Highlights d Global phospho and ubiquitylome analysis of PINK1-Parkin pathway in iNeurons d Dynamics and specificity of Parkin-mediated ubiquitylation revealed in iNeurons d p97-mediated MFN turnover not required for Parkin substrate ''gating'' in iNeurons d USP30 acts primarily on translocon and supports import quality control in iNeurons

Dopaminergic signaling plays a critical role in the nervous system, but little is known about its potential role in breast cancer and bone metabolism. A screening of ~1,000 biologically active compounds revealed that a selective agonist of dopamine receptor D1 (DRD1), A77636, inhibited proliferation of 4T1.2 mammary tumor cells as well as MDA-MB-231 breast cancer cells. Herein, we examined the effect of A77636 on bone quality using a mouse model of bone metastasis from mammary tumor. A77636 inhibited migration of cancer cells in a DRD1-dependent fashion and suppressed development of bone-resorbing osteoclasts by downregulating NFATc1 through the elevation of phosphorylation of eIF2α. In the mouse model of bone metastasis, A77636 reduced osteolytic lesions and prevented mechanical weakening of the femur and tibia. Collectively, we expect that dopaminergic signaling might provide a novel therapeutic target for breast cancer and bone metastasis.

Bone loss in postmenopausal osteoporosis is induced chiefly by an imbalance of bone-forming osteoblasts and boneresorbing osteoclasts. Salubrinal is a synthetic compound that inhibits de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Phosphorylation of eIF2α alleviates endoplasmic reticulum (ER) stress, which may activate autophagy. We hypothesized that eIF2α signaling regulates bone homeostasis by promoting autophagy in osteoblasts and inhibiting osteoclast development. To test the hypothesis, we employed salubrinal to elevate the phosphorylation of eIF2α in an ovariectomized (OVX) mouse model and cell cultures. In the OVX model, salubrinal prevented abnormal expansion of rough ER and decreased the number of acidic vesiculars. It regulated ER stressassociated signaling molecules such as Bip, p-eIF2α, ATF4 and CHOP, and promoted autophagy of osteoblasts via regulation of eIF2α, Atg7, LC3, and p62. Salubrinal markedly alleviated OVX-induced symptoms such as reduction of bone mineral density and bone volume fraction. In primary bone-marrow-derived cells, salubrinal increased the differentiation of osteoblasts, and decreased the formation of osteoclasts by inhibiting nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Live cell imaging and RNA interference demonstrated that suppression of osteoclastogenesis is in part mediated by Rac1 GTPase. Collectively, this study demonstrates that ER stress-autophagy axis plays an important role in OVX mice. Bone-forming osteoblasts are restored by maintaining phosphorylation of eIF2α, and bone-resorbing osteoclasts are regulated by inhibiting NFATc1 and Rac1 GTPase.

Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts.