To investigate the interconnection of apoptosis and autophagy in trophoblastic cells, we treated JEG-3 cells with tumor necrosis factor a (TNF-a) after transfecting LC3 or Beclin 1 or calpain small interfering RNA (siRNA), which blocks cleavage of autophagyrelated gene 5 (Atg5) into N-terminal truncated Atg5 (tAtg5), a mediator between apoptosis and autophagy, and assessed the changes in LC3-II, caspase 9, caspase 3, and tAtg5. We also assessed the TNF-a-induced changes in LC3-II, caspase 9, and caspase 3 in primary trophoblasts from term placentae after transfecting siRNA for LC3 or Beclin 1. In both types of cells, transfection of LC3 or Beclin 1 siRNA significantly attenuated TNF-a-induced increases in LC3-II and activations of caspase 9 and caspase 3. There was significant abrogation of TNF-a-induced expression of tAtg5 after transfection with LC3 or Beclin 1 siRNA. Moreover, transfection with calpain siRNA significantly decreased TNF-a-induced changes in caspase 3 and caspase 9 in addition to tAtg5 in JEG-3 cells. Our data suggest that TNF-a-induced autophagy mediates intrinsic apoptosis, probably through tAtg5, in trophoblastic cells.

Normal human epidermal keratinocytes (NHEK) respond to the autocrine activated extracellular signal-regulated kinase (ERK) signaling pathway, which contributes to the survival of keratinocytes. However, during the condition of calcium-induced differentiation, how the autocrine ERK signaling is regulated and affected is poorly understood. The purpose of this study was to understand and to obtain clues to the possible function of the autocrine ERK activation during the calcium-induced differentiation of NHEK. We demonstrated that the autocrine activated ERK was not interrupted by calcium triggering and that it was sustained for at least one day after changing the medium. We also found that the autocrine ERK activation was associated with the expression of cyclin D1 and the cell cycle regulation at the early stage of calcium triggering by treating the cells with the mitogen-activated protein kinase inhibitor PD98059. However, the PD98059 treatment did not have a significant influence on the expression of involucrin and loricrin. In addition, we demonstrated that autocrine ERK activation was associated with protein kinase C and p38MAPK signaling. We suggest that the activation of autocrine ERK is not interrupted by calcium triggering and it might participate in cell growth during the early stage of calcium-induced differentiation in NHEK.