βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathw... more βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of βcatenin in the cytoplasm has been well studied but βcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of βcatenin destruction complex element GSK3β (glycogen synthase kinase 3β) to increase nuclear βcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3β due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, βcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of βcatenin, resulting in decreased nuclear βcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in βcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of βcatenin.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2015
Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and... more Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2008
Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the mole... more Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the molecular events involved in mechanical signal transduction are not well understood. Wnt/ -catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce -catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on -catenin signaling. We found that mechanical strain caused a rapid, transient accumulation of active -catenin in the cytoplasm and its translocation to the nucleus. This was followed by up-regulation of the Wnt/-catenin target genes Wisp1 and Cox2, with peak responses at 4 and 1 h of strain, respectively. The increase of -catenin was temporally related to the activation of Akt and subsequent inactivation of GSK3, and caveolin-1 was not required for these molecular events. Application of Dkk-1, which disrupts canonical Wnt/LRP5 signaling, did not block strain-induced nuclear translocation of -catenin or upregulation of Wisp1 and Cox2 expression. Conditions that increased basal -catenin levels, such as lithium chloride treatment or repression of caveolin-1 expression, were shown to enhance the effects of strain. In summary, mechanical strain activates Akt and inactivates GSK3 to allow -catenin translocation, and Wnt signaling through LRP5 is not required for these strain-mediated responses. Thus, -catenin serves as both a modulator and effector of mechanical signals in bone cells.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2015
A cell's ability to recognize and adapt to the physical environment is central to its survival an... more A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSC), high magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of FAK/mTORC2/Akt signaling generated at focal adhesions [1]. Physiologic systems also rely on a persistent barrage of low level signals to regulate behavior [2]. Exposing MSC to extremely low magnitude mechanical signals (LMS) suppresses adipocyte formation [3] despite the virtual absence of substrate strain (<0.001%) [2], suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating focal adhesion kinase (FAK) and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the focal adhesions, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting LINC (Linker of Nucleoskeleton and Cytoskeleton) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by high magnitude strain (HMS) was unaffected by LINC decoupling, consistent with signal initiation at the focal adhesion
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2011
Mechanical signals can inactivate glycogen synthase kinase 3 (GSK3), resulting in stabilization... more Mechanical signals can inactivate glycogen synthase kinase 3 (GSK3), resulting in stabilization of -catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3 in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3 at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3 at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but straininduced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3 inactivation was prevented, whereas insulin inhibition of GSK3 was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3. Thus, mechanical regulation of GSK3 downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2011
Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, ... more Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, which may include regulation of transcription factors that support osteoblast differentiation and function. Flow effects on osteoblast transcription factors have generally been evaluated after short exposures. In this work, we assayed flow effects on osteogenic genes at early and late time points in a preosteoblast (CIMC-4) cell line and evaluated both steady and oscillatory flows. Four hours of steady unidirectional flow decreased the level of RANKL mRNA 53 ± 7% below that of nonflowed cells, but increases in Runx2 and osterix mRNA (44 ± 22% and 129 ± 12%, respectively) were significant only after 12-19 h of continuous flow. Late flow effects on RANKL and osterix were also induced by an intermittent flow-rest protocol (four cycles of 1 h on/1 h off + overnight rest). Four hours of oscillatory flow decreased RANKL mRNA at this early time point (63 ± 2%) but did not alter either osterix or Runx2. When oscillatory flow was delivered using the intermittent flow-rest protocol, Runx2 and osterix mRNA increased significantly (85 ± 19% and
The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cell... more The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cells (MSCs), where adipogenic commitment depletes the available pool for osteogenesis. Cell architecture influences lineage decisions, where interfering with cytoskeletal structure promotes adipogenesis. Mechanical strain suppresses MSC adipogenesis partially through RhoA driven enhancement of cytoskeletal structure. To understand the basis of force-driven RhoA activation, we considered critical GEFs (activators) and GAPs (inactivators) on bone marrow MSC lineage fate. Knockdown of LARG accelerated adipogenesis and repressed basal RhoA activity. Importantly, mechanical activation of RhoA was almost entirely inhibited following LARG depletion, and the ability of strain to inhibit adipogenesis was impaired. Knockdown of ARHGAP18 increased basal RhoA activity and actin stress fiber formation, but did not enhance mechanical strain activation of RhoA. ARHGAP18 null MSCs exhibited suppressed adipogenesis assessed by Oil-Red-O staining and Western blot of adipogenic markers. Furthermore, ARHGAP18 knockdown enhanced osteogenic commitment, confirmed by alkaline phosphatase staining and qPCR of Sp7, Alpl, and Bglap genes. This suggests that ARHGAP18 conveys tonic inhibition of MSC cytoskeletal assembly, returning RhoA to an "off state" and affecting cell lineage in the static state. In contrast, LARG is recruited during dynamic mechanical strain, and is necessary for mechanical suppression of adipogenesis. In summary, mechanical activation of RhoA in mesenchymal progenitors is dependent on LARG, while ARHGAP18 limits RhoA delineated cytoskeletal structure in static cultures. Thus, on and off GTP exchangers work through RhoA to influence MSC fate and responses to static and dynamic physical factors in the microenvironment.
