ABSTRACT
Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR), and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates transforming growth factor beta (TGF-β) expression, whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions, and EMT induction is unknown. We hypothesized that the EGFR and TGF-β signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-β expression as early as 6 h postinfection of epithelial cells and stimulated both the EGFR and TGF-β signaling pathways. Inhibition of either the EGFR or TGF-βR1 signaling substantially reduced inclusion development; however, the combined inhibition of both EGFR and TGF-βR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-β expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-β during infection. Finally, TGF-βR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3), which stabilizes EGFR signaling, suggesting reciprocal regulation between TGF-β and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-β expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. This finding may provide new targets for chlamydial disease biomarkers and prevention.
INTRODUCTION
Human genital infections by Chlamydia trachomatis are rising worldwide, and the attendant high health care cost constitutes a major burden on the public health care system. The serious complications include reproductive system fibrosis, pelvic inflammatory disease (PID), tubal factor infertility, and ectopic pregnancy (1). Genital chlamydial infection is also a significant cofactor in human papillomavirus (HPV)-related cervical carcinoma (2). The rampant asymptomatic infections usually make the onset of complications the first evidence of exposure to chlamydia, during which antibiotic treatment alone may be inadequate. Therefore, a better understanding of the molecular pathogenesis of chlamydial complications will aid the design of therapeutic measures as adjunctive or alternatives to antibiotics. The development of chlamydial complications requires a productive infection, characterized by inclusion formation in the host’s eukaryotic cell. Extracellular cues, including infections, growth factors, and hormones, activate eukaryotic cellular signaling networks that are important for normal cell differentiation, function, defense, and tissue/organ development but are also exploited, hijacked, or appropriated by microbial pathogens and other agents to facilitate infectious and noninfectious diseases (3, 4). Accordingly, Chlamydia has evolved strategies to induce, redirect, or usurp the host’s signaling pathways to facilitate a productive infection with inclusion development that ensures its survival and propagation and drives pathological outcomes. The major host cell signaling pathways and responses that are induced by Chlamydia during a productive infection of epithelial cells are the activation of receptor tyrosine kinases (RTKs) and integrin-related signaling, stimulation of unfolded protein response (UPR), and induction of epithelial-mesenchymal transition (EMT), all requiring cytoskeletal reorganizations for manifestation and being involved in infectivity and/or pathogenesis (5–13).
The RTKs are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones that play key roles in regulating cell proliferation, differentiation, survival, metabolism, migration, and cell cycle control. All 58 RTKs identified in the human genome belong to 20 distinct subfamilies and share similar structural organizations, comprising an extracellular ligand-binding domain, a single transmembrane helix, and a cytoplasmic region harboring the protein tyrosine kinase domain (3). Key members of the RTK families include the fibroblast growth factor receptors (FGFRs), the platelet-derived growth factor receptors (PDGFRs), epidermal growth factor receptors (EGFRs), the vascular endothelial growth factor receptors (VEGFRs), neuronal growth factor receptors (NGFRs), and insulin growth factor receptors (IGFRs). The engagement of RTKs by extracellular ligands triggers endocytic processes involving actin cytoskeleton remodeling. It also triggers processes involving the generation of intracellular endosomes with activated RTKs that initiate signaling cascades concerning the regulation of normal cellular processes and functions, as well as the development and progression of several pathological conditions, including EMT, tissue or organ fibrosis, and tumorigenesis (3, 14). RTK activation upon ligand binding involves receptor dimerization (homo- or heterodimeric) and phosphorylation at intracellular tyrosine residues, which triggers recruitment and binding of the signaling proteins that further activate downstream effectors in the signaling pathways. Generally, different RTKs signal via the same downstream signaling effectors, such as phospholipase Cγ1 (PLCγ1), phosphatidylinositol 3-kinase (PI3K)-AKT, and the mitogen-activated protein kinase (MAPK) pathways, although they produce distinct cellular outcomes in many different cell types due to differences in signal timing, magnitude, duration, and pathway cross talk (3). Thus, ligand-activated EGFRs can escape lysosomal degradation and transmit the activation signal through interaction with other molecules and transcription factors (e.g., protein kinase B, or Atk; the mammalian target of rapamycin complex, or mTORC; and the extracellular signal-regulated kinase 1 and 2, or ERK 1/2) and their translocation into the nucleus to mediate gene expression, cellular activities, and biological functions, including cytoskeletal remodeling, cell migration, proliferation, EMT, and diseases (13, 15–20). In fact, EGFR signaling is commonly exploited for tumor promotion as potent inducers of EMT in several cancers, including cervical carcinoma, where it is overexpressed, causing cervical stromal invasion and nodal metastasis (21–25). In addition, the EGFR signaling pathway also interfaces or cross talks with other cell signaling pathways, such as the transforming growth factor β1 (TGF-β1) and integrin/focal adhesion kinase (FAK) signaling pathways, for robust cellular response, function, and pathological outcome (26, 27). The combined EGF and TGF-β1 signaling is a potent inducer of cytoskeletal reorganization, stable EMT, and tumor promotion (28).
