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Osteocytes act as mechanosensors in bone; however, the underlying mechanism remains poorly understood. Here we report that deleting Kindlin-2 in osteocytes causes severe osteopenia and mechanical property defects in weight-bearing long bones, but not in non-weight-bearing calvariae. Kindlin-2 loss in osteocytes impairs skeletal responses to mechanical stimulation in long bones. Control and cKO mice display similar bone loss induced by unloading. However, unlike control mice, cKO mice fail to restore lost bone after reloading. Osteocyte Kindlin-2 deletion impairs focal adhesion (FA) formation, cytoskeleton organization and cell orientation in vitro and in bone. Fluid shear stress dose-dependently increases Kindlin-2 expression and decreases that of Sclerostin by downregulating Smad2/3 in osteocytes; this latter response is abolished by Kindlin-2 ablation. Kindlin-2-deficient osteocytes express abundant Sclerostin, contributing to bone loss in cKO mice. Collectively, we demonstrate an indispensable novel role of Kindlin-2 in maintaining skeletal responses to mechanical stimulation by inhibiting Sclerostin expression during osteocyte mechanotransduction., Qin et al. show the involvement of Kindlin-2 in osteocyte mechanotransduction. They show Kindlin-2 mediates skeletal responses to mechanical stimulation through Smad 2/3 mediated inhibition of Sclerostin.

Anaplastic lymphoma kinase (ALK) has been described in a range of human cancers and is involved in cancer initiation and progression via activating multiple signaling pathways, such as the PI3K-AKT, CRKL-C3G, MEKK2/3-MEK5-ERK5, JAK-STAT and MAPK signal pathways. Recently ALK and LTK ligand 1 (ALKAL1) also named “augmentor-β” or “FAM150A” is identified as a potent activating ligands for human ALK that bind to the extracellular domain of ALK. However, due to its poor stability, the mechanisms of ALKAL1 underlying the tumor progression in the human cancers including colorectal cancer have not been well documented. Herein, ALKAL1 expression was evaluated by RNA sequencing datasets from The Cancer Genome Atlas (TCGA) of 625 cases colorectal cancer, immunohistochemical analysis of 377 cases colorectal cancer tissues, and Western blotting even Real-time PCR of 10 pairs of colorectal cancer tissues and adjacent normal tissues, as well as 8 colorectal cancer cell lines. Statistical analysis was performed to explore the correlation between ALKAL1 expression and clinicopathological features in colorectal cancer. Univariate and multivariate Cox regression analysis were performed to examine the association between ALKAL1 expression and overall survival. In vitro and in vivo assays were performed to assess the biological roles of ALKAL1 in colorectal cancer. Gene set enrichment analysis (GSEA), Western blotting and luciferase assays were used to identify the underlying signal pathway involved in the tumor progression role of ALKAL1. As a result, we showed that ALKAL1 was upregulated in colorectal cancer tissues and cell lines. Upregulation of ALKAL1 correlated with tumor malignancy and poor prognosis in colorectal cancer. ALKAL1 silencing inhibited tumorigenesis, metastasis and invasion of colorectal cancer cells, and inhibited SHH signaling pathway, which is essential for ALKAL1 induced migration. Our findings reveal a new mechanism by which ALKAL1 participates in colorectal cancer migration and invasion via activating the SHH signaling pathway.

In vertebrates, the type 1 parathyroid hormone receptor (PTH1R) is a critical regulator of skeletal development and homeostasis; however, how it is modulated is incompletely understood. Here we report that deleting Kindlin-2 in osteoblastic cells using the mouse 10-kb Dmp1-Cre largely neutralizes the intermittent PTH-stimulated increasing of bone volume fraction and bone mineral density by impairing both osteoblast and osteoclast formation in murine adult bone. Single-cell profiling reveals that Kindlin-2 loss increases the proportion of osteoblasts, but not mesenchymal stem cells, chondrocytes and fibroblasts, in non-hematopoietic bone marrow cells, with concomitant depletion of osteoblasts on the bone surfaces, especially those stimulated by PTH. Furthermore, haploinsufficiency of Kindlin-2 and Pth1r genes, but not that of either gene, in mice significantly decreases basal and, to a larger extent, PTH-stimulated bone mass, supporting the notion that both factors function in the same genetic pathway. Mechanistically, Kindlin-2 interacts with the C-terminal cytoplasmic domain of PTH1R via aa 474–475 and Gsα. Kindlin-2 loss suppresses PTH induction of cAMP production and CREB phosphorylation in cultured osteoblasts and in bone. Interestingly, PTH promotes Kindlin-2 expression in vitro and in vivo, thus creating a positive feedback regulatory loop. Finally, estrogen deficiency induced by ovariectomy drastically decreases expression of Kindlin-2 protein in osteocytes embedded in the bone matrix and Kindlin-2 loss essentially abolishes the PTH anabolic activity in bone in ovariectomized mice. Thus, we demonstrate that Kindlin-2 functions as an intrinsic component of the PTH1R signaling pathway in osteoblastic cells to regulate bone mass accrual and homeostasis.

