TGF-β Signaling In Epithelial Cells Research Articles

Expansion and maintenance of primary corneal epithelial stem/progenitor cells by inhibition of TGFβ receptor I-mediated signaling

Transforming growth factor β (TGFβ) signaling is one of the most important signaling pathways regulating cell behavior in ocular tissues. Its functions are mainly linked to tissue fibrosis and inflammatory responses in ophthalmology. In epithelial cells, however, the growth inhibitory activity of TGFβ was reported in both non-ocular and ocular tissues. Since TGFβ is a bifunctional regulator that either inhibits or stimulates cell proliferation according to the specific context, we examined the effect of inhibition of TGFβ receptor (TβR) I-mediated signaling on primary corneal epithelial cells (CECs) in serum- and feeder-free conditions. The mouse CECs were isolated from the eyeballs of 6–8 weeks old female C57BL/6 mice using dispase and trypsin separately, cultivated in defined Keratinocyte serum-free medium (KSFM) with supplements (the complete medium) without feeder layer. Cells were divided into three groups, those cultured in complete medium additionally supplemented with 10 μM SB-431542, a specific inhibitor of TβR-I, were SB-CECs; those cultured in complete medium additionally supplemented with 10 ng/ml SRI-011381, a TGF-beta signaling agonist, were SRI-CECs; those cultured in complete medium without SB-431542 or SRI-011381 were control CECs. The growth rate and morphology were analyzed by light microscopy. The identity and stemness of cells was investigated through marker staining of p63, inhibitor of differentiation 1 (ID1), cytokeratin 12 (K12), cytokeratin 14 (K14), PAX6, pSmad3, alpha smooth muscle Actin (αSMA) and E-cadherin (E-cad); Real-time quantitative (RT-PCR) analysis of p63; Western blot analysis of ID1; as well as colony forming assay, sphere forming assay, healing wound in vitro assay and air-lifting interface assay. The results showed SB-CECs subcultured steadily, achieved sustained expansion, and expanded almost thrice faster than control CECs. Expanded SB-CECs exhibited smaller and more compact morphology, up-regulated p63 and ID1, as well as better performed colony-forming capacity, sphere-forming capacity, in vitro wound healing capacity, and the capacity to stratify and differentiate on air-lifting interface. Preliminary tests on human limbal epithelial cells (HLECs) showed the same results as mouse CECs. Interestingly, the ID1 expression pattern was almost identical to p63, the typical marker for corneal epithelial stem/progenitor cell (CESC/CEPC), in cultured CECs and normal corneal sections. Since ID1 has been proven to be regulated negatively by TGFβ signaling in epithelial cells and plays a role in blocking cell differentiation, its derepression by TβR-I inhibitor could be, at least in part, the underlying cause of CESC/CEPC expansion and the synchronously up-regulated expression of p63 in SB-CECs. In conclusion, inhibition of TβR-I-mediated signaling, CESCs/CEPCs achieved efficient long-term expansion in a feeder- and serum-free condition in vitro. And derepression of ID1 could be the underlying cause. Meanwhile, ID1 could serve as a marker for CESC/CEPC. These results may advance the basic and clinical CESC/CEPC research.

TGFBR1M318R+/−: A monogenic presentation of eosinophilic esophagitis.

Abstract Eosinophilic esophagitis (EoE) is a chronic immune-mediated disease characterized by esophageal dysfunction and accumulation of tissue eosinophils, T cells, and mast cells. The exact causes of EoE are not known. Sequence variants associated with increased EoE susceptibility have been identified, suggesting that there can be a genetic component to the disease. Several of the genes encode proteins involved in TGFβ signaling. Patients and mice heterozygous for a mutation in the kinase domain of TGFbR1 (TGFBR1M318R+/−) exhibit cardiovascular, skeletal, and immune manifestations of Loeys-Dietz syndrome (LDS). Immune symptoms include elevated serum IgE and a propensity to develop EoE. We used TGFBR1M318R+/− mice to determine which cells are responsible for the initiation and perpetuation of EoE. Current dogma posits that EoE occurs when Th2 cells are stimulated by inhaled or ingested antigens to secrete cytokines that then recruit and stimulate eosinophils and mast cells. Thus we were surprised to find that RAG2−/−R1 mice developed disease, illustrating that EoE associated with the TGFBR1M318R mutation was not lymphocyte-dependent. Bone marrow (BM) chimeras were made to confirm that altered TGFβ signaling in radio-resistant cells was sufficient to cause EoE. Lethally irradiated R1mice reconstituted with WT BM developed EoE. In contrast, eosinophils and IgE were not elevated in WT mice reconstituted with R1BM. We looked for other potential sources of type 2 cytokines and discovered that type 2 innate lymphoid cells (ILC2) were increased in the esophagus of R1 mice. We propose that altered TGFβ signaling in epithelial cells leads to EoE by stimulating ILC2 to proliferate and secrete cytokines that result in accumulation of eosinophils.

