Kinases in bone homeostasis: Studies on the roles of AMPKalpha2 and PI3Kgamma in bone homeostasis

AMPKα2 is a catalytic subunit of AMPK, which is a key regulator for cellular and whole body energy homeostasis. Bone is a dynamic organ and susceptible to metabolic changes. To assess the physiological role of AMPKα2 in bone homeostasis, we characterized bone phenotype of AMPKα2 KO mice. We found that AMPKα2 KO mice have lower bone mass than WT littermates and it was accounted for by the increased osteoclast development. The increased osteoclastogenesis of AMPKα2deficient macrophages in vitro was associated with the up-regulated expression of osteoclast-associated marker genes. To further elucidate how AMPKα2 modulates RANKL-mediated osteoclast formation, we examined global gene expression profiles of BMM-OCL via microarray analysis. Surprisingly, many of the genes that were up-regulated by AMPKα2 deficiency were associated with bone marrow stromal cells. Smooth muscle α-actin (SMAA), which was among the up-regulated genes in AMPKα2 KO, was utilized for our further investigation on the role of AMPKα2-deficient stromal cells in the increased osteoclastogenesis of AMPKα2-deficient BMM-OCL. Experiments with SMAA-GFP;AMPKα2 KO mice exhibited that the increased growth rate of AMPKα2-deficient stromal cells was a contributing factor for the elevated osteoclastogenesis in AMPKα2-deficent BMM-OCL. ^ G protein-coupled receptor-regulated PI3Kγ is abundantly expressed in myeloid cells and became a promising drug target to treat various inflammatory diseases. However, its role in bone homeostasis has not been documented. We therefore characterized bone phenotype of PI3Kγ-deficient mice and found that PI3Kγ-deficient mice had higher bone mass than WT littermates. Our analyses further revealed that PI3Kγ deficiency did not affect the bone formation because no significant changes in osteoblast number and bone formation rate were observed. Instead, the lack of PI3Kγ was associated with decreased bone resorption as evidenced by decreased osteoclast number in vivo and impaired osteoclast formation in vitro. The decreased osteoclast formation was accompanied by down-regulated expression of osteoclastogenic genes, compromised chemokine receptor signaling, and an increase in apoptosis during osteoclast differentiation. These data suggest that PI3Kγ regulates bone homeostasis by modulating osteoclastogenesis. Our study also suggests that inhibition of PI3Kγ, which is being considered as a potential therapeutic strategy for treating chronic inflammatory disorders, may result in an increase in bone mass.

Control of Mammalian Circadian Rhythm by CKIɛ-Regulated Proteasome-Mediated PER2 Degradation

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIɛ (casein kinase Iɛ) has been postulated to prime PER2 for degradation. Supporting this idea, CKIɛ inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIɛ inhibition also slows the degradation of PER2 in cells. CKIɛ-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein β-TrCP to a specific site, and dominant negative β-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock

SMAD3 mutation in LDS3 causes bone fragility by impairing the TGF-β pathway and enhancing osteoclastogenesis

Loss-of-function mutations in SMAD3 cause Loeys-Dietz syndrome type 3 (LDS3), a rare autosomal-dominant connective tissue disorder characterized by vascular pathology and skeletal abnormalities. Dysregulation of TGF-β/SMAD signaling is associated with abnormal skeletal features and bone fragility. To date, histomorphometric and ultrastructural characteristics of bone with SMAD3 mutations have not been reported in humans and the exact mechanism by which SMAD3 mutations cause the LDS3 phenotype is poorly understood. Here, we investigated bone histomorphometry and matrix mineralization in human bone with a SMAD3 mutation and explored the associated cellular defect in the TGF-β/SMAD pathway in vitro. The index patient had recurrent fractures, mild facial dysmorphism, arachnodactyly, pectus excavatum, chest asymmetry and kyphoscoliosis. Bone histomorphometry revealed markedly reduced cortical thickness (−68 %), trabecular thickness (−32 %), bone formation rate (−50 %) and delayed mineralization. Quantitative backscattered electron imaging demonstrated undermineralized bone matrix with increased heterogeneity in mineralization. The patient's SMAD3 mutation (c.200 T > G; p.I67S), when expressed from plasmid vectors in HEK293 cells, showed reduced phosphorylation and transcription factor activity compared to normal control and SMAD3 (p.S264Y), a gain-of-function mutation, somatic mosaicism of which causes melorheostosis. Transfection study of the patients' SMAD3 (p.I67S) mutation displayed lower luciferase reporter activity than normal SMAD3 and reduced expression of TGF-β signaling target genes. Patient fibroblasts also demonstrated impaired SMAD3 protein stability. Osteoclastogenic differentiation significantly increased and osteoclast-associated genes, including ACP5 (encoding TRAP), ATP6V0D2, and DCSTAMP, were up-regulated in CD14 (+) peripheral blood mononuclear cells (PBMCs) with the SMAD3 (p.I67S) mutation. Upregulation of osteoclastogenic genes was associated with decreased expression of TGF-β signaling target genes. We conclude that bone with the SMAD3 (p.I67S) mutation features reduced bone formation, and our functional studies revealed decreased SMAD3 activation and protein stability as well as increased osteoclastogenesis. These findings enhance our understanding of the pathophysiology of LDS3 caused by SMAD3 mutations. Emerging therapies targeting in the TGF-β/SMAD pathway also raise hope for treatment of LDS3