Smurf1 Inhibits Osteoblast Differentiation, Bone Formation, and Glucose Homeostasis through Serine 148.

The E3 ubiquitin ligase Smurf1 targets the master regulator of osteoblast differentiation, Runx2, for degradation, yet the function of Smurf1, if any, during osteoblast differentiation in vivo is ill defined. Here, we show that Smurf1 prevents osteoblast differentiation by decreasing Runx2 accumulation in osteoblasts. Remarkably, mice harboring a substitution mutation at serine 148 (S148) in Smurf1 that prevents its phosphorylation by AMPK (Smurf1(ki/ki)) display a premature osteoblast differentiation phenotype that is equally severe as that of Smurf1(-/-) mice, as well as a high bone mass, and are also hyperinsulinemic and hypoglycemic. Consistent with the fact that Smurf1 targets the insulin receptor for degradation, there is, in Smurf1(ki/ki) mice, an increase in insulin signaling in osteoblasts that triggers a rise in the circulating levels of osteocalcin, a hormone that favors insulin secretion. These results identify Smurf1 as a determinant of osteoblast differentiation during the development of bone formation and glucose homeostasis post-natally and demonstrate the necessity of S148 for these functions.

Glucose Uptake and Runx2 Synergize to Orchestrate Osteoblast Differentiation and Bone Formation.

The synthesis of type I collagen, the main component of bone matrix, precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. We hypothesized that the energetic needs of osteoblasts might explain this apparent paradox. We show here that glucose, the main nutrient of osteoblasts, is transported in these cells through Glut1, whose expression precedes that of Runx2. Glucose uptake favors osteoblast differentiation by suppressing the AMPK-dependent proteasomal degradation of Runx2 and promotes bone formation by inhibiting another function of AMPK. While RUNX2 cannot induce osteoblast differentiation when glucose uptake is compromised, raising blood glucose levels restores collagen synthesis in Runx2-null osteoblasts and initiates bone formation in Runx2-deficient embryos. Moreover, RUNX2 favors Glut1 expression, and this feedforward regulation between RUNX2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. These results reveal an unexpected intricacy between bone and glucose metabolism.

Loss of catalytically inactive lipid phosphatase myotubularin-related protein 12 impairs myotubularin stability and promotes centronuclear myopathy in zebrafish.

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., "MTMRs"). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.

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

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 PKCß 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 PKCß--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.