Several species of Chlamydia gain entry into their hosts by hijacking the host cell’s RTK signaling proteins, which promote endocytosis (3). Therefore, RTK-mediated pathogen uptake and activation of cell signaling play an important role in chlamydial pathogenesis, being required for a productive infection and inclusion development (3, 8, 11). The FGFRs and PDGFRs are important for binding of the chlamydial elementary bodies (EBs) to host cell and uptake (12, 29), and EGFR (8), with an F-actin binding/reorganization property (which is absent in FGFR or PDGFRβ [8, 30]), is involved in both chlamydial entry (attachment/internalization) and optimal-sized inclusion development (8). While some Chlamydia spp. use members of the polymorphic outer membrane protein (Pmp) family, which directly bind RTKs on host cells (31), others use indirect ligand binding (12). Specifically, pmp21 serves as a binding ligand of EGFR for host cell invasion and internalization (9), and EGFR inhibition causes a decrease in the number and size of inclusions after infection (8). Chlamydial binding and activation of EGFR signaling involve upregulation of the downstream signaling/effector molecules, including STAT5 and the antiapoptotic BAD protein, as vital intracellular calcium mobilization events for inclusion development and release (8, 32–35). Cytoskeletal reorganization occurs from the initial attachment of the EB to the host cell to the final egress stage of chlamydial inclusion development. This involves the assembly of filamentous actin at the base of EB adhesion points, formation of actin-rich cell surface microvillar projections that surround the invading EB, microtubule transport of nascent inclusions to the microtubule organizing center (MTOC) for homotypic fusion into a single large inclusion that is surrounded by nutrient-laden vesicles, and the formation of a dynamic coat of F-actin and intermediate filaments around the surface periphery of the growing inclusion to maintain the structural integrity (8, 36). There is evidence for both RTK-mediated activation of actin-nucleating factors (e.g., WAVE2, Arp2/3, and cortactin) and the chlamydial, actin-nucleating Tarp protein being the initiating signals for cytoskeletal remodeling during chlamydial infection (18). Thus, EGFR activation and cytoskeletal remodeling play a key role in the infectivity and pathogenesis of Chlamydia as well as several other microbial pathogens (3).
Like the EGF/EGFR system, the serine/threonine kinase TGF-β/TGF-βR signaling cascade induces cytoskeletal remodeling and cellular functions, and it synergizes with the EGFR pathway in cellular regulation, which leads to stable EMT and other pathological consequences, such as fibrosis and tumor progression and invasiveness (37–39). Although Chlamydia also upregulates TGF-β expression in both animals and humans, it is thought to play an immunoregulation function predominantly (40, 41), so its role in infectivity or inclusion development and Chlamydia-induced EMT is unknown. In this study, we extended our previous observation relating to Chlamydia-induced EMT (6) by testing the hypothesis that the TGF-βR and EGFR signaling pathways cooperate to ensure a productive Chlamydia infection, leading to EMT induction and development of complications.
RESULTS
Upregulation of TGF-β expression and activation of the signaling pathway during Chlamydia infection and role of EGFR.It has been established that Chlamydia binds EGFR and activates the EGFR signaling pathway, which plays a crucial role in host cell invasion and cytoskeletal reorganization for a productive infection with optimal inclusion development (8, 9, 32–36, 42). The EGFR and TGF-β signaling pathways also often synergize to mediate cytoskeletal reorganization, leading to stable EMT induction (20, 43), and Chlamydia induces EMT (6) as well as upregulates TGF-β expression in humans and animals (40, 41). However, the involvement of TGF-β in chlamydial inclusion development and chlamydia-induced EMT is unknown. We investigated the hypothesis that TGF-β signaling cooperates with the EGFR signaling pathway for the cytoskeletal reorganization that supports optimal chlamydial inclusion development and for stable EMT induction. Figure 1a confirms previous reports (40, 41) that upregulated TGF-β expression is induced during chlamydial infection (P > 0.0001). The induction occurred within 6 h of infection of epithelial cells.
