Apoptosis and autophagy in involuting bovine mammary gland is accompanied by up-regulation of TGF-beta1 and suppression of somatotropic pathway.

Regulation of mammary gland remodeling during the lactation cycle in cattle still remains unclear. The present study focused on the role of TGF-beta1 and somatotropic pathways proteins in control of the switch between survival and death of bovine mammary epithelial cells (MEC). Expressions of TGF-beta1, TGF-betaRII, IGF-IRalpha, IGF-IRbeta, GH-R, IGFBP-3, -4, and -5 in mammary tissue explants in Holstein-Fresian heifers (n = 7) and cows (n = 23) in early lactation (1-100 day), late lactation (200-260 day) and drying off (280-340 day) were compared with biochemical indices of apoptosis (caspase 3, 89 kDa fragment of PARP) and autophagy (Beclin1). The results revealed that an increase in apoptosis during the dry period was accompanied by highly significant increases in TGF-beta1 and TGF-betaRII expression. Beside biochemical markers, typical morphological features of apoptosis, such as cell shrinkage, separation from the neighboring cells and condensation of chromatin were observed. TGF-beta1 expression and induction of apoptosis was facilitated by the suppression of somatotropic pathway during drying off, manifested with down-regulation of GH-R and IGF-IRalpha, and up-regulation of IGFBP-4 and -5. This is the first report describing autophagy in the bovine mammary gland. Similarly to apoptosis, the intensity of autophagy was the highest in the dry period, as shown by increased expression of Beclin1 and morphological features, e.g. autophagosomes, autophagic vacuoles. Autophagy observed in the involuting mammary tissue could be the natural cell defense against transient undernourishment and action of apoptogenic peptides (e.g. TGF-beta1, IGFBPs), thus maintaining cellular homeostasis in the dry period.

Apoptosis and autophagy in involuting bovine mammary gland.

Successful lactation depends on a controlled process of cell proliferation and mammary gland growth during pregnancy and cell death during involution. The main type of cell death responsible for involution of bovine mammary gland is apoptosis, but programmed cell death type II (PCD II)--autophagic cell death is also observed. The regulation of mammary epithelial cells (MEC) apoptosis occurs at three levels, the molecular regulators of autophagy have not been identified yet. There are possible correlations between both processes, because cells sharing morphological features of apoptosis and autophagy were found in involuting mammary gland. Autophagy seems to be cellular defense against starvation, when it fails, a secondary response of apoptotic cell death is triggered.

Apoptosis and autophagy in mammary gland remodeling and breast cancer chemotherapy.

Apoptosis - programmed cell death (PCD) type I is physiological process responsible for cell loss during mammary gland involution after natural weaning or litter removal in rodents, after weaning in sow and during drying off in goat and cow. The regulation of mammary epithelial cell (MEC) apoptosis in bovine mammary gland occurs at three levels. The first level comprises intracellular regulatory proteins, e.g. Bcl-2 family death promoters and inhibitors. The second level is represented by intramammary inductors of apoptosis, e.g. FIL, IGFBPs, Fas ligand, TGF-betas. The expression and activity of these auto/paracrine inductors of apoptosis is controlled and modulated by the third level factors, e.g. systemic galactopoetic hormones, nutrition, reproductive status and milking management. Our recent study proved that apoptosis in involuting bovine mammary gland is accompanied by increased intensity of autophagy, regarded as a cytoprotective process but in advanced stage as a PCD type II. Moreover, we have reported for the first time the ability of TGF-beta(1) to induce both apoptosis and autophagy in bovine BME-UV1 MEC. Much more pronounced heterogeneity of PCD was observed when breast cancer cells were exposed to anticancer drugs. The primary responses of breast cancer MCF-7 cells to camptothecin (CPT) are apoptosis and autophagy (as a cytoprotective process). In this case autophagy occurs in cells which are resistant to apoptosis as a tool of cancer cell survival. The fail-safe responses of breast cancer cells to persisting CPT-induced stress are apoptosis accompanied by morphological and biochemical features of autophagy or type II PCD with advanced subcellular degradation. The threshold between autophagy as a cytoprotective process (reversible) or PCD (irreversible) is difficult to establish and probably depends on the extent of degradation of cellular components. Proapoptotic protein Bid may serve as a molecular switch between apoptosis and autophagy. Bid knock down in MCF-7 cells exposed to CPT leads to a shift of cell death from apoptosis to autophagy. Since bid and other proapoptotic genes undergo mutations in malignant cells, the ability of cancer cells commitment to autophagy may have important therapeutic implications.

Dissimilar effects of LY 294002 and PD 098059 in IGF-I-mediated inhibition of TGF-beta1 expression and apoptosis in bovine mammary epithelial cells.

In mammary epithelial cells (MEC) TGF-beta(1) is the auto-/paracrine growth inhibitor and inducer of apoptosis and therefore is considered as an important local regulator of mammary tissue involution. However, the mechanisms of controlled TGF-beta(1) expression in the course of bovine mammary gland remodelling are still unclear. Recent study performed in this laboratory support the evidence that TGF-beta(1) expression in bovine MEC is regulated by hormones of somatotropic axis (GH, IGF-I and somatostatin). Present study was focused on the contribution of IGF-I-induced signaling pathways in anti-TGF-beta(1) and anti-apoptotic effects of IGF-I. Laser scanning cytometry was applied for the measurement of TGF-beta(1) content and apoptotic cell number in bovine BME-UV1 MEC. Involution of the bovine mammary gland in vitro was modeled by decreasing the availability of FBS for bovine MEC. Reducing FBS content in the medium from 10% to 0.5% evoked highly significant increase of TGF-beta(1) expression and increase of apoptotic cell number. IGF-I (50 ng/ml) completely abrogated FBS deficiency-induced TGF-beta(1) expression and apoptosis in bovine MEC. In order to establish which of the IGF-I signaling pathways contributed to anti-TGF-beta(1) and anti-apoptotic effects, the inhibitors of PI3-kinase - (LY 294002) and MEK- (MAPKK for ERK) (PD 098059) mediated signaling pathways were applied to our model. The results clearly showed that inhibition of PI3-K reverses the ability of IGF-I to suppress TGF-beta(1) expression and apoptosis. An inhibition of ERK1/2 pathway even potentiated inhibitory effect of IGF-I on TGF-beta(1) expression, but partially abrogated anti-apoptotic effect of IGF-I. In conclusion, the results of the study indicate that PI3-K/Akt pathway contributed significantly to the inhibition of TGF-beta(1) expression by IGF-I, whereas both PI3-K/Akt and ERK1/2 pathways are involved in the anti-apoptotic effect of IGF-I in bovine MEC.