Dataset Information

Epidermal Growth Factor Stimulates Transforming Growth Factor-Beta Receptor Type II Expression In Corneal Epithelial Cells.

ABSTRACT: We previously demonstrated that inhibition of epidermal growth factor receptor (EGFR) slowed corneal epithelial migration. Here we examine the effect of EGF on transforming growth factor-beta receptor II (TGF-βRII) in a corneal wound-healing model and primary human corneal epithelial cells (pHCE). Corneal debridement wounds were made and allowed to heal ± Tyrphostin AG1478 (EGFR inhibitor), and assayed for EGFR activation and EGFR and TGF-βRII localization. Primary HCE were treated with EGF ± U0126 (MEK inhibitor) and assayed for TGF-βRII expression. EGFR activation was maximal 15 minutes after wounding and localized in the migrating epithelial cells. TGF-βRII localization was also observed in the migrating epithelium and was reduced when EGFR was blocked. When pHCE were treated with EGF for 6 hours, the cells produced enhanced levels of TGF-βRII, which was blocked by U0126. Downstream signaling pathways of MEK (p38^MAPK and ERK1/2^MAPK) were then examined, and TGF-β1 and EGF were found to have differential effects on the phosphorylation of p38 and ERK1/2, with TGF-β1 upregulating p-p38 but not pERK1/2 and EGF upregulating pERK1/2 but not p-p38. Taken together, these data indicate that EGF stimulates TGF-βRII through ERK1/2 and EGFR signaling, suggesting interplay between EGF- and TGF-β-signaling pathways during corneal wound repair.

SUBMITTER: Shu DY

PROVIDER: S-EPMC6542834 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Epidermal Growth Factor Stimulates Transforming Growth Factor-Beta Receptor Type II Expression In Corneal Epithelial Cells.

Shu Daisy Y DY Hutcheon Audrey E K AEK Zieske James D JD Guo Xiaoqing X

Scientific reports 20190530 1

We previously demonstrated that inhibition of epidermal growth factor receptor (EGFR) slowed corneal epithelial migration. Here we examine the effect of EGF on transforming growth factor-beta receptor II (TGF-βRII) in a corneal wound-healing model and primary human corneal epithelial cells (pHCE). Corneal debridement wounds were made and allowed to heal ± Tyrphostin AG1478 (EGFR inhibitor), and assayed for EGFR activation and EGFR and TGF-βRII localization. Primary HCE were treated with EGF ± U0 ...[more]

PMID: 31147562

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Project description:BackgroundNerve growth factor (NGF) contributes to pain in knee osteoarthritis (KOA) patients. Transforming growth factor-beta (TGF-β) stimulates NGF expression in chondrocytes from KOA patients. However, the correlation between synovial TGF-β and NGF levels has not been sufficiently studied in human KOA patients. Further, the mechanism governing NGF regulation by TGF-β in synovial cells is unclear.MethodsDuring total knee arthroplasty, we extracted the synovial tissue (SYT) of 107 subjects with unilateral Kellgren/Lawrence grade 3-4 KOA confirmed by radiography. We examined the distribution of TGF-β and NGF using immunohistochemistry, and analyzed the relationship between NGF and TGFB mRNA levels. Cultured synovial cells extracted from SYT were exposed to culture medium (control), human recombinant TGF-β (rhTGF-β), rhTGF-β + ALK5 inhibitor SB505124, rhTGF-β + transforming growth factor activating kinase 1 (TAK1) inhibitor (5Z)-7-oxozeaenol, or rhTGF-β + p38 inhibitor SB203580 for 30 min, 6 h and 24 h. NGF mRNA expressed by the cultured cells and NGF protein levels in the cell supernatant were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Phosphorylation of p38 was evaluated by western blotting.ResultsNGF mRNA levels were positively correlated with those of TGFB. Cells expressing TGF-β and NGF protein were observed in the lining layer of SYT. TGF-β stimulated increased NGF mRNA expression and NGF protein production. The ALK5 inhibitor completely suppressed the TGF-β-mediated increase in NGF expression and NGF production in synovial cells. ALK5, TAK1 and p38 inhibitors inhibited the TGF-β-induced phosphorylation of p38, and TAK1 and p38 inhibitors partially inhibited the TGF-β-mediated increase in NGF expression and NGF production in synovial cells.ConclusionTGF-β regulates NGF production via the TGF-β/ALK5 signaling pathway in osteoarthritic synovium. This effect may partially occur through inhibition of the TAK1/p38 pathway in the SYT of KOA patients.