(a) Chlamydia induced the upregulation of TGF-β expression and role of EGFR. Monolayers of the murine oviduct epithelial cells (C57epi.1) were infected with MoPn (MOI of 1) in the presence or absence of 50 mM Z-VAD-fmk (caspase inhibitor) and 10 μM gefitinib (ZD-1839; EGFR inhibitor; EGF-I), and Z-FA-fmk was used in control cultures. After 48 h, immunofluorescence staining of the cells for murine TGF-β was performed on the cells by standard procedures (44) using a specific primary antibody. Quantification of fluorescence was performed by scanning fluorescence-stained cells with a 20× objective on a Nikon fluorescence microscope using NIS-Elements imaging software, version 3.20 (Nikon Instruments Inc., Melville, NY). Images were acquired for experimental and control cultures with the same microscope settings, exposure time, and background. The mean fluorescence intensity per cell and standard deviations were calculated from 6 scanned fields per slide. Plotted data were derived from 3 independent experiments. NI, noninfected; Inf, MoPn infected; ZVAD, pancaspase z-VAD-fmk treated; EGFR-I, EGFR inhibitor treated. (b) Chlamydia induced the upregulation of the Smad transcription factors that mediate the canonical TGF-βR signaling pathway. Monolayers of C57epi.1 cells were infected with MoPn (MOI of 1) in the presence or absence of a mixture of inhibitors (10 μM SB-431542, TGF-βR inhibitor; 10 μM gefitinib/ZD-1839, EGFR inhibitor; and 10 μM PD98059, MAPK/MEK inhibitor). After 24 h, immunofluorescence staining of the cells for Smad2/3, phospho-Smad2/3, and Smad4 was performed on the cells by standard procedures, as previously described (44), using the specific primary antibodies as described for panel a. Plotted data were derived from 3 independent experiments. NI, noninfected; MoPn, MoPn infected; combined TGF-βR, EGFR, MAPK/MEK EGFR inhibitor treated.
EGFR is a key receptor tyrosine kinase (RTK) that is activated early during chlamydial invasion, leading to inclusion formation (8, 9). Using specific EGFR inhibitors, we investigated the role of EGFR activation by chlamydia in early or late TGF-β expression. Figure 1a also shows that the specific EGFR inhibitor (gefitinib) that prevents EGFR signaling through binding to the ATP-binding site significantly inhibited chlamydia-induced TGF-β expression upregulation (P > 0.00015), but the caspase inhibitor (Z-VAD-fmk) that inhibits EMT induction (6, 44) did not suppress the enhanced TGF-β expression (P > 0.34). The increase in background TGF-β expression could be due to cell tension and stretching, known activators of latent TGF-β (45, 46). Figure 1b provides evidence of TGF-β signaling during chlamydial infection by the upregulation of Smad2, Smad3, the phosphorylated forms of SMAD 2/3, and Smad4, which are the transcription factors responsible for TGF-β gene regulation for cytoskeletal remodeling and EMT induction. The results indicate that Chlamydia upregulates TGF-β production, requiring EGFR signaling, and activates the canonical TGF-β signaling pathway during infection of epithelial cells. The ability of EGFR signaling inhibition to suppress TGF-β expression indicated that the initial chlamydia-EGFR interaction during invasion is required for TGF-β upregulation, suggesting an interplay between the TGF-β and EGFR signaling pathways during chlamydial infection.