Osteoarthritis (OA) is an aging-related degenerative joint disease, which has no cure partly due to limited understanding of its pathological mechanism(s). Here we report that the focal adhesion protein Kindlin-2, but not Kindlin-1 or −3, is highly expressed in articular chondrocytes of the hyaline cartilage, which is dramatically decreased in the degenerated articular cartilage of aged mice and patients with OA. Inducible deletion of Kindlin-2 in chondrocytes at adult stage leads to spontaneous OA and much severe OA lesions in the mice receiving the surgery of destabilization of the medial meniscus. Mechanistically, Kindlin-2 deficiency promotes mitochondrial oxidative stress and activates Stat3 in articular chondrocytes, leading to Runx2-mediated chondrocyte hypertrophic differentiation and catabolism. In vivo, systemic pharmacological blockade of Stat3 activation or genetic ablation of Stat3 in chondrocytes reverses aberrant accumulation of Runx2 and ECM-degrading enzymes and limits OA deteriorations caused by Kindlin-2 deficiency. Furthermore, genetic inactivation of Runx2 in chondrocytes reverses structural changes and OA lesions caused by Kindlin-2 deletion without down-regulating p-Stat3 in articular chondrocytes. Of translational significance, intraarticular injection of Kindlin-2-expressing adeno-associated virus decelerates progression of aging- and instability-induced knee joint OA in mice. Collectively, we identify a novel pathway comprising of Kindlin-2, Stat3 and Runx2 in articular chondrocytes responsible for maintaining integrity of the articular cartilage and define a potential therapeutic target for OA.

The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion. Here, we report that deleting Pinch1 in limb mesenchymal stem cells (MSCs) and Pinch2 globally (double knockout; dKO) in mice causes severe chondrodysplasia, while single mutant mice do not display marked defects. Pinch deletion decreases chondrocyte proliferation, accelerates cell differentiation and disrupts column formation. Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone (PZ) chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone (HZ) chondrocytes. Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes, reduces bone formation, and increases bone resorption, leading to low bone mass. In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes. Through its C-terminal region, Pinch1 interacts with Smad2/3 proteins. Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells (BMSCs). Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs. Interestingly, compared to those from single mutant mice, BMSCs from dKO mice express dramatically lower protein levels of β-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity. Finally, ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening. Collectively, our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.

Biological macromolecules are the basis of life activities. There is a separation of spatial dimension between DNA replication and RNA biogenesis, and protein synthesis, which is an interesting phenomenon. The former occurs in the cell nucleus, while the latter in the cytoplasm. The separation requires protein to transport across the nuclear envelope to realize a variety of biological functions. Nucleocytoplasmic transport of protein including import to the nucleus and export to the cytoplasm is a complicated process that requires involvement and interaction of many proteins. In recent years, many studies have found that proteins constantly shuttle between the cytoplasm and the nucleus. These shuttling proteins play a crucial role as transport carriers and signal transduction regulators within cells. In this review, we describe the mechanism of nucleocytoplasmic transport of shuttling proteins and summarize some important diseases related shuttling proteins.

Citrate is essential to biomineralization of the bone especially as an integral part of apatite nanocomposite. Citrate precipitate of apatite is hypothesized to be derived from mesenchymal stem/stromal cells (MSCs) upon differentiation into mature osteoblasts. Based on 13C‐labeled signals identified by solid‐state multinuclear magnetic resonance analysis, boosted mitochondrial activity and carbon‐source replenishment of tricarboxylic acid cycle intermediates coordinate to feed forward mitochondrial anabolism and deposition of citrate. Moreover, zinc (Zn2+) is identified playing dual functions: (i) Zn2+ influx is influenced by ZIP1 which is regulated by Runx2 and Osterix to form a zinc‐Runx2/Osterix‐ZIP1 regulation axis promoting osteogenic differentiation; (ii) Zn2+ enhances citrate accumulation and deposition in bone apatite. Furthermore, age‐related bone loss is associated with Zn2+ and citrate homeostasis; whereas, restoration of Zn2+ uptake alleviates age‐associated declining osteogenic capacity and amount of citrate deposition. Together, these results indicate that citrate is not only a key metabolic intermediate meeting the emerging energy demand of differentiating MSCs but also participates in extracellular matrix mineralization, providing mechanistic insight into Zn2+ homeostasis and bone formation.

