Molecular mechanosensors in osteocytes

Osteocytes, the most abundant and long-lived cells in bone, are the master regulators of bone remodeling. In addition to their functions in endocrine regulation and calcium and phosphate metabolism, osteocytes are the major responsive cells in force adaptation due to mechanical stimulation. Mechanically induced bone formation and adaptation, disuse-induced bone loss and skeletal fragility are mediated by osteocytes, which sense local mechanical cues and respond to these cues in both direct and indirect ways. The mechanotransduction process in osteocytes is a complex but exquisite regulatory process between cells and their environment, between neighboring cells, and between different functional mechanosensors in individual cells. Over the past two decades, great efforts have focused on finding various mechanosensors in osteocytes that transmit extracellular mechanical signals into osteocytes and regulate responsive gene expression. The osteocyte cytoskeleton, dendritic processes, Integrin-based focal adhesions, connexin-based intercellular junctions, primary cilium, ion channels, and extracellular matrix are the major mechanosensors in osteocytes reported so far with evidence from both in vitro and in vitro studies. This review aims to give a systematic introduction to osteocyte mechanobiology, provide details of osteocyte mechanosensors, and discuss the roles of osteocyte mechanosensitive signaling pathways in the regulation of bone homeostasis.

Roles of mechanosensitive channel Piezo 1/2 proteins in skeleton and other tissues

Mechanotransduction is a fundamental ability that allows living organisms to receive and respond to physical signals from both the external and internal environments.The mechanotransduction process requires a range of special proteins termed mechanotransducers to convert mechanical forces into biochemical signals in cells.The Piezo proteins are mechanically activated nonselective cation channels and the largest plasma membrane ion channels reported thus far.The regulation of two family members,Piezol and Piezo2#has been reported to have essential functions in mechanosensation and transduction in different organs and tissues.Recently,the predominant contributions of the Piezo family were reported to occur in the skeletal system,especially in bone development and mechano-stimulated bone homeostasis.Here we review current studies focused on the tissue-specific functions of Piezol and Piezo2 in various backgrounds with special highlights on their importance in regulating skeletal cell mechanotransduction.In this review,we emphasize the diverse functions of Piezol and Piezo2 and related signaling pathways in osteoblast lineage cells and chondrocytes.We also summarize our current understanding of Piezo channel structures and the key findings about PIEZO gene mutations in human diseases.

Kindlin-2 inhibits Nlrp3 inflammasome activation in nucleus pulposus to maintain homeostasis of the intervertebral disc

Intervertebral disc(IVD) degeneration(IVDD) is the main cause of low back pain with major social and economic burdens;however, its underlying molecular mechanisms remain poorly defined. Here we show that the focal adhesion protein Kindlin-2 is highly expressed in the nucleus pulposus(NP), but not in the anulus fibrosus and the cartilaginous endplates, in the IVD tissues. Expression of Kindlin-2 is drastically decreased in NP cells in aged mice and severe IVDD patients. Inducible deletion of Kindlin-2 in NP cells in adult mice causes spontaneous and striking IVDD-like phenotypes in lumbar IVDs and largely accelerates progression of coccygeal IVDD in the presence of abnormal mechanical stress. Kindlin-2 loss activates Nlrp3 inflammasome and stimulates expression of IL-1β in NP cells, which in turn downregulates Kindlin-2. This vicious cycle promotes extracellular matrix(ECM) catabolism and NP cell apoptosis. Furthermore, abnormal mechanical stress reduces expression of Kindlin-2, which exacerbates Nlrp3 inflammasome activation, cell apoptosis, and ECM catabolism in NP cells caused by Kindlin-2 deficiency. In vivo blocking Nlrp3 inflammasome activation prevents IVDD progression induced by Kindlin-2 loss and abnormal mechanical stress. Of translational significance, adeno-associated virus-mediated overexpression of Kindlin-2 inhibits ECM catabolism and cell apoptosis in primary human NP cells in vitro and alleviates coccygeal IVDD progression caused by mechanical stress in rat. Collectively, we establish critical roles of Kindlin-2 in inhibiting Nlrp3 inflammasome activation and maintaining integrity of the IVD homeostasis and define a novel target for the prevention and treatment of IVDD.

Focal adhesion protein Kindlin-2 regulates bone homeostasis in mice

Our recent studies demonstrate that the focal adhesion protein Kindlin-2 is critical for chondrogenesis and early skeletal development. Here, we show that deleting Kindlin-2 from osteoblasts using the 2.3-kb mouse Col1 a1-Cre transgene minimally impacts bone mass in mice, but deleting Kindlin-2 using the 10-kb mouse Dmp1-Cre transgene, which targets osteocytes and mature osteoblasts, results in striking osteopenia in mice. Kindlin-2 loss reduces the osteoblastic population but increases the osteoclastic and adipocytic populations in the bone microenvironment. Kindlin-2 loss upregulates sclerostin in osteocytes,downregulates β-catenin in osteoblasts, and inhibits osteoblast formation and differentiation in vitro and in vivo. Upregulation ofβ-catenin in the mutant cells reverses the osteopenia induced by Kindlin-2 deficiency. Kindlin-2 loss additionally increases the expression of RANKL in osteocytes and increases osteoclast formation and bone resorption. Kindlin-2 deletion in osteocytes promotes osteoclast formation in osteocyte/bone marrow monocyte cocultures, which is significantly blocked by an anti-RANKLneutralizing antibody. Finally, Kindlin-2 loss increases osteocyte apoptosis and impairs osteocyte spreading and dendrite formation.Thus, we demonstrate an important role of Kindlin-2 in the regulation of bone homeostasis and provide a potential target for the treatment of metabolic bone diseases.

