Anxa2 attenuates osteoblast growth and is associated with hip BMD and osteoporotic fracture in Chinese elderly.

Low bone mineral density (BMD) is a risk factor of osteoporotic fracture (OF). Peripheral blood monocytes (PBM) can differentiate into osteoclasts to resorb bone. It was known that PBM-expressed Anxa2 protein is associated with BMD, and extracellular Anxa2 protein promotes osteoclastogenesis. This study aimed to test 1) whether Anxa2 protein level in PBM differs significantly between subjects with OF and without fracture history (NF); 2) whether Anxa2 level in plasma is associated with BMD; 3) how Anxa2 protein at various concentrations would affect osteoblastic activity in vitro. All the study subjects were Chinese Han elderly. Firstly, Anxa2 protein in PBM was identified and quantitated by LC-MS/MS and compared between 45 OF cases and 42 healthy controls. Secondly, plasma Anxa2 protein level was quantitated by ELISA and compared between unrelated subjects with extremely low vs. high hip BMD (0.63±0.10 vs. 1.05±0.10 g/cm2, n = 75). Furthermore, in vitro functional assay was utilized to test the effects of extracellular Anxa2 protein on osteoblastic growth. We found that Anxa2 protein expression in PBM was significantly up-regulated in OF vs. NF subjects (fold change [FC)] = 1.16, P<0.05). Plasma Anxa2 protein concentration (range: 31.69-227.35ng/ml) was significantly elevated in low vs. high BMD subjects (84.85 vs. 66.15ng/ml, FC = 1.28, P<0.05). Cellular dynamical monitoring demonstrated that the general shape of dose-response relationship is the inverse U-shaped curve. Specifically, lower dose of Anxa2 protein may promote osteoblast growth and the optimal concentration for osteoblastic growth was around 50ng/ml, but even higher concentration could attenuate hFOB1.19 osteoprogenitor cell growth. We concluded that Anxa2 protein could attenuate osteoblast growth and be associated with hip BMD and OF in Chinese elderly.

PKR and PKR-like endoplasmic reticulum kinase induce the proteasome-dependent degradation of cyclin D1 via a mechanism requiring eukaryotic initiation factor 2alpha phosphorylation.

Cyclin D1 plays a critical role in controlling the G(1)/S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases. We examined the effect of activation of the eIF2alpha kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2alpha phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3beta and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2alpha phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2alpha phosphorylation during short, but not prolonged, periods of stress.

Modulation of the eukaryotic initiation factor 2 alpha-subunit kinase PERK by tyrosine phosphorylation.

The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2alpha at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2alpha. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2alpha in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress.

Chlorolissoclimides: new inhibitors of eukaryotic protein synthesis.

Lissoclimides are cytotoxic compounds produced by shell-less molluscs through chemical secretions to deter predators. Chlorinated lissoclimides were identified as the active component of a marine extract from Pleurobranchus forskalii found during a high-throughput screening campaign to characterize new protein synthesis inhibitors. It was demonstrated that these compounds inhibit protein synthesis in vitro, in extracts prepared from mammalian and plant cells, as well as in vivo against mammalian cells. Our results suggest that they block translation elongation by inhibiting translocation, leading to an accumulation of ribosomes on mRNA. These data provide a rationale for the cytotoxic nature of this class of small molecule natural products.