Energy deprivation activates the cellular energy sensor AMP-activated protein kinase (AMPK), which in turn induces macroautophagy/autophagy. The mitochondrial-associated ER membrane (MAM) plays a key role in mitochondrial division and autophagy, and the mitochondrial fusion protein MFN2 (mitofusin 2) tethers the MAM, but the mechanism by which AMPK and MFN2 regulate autophagy in response to energy stress remains unclear. Here, we found that energy stress not only triggers mitochondrial fission and autophagy, but more importantly increases the number of MAMs, a process that requires AMPK. Interestingly, under energy stress, considerable amounts of AMPK translocate from cytosol to the MAM and the mitochondrion as mitochondrial fission occurs. Unexpectedly, AMPK interacts directly with MFN2. The autophagic ability of mouse embryonic fibroblasts (MEFs) lacking MFN2 (mfn2^−/-) is significantly attenuated in response to energy stress as compared to wild-type MEFs (WT MEFs), while re-expression of MFN2 in mfn2^−/- cells rescues the autophagy defects of these cells. The abundance of MAMs is also greatly reduced in MFN2-deficient cells. Functional experiments show that the oxygen consumption rate and the glycolytic function of cells lacking MFN2 but not MFN1 are obviously attenuated, and MFN2 is important for cell survival under energy stress. In conclusion, our study establishes the molecular link between the energy sensor AMPK and the MAM tether MFN2, and reveals the important role of AMPK and MFN2 in energy stress-induced autophagy and MAM dynamics.

Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment. In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1^−/− mouse aortic tissue. This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention.

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PP_i), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1^−/−) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1^−/− mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1^−/− mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1^−/− mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1^−/− tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1^−/− null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1^−/− mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1^−/− mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1^−/− mice compared to controls. These results indicate that Enpp1^−/− mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization.

Vascular calcification has severe clinical consequences and is considered an accurate predictor of future adverse cardiovascular events, including myocardial infarction and stroke. Previously vascular calcification was thought to be a passive process which involved the deposition of calcium and phosphate in arteries and cardiac valves. However, recent studies have shown that vascular calcification is a highly regulated, cell-mediated process similar to bone formation. In this article, we outline the current understanding of key mechanisms governing vascular calcification and highlight the clinical consequences. By understanding better the molecular pathways and genetic circuitry responsible for the pathological mineralization process novel drug targets may be identified and exploited to combat and reduce the detrimental effects of vascular calcification on human health.

ENPP1 is well known for its role in regulating skeletal and soft tissue mineralization. It primarily exerts its function through the generation of pyrophosphate, a key inhibitor of hydroxyapatite formation. Several previous studies have suggested that ENPP1 also contributes to a range of human diseases including diabetes, cancer, cardiovascular disease, and osteoarthritis. In this review, we summarize the pathological roles of ENPP1 in mineralization and these soft tissue disorders. We also discuss the underlying mechanisms through which ENPP1 exerts its pathological effects. A fuller understanding of the pathways through which ENPP1 acts may help to develop novel therapeutic strategies for these commonly diagnosed morbidities.

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein ( BMP ‐9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP ‐9 in vascular smooth muscle cell ( VSMC ) calcification. Vessel calcification in chronic kidney disease ( CKD ) begins pre‐dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP ‐9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP ‐9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase ( ALP ) activity and m RNA expression of osteogenic markers. BMP ‐9‐induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5‐Dimethoxy‐ N ‐(quinolin‐3‐yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK 1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP ‐9 is a physiological ALK 1 ligand. Signal transduction studies revealed that BMP ‐9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, si RNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4‐si RNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP ‐9 induces VSMC osteogenic differentiation and calcification via ALK 1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.