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 PKCb 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 PKCb-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.[Keywords: osteoclast; RANKL; lysosomal biogenesis; TFEB; PKCb] Supplemental material is available for this article. Received January 11, 2013; revised version accepted March 21, 2013. Bone is constantly remodeled through the coordinated action of two cell types: the osteoblast and the osteoclast (Ducy et al. 2000;Teitelbaum 2000). While the osteoblast synthesizes and mineralizes the bone extracellular matrix (ECM), the osteoclast is responsible for resorbing this mineralized ECM. To achieve this specialized task, differentiated and multinucleated osteoclasts attach tightly to the bone surface and generate closed resorption lacunae. These resorption lacunae are characterized by an acidic pH (;4.5) and contain numerous proteases that are exported by the large number of lysosomes present in this cell type (Coxon and Taylor 2008). Proteases and acidification of the lacunae are both necessary for efficient bone resorption. The importance of lysosomal biogenesis for osteoclast function and optimum bone resorption underscores the need to elucidate how this aspect of osteoclast biology is regulated.Over the last two decades, our understanding of the mechanisms by which cells of the myeloid lineage differentiate into functional multinucleated osteoclasts has made considerable progress (Teitelbaum and Ross 2003;Takayanagi 2007). This led to the identification of two cytokines, RANKL and M-CSF (Yoshida et al. 1990;Lacey et al. 1998), as critical determinants of this process and the identification of numerous transcription factors acting downstream from these cytokines. Some of these factors-PU.1, c-FOS, JunB, Fra-1, NFkB, and PPARg-act early in the differentiation process within the myeloid precursor cells (Grigoriadis et al. 1994;Iotsova et al. 1997;Tondravi et al. 1997;Matsuo et al. 2000;Kenner et ...

Activation of Peroxisome Proliferator-activated Receptor α Induces Lysosomal Biogenesis in Brain Cells

“…Semi-quantitative RT-PCR was carried out as described earlier (28) using oligo(dT) [12][13][14][15][16][17][18] as primer and Moloney murine leukemia virus-RT in a 20-l reaction mixture. The resulting cDNA was appropriately amplified using Promega Master Mix (Madison, WI) and the following primers (Life Technologies) for murine genes: mouse Tfeb, sense, 5Ј-AAC AAA GGC ACC ATC CTC AA-3Ј, and antisense, 5Ј-CAG CTC GGC CAT ATT CAC AC-3Ј; mouse Lamp2, sense, 5Ј-GGT GCT GGT CTT TCA GGC TTG ATT-3Ј, and antisense, 5Ј-ACC ACC CAA TCT AAG AGC AGG ACT-3Ј; mouse Limp2, sense, 5Ј-TGT TGA AAC GGG AGA CAT CA-3Ј, and antisense, 5Ј-TGG TGA CAA CCA AAG TCG TG-3Ј; mouse Npc1, sense, 5Ј-GGG ATG CCC GTG CCT GCA AT-3Ј, and antisense, 5Ј-CTG GCA GCT ACA TGG CCC CG-3Ј; mouse Gapdh, sense, 5Ј-GCA CAG TCA AGG CCG AGA AT-3Ј, and antisense, 5Ј-GCC TTC TCC ATG GTG GTG AA-3Ј. Amplified products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.…”

“…The regulation of Tfeb appears to be complex and dependent on cell type and stimuli. In differentiated osteoclasts, a receptor activator of NF-B ligand-dependent signaling pathway induces TFEB activation and lysosomal biogenesis (13). Starvation or stress conditions may also activate TFEB, which otherwise is maintained in an inactivated state by mTORC1 (14,15).…”

Regulation of lysosome biogenesis and functions in osteoclasts

“…The lysosomal biogenesis dysfunction observed in Tfeb −/− osteoclasts is probably caused by a direct effect on lysosomal gene transcription, since TFEB binds the promoter region of several lysosomal genes (Atp6v1c1, Clcn7, and Ctsk), and loss of Tfeb in osteoclasts is characterized by lower expression levels of Acp5, Atp6v1c1, Clcn7, Ctsk, and Tcirg1. 86 Altogether, these data point to TFEB as a critical regulator of lysosomal biogenesis directly modulating the expression of key effectors in this process.…”

“…Importantly, this occurs without affecting osteoclasts number. 86 The dissociation of the role of RANKL in osteoclasts differentiation from the one it has in lysosomal biogenesis suggested that RANKL might control osteoclasts differentiation and activity through distinct mechanisms.…”

“…Our recent work has linked lysosomal biogenesis to this function of RANKL in differentiated osteoclasts. 86 Stimulation of fully differentiated osteoclasts with RANKL increases the size and number of lysosomes in these cells because of the activation of a specific transcriptional program increasing the expression of lysosomal genes implicated in bone resorption. Importantly, this occurs without affecting osteoclasts number.…”