Airway smooth muscle (ASM) remodeling is a key feature in asthma and includes changes in smooth muscle-specific gene and protein expression. Despite this being a major contributor to asthma pathobiology, our understanding of the mechanisms governing ASM remodeling remains poor. Here, we studied the functional interaction between WNT-11 and TGF-β1 in ASM cells. We demonstrate that WNT-11 is preferentially expressed in contractile myocytes and is strongly upregulated following TGF-β1-induced myocyte maturation. Knock-down of WNT-11 attenuated TGF-β1-induced smooth muscle (sm)-α-actin expression in ASM cells. We demonstrate that TGF-β1-induced sm-α-actin expression is mediated by WNT-11 via RhoA activation and subsequent actin cytoskeletal remodeling, as pharmacological inhibition of either Rho kinase by Y27632 or actin remodeling by latrunculin A attenuated sm-α-actin induction. Moreover, we show that TGF-β1 regulates the nuclear expression of myocardin-related transcription factor-A (MRTF-A) in a Rho kinase-dependent fashion, which in turn mediates sm-α-actin expression. Finally, we demonstrate that TGF-β1-induced MRTF-A nuclear translocation is dependent on endogenous WNT-11. The present study thus demonstrates a WNT-11-dependent Rho kinase-actin-MRTF-A signaling axis that regulates the expression of sm-α-actin in ASM cells.
- airway smooth muscle
airway remodeling is a hallmark pathological feature of individuals with asthma and is associated with airway obstruction, airway hyperresponsiveness, and declining lung function in severe disease (4, 19, 22). Increased ASM mass and increased expression of contractile and contraction regulatory proteins are important features of the asthmatic airway (3, 23) and correlate with the severity of disease (3, 18, 44). ASM cells show a remarkable phenotypic plasticity, as they can exist in a synthetic proliferative phenotype actively undergoing cell divisions and releasing a number of inflammatory mediators and growth factors (17). In contrast, mature ASM cells have a low proliferation index and high abundance of contractile proteins like smooth muscle myosin heavy chain (sm-MHC), SM-22, and smooth muscle-α-actin (sm-α-actin) (15, 45).
WNT signaling regulates a myriad of functions from embryonic development extending throughout the lifespan of humans (1, 26). Intracellular WNT signaling is broadly classified into β-catenin-dependent and β-catenin-independent branches. The activation of WNT/β-catenin signaling results in the cytosolic accumulation of the transcriptional coactivator β-catenin which, in turn, translocates to the nucleus and activates WNT-target gene transcription. β-Catenin-independent, or non-canonical WNT signaling, on the other hand, activates various signaling cascades regulating cell polarity, cell movements, cytoskeletal reorganization, and gene transcription. The downstream effectors in these pathways include Ca2+ and RhoA (1).
WNT-11 is a member of the family of WNT ligands that consists of 19 members in humans. Although not studied in great detail, recent studies highlight a key role for WNT-11 in the regulation of myocyte differentiation via β-catenin-independent, or noncanonical signaling. For example, WNT-11 engages β-catenin-independent signaling, drives skeletal muscle morphogenesis (13), and promotes cardiac development and cardiomyocyte differentiation (6, 33, 37). In the developing lung, WNT-11 is preferentially expressed in the lung mesenchyme (21) suggesting a physiological role beyond cardiomyocyte differentiation. WNT-11 has been shown to mediate TGF-β1-induced sm-α-actin expression in renal epithelial cells (47). Based on these findings, we hypothesized a functional role for WNT-11 in the regulation of contractile phenotype maturation of smooth muscle in the airway.
Here, we demonstrate that WNT-11 expression increases during human ASM maturation. Furthermore, we show that WNT-11 mediates TGF-β1-induced actin remodeling via Rho kinase. Finally, we demonstrate that WNT-11 regulates myocardin-related transcription factor-A (MRTF-A) dynamics and mediates sm-α-actin expression.