Inhibition of TGF-β activation during Chlamydia infection limited inclusion development.To investigate the possible interplay between the TGF-β and EGFR signaling pathways during chlamydial infection, we assessed the effect of inhibiting TGF-β signaling on chlamydial inclusion development and EMT induction. TGF-β is produced in the latent (inactive) form and stored in the extracellular matrix (ECM), and thrombospondin 1 (Tsp1), which we have previously shown to be highly upregulated during chlamydial infection (6), is a major activator of TGF-β (45, 46). Figure 2 shows that the TGF-β inhibitor (TGF-β RI kinase inhibitor VI, SB431542) alone inhibited chlamydia (the agent of mouse pneumonitis, or MoPn) inclusion development by 56% (P < 0.00001). The SMAD3 inhibitor (SIS3), which acts downstream of TGF-βR, inhibited inclusion development by 80% (P < 0.00001). The MAPK/MEK inhibitor (PD98059) alone, which acts downstream of the EGFR/FAK signaling (19), inhibited inclusion development by 46% (P < 0.00001). Interestingly, the combination of the inhibitors of EGFR (gefitinib; ZD-1839) and TGF-β (SB431542) (20) reduced inclusion development by 77% (P < 0.00001). Combining these inhibitors of EGFR, TGF-β, and MAPK/MEK signaling, inclusion development was inhibited by 95% (P < 0.00001). The results indicated that both EGFR and TGF-β, along with their signaling pathways, cooperate to support chlamydial inclusion development.
Inhibition of TGF-β activation during Chlamydia infection limited inclusion development. Monolayers of C57epi.1 cells were infected with MoPn (MOI of 1) in the presence or absence of individual or a combination of inhibitors as indicated (10 μM SB-431542, TGF-βR inhibitor; 10 μM Gefitinib/ZD-1839, EGFR inhibitor; 3 μM SIS3, Smad3 inhibitor; and 10 μM PD98059, MAPK/MEK inhibitor). After 48 h, immunofluorescence staining of the cells for chlamydial inclusions was performed by standard procedures using a specific primary antibody against chlamydial LPS, and the inclusions were enumerated as previously described (59, 60). Plotted data were derived from 4 independent experiments. MoPn, MoPn infected.
Inhibition of TGF-β activation during Chlamydia infection prevents EMT induction.Just like chlamydia inclusion development, chlamydia-induced EMT was also inhibited by inhibitors of TGF-βR and EGFR signaling (Fig. 3a). Specifically, the results showed that chlamydial infection-induced EMT was marked by a significant reduction of the expression of the specific epithelial marker E-cadherin (the hallmark of EMT) (P > 0.002); however, the presence of inhibitors of TGF-β and/or EGFR signaling prevented EMT induction. In addition, the peptide mimic of Tsp-1 that inhibits latent TGF-β activation (47, 48) prevented chlamydia-induced EMT (Fig. 3b). The results indicated that the activation of TGF-β plays a key role in chlamydia-induced EMT, and both TGF-β and EGFR signaling cooperate during chlamydia infection, leading to EMT induction.
(a) Inhibition of TGF-β activation during Chlamydia infection blocks EMT induction. Monolayers of C57epi.1 cells were infected with MoPn (MOI of 1) in the presence or absence of individual or a combination of inhibitors as indicated (10 μM SB-431542, TGF-βR inhibitor; 10 μM Gefitinib/ZD-1839, EGFR inhibitor). After 48 h, immunofluorescence staining of the cells for E-cadherin (the epithelial hallmark of EMT) was performed on the cells by standard procedures (44) using the specific primary antibody. Plotted data were derived from 3 independent experiments. NI, noninfected; MoPn, MoPn infected. (b) Inhibition of TGF-β activation by a Tsp1 peptide mimetic blocked chlamydia-induced EMT. Monolayers of C57epi.1 cells were infected with MoPn (MOI of 1) after treatment with 100 μM inhibitory peptide (TROM2) or control peptide (TROMC2). Cultures were incubated for 48 h and assayed for EMT (E-cadherin expression) by standard procedures (44) using the specific primary antibody described for panel a. Plotted data were derived from 3 independent experiments.
TGF-βR signaling stabilizes EGFR signaling via TACC3 during Chlamydia infection.To further explore the mechanism of the cross talk between TGF-β and EGFR signaling during chlamydial infection, we examined the regulatory effect of TGF-β on the promoters of EGFR signaling leading to EMT. The transforming acidic coiled-coil protein 3 (TACC3) is a microtubule-associated protein that functions as a binding partner of EGFR, stabilizing the receptor on the membrane to sustain EGFR signaling for achieving the cellular functions or differentiation effect, including cell motility, myofibroblast generation, and EMT phenotype (25, 49). We investigated the hypothesis that a major role of TGF-β during chlamydial infection is to regulate TACC3 expression to sustain EGFR signaling. Figure 4a shows that TACC3 is upregulated during chlamydial infection of epithelial cells. In addition, the inhibition of TGF-β eliminated TACC3 expression (Fig. 4b). The results suggested that TGF-β regulates EGFR activation and signaling at least via TACC3, which corroborates previous reports that TGF-β promotes the expression of EGFR on the cell membrane (19).
