Figure 8.

RANKL induces accumulation of TFEB but not MITF. (A) Sequence alignment of MITF/TFE mouse proteins. The positions of three putative conserved PKCβ phosphorylation sites are indicated by asterisks. (B) Kinase assay was performed on recombinant GST fusion proteins as described in the Materials and Methods. (C) RAW 264.7 cells stably transfected with TFEB-Flag, TFEBΔCT-Flag, MITF, or a MITF construct lacking the last 16 amino acids (MITFΔCT-Flag) were treated or not with RANKL (50 ng/mL) for 16 h. TFEB and MITF proteins in total cell extracts were revealed by Western blotting using Flag antibodies. Two exposure times of the same blot are included. (D) Quantification of the results presented in C. The levels of the Flag proteins were quantified, normalized to actin levels, and expressed as a fold induction normalized to the protein level in the absence of RANKL. Three independent experiments were analyzed. (E) qPCR expression analysis in RAW 264.7 cells stably transfected with the empty vector or TFEB-Flag or MITF-Flag construct and treated for 72 h with RANKL (30 ng/mL). (#) P < 0.05 when comparing TFEB-Flag and MITF-Flag. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 when compared with vector. (F) Model. To promote lysosomal biogenesis, RANKL signaling uses PKCβ that phosphorylates TFEB on at least three serine residues located in its C terminus. This phosphorylation stabilizes TFEB, leading to its accumulation and the activation of target genes implicated in lysosomal biogenesis and bone resorption.