Role of Areca Nut Induced TGF-β and Epithelial-Mesenchymal Interaction in the Pathogenesis of Oral Submucous Fibrosis.

Areca nut consumption has been implicated in the progression of Oral Submucous fibrosis (OSF); an inflammatory precancerous fibrotic condition. Our previous studies have demonstrated the activation of TGF-β signaling in epithelial cells by areca nut components and also propose a role for epithelial expressed TGF-β in the pathogenesis of OSF. Although the importance of epithelial cells in the manifestation of OSF has been proposed, the actual effectors are fibroblast cells. However, the role of areca nut and TGF-β in the context of fibroblast response has not been elucidated. Therefore, to understand their role in the context of fibroblast response in OSF pathogenesis, human gingival fibroblasts (hGF) were treated with areca nut and/or TGF-β followed by transcriptome profiling. The gene expression profile obtained was compared with the previously published transcriptome profiles of OSF tissues and areca nut treated epithelial cells. The analysis revealed regulation of 4666 and 1214 genes by areca nut and TGF-β treatment respectively. The expression of 413 genes in hGF cells was potentiated by areca nut and TGF-β together. Further, the differentially expressed genes of OSF tissues compared to normal tissues overlapped significantly with areca nut and TGF-β induced genes in epithelial and hGF cells. Several positively enriched pathways were found to be common between OSF tissues and areca nut +TGF-β treated hGF cells. In concordance, areca nut along with TGF-β enhanced fibroblast activation as demonstrated by potentiation of αSMA, γSMA and collagen gel contraction by hGF cells. Furthermore, TGF-β secreted by areca nut treated epithelial cells influenced fibroblast activation and other genes implicated in fibrosis. These data establish a role for areca nut influenced epithelial cells in OSF progression by activation of fibroblasts and emphasizes the importance of epithelial-mesenchymal interaction in OSF.

Curtailing endothelial TGF-β signaling is sufficient to reduce endothelial-mesenchymal transition and fibrosis in CKD.

Excessive TGF-β signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-β signaling on development of fibrosis, there is a lack of information on ablating TGF-β signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-β receptor knockout (TβRII(endo+/-)) mice to explore whether curtailed TGF-β signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TβRII(endo+/+) mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TβRII(endo+/-) mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TβRII(endo+/+) counterparts. In addition, partial deletion of TβRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-β-induced canonical Smad2 signaling was reduced in TβRII(+/-) ECs; however, activin receptor-like kinase 1 (ALK1)-mediated Smad1/5 phosphorylation in TβRII(+/-) ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TβRII(+/-) ECs compared with TβRII(+/+) ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-β signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-β signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.

TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate

Clefting of the soft palate occurs as a congenital defect in humans and adversely affects the physiological function of the palate. However, the molecular and cellular mechanism of clefting of the soft palate remains unclear because few animal models exhibit an isolated cleft in the soft palate. Using three-dimensional microCT images and histological reconstruction, we found that loss of TGFβ signaling in the palatal epithelium led to soft palate muscle defects in Tgfbr2(fl/fl);K14-Cre mice. Specifically, muscle mass was decreased in the soft palates of Tgfbr2 mutant mice, following defects in cell proliferation and differentiation. Gene expression of Dickkopf (Dkk1 and Dkk4), negative regulators of WNT-β-catenin signaling, is upregulated in the soft palate of Tgfbr2(fl/fl);K14-Cre mice, and WNT-β-catenin signaling is disrupted in the palatal mesenchyme. Importantly, blocking the function of DKK1 and DKK4 rescued the cell proliferation and differentiation defects in the soft palate of Tgfbr2(fl/fl);K14-Cre mice. Thus, our findings indicate that loss of TGFβ signaling in epithelial cells compromises activation of WNT signaling and proper muscle development in the soft palate through tissue-tissue interactions, resulting in a cleft soft palate. This information has important implications for prevention and non-surgical correction of cleft soft palate.