Poly (ADP-ribose) polymerase 1 (PARP1), the best-studied isoform of the nuclear enzyme PARP family, plays a pivotal role in cellular biological processes, such as DNA repair, gene transcription, and so on. PARP1 has been found to be overexpressed in various carcinomas. These all indicate the clinical potential of PARP1 as a therapeutic target of human malignancies. Additionally, multiple preclinical research studies and clinical trials demonstrate that inhibition of PARP1 can repress tumor growth and metastasis. Up until now, PARP1 inhibitors are clinically used not only for monotherapy to suppress various tumors, but also for adjuvant therapy, to maintain or enhance therapeutic effects of mature antineoplastic drugs, as well as protect patients from chemotherapy and surgery-induced injury. To supply a framework for understanding recent research progress of PARP1 in carcinomas, we review the structure, expression, functions, and mechanisms of PARP1, and summarize the clinically mature PARP1-related anticancer agents, to provide some ideas for the development of other promising PARP1 inhibitors in antineoplastic therapy.

A series of biodegradable Mg-Cu alloys is designed to induce osteogenesis, stimulate angiogenesis, and provide long-lasting antibacterial performance at the same time. The Mg-Cu alloys with precipitated Mg2Cu intermetallic phases exhibit accelerated degradation in the physiological environment due to galvanic corrosion and the alkaline environment combined with Cu release endows the Mg-Cu alloys with prolonged antibacterial effects. In addition to no cytotoxicity towards HUVECs and MC3T3-E1 cells, the Mg-Cu alloys, particularly Mg-0.03Cu, enhance the cell viability, alkaline phosphatase activity, matrix mineralization, collagen secretion, osteogenesis-related gene and protein expressions of MC3T3-E1 cells, cell proliferation, migration, endothelial tubule forming, angiogenesis-related gene, and protein expressions of HUVECs compared to pure Mg. The favorable osteogenesis and angiogenesis are believed to arise from the release of bioactive Mg and Cu ions into the biological environment and the biodegradable Mg-Cu alloys with osteogenesis, angiogenesis, and long-term antibacterial ability are very promising in orthopedic applications.

Aging deteriorates osteogenic capacity of mesenchymal stem/stromal cells (MSCs), contributing to imbalanced bone remodeling and osteoporosis. Glutaminase (Gls) catabolizes glutamine into glutamate at the first step of mitochondrial glutamine (Gln)-dependent anaplerosis which is essential for MSCs upon osteogenic differentiation. Estrogen-related receptor α (ERRα) regulates genes required for mitochondrial function. Here, we found that ERRα and Gls are upregulated by osteogenic induction in human MSCs (hMSCs). In contrast, osteogenic differentiation capacity and glutamine consumption of MSCs, as well as ERRα, Gls and osteogenic marker genes are significantly reduced with age. We demonstrated that ERRα binds to response elements on Gls promoter and affects glutamine anaplerosis through transcriptional induction of Gls. Conversely, mTOR inhibitor rapamycin, ERRα inverse agonist compound 29 or Gls inhibitor BPTES leads to reduced Gln anaplerosis and deteriorated osteogenic differentiation of hMSCs. Importantly, overexpression of ERRα or Gls restored impairment by these inhibitors. Finally, we proved that compensated ERRα or Gls expression indeed potentiated Gln anaplerosis and osteogenic capability of elderly mice MSCs in vitro. Together, we establish that Gls is a novel ERRα target gene and ERRα/Gls signaling pathway plays an important role in osteogenic differentiation of MSCs, providing new sights into novel regenerative therapeutics development. Our findings suggest that restoring age-related mitochondrial Gln-dependent anaplerosis may be beneficial for degenerative bone disorders such as osteoporosis.

As a novel mediator of hepatic very low-density lipoproteins (VLDL) secretion, phospholipase A2 G12B (PLA2G12B) is transcriptionally regulated by hepatocyte nuclear factor-4 alpha (HNF-4α). Farnesoid X receptor (FXR) plays a critical role in maintaining bile acids and triglycerides (TG) homeostasis. Here we report that FXR regulates serum TG level in part through PLA2G12B. Activation of FXR by chenodeoxycholic acid (CDCA) or GW4064 significantly decreased PLA2G12B expression in HepG2 cells. PLA2G12B expression was transcriptionally repressed due to an FXR-mediated up-regulation of small heterodimer partner (SHP) which functionally suppresses HNF-4α activity. We found that hepatic PLA2G12B expression was suppressed and serum TG level reduced in high fat diet mice treated with CDCA. Concurrently, CDCA treatment lowered hepatic VLDL-TG secretion. Our data demonstrate that activation of FXR promotes TG lowering, not only by decreasing de novo lipogenesis but also reducing hepatic secretion of TG-rich VLDL particles in part through suppressing PLA2G12B expression.