MATERIALS AND METHODS
Recombinant human TGF-β1 and recombinant human WNT-11 were from R&D Systems (Abingdon, UK). siRNA specific for human WNT-11 and human MRTF-A, rabbit anti-MRTF-A antibody, mouse anti-Lamin A/C antibody and mouse anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-MYPT1 (Thr850) antibody was obtained from Cell Signaling Technology (Beverly, MA) and anti-β-actin antibody, anti-sm-α-actin antibody, HRP-conjugated goat anti-mouse antibody and HRP-conjugated goat anti-rabbit antibody were obtained from Sigma (St. Louis, MO). Alexa Fluor 488-conjugated phalloidin, Alexa Fluor 594-conjugated DNase I, Hoechst 33342 stain and ProLong Gold Antifade reagent were from Life Technologies (Bleiswijk, The Netherlands). Fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibody and Cy3-conjugated donkey anti-mouse antibody were procured from Jackson Immunoresearch Europe (Suffolk, UK). Human nonspecific control siRNAs were procured from Qiagen (Venlo, The Netherlands) and X-tremeGENE siRNA transfection reagent was purchased from Roche Applied Science (Mannheim, Germany). Y27632 dihydrochloride and latrunculin A were from Tocris (Bristol, UK). All other chemicals were of analytical grade.
Human ASM cell lines, immortalized by human telomerase reverse transcriptase (hTERT) and primary ASM cells, were used for all the experiments. The primary cultured human ASM cells used to generate each hTERT immortalized cell line were prepared as described previously (12). All procedures were approved by the Human Research Ethics Board (University of Manitoba). hTERT-ASM cell lines were maintained on uncoated plastic dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics (50 U/ml streptomycin, 50 μg/ml penicillin) and 10% (vol/vol) fetal bovine serum (FBS). For each experiment, hTERT-ASM cell lines (ASM cells) derived from two to three different donors were used for repeated measurements. Cells were serum-deprived in DMEM supplemented with antibiotics and ITS (5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenium) before each experiment. When applied, inhibitors were added 30 min before the TGF-β1 stimulation.
Primary ASM tissue and cells were derived from trachea and main stem bronchus obtained from healthy transplant donors at the University Medical Center Groningen. The study protocol followed national ethical and professional guidelines (“Code of conduct; Dutch federation of biomedical scientific societies”; http://www.federa.org) for all lung tissue and explant cell culture studies in Groningen. Selection criteria for healthy lung transplant donors are listed in the Eurotransplant guidelines and include the absence of primary lung disease such as asthma and COPD and no more than 20 pack years of smoking history. The ASM tissue was snap frozen after isolation and stored at −80°C until further use. The primary ASM cell cultures derived from the trachea and mainstem bronchus were cultured on uncoated plastic dishes in DMEM supplemented with antibiotics, 1% MEM essential vitamins, and 10% FBS.
ASM cells were grown to ∼90% confluence in 6-well cluster plates and transfected with 200 pmol of specific siRNA in serum and antibiotic free DMEM with X-tremeGENE siRNA transfection reagent. Control transfections were performed using a nontargeting control siRNA. After 6 h of transfection, medium was replaced with DMEM supplemented with antibiotics and ITS for a period of 42 h before TGF-β1 stimulation.
RNA isolation and real-time PCR.
Total RNA was extracted using the Nucleospin RNAII kit (Macherey-Nagel, Duren, Germany) as per the manufacturer's instructions. Equal amounts of total RNA were then reverse transcribed using the Reverse Transcription System (Promega, Madison, WI). One microliter of 1:2 diluted cDNA was subjected to real-time PCR, which was performed with the Illumina Eco Personal QPCR System (Westburg, Leusden, the Netherlands) using FastStart Universal SYBR Green Master Mix from Roche Applied Science (Mannheim, Germany). Real-time PCR was performed with denaturation at 94°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 30 s for 40 cycles followed by 10 min at 72°C. Real-time PCR data were analyzed using the comparative cycle threshold (Cq: amplification cycle number) method. The amount of target gene was normalized to the endogenous reference gene 18S ribosomal RNA (ΔCq). Relative differences were determined using the equation 2(−ΔΔCq). Primers used to analyze gene expression are WNT-11 forward (Fwd) 5′-ACTCTGCTCAAGGACCCTCA-3′ and reverse (Rev) 5′-GCTTCCAAGTGAAGGCAAAG-3′; sm-α-actin Fwd 5′-GACCCTGAAGTACCCGATAGAAC-3′ and Rev 5′-GGGCAACACGAAGCTCATTG-3′; MRTF-A Fwd 5′-GCCAGGTGAACTATCCCAAA-3′ and Rev 5′-CACAGAACCCTGGGACTCAT-3′; and 18S rRNA Fwd 5′-CGCCGCTAGAGGTGAAATTC-3′and Rev 5′-TTGGCAAATGCTTTCGCTC-3′.
