A RANKL-PKCβ-TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts

Abstract

Bone resorption by osteoclasts requires a large number of lysosomes that release proteases in the resorption lacuna. Whether lysosomal biogenesis is a consequence of the action of transcriptional regulators of osteoclast differentiation or is under the control of a different and specific transcriptional pathway remains unknown. We show here, through cell-based assays and cell-specific gene deletion experiments in mice, that the osteoclast differentiation factor RANKL promotes lysosomal biogenesis once osteoclasts are differentiated through the selective activation of TFEB, a member of the MITF/TFE family of transcription factors. This occurs following PKCβ phosphorylation of TFEB on three serine residues located in its last 15 amino acids. This post-translational modification stabilizes and increases the activity of this transcription factor. Supporting these biochemical observations, mice lacking in osteoclasts--either TFEB or PKCβ--show decreased lysosomal gene expression and increased bone mass. Altogether, these results uncover a RANKL-dependent signaling pathway taking place in differentiated osteoclasts and culminating in the activation of TFEB to enhance lysosomal biogenesis-a necessary step for proper bone resorption.

Figure 1.

Transcriptional regulation of lysosomal biogenesis by RANKL in osteoclasts. (A) LAMP1 immunofluorescence staining of differentiated osteoclasts cultured in the presence (right panels) or absence (left panels) of RANKL (30 ng/mL) for 18 h. The top panels show 63× magnification pictures, and the boxed area is further magnified in the bottom panels. Bar, 10 μm; (N) Nucleus. (B) Quantification of the percentage of the osteoclast area covered by lysosomes in individual osteoclasts and of the number of lysosomes per osteoclast relative to −RANKL, normalized by the osteoclast area and the number of nuclei per osteoclast. (C) TRAP staining of osteoclasts treated or not with RANKL for 18 h (2.5× magnification). (D) Quantitative PCR (qPCR) expression analysis of differentiated osteoclasts treated or not with RANKL for 6 h and 18 h. In all experiments, the cells were cultured in the presence of FBS (10%) and M-CSF (10 ng/mL).

Figure 2.

TFEB is required for normal osteoclast function in vitro and in vivo. (A) Expression pattern of Tfeb and Ctsk in mouse tissues and cell types by qPCR. (SM) Skeletal muscle; (WAT) white adipose tissue; (Liv) liver; (OSB) osteoblasts; (OCL) osteoclasts. (B) qPCR expression analysis in RAW 264.7 cells stably transfected with empty vector or TFEB-Flag and treated for 72 h with RANKL (30 ng/mL). (C) Resorption assay. RAW 264.7 cells expressing TFEB-Flag or not were treated for 96 h with RANKL (30 ng/mL), and the percentage of resorbed area over total area was quantified. (D) qPCR expression analysis in RAW 264.7 cells transfected with control nontargeting siRNA (Con siRNA) or siRNA targeting Tfeb (Tfeb siRNA) and treated for 72 h with RANKL (30 ng/mL). (E) ChIP assays performed on RAW 264.7 cells transfected with vector or TFEB-Flag using Flag antibodies demonstrate binding of TFEB to indicated genes but not to the coding region of Actin (see also Supplemental Fig. S1D). (F) Bone histomorphometric analysis of lumbar vertebrae in 6-wk-old control and Tfeb_osc^−/− female mice. (BV/TV) Bone volume over tissue volume; (OcS/BS) osteoclast surface over bone surface; (NOc/Tar) number of osteoclasts per tissue area; (ObS/BS) osteoblast surface over bone surface; (NOb/BPm) number of osteoblasts per bone perimeter; (MAR) mineral apposition rate; (BFR/BS) bone formation rate over bone surface. Fasting serum CTx levels are also included. (G) TRAP staining of control and Tfeb_osc^−/− bone marrow-derived osteoclasts (5× magnification). The number of osteoclasts per well and the relative TRAP staining intensity are indicated. (H) Resorption assay. Control and Tfeb_osc^−/− primary monocytes were differentiated into osteoclasts on Osteo assay for 6 d. The percentage of the resorbed area over the total area is indicated. All experiments were performed at least in quadruplicate. Four to six animals of each genotype were analyzed in F.