TGF-β Signaling in Myeloid Cells Is Required for Tumor Metastasis

TGF-β is overexpressed in advanced human cancers. It correlates with metastasis and poor prognosis. However, TGF-β functions as both a tumor suppressor and a tumor promoter. Here, we report for the first time that genetic deletion of Tgfbr2 specifically in myeloid cells (Tgfbr2(MyeKO)) significantly inhibited tumor metastasis. Reconstitution of tumor-bearing mice with Tgfbr2(MyeKO) bone marrow recapitulated the inhibited metastasis phenotype. This effect is mediated through decreased production of type II cytokines, TGF-β1, arginase 1, and inducible nitric oxide synthase, which promoted IFN-γ production and improved systemic immunity. Depletion of CD8 T cells diminished the metastasis defect in the Tgfbr2(MyeKO) mice. Consistent with animal studies, myeloid cells from patients with advanced-stage cancer showed increased TGF-β receptor II expression. Our studies show that myeloid-specific TGF-β signaling is an essential component of the metastasis-promoting puzzle of TGF-β. This is in contrast to the previously reported tumor-suppressing phenotypes in fibroblasts, epithelial cells, and T cells.

Identification of a Novel Link between the Protein Kinase NDR1 and TGFβ Signaling in Epithelial Cells.

Transforming growth factor-beta (TGFβ) is a secreted polypeptide that plays essential roles in cellular development and homeostasis. Although mechanisms of TGFβ-induced responses have been characterized, our understanding of TGFβ signaling remains incomplete. Here, we uncover a novel function for the protein kinase NDR1 (nuclear Dbf2-related 1) in TGFβ responses. Using an immunopurification approach, we find that NDR1 associates with SnoN, a key component of TGFβ signaling. Knockdown of NDR1 by RNA interference promotes the ability of TGFβ to induce transcription and cell cycle arrest in NMuMG mammary epithelial cells. Conversely, expression of NDR1 represses TGFβ-induced transcription and inhibits the ability of TGFβ to induce cell cycle arrest in NMuMG cells. Mechanistically, we find that NDR1 acts in a kinase-dependent manner to suppress the ability of TGFβ to induce the phosphorylation and consequent nuclear accumulation of Smad2, which is critical for TGFβ-induced transcription and responses. Strikingly, we also find that TGFβ reciprocally regulates NDR1, whereby TGFβ triggers the degradation of NDR1 protein. Collectively, our findings define a novel and intimate link between the protein kinase NDR1 and TGFβ signaling. NDR1 suppresses TGFβ-induced transcription and cell cycle arrest, and counteracting NDR1's negative regulation, TGFβ signaling induces the downregulation of NDR1 protein. These findings advance our understanding of TGFβ signaling, with important implications in development and tumorigenesis.

TGFβ signaling in head and neck squamous cell carcinoma

Transforming growth factor beta (TGFβ) is a key regulator of epithelial cell proliferation, immune function and angiogenesis. Because TGFβ signaling maintains epithelial homeostasis, dysregulated TGFβ signaling is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Defective TGFβ signaling in epithelial cells causes hyperproliferation, reduced apoptosis and increased genomic instability, and the compensatory increase in TGFβ production by tumor epithelial cells with TGFβ signaling defects further promotes tumor growth and metastases by increasing angiogenesis and inflammation in tumor stromal cells. Here, we review the mouse models that we used to study TGFβ signaling in HNSCC.

Abstract 3848: Abrogation of TGF-β signaling promotes mammary carcinoma progression by enhanced Th17 response