Phalloidin was used to stain F-actin, and DNAse I was used to stain G-actin. ASM cells were cultured on coverslips or in Labtek 8-chamber slides. Poststimulation, cells were washed in warm PBS and fixed in 4% paraformaldehyde (PFA) plus 4% sucrose in PBS for 15 min. Cells were then incubated with 0.3% Triton X-100 in PBS for 2 min and blocked in 5% BSA in PBS for 1 h after which cells were incubated with Alexa Fluor 488-conjugated Phalloidin (1:500) plus Alexa Fluor 594-conjugated DNAse I (1:500) in 1% BSA for 1 h. Nuclei were stained with Hoechst stain (1:10,000) diluted in ddH2O. After staining, coverslips were mounted in ProLong Gold Antifade reagent. Immunofluorescence was analyzed using a Leica microscope. For MRTF-A and sm-α-actin, immunofluorescence was done as described earlier (2) using specific antibodies (dilution 1:100) and donkey anti-rabbit FITC or donkey anti-mouse cy3 antibodies (dilution: 1:50).
Nuclear extract preparation.
Nuclear extracts were prepared as described previously (20).
Preparation of cell lysates.
The whole cell extracts were either prepared as described previously (20) using SDS lysis buffer or by direct lysis in 2X Laemmli loading buffer.
Protein samples were subjected to electrophoresis, transferred to nitrocellulose membranes, and analyzed for the proteins of interest using specific primary and HRP-conjugated secondary antibodies. Bands were subsequently visualized using the G-box gel documentation system (Syngene, Cambridge, UK) using enhanced chemiluminescence reagents and were quantified by densitometry using Genetools software.
Values reported for all data are represented as means ± SE. The statistical significance of differences between means was determined by Student's t-test or by 1-way ANOVA followed by Student-Newman-Keuls multiple comparisons test. Differences were considered to be statistically significant when P < 0.05.
WNT-11 expression in contractile ASM cells.
We first investigated whether there is any link between WNT-11 expression and the contractile phenotype of ASM cells, using human tracheal smooth muscle tissue, cultured hTERT-ASM cells, and a previously reported model of contractile phenotype maturation in primary human ASM cells (11). We cultured cells in serum-containing medium until 50% confluence to represent myocyte cultures of a proliferative phenotype, whereas cultures were grown to 100% confluence then subjected to serum-deprivation for 7 days to generate myocytes that express a contractile phenotype (14, 16). We found that serum-starvation induced accumulation of sm-α-actin and calponin in primary human ASM cells, which possessed relatively low levels of these proteins in serum-fed conditions (Fig. 1A). Of note, WNT-11 also accumulated significantly in serum-starved cells compared with serum-fed cells (Fig. 1A).
ASM cells in culture tend to lose contractile characteristics and are less contractile than fresh ASM tissue. In line with this, we found that the abundance of sm-α-actin and calponin was significantly higher in fresh human tracheal smooth muscle tissue compared with serum-fed hTERT-ASM cell cultures (Fig. 1B). Gene expression of WNT-11 was much higher in human tracheal smooth muscle tissue than in the hTERT-ASM cells in culture, with a reduction in Cq value of 8.1 units, corresponding to a 280-fold higher gene expression (Fig. 1B).
To further investigate the link between the expression of sm-α-actin and WNT-11, we analyzed the effects of TGF-β1 on these genes in hTERT-ASM cells. TGF-β1 effectively induces contractile phenotype marker expression in ASM cells (8). Our data confirm this and demonstrate that TGF-β1 induces concomitant expression of sm-α-actin and WNT-11 in a time- and concentration-dependent manner. Of note, for WNT-11 induction, TGF-β1 exhibited an EC50 of 0.58 ng/ml and reached maximal effect in 12 h, an effect that clearly preceded maximum expression of sm-α-actin (Fig. 1, D–F).
WNT-11 regulates expression of sm-α-actin.
