The actin-bundling protein, PLS3, is part of the mechanoresponsive machinery that regulates osteoblast mineralization.

Plastin-3 (PLS3) is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis. Clinical data suggest that PLS3 mutations lead to a defect in osteoblast function, however the underlying mechanism remains elusive. To investigate the role of PLS3 in bone mineralization, we generated MC3T3-E1 preosteoblast cells that are stably depleted of PLS3. Analysis of osteogenic differentiation of control and PLS3 knockdown (PLS3 KD) cells showed that depletion of PLS3 does not alter the first stage of osteoblast mineralization in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. During this phase, osteoblasts heavily rely on mechanosensitive signaling pathways to sustain mineral deposition in response to increasing stiffness of the extracellular matrix (ECM). PLS3 prominently localizes to focal adhesions (FAs), which are intricately linked to mechanosensation. In line with this, we observed that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness and showed the same cell size, FA lengths and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutations that were identified in patients with early onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.

Dual role of endothelial Myct1 in tumor angiogenesis and tumor immunity.

The cross-talk between angiogenesis and immunity within the tumor microenvironment (TME) is critical for tumor prognosis. While pro-angiogenic and immunosuppressive TME promote tumor growth, anti-angiogenic and immune stimulatory TME inhibit tumor progression. Therefore, there is a great interest in achieving vascular normalization to improve drug delivery and enhance antitumor immunity. However, anti-vascular endothelial growth factor (VEGF) mechanisms to normalize tumor vessels have offered limited therapeutic efficacies for patients with cancer. Here, we report that Myct1, a direct target of ETV2, was nearly exclusively expressed in endothelial cells. In preclinical mouse tumor models, Myct1 deficiency reduced angiogenesis, enhanced high endothelial venule formation, and promoted antitumor immunity, leading to restricted tumor progression. Analysis of The Cancer Genome Atlas (TCGA) datasets revealed a significant (P < 0.05) correlation between MYCT1 expression, angiogenesis, and antitumor immunity in human cancers, as suggested by decreased FOXP3 expression and increased antitumor macrophages in patients with low MYCT1 expression. Mechanistically, MYCT1 interacted with tight junction protein Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, thereby promoting endothelial motility in the angiogenic environment. Myct1-deficient endothelial cells facilitated trans-endothelial migration of cytotoxic T lymphocytes and polarization of M1 macrophages. Myct1 targeting combined with anti-PD1 treatment significantly (P < 0.05) increased complete tumor regression and long-term survival in anti-PD1-responsive and -refractory tumor models in mice. Our data collectively support a critical role for Myct1 in controlling tumor angiogenesis and reprogramming tumor immunity. Myct1-targeted vascular control, in combination with immunotherapy, may become an exciting therapeutic strategy.

TorsinB--perinuclear location and association with torsinA.

The torsins comprise a four-member family of AAA+ chaperone proteins, including torsinA, torsinB, torp2A and torp3A in humans. Mutations in torsinA underlie early onset torsion dystonia, an autosomal dominant, neurologically based movement disorder. TorsinB is highly homologous to torsinA with its gene adjacent to that for torsinA on human chromosome 9q34. Antibodies have been generated which can distinguish torsinA and torsinB from each other, and from the torps in human and rodent cells. TorsinB (approximately MW 38 kDa), like torsinA ( approximately MW 37 kDa), is an N-glycosylated protein and both reside primarily in the endoplasmic reticulum (ER) and nuclear envelope in cultured cells. Immunoprecipitation studies in cultured cells and human brain tissue indicate that torsinA and torsinB are associated with each other in cells. Overexpression of both wild-type torsinB and mutant torsinA lead to enrichment of the protein in the nuclear envelope and formation of large cytoplasmic inclusions. We conclude that torsinB and torsinA are localized in overlapping cell compartments within the same protein complex, and thus may carry out related functions in vivo.

TorsinA in PC12 cells: localization in the endoplasmic reticulum and response to stress.

Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA(+)) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress.