ml) and IFNγ (500U/ml) 16 hours. A) Nuclei (blue), ZO-1 (green), and FoxO1 (red) were detected via confocal microscopy. B) Quantitation of the ratio of ZO-1 intensity in the cytoplasm over cell-cell junction from Fig 3A. C) Quantitation for nuclear FoxO1 intensity of FoxO1 stain in Fig 3A. D) Western blot indicating expression levels of ZO-1 in total cell lysates (please see
In order to identify the pathways PTK6 may regulate in order to increase permeability in response to inflammation, we treated cells expressing siRNA against PTK6 or PTK6 knockout cells and identified changes in markers of epithelial inflammation relative to the status of PTK6 expression. As shown in
A) YAMC cultures were transfected with vehicle control or PTK6 siRNA then treated with vehicle control or TNFα/IFNγ for 16 hours. Nuclear fractions were harvested then assayed for presence of FoxO1. PTK6 was assayed in whole cell lysate to ensure knockdown. Densitometric calculations were based on 3 separate experiments (please see
Since microRNA often target multiple genes in a pathway in a manner that yields a physiological change, we sought to identify putative microRNA that target PTK6 and assess its efficacy in mitigating poor barrier function due to inflammation. Therefore, we utilized a number of sequence analysis suites to determine which miRNA may be appropriate to study. After considering 4 levels of criteria (site binding prediction, literature support, conservation of binding site, and number of available binding sites in the 3’UTR), miR-93 was identified as a candidate most worthy to pursue in improving barrier function and targeting PTK6 (
A) YAMC were co-transfected with the indicated construct and scrambled sequence control or miR-93 then inhibition of luciferase excitation was assessed. B) YAMC were co-transfected with the indicated combinations of reporter/ oligo then luciferase excitation was assessed. C,D) YAMC were transfected with miR-93 mimic C) or miR-93 inhibitor D) then expression of PTK6 was determined by Western blot (please see
Since it was clear that PTK6 plays a significant role in inflammatory epithelial barrier dysfunction and miR-93 targets PTK6, we were interested in determining whether miR-93 treatment of YAMC improved cytokine mediated epithelial barrier dysfunction. To assess paracellular permeability, miR-93 transfected WT YAMC cells (2 X 105 cells/ml) or mock transfected were seeded on collagen I coated transwell inserts then grown to 2 days post-confluence. Sodium fluorescein flux was measured in presence of either vehicle control or TNFα/IFNγ Results showed that miR-93 transfection of YAMC significantly attenuated TNFα/IFNγ mediated increase in sodium fluorescein flux while the cells transfected with mock displayed significant increase in permeability upon TNFα/IFNγ (
A) YAMC were electroporated with miR-93 or vehicle control (TE) and allowed to express for 48 hours. Monolayers were then treated with vehicle control or indicated cytokines for 24 hours then sodium fluorescein flux was assayed. Cell lysates were analyzed for PTK6 expression by Western blot. (*p<0.05 compared to treated mock, #p<0.05 compared to VC-mock. Error bars represent standard error, n = 4) C) YAMC were transfected as indicated then seeded on ECIS arrays at 2X105 cells/ml followed by treatment with either vehicle control or TNFα/IFNγ. Resistance measurements were recorded every 90 seconds and values were normalized to timepoint zero. Shaded area represents standard error. C) YAMC were transfected with the indicated sequences for 48 hours then treated with vehicle control or TNFα/IFNγ as indicated in Methods. Flux to sodium fluorescine was measured by comparing fluorescence in luminal vs. abluminal compartments. D) YAMC cultures were transfected with vehicle control or mature miR-93 then treated with vehicle control or TNFα/IFNγ 16 hours. Nuclear fractions were harvested then assayed for presence of FoxO1. E) PTK6 was assayed in whole cell lysate to ensure adequate targeting of PTK6. F) qPCR results indicating successful transfection of miR-93 mimic in YAMC. Densitometric calculations were based on 3 separate experiments. (please see
Aberrant intestinal epithelial barrier function is a critical factor in several inflammatory diseases in the gut. Since PTK6 has been shown to play a role in basal epithelial barrier function, we sought to determine whether PTK6 also plays a role in mediating the response of inflammatory cytokines TNFα and IFNγ induced epithelial barrier dysfunction, and whether miR-93 targeting of PTK6 improved barrier function under such conditions. In this study, we provided evidence in support of 1) TNFα/ IFNγ induced expression and post-translational modification of PTK6; 2) PTK6-/- intestinal epithelial cells showed enhanced ZO-1 localization to cell-cell borders, and an attenuated response to TNFα/ IFNγ in ECIS and permeability assays; 3) Targeting PTK6 attenuated TNFα/ IFNγ driven JNK activation and claudin-3 downregulation 4) MiR-93 targeted PTK6 in YAMC and improved TNFα/ IFNγ mediated barrier dysfunction; and 5) FoxO1 nuclear accumulation resulting from TNFα/ IFNγ treatment was attenuated by PTK6 siRNA or miR-93.