Figure 3.

TFEB regulates lysosomal biogenesis in osteoclasts. (A) qPCR expression analysis in control and Tfeb_osc^−/− flushed long bones. (B) LAMP1 immunofluorescence staining of control (left panels) and Tfeb_osc^−/− (right panels) primary osteoclasts. The top panels show 40× magnification pictures, and the boxed area is further magnified in the bottom panels. Bar, 10 μm. (N) Nucleus. (C) Quantification of the percentage of the osteoclast area covered by lysosomes in individual osteoclasts. (D) Quantification of the number of lysosomes per osteoclast relative to control, normalized by the osteoclast area and the number of nuclei per osteoclast. (E) Acridine orange staining of live osteoclasts differentiated on bone slices imaged in a Z-stack using a confocal microscope. The ratio of red (acidic) over green (neutral) signal was quantified in several osteoclasts (n = 10–12) within and underneath the cells. The bottom panels display an orthogonal projection of the top panels. All experiments were performed at least in quadruplicate, and four to six animals of each genotype were analyzed in A.

Figure 4.

TFEB is regulated post-translationally by RANKL. (A) qPCR expression analysis of the indicated genes in bone marrow-derived osteoclasts differentiated in the presence of M-CSF and RANKL for 0–6 d. The expression level of each gene is normalized to the expression at day 6. (B) RAW 264.7 cells stably transfected with TFEB-Flag were treated for the indicated times with RANKL (50 ng/mL). Nucleus and cytosol extracts were prepared as described in the Materials and Methods, and TFEB protein was revealed by Western blotting using Flag antibodies. Lamin C and tubulin were used as nuclear- and cytosolic-specific markers, respectively. (C) Bone marrow-derived monocytes were treated with RANKL (50 ng/mL) for the indicated times, and endogeous TFEB protein was detected by Western blotting. (D) RAW 264.7 cells stably transfected with TFEB-Flag were treated with cycloheximide (CHX at 100 μg/mL) and/or RANKL (50 ng/mL) for the indicated times, and TFEB protein in total cell extracts was revealed by Western blotting using Flag antibodies. (E) RAW 264.7 cells stably transfected with TFEB-Flag were treated with RANKL (50 ng/mL) for the indicated times, and TFEB protein in total cell extracts was revealed by Western blotting using Flag antibodies. (F) RAW 264.7 cells stably transfected with TFEB-Flag were treated with RANKL (50 ng/mL) for 30 min. The total cell extracts were next subjected to λPPase treatment as described in the Materials and Methods. In D–G, actin was used as a loading control. In all experiments, the cells were cultured in the presence of FBS (10%).

Figure 5.

RANKL-induced TFEB stabilization is PKCβ-dependent. (A) RAW 264.7 cells stably transfected with TFEB-Flag were treated with vehicle (DMSO) or the indicated inhibitors as described in the Materials and Methods and with or without RANKL (50 ng/mL) for 16 h. TFEB protein in total cell extracts was determined by Western blotting using Flag antibody. (B) Relative qPCR expression analysis of PKCs encoding genes in primary monocytes and osteoclasts. Values are expressed as fold of Prcka expression levels in monocytes. (C,D) RAW 264.7 cells stably transfected with TFEB-Flag were treated with vehicle (DMSO) or the indicated inhibitors and with or without RANKL (50 ng/mL) for 16 h. TFEB accumulation in cell extracts was measured by Western blotting using Flag antibody. In all experiments, the cells were cultured in the presence of FBS (10%). (E) Bone marrow-derived monocytes were serum-starved for 3 h and treated for the indicated times with RANKL (50 ng/mL), and PKCβ phosphorylation was assessed by Western blotting using an antibody previously validated using Prkcb^−/− cell extracts (see Supplemental Fig. S5C).