(a) Chlamydia infection upregulated TACC3 expression. Monolayers of C57epi.1 cells were infected with MoPn (MOI of 1) and incubated for 24 or 48 h. Immunofluorescence staining of the cells for TACC3 was performed by standard procedures (44) using the specific primary antibody. Plotted data were derived from 3 independent experiments. (b) Chlamydia infection upregulated TACC3 expression. Monolayers of C57epi.1 cells were infected with MoPn (MOI of 1) in the presence or absence of 10 μM SB-431542, the TGF-βR inhibitor. After 48 h, immunofluorescence staining of the cells for TACC3 was performed on the cells by standard procedures (44) using the specific primary antibody. Plotted data were derived from 3 independent experiments.
DISCUSSION
We had proposed that Chlamydia-induced EMT underlies the pathogenesis of genital chlamydial infection complications, including fibrosis, infertility, and tumor promotion (6). Interestingly, several host cell signaling pathways, especially the EGFR, TGF-βR, and integrin cascades, cooperate for a stable EMT induction that drives the pathogenesis of certain serious diseases, including tissue/organ fibrosis and metastatic cancers (20, 50, 51). Since Chlamydia activates the EFGR and integrin signaling pathways (8, 10), induces EMT (6), and upregulates TGF-β (40, 41), we investigated the contribution of TGF-β to the host cell signaling events that support inclusion development and EMT induction. Specifically, we investigated the cross talk between EGFR and TGF-βR signaling during chlamydial infection of epithelial cells and the effect on chlamydial inclusion formation and EMT induction. We tested the hypothesis that the TGF-βR and EGFR signaling pathways cooperate to ensure a productive Chlamydia infection, leading to inclusion development and EMT that drives infection complications. Our results revealed that Chlamydia upregulates TGF-β expression early during infection of epithelial cells and requires EGFR signaling. Chlamydia also activates the canonical TGF-β signaling pathway along with the known EGFR (8) and integrin (10) signaling pathways. The activation of TGF-βR signaling plays a major role in chlamydial inclusion development and EMT induction, and both TGF-β and EGFR signaling cooperate during Chlamydia infection, leading to EMT induction. Our results and previous reports indicate that Chlamydia activates at least three important host cell signaling pathways (i.e., EGFR, integrins, and TGF-βR), all involving cytoskeletal remodeling that supports a productive infection and induces EMT to drive infection complications. The role of cytoskeletal remodeling is underscored by the fact that agents that cause disruption of the cytoskeleton inhibit chlamydial infection (18).
In addition, our results suggest that EGFR activation following chlamydial binding stimulates TGF-β-mediated signaling. Members of the chlamydial Pmp family (9, 31) and certain invasins, such as the chlamydial Ctad1 (10), have been established to mediate EB adhesion, invasion, and internalization into host cells via direct interaction with EGFR and integrins. In addition to RTK and TGF-βR-mediated signaling pathways, Chlamydia also activates the integrin receptor signaling pathway (10). Coupling of integrin receptors to the cytoskeletal apparatus during remodeling via FAK drives cell migration during EMT (51). Therefore, the observation in this study that inhibition of the EGFR and TGF-βR signaling pathways produced less than 100% inhibition of inclusion formation suggests that all three signaling cascades contribute to the cytoskeletal remodeling and gene expression regulation, culminating in optimal support for chlamydial inclusion development and stable EMT induction. Beyond the indirect transactivation of TGF-βR by EGFR and integrins following interaction with chlamydial EBs, the possibility that Chlamydia also interacts with TGF-βR or TGF-β directly has not been ruled out. Furthermore, our results suggested that Tsp1-mediated activation of latent TGF-β in the extracellular matrix (ECM) (46) is a major source of TGF-β that activates TGF-βR signaling during chlamydial infection. We had reported that Tsp1 was dramatically upregulated in the genital tract tissues of chlamydia-infected animals (6), but the mechanism of chlamydial upregulation of Tsp1 is presently unknown. However, the integrins activated by Chlamydia (10, 18) are also known activators of latent TGF-β in the ECM (46, 52). Therefore, as illustrated in Fig. 5, Chlamydia induces two key activators of the TGF-β (Tsp1 and integrins) as well as the EGFR signaling pathways to orchestrate three cooperative signaling cascades that support the cytoskeletal remodeling required for inclusion development as well as stable EMT induction.
