Metabolic remodeling precedes mTORC1-mediated cardiac hypertrophy.

RATIONALE: The nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown. OBJECTIVE: To examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart. METHODS AND RESULTS: We developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1iSA) through deletion of Tsc2. Prior to hypertrophy, rates of glucose uptake and oxidation, as well as protein and enzymatic activity of glucose 6-phosphate isomerase (GPI) were decreased, while intracellular levels of glucose 6-phosphate (G6P) were increased. Subsequently, hypertrophy developed. Transcript levels of the fetal gene program and pathways of exercise-induced hypertrophy increased, while hypertrophy did not progress to heart failure. We therefore examined the hearts of wild-type mice subjected to voluntary physical activity and observed early changes in GPI, followed by hypertrophy. Rapamycin prevented these changes in both models. CONCLUSION: Activation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2.

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys(370) at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes.

Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species.

Signal transducers and activators of transcription 3 (Stat3) is a transcription factor expressed in many cell types including osteoblasts, osteocytes, and osteoclasts. STAT3 mutations cause a rare human immunodeficiency disease that presents reduced bone mineral density and recurrent pathological fractures. To investigate the role of Stat3 in load-driven bone metabolism, two strains of osteoblast/osteocyte-selective Stat3 knockout (KO) mice were generated. Compared to age-matched littermate controls, this selective inactivation of Stat3 significantly lowered bone mineral density (7-12%, p<0.05) as well as ultimate force (21-34%, p<0.01). In ulna loading (2.50-2.75N with 120 cycles/day at 2Hz for 3 consecutive days), Stat3 KO mice were less responsive than littermate controls as indicated by reduction in relative mineralizing surface (rMS/BS, 47-59%, p<0.05) and relative bone formation rate (rBFR/BS, 64-75%, p<0.001). Furthermore, inactivation of Stat3 suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Taken together, the results support the notion that the loss-of-function mutation of Stat3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.

Parathyroid hormone and bisphosphonate have opposite effects on stress fracture repair.

This study was aimed to investigate the effects of Parathyroid hormone (PTH) and alendronate (ALN) on stress fracture repair. Stress fractures were induced in the ulnae of female adult rats. Animals were treated daily with vehicle, PTH (40 microg/kg) or alendronate (2 microg/kg), respectively. Bone mineral content (BMC) and bone mineral density (BMD) of bilateral ulnae were measured at two, four and eight weeks following induction of stress fracture. Histology at the ulna midshaft was undertaken at 2 and 4 weeks and mechanical testing was done at 8 weeks after stress fracture. PTH increased BMC significantly by 7% at 4 weeks and BMD and BMC significantly by 10% and 7% at 8 weeks compared to the control. Alendronate did not change BMD or BMC in comparison with the control. PTH significantly stimulated bone formation by 114% at 2 weeks, increased intracortical resorption area by 23% at 4 weeks, and enhanced the ultimate force of the affected ulnae by 15% at 8 weeks compared to the control. Alendronate significantly suppressed bone formation rate by 44% compared to the control at 4 weeks. These data indicate that PTH may accelerate intracortical bone remodeling induced by microdamage and alendronate may delay intracortical bone remodeling during stress fracture repair in rats. This study suggests that PTH may be used to facilitate stress fracture repair whereas bisphosphonates may delay tissue level repair of stress fractures.