Project description:PurposeTransforming growth factor-β (TGF-β) is considered to be essential to induce epithelial-to-mesenchymal transition (EMT) which plays central roles in wound healing in ocular fibrotic complication. The present study investigates whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-β (TβRII) could be used to minimize the TGF-β action.MethodsTGF-β receptor type II (TβRII) specific siRNAs designed from the Nakamura human gene sequence were used to transfect the cultured lens epithelial cells (LECs). The optimal transfection of scramble siRNA-Cy3 labeled duplexes in cultured LECs were examined by laser scanning confocal microscope and flow cytometry. TβRII protein expression and transcript levels were analyzed by immunofluorescence, western blotting, and real time PCR, respectively. Western blotting was performed to examine protein expression of fibronectin and alpha-smooth muscle actin (α-SMA). Scratch assay was used to determine cell migration. Cell morphology was observed after transfection by inverted microscope.ResultsThe optimal transfection rate of scramble siRNA-Cy3 labeled duplexes was efficient in that nearly to 50% in cultured LECs. TβRII specific siRNAs significantly reduced the receptor transcript and protein expression in cultured LECs. The gene knockdown inhibited LECs transdifferentiation, as it abrogated the expression of fibronectin and α-SMA, and retarded cell migration on the scratch assay. In addition, after transfection with TβRII specific siRNA, the cultured LECs did not show fibroblast-like shape which was one of the feature signs of EMT. Wound scratch assays indicated that the number of cultured LECs migrated into the wounded area was significantly lower in TβRII specific siRNA treated group (12.8 ± 3.27/7.85 mm(2)), compared with normal (57.8 ± 3.06/7.85 mm(2)) and scrambled RNA transfected group (50.8 ± 3.64/7.85 mm(2); p<0.0001).ConclusionsOur results provided additional evidence to support that TGF-β pathway was involved in the development of EMT of human posterior capsule opacification, while how TβRII was involved should be further investigated.

Project description:Transforming growth factor-? (TGF-?) and epidermal growth factor (EGF) have critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-? in altering the response of breast cancer cells to EGF remains unknown. We show in this study that murine metastatic 4T1 breast cancer cells formed compact and dense spheroids when cultured under three-dimensional (3D) conditions, which was in sharp contrast to the branching phenotypes exhibited by their nonmetastatic counterparts. Using the human MCF10A series, we show that epithelial-type and nonmetastatic breast cancer cells were unable to invade to EGF, whereas their mesenchymal-type and metastatic counterparts readily invaded to EGF. Furthermore, EMT induced by TGF-? was sufficient to manifest dense spheroid morphologies, a phenotype that increased primary tumor exit and invasion to EGF. Post-EMT invasion to EGF was dependent on increased activation of EGF receptor (EGFR) and p38 mitogen-activated protein kinase, all of which could be abrogated either by pharmacologic (PF-562271) or by genetic (shRNA) targeting of focal adhesion kinase (FAK). Mechanistically, EMT induced by TGF-? increased cell-surface levels of EGFR and prevented its physical interaction with E-cadherin, leading instead to the formation of oncogenic signaling complexes with T?R-II. Elevated EGFR expression was sufficient to transform normal mammary epithelial cells, and to progress their 3D morphology from that of hollow acini to branched structures characteristic of nonmetastatic breast cancer cells. Importantly, we show that TGF-? stimulation of EMT enabled this EGFR-driven breast cancer model to abandon their inherent branching architecture and form large, undifferentiated masses that were hyperinvasive to EGF and showed increased pulmonary tumor growth upon tail vein injection. Finally, chemotherapeutic targeting of FAK was sufficient to revert the aggressive behaviors of these structures. Collectively, this investigation has identified a novel EMT-based approach to neutralize the oncogenic activities of EGF and TGF-? in aggressive and invasive forms of breast cancer.

Dataset Information

Epidermal Growth Factor Stimulates Transforming Growth Factor-Beta Receptor Type II Expression In Corneal Epithelial Cells.

Publications

Epidermal Growth Factor Stimulates Transforming Growth Factor-Beta Receptor Type II Expression In Corneal Epithelial Cells.

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