Abstract We have shown that TGF-β signaling in carcinoma cells can suppress the expression of chemokines that are responsible for the expansion and recruitment of myeloid derived suppressor cells (MDSCs) in tumor bearing mice. Using mice with conditional deletion of the type II TGF-β receptor gene (Tgfbr2) in mammary epithelial cells we have shown that derivative MMTV-PyMT induced carcinomas were better able to recruit tumor-promoting MDSCs than carcinomas with intact TGF-β signaling. This recruitment correlated with higher levels of Cxcl1/5 expression in tumors lacking intact TGF-β signaling. Cxcl1/5 signal through the CXCR2 receptor and it has been shown that CXCR2 signaling in vitro was able to promote migration and invasion of MDSC. IL-17 can up-regulate secretion of Cxc chemokines by epithelial cells. Differentiation of Th17 cells in the tumor microenvironment depends on the balance of cytokines such as TGF-β, IL-6 and IL-23, which are primarily produced by tumor infiltrating myeloid cells. Based on this information, we hypothesize that loss of TGF-β signaling in carcinoma cells results in increased tumor volume through an enhanced Th17 response. In vivo studies were performed using a spontaneous tumor model MMTV-PyMT and MMTV-PyMT/Tgfbr2 KO. On 7-9 days after tumor formation we isolated tumor tissue and prepared single cell suspensions which were used for flow cytometry analysis and for FACS sorting. CD4+ cells were isolated by CD4 Microbeads from Miltenyi Biotec. For in vitro studies we used immortalized epithelial cell lines which were prepared from PyMT tumors of MMTV-WT and MMTV-KO mice. Levels of cytokines were measured by ELISA. We found that MDSCs don't express CXCR2 in the lung and tumor tissue compared with MDSCs from the spleen and bone marrow. Carcinoma cells without Tgbr2 produce more CXCL1/5 and ELISA data showed that release of these two chemokines is upregulated by IL-17. Also, we have found increased expression of IL-17r on knockout epithelial cells and these cells are 4 times more sensitive to low concentrations of IL-17. In parallel, we found that TGF-β decreases secretion of CXCL1/5 from normal epithelial cells but not from cells without Tgbr2. Levels of TGF-β and IL-6, which are important for differentiation of Th17 cells, are increased in tumor tissue without Tgbr2. These data are paralleled with an increased number of Th-17 cells and level of IL-17. In addition, we discovered a secondary effect of IL-17 in increasing the suppressive function of MDSC's on T cells through the upregulation of Arg, IDO and COX2. We conclude that the tumor microenvironment of mammary carcinomas with defective TGF-β signaling in epithelial cells have increased levels of IL-17 which enhance tumor progression thru increased MDSCs migration and enhanced suppressive function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3848.

Role of p38 MAP Kinase and Transforming Growth Factor-β Signaling in Transepithelial Migration of Invasive Bacterial Pathogens

Streptococcus pneumoniae and Haemophilus influenzae are human pathogens that often asymptomatically colonize the mucosal surface of the upper respiratory tract, but also occasionally cause invasive disease. The ability of these species to traverse the epithelium of the airway mucosa was modeled in vitro using polarized respiratory epithelial cells in culture. Migration across the epithelial barrier was preceded by loss of transepithelial resistance. Membrane products of S. pneumoniae that included lipoteichoic acid induced disruption of the epithelial barrier in a Toll-like receptor 2-dependent manner. This result correlates with a recent genetic study that associates increased TLR2 signaling with increased rates of invasive pneumococcal disease in humans. Loss of transepithelial resistance by the TLR2 ligand correlated with activation of p38 MAP kinase and transforming growth factor (TGF)-beta signaling. Activation of p38 MAPK and TGF-beta signaling in epithelial cells upon nasal infection with S. pneumoniae was also demonstrated in vivo. Inhibition of either p38 MAPK or TGF-beta signaling was sufficient to inhibit the migration of S. pneumoniae or H. influenzae. Our data shows that diverse bacteria utilize common mechanisms, including MAPK and TGF-beta signaling pathways to disrupt epithelial barriers and promote invasion.

Inhibition of Transforming Growth Factor-β1 Signaling Attenuates Ataxia Telangiectasia Mutated Activity in Response to Genotoxic Stress

Abstract Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor β (TGFβ)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGFβ inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgfβ1 null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGFβ type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced γH2AX radiation-induced foci; and increased radiosensitivity compared with TGFβ competent cells. We determined that loss of TGFβ signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGFβ restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgfβ1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGFβ may be used to advantage in cancer therapy. (Cancer Res 2006; 66(22): 10861-9)

Inhibition of Transforming Growth Factor-β Signaling in Human Cancer: Targeting a Tumor Suppressor Network as a Therapeutic Strategy

Transforming growth factor-β (TGFβ) is both a tumor suppressor and tumor promoter. The loss or attenuation of TGFβ signaling in epithelial cells and stroma is permissive for epithelial cell transformation, whereas the introduction of dominant-negative TGFβ receptors into metastatic cancer cells