Summary of signaling pathways and molecular events induced by Chlamydia to promote inclusion development and EMT induction, leading to complications. Chlamydial elementary body (EB) binding to RTKs, such as EGFR, initiates host cell invasion and stimulation of cellular signaling cascades that include the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways, leading to enhanced transcriptional and translational activities relating to cytoskeletal remodeling, initial inhibition of apoptosis (cell survival), and proliferation. Simultaneously, chlamydial EBs also bind the integrins on host cells via SH3-containing ligands, such as Ctad1, to activate the host cell signaling cascade involving the focal adhesion kinase (FAK) and MEK/ERK pathway. In addition, chlamydia-induced Tsp1 and activated integrins convert latent TGF-β in the ECM into active TGF-β that binds the TGF-βR to activate the canonical Smad signaling pathways, leading to enhanced transcriptional and translational activities relating to cytoskeletal remodeling, cell survival, motility, and proliferation. The cross-talk among the different signaling pathways due to the transactivation between the effectors in the signaling cascades results in synergy and a boost in the cellular response. Thus, for successful infectivity with adequate inclusion development leading to disease, chlamydia orchestrates these multiple host cell signaling pathways to provide a supportive cytoskeletal environment, adequate nutrients, and cellular differentiation that culminates in pathological EMT underlying the infection complications.
While EGFR was previously known to regulate TGF-βR signaling through the AKT-mediated phosphorylation of ser208 at the linker region of Smad3 (20), our results revealed that the reciprocal TGF-β–EGFR regulation (19) also involves TACC3, which stabilizes EGFR on the membrane to sustain cell signaling (49). Therefore, chlamydia-induced TGF-β regulates EGFR activation and signaling at least via TACC3, which corroborates previous reports that TGF-β promotes the expression of EGFR on the cell membrane (19). The TACC family members (TACC1, TACC2, and TACC3) are centrosomal proteins with a highly conserved C-terminal coiled-coil domain that interact with tubulin and microtubules to regulate centrosome and microtubule dynamics during cell differentiation and division (25, 53). TACC3 promotes EMT and stemness, with high-level expression causing defective cell cycle checkpoints and repair systems, leading to genomic instability and tumorigenesis (54, 55). TACC3 is essential for EGFR-mediated EMT induction (25, 55), since EGFR-TACC3 interaction stabilizes and maintains EGFR on the cell surface to sustain the downstream signaling involving MAPK/Akt effectors and transcription factors that activate Snail and Slug, which are the direct E-cadherin suppressors and EMT inducers (25, 49, 55). Pharmacological inhibition of TACC3 expression resulted in inhibition of tumor growth and invasiveness or metastasis (56–58). Chlamydia appears to induce EMT through a combination of EGFR activation and TGF-β-mediated TACC-3 upregulation, leading to the stimulation of mesenchymal proteins and cytoskeletal reorganization. The involvement of TACC3 in chlamydia-induced EMT may offer several specific therapeutic targets for controlling chlamydial complications as well as the purported tumor-promoting properties, especially human papillomavirus-related cervical carcinoma.
Finally, this study provides greater insights into chlamydial pathogenesis, especially the cooperation among the cell signaling pathways associated with cytoskeletal remodeling that are induced by Chlamydia to ensure productive infection with inclusion development and induce EMT to drive disease complications. The multiple host cell signaling pathways may offer a robust and interdependent network of synergizing processes that ensure complete or stable EMT with the pathological outcomes. The host cell signaling pathway cross talk finding is significant because it offers a broader approach to defining new biomarkers or therapeutic targets and strategies for prevention of Chlamydia infection or development of complications when antibiotic treatment is limiting.
