Abstract
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 extraction of tissues. Analysis of multiple tissues and plasma from four strains of adult mice showed that TGF-β1 is the predominant isoform with TGF-β2 being ~10-fold lower. There were no sex-specific differences in isoform expression, but some tissues showed inter-strain variation, particularly for TGF-β2. The only adult tissue expressing appreciable TGF-β3 was the mammary gland, where its levels were comparable to TGF-β1. In situ hybridization showed the luminal epithelium as the major source of all TGF-β isoforms in the normal mammary gland. TGF-β1 protein was 3-8-fold higher in three murine mammary tumor models than in normal mammary gland, while TGF-β3 protein was 2-3-fold lower in tumors than normal tissue, suggesting reciprocal regulation of these isoforms in mammary tumorigenesis.
Highlights
Transforming growth factor-β (TGF-β) proteins are widely expressed and are critical regulators of embryonic development and normal adult tissue maintenance [1]
Since TGF-β levels differ widely between tissues, we believe the acidethanol step is necessary for accurate TGF-β quantitation, as different tissues must be assayed at very different dilutions to fall within range of the standard curve
The amount of TGF-β1 in frozen kidney homogenized in RIPA and extracted in acid-ethanol (38.2 ng/g tissue) was similar to that found in fresh kidney extracted directly into acid-ethanol (42.4 ng/g tissue) suggesting that prior homogenization in RIPA buffer overcomes the sample losses previously associated with direct homogenization of frozen tissues in acid-ethanol
Summary
Transforming growth factor-β (TGF-β) proteins are widely expressed and are critical regulators of embryonic development and normal adult tissue maintenance [1]. All TGF-βs are synthesized as larger precursor proteins that are cleaved intracellularly, and the upstream pro-regions (known as the “latency-associated peptides” or LAPs) remain non-covalently associated with the mature TGF-βs after secretion This feature renders them biologically latent until an activation signal is received which releases the mature protein in a form that can bind to the signaling receptors [4]. NMR studies show that the α3-helical region of TGF-β3 is more disordered than it is in TGF-β1, with TGF-β3 adopting a more “open” flexible conformation in solution, while TGF-β1 predominantly exists in a “closed”, more rigid state [6] These conformational differences may lead to differential interactions of TGF-β1 and TGF-β3 with unidentified binding proteins, that affect binding to the signaling receptors [6].
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