We next investigated whether WNT-11 may orchestrate sm-α-actin expression in ASM cells. Exogenous, recombinant human WNT-11 alone did not induce accumulation of sm-α-actin, neither at the mRNA level nor the protein level (not shown). We next employed siRNA-mediated knock-down of WNT-11 (knockdown efficiency 58 ± 18% at the mRNA level). WNT-11 knock-down had no effect on baseline sm-α-actin levels, but significantly attenuated TGF-β-induced expression of sm-α-actin (Fig. 2). These findings show that WNT-11 is required for expression of sm-α-actin in the conditions used for our studies.
TGF-β-induced actin cytoskeleton remodeling is required for sm-α-actin expression.
As both TGF-β1 and noncanonical WNT signaling can induce cytoskeleton reorganization, we hypothesized that actin remodeling is required for TGF-β1-induced sm-α-actin expression in ASM cells. To test this hypothesis, we pretreated hTERT-ASM cells with latrunculin A, an inhibitor of actin polymerization, which binds to and stabilizes the G-actin fraction of actin pool (24). We performed immunofluorescence microscopy to visualize actin polymerization and stained F-actin using fluorescently labeled phalloidin and G-actin using fluorescently labeled DNase I. As expected, TGF-β1 increased actin polymerization, an effect that was abrogated by latrunculin A, as reflected by increased staining of filamentous structures with phalloidin (Fig. 3A).
We next addressed the requirement of actin polymerization in sm-α-actin expression in hTERT-ASM cells. Interestingly, the presence of latrunculin A completely abrogated TGF-β1-induced sm-α-actin expression in hTERT-ASM cells as demonstrated by both Western blotting and microcopy analysis (Fig. 3, B and C). In contrast, the inhibition of actin polymerization had no effect on the abundance of β-actin (data not shown). Collectively, our data suggest that TGF-β1-induced cytoskeletal remodeling is essential for sm-α-actin expression in ASM cells.
WNT-11 mediates TGF-β-induced actin remodeling via Rho kinase.
Next, we investigated the role of WNT-11 in the regulation of TGF-β1-induced cytoskeletal remodeling in ASM cells. We first performed siRNA knock down of WNT-11 and analyzed actin polymerization in response to TGF-β1. Interestingly, silencing of WNT-11 was sufficient to attenuate TGF-β1-induced actin stress fiber formation, thus confirming a role for WNT-11 in TGF-β1-induced actin remodeling in hTERT-ASM cells (Fig. 4).
Rho kinase signaling has been intrinsically linked to both cytoskeletal remodeling and sm-α-actin expression (10). We next confirmed the essential role of the Rho kinase cascade in TGF-β1-induced actin remodeling, as microscopy analysis showed that Y27632 attenuated TGF-β1-induced appearance of F-actin filaments (Fig. 5A). Consistent with this and our earlier findings, Rho kinase inhibition also attenuated TGF-β1-induced sm-α-actin accumulation (Fig. 5C).
Noncanonical WNT signaling can activate RhoA as part of the planar cell polarity pathway (1). Interestingly, we observed that exogenous WNT-11 was sufficient to induce actin cytoskeletal remodeling, as revealed by increased phalloidin labeling of F-actin (Fig. 5B). Notably, pharmacological inhibition of Rho kinase attenuated WNT-11-induced actin remodeling (Fig. 5B). Thus our data show that WNT-11 mediates TGF-β1-induced actin remodeling in ASM cells in which Rho kinase plays a major role.
WNT-11-mediates MRTF-A nuclear translocation.
Our observations linking actin remodeling to sm-α-actin expression prompted us to investigate the underlying mechanistic link. MRTF-A is a transcription factor released from G-actin following actin polymerization and it translocates to the nucleus, promoting smooth muscle-specific gene expression, including sm-α-actin (28). Our studies revealed that TGF-β1 had no effect on total MRTF-A gene or protein expression (Fig. 6, A and B) but increased MRTF-A abundance in the nuclear lysate (Fig. 6D). To elucidate whether MRTF-A is required for TGF-β1-induced sm-α-actin expression, we performed siRNA knock down of MRTF-A. MRTF-A expression was markedly reduced in cells pretreated with MRTF-A-specific siRNA (Fig. 6E), and this effect was associated with a reduction of TGF-β1-induced sm-α-actin expression (Fig. 6C).