MATERIALS AND METHODS
Chlamydia strains and cell cultures.Chlamydia muridarum Nigg (the agent of mouse pneumonitis, or MoPn) and C. trachomatis (human-specific strain) serovars L2/LGV-434 and D/UW-3 were grown in HeLa 229 cells (ATCC, Rockville, MD), and titers of purified elementary bodies (EBs) were determined as infection-forming units per millimeter (IFU/ml) using standard procedures previously described (59, 60).
TGF-βR, EGFR, and cell signaling pathway inhibitors.Table 1 shows the TGF-βR, EGFR, and cell signaling pathway inhibitors used in this study. The nontoxic concentrations of these inhibitors used in cultures have been established in other studies (6, 19, 20). Accordingly, SIS3 was used at 3 μM in culture, SB-431542 at 10 μM, PD98059 at 10 μM, and gefitinib (ZD-1839) at 10 μM. The pancaspase inhibitor Z-VAD-FMK and the control Z-FA-FMK were used in culture at 50 μM. No toxicity was observed in cultures with these concentrations.
TGF-βR, EGFR, and cell signaling pathway inhibitors
Assessment of EMT and chlamydial inclusions by immunofluorescence.The chlamydia-susceptible murine oviduct epithelial cell line (C57epi.1) (61) was kindly provided by Raymond Johnson, Yale University, New Haven, CT. C57epi.1 cells were plated on slides in 24-well tissue culture plates and infected with MoPn (at a multiplicity of infection [MOI] of 1), as previously described (6, 44). Chlamydia (MoPn)-infected and control samples were treated with inhibitors of EGFR, TGF-β RI kinase, Smad3, and MAP kinase kinase (MEK), or the EMT inhibitor Z-VAD-FMK and the control Z-FA-FMK at 50 μM, and incubated at 37°C for the indicated periods of time. Cultured cells were analyzed by immunofluorescence staining for EMT markers (E- and T-cadherin, β-catenin, fibronectin, Snail1/2, and α-smooth muscle actin, or α-SMA) after 48 h, as previously described (6, 44). Quantification of fluorescence was performed by scanning fluorescence-stained cells with a 20× objective on a Nikon fluorescence microscope using NIS-Elements Imaging Software version 3.20 (Nikon Instruments, Inc., Melville, NY). Images were acquired for experimental and control cultures with the same microscope settings, exposure time, and background. The mean fluorescence intensity and standard deviations of 6 out of 10 scanned fields per slide were calculated automatically by the software. Enumeration and quantification of chlamydial IFU/ml were performed by using the standard procedures previously described (59, 60).
All animal protocols were approved by the CDC Institutional Animal Care and Use Committee (IACUC) under protocol 2894IGIMOUC-A1. The CDC IACUC is guided by Title 9, Chapter I, Subchapter A–Animal Welfare (USDA Regulations).
TFG-β inhibitory peptide design and assay.A peptide (TROM2) spanning amino acids 54 to 57 of LAP (latency-associated protein) of latent TGF-β1 was designed to mimic the N terminus of LAP that interacts with the type 1 repeat of TSP-1 during TGF-β1 activation in the ECM (47). A nonfunctional peptide (TROMC2) of the same number of amino acids as TROM2, known to neither inhibit nor activate TGF-β1, was also designed (48). The efficacy of the peptides in blocking the functional activation of TGF-β1 was assayed by pretreating infected cells with 100 μM for 1 h, followed by infection with MoPn for 48 h and assaying for EMT (E-cadherin expression).
Statistical analysis.Statistical analyses were performed with SigmaPlot and SigmaStat software. The data derived from different experiments were analyzed and compared by performing a 1- or 2-tailed t test, and the relationship between different experimental groupings was assessed by analysis of variance (ANOVA). Statistical significance was judged at a P value of <0.05.
ACKNOWLEDGMENTS
This work was supported by the Centers for Disease Control and Prevention (CDC) and PHS grants (AI41231, GM 08248, RR03034, and 1SC1GM098197) from the NIH.
We have no financial interests related to this work to declare. All of the authors consented to this submission, and the results are not under consideration for publication elsewhere.
The conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry.
FOOTNOTES
- Received 24 October 2019.
- Returned for modification 25 November 2019.
- Accepted 15 January 2020.
- Accepted manuscript posted online 21 January 2020.
- Copyright © 2020 American Society for Microbiology.
