Goat Rad21 plays a role of protecting caprine mammary epithelial cells from apoptosis

Mammary epithelial cells periodically undergo proliferation, differentiation, growth arrest, and apoptosis during each pregnancy-weaning cycle. Using PCR-select cDNA subtraction strategy, we found that Rad21 mRNA has highly diversity of expression between involution and lactation stage of the murine mammary gland. To begin with, we examined the expression pattern of Rad21 in different developmental stages of mammary gland to establish its correlative function in mammary glands. The Rad21 protein expression profile was demonstrate with western blotting technique. The result showed that Rad21 was highly expressed in pregnancy and lactation stage but low in the beginning of weaning. However, Rad21 was still unclear in goat mammary gland development. We used immortal caparin mammary epithelial cell (CMEC) as a model to study the role of Rad21 and its regulatory mechanism in development of goat mammary gland. Rad21 is a nuclear protein, and Rad21 is one of the major cohesin subunits that holds sister chromatids together until anaphase, when proteolytic cleavage by separase, allows chromosomal separation. In addition, the cleavage of Rad21 also occurs during apoptosis induced by diverse stimuli. This apoptotic cleavage site is distinct from previously described mitotic cleavage sites. Rad21 is cleaved in carboxy-terminal and amino-terminal products. One of cleaved products is translocated to the cytoplasm early in apoptosis before chromatin condensation and nuclear fragmentation. According to goat Rad21 has highly conservation with human, murine and bovine, we cloned goat full-length Rad21 (gRad21) from primary cultured goat mammary gland. To investigate the effect of Rad21 and its cleavage products to the activation of caspase-3 in CMEC, we transiently transfected full length, N-and C-terminal Rad21 into CMEC and treated with etoposide for 36 hours, then observed actived caspase-3 by western blotting. It showed no effect on actived caspase-3 expression profile, suggested that no matter exogenous full length, N and C-terminal Rad21 did not induce apoptosis. Further more, full length Rad21 did not be cleaved into N-and C-terminal products. Using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to investigate apoptosis, after eioposide treatment 36 hours, we found that it was a small amount of cell expressed Rad21 and apoptosis at the same time. We speculated full length, N- and C-terminal Rad21 can inhibit apoptosis. Combined together, these results show that, full length Rad21 protein remain high expression during lactation but reduce expression as entry of weaning. These data hind that expression of full length and C-terminal Rad21 may have a role in inhibition promoting apoptosis of mammary epithelial cell to remain mammary gland lactation and afford early entry of weaning.