As Rho kinase regulates TGF-β1-induced actin remodeling, we investigated the impact of inhibition with Y27632, and found that TGF-β1-induced MRTF-A nuclear presence was attenuated (Fig. 6D). Importantly, knock down of WNT-11 completely abrogated TGF-β1-induced nuclear presence of MRTF-A (Fig. 6F). Altogether, our data suggest that WNT-11 mediates TGF-β1-induced nuclear presence of MRTF-A, directly linking its effects on actin remodeling to sm-α-actin expression.
In the present study, we demonstrate a role for the noncanonical WNT ligand, WNT-11, in mediating TGF-β1-induced sm-α-actin expression in ASM cells. We show that WNT-11 is preferentially expressed in contractile myocytes and is sufficient to induce Rho kinase-dependent actin remodeling in ASM cells, a response that is required for TGF-β1 induction of sm-α-actin expression. Furthermore, TGF-β1 induces nuclear expression of MRTF-A, an effect that is regulated by WNT-11, and required for sm-α-actin expression in ASM cells.
Noncanonical WNT/Ca2+ and WNT/planar cell polarity signaling are involved in cytoskeletal reorganization and cell movements along with the transcriptional regulation of various genes (1). While there is no direct evidence implicating noncanonical WNT signaling in the regulation of contractile protein expression in smooth muscle, two of the mediators of noncanonical WNT signaling, NFAT and JNK, can regulate sm-α-actin expression. For instance, NFAT activation induces sm-α-actin expression in smooth muscle and the inhibition of the calcineurin-NFAT pathway attenuates the expression of the contractile protein (9). Similarly, JNK induces expression of sm-α-actin in response to mechanical strain (43) and arginine vasopressin (7) in vascular smooth muscle cells. Importantly, the small GTPase RhoA, which is both an effector in noncanonical WNT signaling and is activated downstream of TGF-β1 signaling, is an integral regulator of smooth muscle differentiation and expression of sm-α-actin (28). Here, we show that WNT-11 regulates actin remodeling in a Rho kinase-dependent manner in ASM cells. In line with this, pharmacological inhibition of Rho kinase signaling attenuated TGF-β1-induced sm-α-actin expression in ASM cells.
Noncanonical WNT signaling via small GTPases and the Ca2+ pathway regulates cytoskeletal remodeling and stress fiber formation to promote cell migration (1). Studies including those from our group show that inhibition of actin remodeling by latrunculin A or B inhibits contractile protein expression (29, 40). We demonstrate here that WNT-11 promotes actin remodeling by augmenting polymerized filamentous actin (F-actin) with a concomitant decrease in monomeric globular actin (G-actin) labeling in a Rho kinase-dependent manner. Moreover, inhibition of TGF-β1-induced actin stress fiber formation by latrunculin A attenuates sm-α-actin expression suggesting that TGF-β1-induced and noncanonical WNT-mediated actin dynamics are linked to the transcriptional control of sm-α-actin.
Smooth muscle-specific genes contain CArG box DNA elements [CC(A/T)6GG] in their promoters which are regulated by serum response factor (SRF) in association with the myocardin family of transcription factors (myocardin and myocardin-related transcription factors) to drive contractile gene expression programs (30). While myocardin is constitutively nuclear, myocardin-related transcription factors (MRTFs) remain associated with G-actin and stay primarily cytosolic. Actin remodeling depletes the G-actin pool leading to the release of MRTFs and their nuclear translocation where they associate with SRF and other transcriptional coregulators to activate target gene transcription (35, 38). TGF-β1 has been shown to regulate the Rho-actin-MRTF axis (32, 34, 35, 42); however, a direct link between noncanonical WNTs and MRTF signaling has not been documented before our study. We show that TGF-β1 induces the nuclear presence of MRTF-A in ASM cells where it drives TGF-β1-induced sm-α-actin expression. Of note, inhibition of Rho kinase or knock-down of WNT-11 attenuates TGF-β1-induced nuclear presence of MRTF-A validating a Rho kinase-dependent and WNT ligand-mediated axis in MRTF-A nuclear shuttling. We provide the first evidence for the regulation of a Rho kinase-actin-MRTF axis by a noncanonical WNT ligand.
