Effects of transforming growth factor-β on mammary remodeling during the dry period of dairy cows

Abstract

Mammary remodeling in dairy cows involves coordinated changes in stromal and epithelial tissue. Tissue remodeling is characterized by changes in cell proliferation, activation of fibroblasts into myofibroblasts, and changes in extracellular matrix content. Transforming growth factor β-1 (TGF-β1) increases differentiation of fibroblasts to myofibroblasts, regulates expression of extracellular matrix proteins and proteases, and has cell-type dependent effects on proliferation. The objective of this study was to determine whether TGF-β1 treatment of mammary tissue from cows in late lactation and the dry period affects cell proliferation, expression of matrix metalloproteinase-3 (MMP-3) and fibronectin (FN), and the differentiation of fibroblasts into myofibroblasts that express smooth muscle α actin (SMA). Tissue was biopsied from 7 Holstein cows at 4 time points: late lactation, 1 wk after dry-off, 3 wk before expected calving, and 1 wk before expected calving. Explants of biopsied tissue were incubated for 2h in Waymouth's medium containing insulin, hydrocortisone, and 0 or 5 ng of TGF-β1/mL; a subset of cultures was also incubated with bromodeoxyuridine to measure epithelial and stromal cell proliferation. Tissues were fixed, embedded in paraffin, sectioned, and stained by immunohistochemistry. Stage at biopsy had an overall effect on rate of epithelial and stromal cell proliferation, and TGF-β1 treatment increased rate of bromodeoxyuridine incorporation more than 2-fold in both cell types at 1 wk after dry-off. The number of fibroblasts expressing SMA was 19% higher in the intralobular stroma at 1 wk after dry-off compared with that at 1 wk before expected calving, and the percentage of activated fibroblasts tended to be higher in tissue incubated with TGF-β1. Biopsy stage had an overall effect on percentage area of epithelium expressing FN and MMP-3. Incubation with TGF-β1 had no effect on percentage intralobular stroma area expressing FN or MMP-3. Effects of TGF-β1 treatment were most apparent at 1 wk after dry-off, indicating that the first week of dry period may be an ideal target for testing effects in vivo.

Peer Review #3 of "TGF-beta signalling in bovine mammary gland involution and a comparative assessment of MAC-T and BME-UV1 cells as in vitro models for its study (v0.2)"

“…Further, with the strong evidence 145 supporting TGFβ1's effects on both the stromal and parenchymal compartments of the mammary 146 gland, a more holistic approach to studying TGFβ1 signalling incorporating both stromal and 147 epithelial cells may be necessary to study novel approaches to increasing milk production. In 148 vitro treatment of whole tissue explants (De Vries et al, 2011, Magro et al, 2017 is an attractive 149 possibility, as they may convey key mechanistic information impossible to obtain by analysis of 150 biopsies. Another alternative is the use of three-dimensional co-culture models that incorporate 151 ECM and stromal cells of interest in addition to the epithelial cells, as those recently developed 152 by our group (Pallegar et al, 2018).…”

Peer Review #2 of "TGF-beta signalling in bovine mammary gland involution and a comparative assessment of MAC-T and BME-UV1 cells as in vitro models for its study (v0.2)"

“…Whether TGFβ1 disrupts tight junctions in the bovine mammary gland, and whether this is responsible for TGFβ1-induced apoptosis in the involuting bovine mammary gland, remains to be addressed. TGFβ1 and its receptors are upregulated during bovine involution (Plath et al, 1997), and in vitro findings suggest that elevated TGFβ1 expression during involution is due in part to its increased secretion by bovine mammary cells during the transition from lactation to the dry period (Zarzyńska, Gajewska & Motyl, 2005), as well as through enhanced release of TGFβ1 from the extracellular matrix (ECM) (De Vries et al, 2011). TGFβ1 arrests bovine epithelial cell growth (Kolek et al, 2003) and such effect of TGF was shown to be important for the development of fully functional mammary glands in mice.…”

“…Further, with the strong evidence supporting TGFβ1's effects on both the stromal and parenchymal compartments of the mammary gland, a more holistic approach to studying TGFβ1 signalling incorporating both stromal and epithelial cells may be necessary to study novel approaches to increasing milk production. In vitro treatment of whole tissue explants (De Vries et al, 2011, Magro et al, 2017 is an attractive possibility, as they may convey key mechanistic information impossible to obtain by analysis of biopsies. Another alternative is the use of three-dimensional co-culture models that incorporate ECM and stromal cells of interest in addition to the epithelial cells, as those recently developed by our group (Pallegar et al, 2018).…”

“…Contrary to its inhibitory effect on the bovine mammary epithelium, TGFβ1 promotes stromal development in the mammary gland (Musters et al, 2004). TGFβ1 enhances bovine fibroblast proliferation through the MAPK/Erk pathway (Gao et al, 2016), and promotes the transition of fibroblasts to myofibroblasts (De Vries et al, 2011); the latter are key mediators of ECM protein synthesis and tissue fibrosis (Phan, 2008). Furthermore, TGFβ1 is known to cause bovine mammary epithelial cells to switch to a mesenchymal phenotype through a process known as epithelial-to-mesenchymal transition (EMT) (Chen et al, 2017).…”

FATOR DE CRESCIMENTO TRANSFORMADOR BETA (TGF-β) EM LEITE: UMA REVISÃO

“…Os TGF-βs compreendem uma família de polipeptídios que incluem os TGF-β1, TGF-β2 e TGF-β3, possuindo a função de regular o desenvolvimento do tecido mamário (VRIES et al, 2011). A forma predominante no leite é a TGF-β2 (85%), apresentando-se ligada a uma proteína e com dímeros com 224 aminoácidos e 25000 Da, após ati vação ácida, sendo que quatro ligações dis sulfeto estabilizam cada monômero e uma ligação dissulfeto estabiliza o dímero.…”