Figure 6.

PKCβ phosphorylates TFEB in a C-terminal motif. (A) Sequence alignment of TFEB protein sequence from different vertebrates. The positions of three putative PKCβ phosphorylation sites (http://scansite.mit.edu) are indicated by asterisks. In B–E, kinase assays were performed on recombinant GST fusion proteins as described in the Materials and Methods. In D, 293–475 and 293–460 indicate GST-TFEB^293–475 and GST-TFEB^293–460, respectively. S461A/S462A, S465A/S466A, and S468A mutations were all introduced into the GST-TFEB^293–475 protein. In E, wild type (WT) indicates GST-TFEB^293–475. S465A, S466A, and S465A/S466A mutations were introduced into the GST-TFEB^293–475 protein. (F) RAW 264.7 cells stably transfected with TFEB-Flag or with a TFEB construct lacking the last 15 amino acids (TFEBΔCT-Flag) were treated or not with RANKL (50 ng/mL) for 16 h. TFEB protein in total cell extracts was determined by Western blotting using Flag antibody. (G) Quantification of the results presented in F. The levels of the Flag proteins were quantified, normalized to actin levels, and expressed as a fold induction normalized to each protein level in the absence of RANKL. Three independent experiments were analyzed. (H) RAW 264.7 cells transiently transfected with TFEB-Flag, TFEBΔCT-Flag, or TFEB-Flag containing the indicated mutation were treated or not with RANKL (50 ng/mL). (I) RAW 264.7 cells stably transfected with TFEB-Flag or TFEBΔCT-Flag were treated with RANKL (50 ng/mL) and/or cycloheximide (CHX at 100 μg/mL) for the indicated times. (J) qPCR expression analysis in RAW 264.7 cells stably transfected with the empty vector or TFEB-Flag or TFEBΔCT-Flag construct and treated for 72 h with RANKL (30 ng/mL). (K) LAMP1 immunofluorescence staining on RAW 264.7 osteoclast-like cells following 96 h of treatment with RANKL (30 ng/mL). Magnification, 63×. Bar, 10 μm. (N) Nucleus. (L) Quantification of the percentage of the osteoclast area covered by lysosomes in individual osteoclasts and of the number of lysosomes per osteoclast relative to vector transfected cells, normalized by the osteoclast area and the number of nuclei per osteoclast. (#) P < 0.05; (##) P < 0.01; (###) P < 0.001 when comparing TFEB-Flag and TFEBΔCT-Flag. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 when compared with vector.

Figure 7.

PKCβ is required for normal osteoclast function in vivo. (A) LAMP1 immunofluorescence staining of differentiated osteoclasts cultured in the presence of 10 μM PKCβ inhibitor (right panels) or vehicle (DMSO) (left panels) for 18 h. Magnification, 63×. Bar, 10 μm. (N) Nucleus. (B) Quantification of the percentage of the osteoclast area covered by lysosomes in individual osteoclasts and of the number of lysosomes per osteoclast relative to vehicle, normalized by the osteoclast area and the number of nuclei per osteoclast. (C) Bone histomorphometric analysis of lumbar vertebrae in mice transplanted with wild-type (WT + WT) or Prkcb^−/− (WT + Prkcb^−/−) bone marrow cells (n = 6–13). (BV/TV) Bone volume over tissue volume; (OcS/BS) osteoclast surface over bone surface; (NOc/Tar) number of osteoclasts per tissue area; (ObS/BS) osteoblast surface over bone surface; (NOb/BPm) number of osteoblasts per bone perimeter; (MAR) mineral apposition rate; (BFR/BS) bone formation rate over bone surface. Fasting serum CTx levels are also included. (D) qPCR expression analysis in wild-type and Prkcb^−/− flushed long bones. (E) Wild-type or Prkcb^−/− bone marrow-derived monocytes were cultured in the presence of M-CSF and with or without RANKL (50 ng/mL) for the indicated times. Accumulation of endogenous TFEB assessed by Western blotting.

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.