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.

Kindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular joint in mice

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint(TMJ) osteoarthritis(OA);however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4(Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore,Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

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.

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.

Bone marrow adipoq+ cell population controls bone mass via sclerostin in mice

Dear editor,The comorbidity of obesity and osteoporosis illustrates the communication and coordination of adipose and bone tissues.Leptin and adiponectin derived from adipocytes regulate osteoblast formation and function to impact bone mass through direct and indirect mechanisms.1 It is known that bone marrow adipocytes(BMA)can control bone mass by modulating the bone morphogenetic protein(BMP)and other signaling pathways.BMAs can secret soluble factors,which impact osteoblasts,osteoclasts,and osteocytes.2 Sclerostin is a potent inhibitor of bone acquisition that antagonizes Wnt/β-catenin signaling.

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.