Dataset Information

Quantitation of TGF-? proteins in mouse tissues shows reciprocal changes in TGF-?1 and TGF-?3 in normal vs neoplastic mammary epithelium.

ABSTRACT: Transforming growth factor-?s (TGF-?s) regulate tissue homeostasis, and their expression is perturbed in many diseases. The three isoforms (TGF-?1, -?2, and -?3) have similar bioactivities in vitro but show distinct activities in vivo. Little quantitative information exists for expression of TGF-? isoform proteins in physiology or disease. We developed an optimized method to quantitate protein levels of the three isoforms, using a Luminex® xMAP®-based multianalyte assay following acid-ethanol extraction of tissues. Analysis of multiple tissues and plasma from four strains of adult mice showed that TGF-?1 is the predominant isoform with TGF-?2 being ~10-fold lower. There were no sex-specific differences in isoform expression, but some tissues showed inter-strain variation, particularly for TGF-?2. The only adult tissue expressing appreciable TGF-?3 was the mammary gland, where its levels were comparable to TGF-?1. In situ hybridization showed the luminal epithelium as the major source of all TGF-? isoforms in the normal mammary gland. TGF-?1 protein was 3-8-fold higher in three murine mammary tumor models than in normal mammary gland, while TGF-?3 protein was 2-3-fold lower in tumors than normal tissue, suggesting reciprocal regulation of these isoforms in mammary tumorigenesis.

SUBMITTER: Flanders KC

PROVIDER: S-EPMC5122380 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Quantitation of TGF-β proteins in mouse tissues shows reciprocal changes in TGF-β1 and TGF-β3 in normal vs neoplastic mammary epithelium.

Flanders Kathleen C KC Yang Yu-An YA Herrmann Michelle M Chen JinQiu J Mendoza Nerissa N Mirza Amer M AM Wakefield Lalage M LM

Oncotarget 20160601 25

Transforming growth factor-βs (TGF-βs) regulate tissue homeostasis, and their expression is perturbed in many diseases. The three isoforms (TGF-β1, -β2, and -β3) have similar bioactivities in vitro but show distinct activities in vivo. Little quantitative information exists for expression of TGF-β isoform proteins in physiology or disease. We developed an optimized method to quantitate protein levels of the three isoforms, using a Luminex® xMAP®-based multianalyte assay following acid-ethanol ex ...[more]

PMID: 27203217

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Project description:Transforming growth factor β (TGF-β) plays a critical role in wound healing and the pathogenesis of fibrosis (scarring). Three isoforms of TGF-β have been identified in mammals. Previous studies have shown that the addition of TGF-β1 (T1) or -β2 (T2) to human corneal fibroblasts (HCF) cultured in a 3-dimensional construct resulted in a fibrotic matrix, while the addition of TGF-β3 (T3) resulted in the production of enhanced non-fibrotic matrix as compared to control (Vitamin C [VitC] only). In the current investigation, we undertook the molecular comparison of fibrosis-related gene expression in T1 or T3-treated HCF to gain further insights into the regulation and roles of these two isoforms on the fibrotic response. HCF were cultured in 100 mm dishes in basic medium (Eagles minimum essential medium [EMEM] with 10% fetal bovine serum [FBS]). At 70-80% confluency, cells were exposed to basic medium with 0.5 mM 2-O-α-d-glucopyranosyl-l-ascorbic acid (VitC) ± 2 ng/ml of T1 or T3. After 4 h or 3 days, cells were harvested, and mRNA or protein was isolated. Fibrosis related mRNA levels were assayed using a commercial qRT-PCR Array. Selected proteins were examined using Western blotting (WB). Experiments were performed 6 times for the qRT-PCR and 4 times for WB for each condition. qRT-PCR results showed that most of the fibrosis-related genes were up or downregulated in HCF exposed to T1 or T3 as compared with VitC control. At 4 h, only Smad7 expression was significantly altered in T3-treated HCF, compared to T1, and at 3 days, five genes were altered. WB confirmed that T1 significantly decreased Smad7 expression compared to T3 and control, and that the expression of thrombospondin-1 in T3-stimulated HCF was enhanced compared to T1-treated cells. Finally, both T1 and T3 decreased Smad3 expression dramatically at both time points. At early time points, T1 and T3 have similar effects on expression of fibrosis related genes; however, with a longer exposure, an increasing number of genes were differentially expressed. Interestingly, most of the differentially expressed gene products are secreted by the cells and may be related to the modulation of extracellular matrix.

Dataset Information

Quantitation of TGF-? proteins in mouse tissues shows reciprocal changes in TGF-?1 and TGF-?3 in normal vs neoplastic mammary epithelium.

Publications

Quantitation of TGF-β proteins in mouse tissues shows reciprocal changes in TGF-β1 and TGF-β3 in normal vs neoplastic mammary epithelium.

Similar Datasets

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