The expression of several kinases are known to be upregulated by inflammatory cytokines including Src, fyn, and Yes [
After demonstrating that PTK6 mRNA was up-regulated by TNFα/ IFNγ, we then pursued the possibility that PTK6 may be involved in barrier dysfunction resulting from this treatment. By permeability and ECIS assays, we showed that diminishing PTK6 expression, by siRNA or knockout, improved barrier function in response to TNFα/ IFNγ. These findings were also observed in endothelial cells [
After the observation that claudin-3 downregulation was attenuated by targeting PTK6 expression, we investigated known signaling pathways to determine a possible mechanism that PTK6 potentiates barrier dysfunction. We and others have demonstrated that FoxO1 nuclear accumulation is a key factor in tight junction protein down-regulation [
Since the therapeutic potential of microRNA is gaining momentum, we were interested in determining whether a microRNA targeting PTK6 may improve epithelial barrier function in the context of inflammation. Although it is widely known that microRNA have many targets, it is generally accepted that specific microRNA, or even microRNA families, target mRNA that encode proteins in the same pathway. Therefore, in demonstrating miR-93 supported FoxO1 exclusion from the nucleus, PTK6 down-regulation, and improved TNFα/IFNγ intestinal epithelial barrier dysfunction, we expect that this particular microRNA may be worth further exploration as a therapeutic agent with respect to intestinal epithelial permeability. In corroboration with our findings, a recent publication demonstrated that miR-93 activated PI3K/Akt signaling through direct suppression of PTEN [
Overall, these results suggest that PTK6 is involved in inflammation driven intestinal epithelial barrier dysfunction, and miR-93 may serve as a novel intervention strategy to improve intestinal epithelial barrier function.
Monolayers of either cell type were grown on ECIS arrays as detailed in the Methods section. Peak decrease in normalized resistance following treatment is shown. Although 100ng/ml TNFα alone showed a similar response to TNFα/IFNγ (4th column vs. last column), since the combination of TNFα/IFNγ more closely mimics that which is seen in vivo, we conducted subsequent experiments performed in this study with TNFα/IFNγ as stimulus.
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A) First, predicted miRNA were identified using MirWalk “Gene-miRNA” prediction algorithm. This analysis produced 140 unique miRNA sequences. From this list, five microRNA known to be involved in inflammation or barrier function were identified for further analysis (Column 1). Probability scores on these five sequences were considered a “hit” when p<0.05 using MiRanda, PicTar, RNA22, and TargetScan (Column 2). Next, a pairwise alignment of the PTK6 3’UTR for human and mouse was conducted to determine areas of conservation (Column 3). The reverse complement (Column 4) of the seed sequence was searched for in the human (Column 5) or mouse PTK6 3’UTR (Column 6) (Ensemble) as well as in the pairwise alignment. The number of sites that were conserved across humans and mice are listed in Column 7. Two sequences showed binding potential in areas conserved between mice and humans. Mir-93 scored the highest in all categories. B) The MirWalk algorithm was used to predict 140 microRNAs that may bind the 3’UTR of PTK6. A literature search was conducted to determine significance of microRNAs predicted to target PTK6, five were taken for further analysis. Additional prediction algorithms were used to assess liklihood for binding (MiRanda, PicTar, RNA22, and TargetScan). Results of this analysis are shown in A. C) Pairwise alignment of the PTK6 3’UTR in humans and mice. The shaded areas represent alignment score for the indicated region. D) The 4 regions with matching sequences are shown, with the region corresponding to miR-93 shown in green.
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Smaller blots are the result of cutting blot prior to antibody incubation for efficiency.
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We would like to acknowledge the generosity of Dr. Robert Whitehead from Vanderbilt University for giving us the WT YAMC and PTK6-/- YAMC cell lines, as well as Kimberly Watt for culturing and shipping the cells.
protein tyrosine kinase 6
young adult mouse colonic epithelial cells
transepithelial resistance
Src-homology 3 domain
wild type
electric cell-substrate impedance sensing
vehicle control