There are no published reports on the function of WNT-11 in smooth muscle to the best of our knowledge. However, considerably more is known on its role in cardiomyocyte differentiation. WNT-11 was originally reported to be required for cardiogenesis in Xenopus laevis with high expression during cardiac development and a functional role in the regulation of a contractile cardiomyocyte phenotype (37). Studies have extended its role in mammalian cardiac development (e.g., 5, 33; and 6 for review) and demonstrate that WNT-11, in cooperation with WNT-5A, regulates gene expression programs in cardiomyocyte differentiation including the regulation of contractile proteins (5). Moreover, lentivirus-mediated gene transfer of WNT-11 to skeletal muscle-derived stem cells promotes cardiomyocyte differentiation including the expression of myosin heavy chain and troponin (46). While the underlying mechanisms involved are not completely understood, WNT-11 has been extensively shown to engage β-catenin-independent signaling (5, 20, 33, 36, 39, 41). In line with that, our current study demonstrates a role for Rho kinase-dependent signaling and cytoskeletal remodeling in the regulation of TGF-β1-induced and WNT-11-mediated sm-α-actin expression. While WNT-11−/− mice show no developmental defects in the expression of smooth muscle-specific α-actin and SM22 in the embryonic heart (5), TGF-β1/WNT-11 cross-talk has been established in several recent studies. WNT-11 is a TGF-β1 target gene (41), and cooperates with TGF-β1 in chondrocyte differentiation (25). WNT-11 can also mediate TGF-β1-induced sm-α-actin expression in renal epithelial cells (47). These observations support our current findings for TGF-β1/WNT-11 cooperativity and a novel functional role in smooth muscle differentiation. Interestingly, recombinant WNT-11 was not sufficient for phenotype maturation, even though actin polymerization was clearly affected and even though WNT-11 knock-down did reduce TGF-β induced phenotype maturation. Thus it appears that noncanonical WNT signaling is sufficient to drive actin polymerization, but that additional signals (e.g., p-Smad) produced by TGF-β are needed to actually drive the cell toward a more contractile phenotype.
Cooperative regulation by TGF-β1 and WNT-11 of a Rho kinase-actin-MRTF-A axis may have important implications in various processes in airway remodeling. Clearly, noncanonical WNT signaling could contribute to contractile protein expression by ASM. Further, as EMT is considered a contributing factor to the increased mesenchymal cell population in asthmatic airways, TGF-β1 and WNT-11 crosstalk could contribute to this process. Indeed, MRTF-A is a critical mediator of TGF-β1-induced EMT (31, 32) and epithelial-to-myofibroblast transition (EMyoT) (34). Myofibroblasts are a rich source of ECM proteins and MRTF-A is a key mediator of myofibroblast activation and can regulate ECM expression. As demonstrated, MRTF-A induces collagen expression in lung fibroblasts (27). Thus the TGF-β1/WNT-11 axis could also regulate ECM expression in the airways. Indeed, we have previously demonstrated that WNT-5A, another noncanonical WNT, mediates TGF-β1-induced ECM expression via Ca2+-NFAT and JNK signaling, further suggesting a role for noncanonical WNT signaling in airway remodeling. Clearly, it will be of interest in future studies to investigate the functional and pathophysiological role of WNT-11 in more detail.
In conclusion, we demonstrate that noncanonical WNT signaling via WNT-11 plays an important role in contractile protein expression in smooth muscle. WNT-11 is preferentially expressed in contractile myocytes and regulates TGF-β1 induced expression of sm-α-actin via Rho kinase-mediated actin polymerization and MRTF-A nuclear translocation. These findings further support a role for noncanonical WNT signaling in airway smooth muscle remodeling, implying that targeting this pathway may be a therapeutic strategy worth pursuing.
This work was supported by a Vidi Grant (016.126.307) from the Dutch Organization for Scientific Research (NWO) and the University of Groningen.
No conflicts of interest, financial or otherwise, are declared by the author(s).
K.K. and R.G. conception and design of research; K.K., T.K., M.H.M., A.P., and M.S. performed experiments; K.K., T.K., M.H.M., A.P., M.S., and R.G. analyzed data; K.K., T.K., A.P., A.J.H., and R.G. interpreted results of experiments; K.K. and T.K. prepared figures; K.K. and R.G. drafted manuscript; K.K. and R.G. edited and revised manuscript; K.K., T.K., A.J.H., and R.G. approved final version of manuscript.
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