LIM domain proteins Pinch1/2 regulate chondrogenesis and bone mass in mice

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 ofβ-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.

人胃癌裸鼠移植瘤中标记滞留细胞的初步鉴定(英文)

Objective:This study has investigated the existence of label-retaining cell and its distribution in gastric cancer,in the hope that this information will assist investigations on gastric cancer stem cells.Methods:The gastric carcinoma cell line BGC-823 was labeled with BrdU in vitro and then engrafted into the right axilla of nude mice,which developed tumors.Label-retaining cells were quantified by immunohistochemical methods.Results:BrdU positive cells constituted about 96%of the cells in xenograft tumors after 10 days.Subsequently,BrdU positive cells gradually decreased,at the 80th day,labelretaining cells steadily occupied about 0.5%.This set of population cell localized in the margin of cancer nests,which had no difference in cellular morpha.Conclusion:The study demonstrates the presence of label-retaining cells in human gastric cancer xenografts in nude mice and the label-retaining cells may be related with cancer stem cells,which are most likely the cause for spread,metastasis and recurrence.

A novel sprayable thermosensitive hydrogel coupled with zinc modified metformin promotes the healing of skin wound

A novel sprayable adhesive is established(ZnMet-PF127)by the combination of a thermosensitive hydrogel(Pluronic F127,PF127)and a coordination complex of zinc and metformin(ZnMet).Here we demonstrate that ZnMet-PF127 potently promotes the healing of traumatic skin defect and burn skin injury by promoting cell proliferation,angiogenesis,collagen formation.Furthermore,we find that ZnMet could inhibit reactive oxygen species(ROS)production through activation of autophagy,thereby protecting cell from oxidative stress induced damage and promoting healing of skin wound.ZnMet complex exerts better effects on promoting skin wound healing than ZnCl2 or metformin alone.ZnMet complex also displays excellent antibacterial activity against Staphylococcus aureus or Escherichia coli,which could reduce the incidence of skin wound infections.Collectively,we demonstrate that sprayable PF127 could be used as a new drug delivery system for treatment of skin injury.The advantages of this sprayable system are obvious:(1)It is convenient to use;(2)The hydrogel can cover irregular skin defect sites evenly in a liquid state.In combination with this system,we establish a novel sprayable adhesive(ZnMet-PF127)and demonstrate that it is a potential clinical treatment for traumatic skin defect and burn skin injury.

The role of E3 ubiquitin ligases in bone homeostasis and related diseases

The ubiquitin-proteasome system(UPS)dedicates to degrade intracellular proteins to modulate demic homeostasis and functions of organisms.These enzymatic cascades mark and modifies target proteins diversly through covalently binding ubiquitin molecules.In the UPS,E3 ubiquitin ligases are the crucial constituents by the advantage of recognizing and presenting proteins to proteasomes for proteolysis.As the major regulators of protein homeostasis,E3 ligases are indispensable to proper cell manners in diverse systems,and they are well described in physiological bone growth and bone metabolism.Pathologically,classic bone-related diseases such as metabolic bone diseases,arthritis,bone neoplasms and bone metastasis of the tumor,etc.,were also depicted in a UPS-dependent manner.Therefore,skeletal system is versatilely regulated by UPS and it is worthy to summarize the underlying mechanism.Furthermore,based on the current status of treatment,normal or pathological osteogenesis and tumorigenesis elaborated in this review highlight the clinical significance of UPS research.As a strategy possibly remedies the limitations of UPS treatment,emerging PROTAC was described comprehensively to illustrate its potential in clinical application.Altogether,the purpose of this review aims to provide more evidence for exploiting novel therapeutic strategies based on UPS for bone associated diseases.

Targeting Kindlin-2 in adipocytes increases bone mass through inhibiting FAS/PPARγ/FABP4 signaling in mice

Osteoporosis(OP)is a systemic skeletal disease that primarily affects the elderly population,which greatly increases the risk of fractures.Here we report that Kindlin-2 expression in adipose tissue increases during aging and high-fat diet fed and is accompanied by decreased bone mass.Kindlin-2 specific deletion(K2KO)controlled by Adipoq-Cre mice or adipose tissue-targeting AAV(AAV-Rec2-CasRx-sgK2)significantly increases bone mass.Mechanistically,Kindlin-2 promotes peroxisome proliferator-activated receptor gamma(PPARγ)activation and downstream fatty acid binding protein 4(FABP4)expression through stabilizing fatty acid synthase(FAS),and increased FABP4 inhibits insulin expression and decreases bone mass.Kindlin-2 inhibition results in accelerated FAS degradation,decreased PPARγactivation and FABP4 expression,and therefore increased insulin expression and bone mass.Interestingly,we find that FABP4 is increased while insulin is decreased in serum of OP patients.Increased FABP4 expression through PPARγactivation by rosiglitazone reverses the high bone mass phenotype of K2KO mice.Inhibition of FAS by C75 phenocopies the high bone mass phenotype of K2KO mice.Collectively,our study establishes a novel Kindlin-2/FAS/PPARγ/FABP4/insulin axis in adipose tissue modulating bone mass and strongly indicates that FAS and Kindlin-2 are new potential targets and C75 or AAV-Rec2-CasRx-sgK2 treatment are potential strategies for OP treatment.

Kindlin-2 regulates skeletal homeostasis by modulating PTH1R in mice

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.

Kindlin-2 regulates skeletal homeostasis by modulating PTH1R in mice

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.