The transcription factor C/EBP α controls the role of cystatin F during the differentiation of monocytes to macrophages.

Cystatin F is an inhibitor of cysteine peptidases expressed solely in immune cells. It is the only type II cystatin able to enter endosomal/lysosomal vesicles and to regulate directly the activity of intracellular cysteine cathepsins. Its expression in promonocytic U937 and promyeloblastic HL-60 cells is highly upregulated but, after differentiation with phorbol 12-myristate 13-acetate - PMA, its levels drop significantly. In contrast, the activities of intracellular cysteine cathepsins C, L and S are higher in differentiated cells than in non-differentiated ones due, presumably, to the lower inhibitory capacity of cystatin F. Using immunofluorescence confocal microscopy, proximity ligation assay and co-immunoprecipitation, cathepsins C, L and S were confirmed to be the main interacting partners of cystatin F in U937 and HL-60 cells. The promoter region of the cystatin F gene, CST7, contains a unique binding site for transcription factor C/EBP α, one of the main myeloid differentiation instructors. Using the chromatin immunoprecipitation assay, C/EBP α was shown to bind to CST7 gene in U937 cells. Following cell differentiation with PMA, the binding of C/EBP α was decreased significantly. The protein level of C/EBP α was also significantly lower in differentiated than in non-differentiated cells. It was shown that, during monocyte to macrophage differentiation, the endosomal/lysosomal proteolytic activity can be regulated by cystatin F whose expression is under the control of transcriptional factor C/EBP α.