Mesenchymal stem cells (MSCs) respond to environmental forces with both cytoskeletal re-structuri... more Mesenchymal stem cells (MSCs) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, β-catenin, and yes-associated protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles × 2%) and static (1 × 2% hold) strain on nuclear translocation of β-catenin and YAP1 at 3 hours after force application. Dynamic strain induced nuclear accumulation of β-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced β-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid did not increase nuclear content of β-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds β-catenin, we considered whether β-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both β-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of β-catenin via mechanisms used for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, β-catenin, and YAP1. Open in a separate windowThe symmetry breaking of autophagic activity in CD133-positive human neuroblastoma cells. During cytokinesis, the recycling endosomal CD133 localizes to the pericentrosomal region asymmetrically to suppress autophagy. The asymmetric distribution of pericentrosomal CD133 endosomes then promotes the localization of β-catenin to the nucleus. β-catenin directly repressed p62/SQSTM1 expression. This suggests that pericentrosomal CD133 and β-catenin cooperatively suppress autophagy. In addition, nuclear β-catenin may inhibit pericentrosomal CD133 degradation to repress p62 gene expression.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2017
The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We use... more The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be attenuated via exercise and whether this correlates with gains in bone quantity and quality. C57BL/6 mice were divided into diet-induced obesity (DIO, n = 14) versus low-fat diet (LFD, n = 14). After 3 months, 16-week-old mice were allocated to an exercise intervention (LFD-E, DIO-E) or a control group (LFD, DIO) for 6 weeks (4 groups, n = 7/group). Marrow adipocyte area was 44% higher with obesity (p<0.0001) and after exercise 33% lower in LFD (p<0.0001) and 39% lower in DIO (p<0.0001). In LFD, exercise did not affect adipocyte number; however, in DIO, the adipocyte number was 56% lower (p<0.0001). MAT was 44% higher in DIO measured by osmium-µCT, whereas exercise associated with reduced MAT (-23% in LFD,-48% in DIO, p<0.05). MAT was additionally quantified by 9.4TMRI, and correlated with osmium-µCT (r = 0.645; p<0.01). Consistent with higher lipid beta oxidation, perilipin 3 (PLIN3) rose with exercise in tibial mRNA (+92% in LFD, +60% in DIO, p<0.05). Tibial µCT-derived trabecular bone volume (BV/TV) was not influenced by DIO but responded to exercise with an increase of 19% (p<0.
Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle ... more Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle lipid is not associated with insulin resistance, a phenomenon known as the athlete's paradox. To understand if exercise altered the phenotype of muscle lipid, female C57BL/6 mice fed CTL or high-fat diet (HFD for 6 or 18 weeks) were further divided into sedentary or exercising groups (CTL-E or HFD-E) with voluntary access to running wheels for the last 6 weeks of experiments, running 6 h/night. Diet did not affect running time or distance. HFD mice weighed more than CTL after 18 weeks ( p < 0.01). Quadriceps muscle TG was increased in running animals and in sedentary mice fed HFD for 18 weeks ( p < 0.05). In exercised animals, markers of fat, Plin1, aP2, FSP27, and Fasn, were increased significantly in HFD groups. Ucp1 and Pgc1a, markers for brown fat, increased with exercise in the setting of high fat feeding. Fndc5, which encodes irisin, and CytC were sensitive to exercise regardless of diet. Plin5 was increased with HFD and unaffected by exercise; the respiratory exchange ratio was 15% lower in the 18-week HFD group compared with CTL ( p < 0.001) and 10% lower in 18 weeks HFD-E compared with CTL-E ( p < 0.001). Increased Ucp1 and Pgc1a in exercised muscle of running mice suggests that a beige/brown fat phenotype develops, which differs from the fat phenotype that induces insulin resistance in high fat feeding. This suggests that increased muscle lipid may develop a "brown" phenotype in the setting of endurance exercise training, a shift that is further promoted by HFD.
Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a ... more Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a condition characterized by paucity of white adipose tissue, with eventual diabetes, and steatosis. We applied a mouse model of global deficiency of Bscl2 (SEIPIN), required for lipid droplet formation. Male twelve-week-old B6 knockouts (KO) and wild type (WT) littermates were assigned six-weeks of voluntary, running exercise (E) versus non-exercise (N=5-8). KO weighed 14% less than WT (p=0.01) and exhibited an absence of epididymal adipose tissue; KO liver Plin1 via qPCR was 9-fold that of WT (p=0.04), consistent with steatosis. Bone marrow adipose tissue (BMAT), unlike white adipose, was measurable, although 40.5% lower in KO vs WT (p=0.0003) via 9.4T MRI/advanced image analysis. SEIPIN ablation’s most notable effect marrow adiposity was in the proximal femoral diaphysis (-56% KO vs WT, p=0.005), with relative preservation in KO-distal-femur. Bone via μCT was preserved in SEIPIN KO, tho...
ABSTRACTUse of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce ... more ABSTRACTUse of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β‐cateninfl/fl in 28‐day‐old male and female Prrx1‐CreER/β‐cateninfl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β‐catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild‐type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose‐dependent manner analyzed by micro–computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) an...
Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In ... more Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentia...
ABSTRACTMarrow adipose tissue (MAT) and its relevance to skeletal health during caloric restricti... more ABSTRACTMarrow adipose tissue (MAT) and its relevance to skeletal health during caloric restriction (CR) is unknown: It remains unclear whether exercise, which is anabolic to bone in a calorie‐replete state, alters bone or MAT in CR. We hypothesized that response of bone and MAT to exercise in CR differs from the calorie‐replete state. Ten‐week‐old female B6 mice fed a regular diet (RD) or 30% CR diet were allocated to sedentary (RD, CR, n = 10/group) or running exercise (RD‐E, CR‐E, n = 7/group). After 6 weeks, CR mice weighed 20% less than RD, p < 0.001; exercise did not affect weight. Femoral bone volume (BV) via 3D MRI was 20% lower in CR versus RD (p < 0.0001). CR was associated with decreased bone by μCT: Tb.Th was 16% less in CR versus RD, p < 0.003, Ct.Th was 5% less, p < 0.07. In CR‐E, Tb.Th was 40% less than RD‐E, p < 0.0001. Exercise increased Tb.Th in RD (+23% RD‐E versus RD, p < 0.003) but failed to do so in CR. Cortical porosity increased after exerci...
The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cell... more The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cells (MSCs), where adipogenic commitment depletes the available pool for osteogenesis. Cell architecture influences lineage decisions, where interfering with cytoskeletal structure promotes adipogenesis. Mechanical strain suppresses MSC adipogenesis partially through RhoA driven enhancement of cytoskeletal structure. To understand the basis of force-driven RhoA activation, we considered critical GEFs (activators) and GAPs (inactivators) on bone marrow MSC lineage fate. Knockdown of LARG accelerated adipogenesis and repressed basal RhoA activity. Importantly, mechanical activation of RhoA was almost entirely inhibited following LARG depletion, and the ability of strain to inhibit adipogenesis was impaired. Knockdown of ARHGAP18 increased basal RhoA activity and actin stress fiber formation, but did not enhance mechanical strain activation of RhoA. ARHGAP18 null MSCs exhibited suppressed adip...
Actin structure contributes to physiologic events within the nucleus to control mesenchymal strom... more Actin structure contributes to physiologic events within the nucleus to control mesenchymal stromal cell (MSC) differentiation. Continuous cytochalasin D (Cyto D) disruption of the MSC actin cytoskeleton leads to osteogenic or adipogenic differentiation, both requiring mass transfer of actin into the nucleus. Cyto D remains extranuclear, thus intranuclear actin polymerization is potentiated by actin transfer: we asked whether actin structure affects differentiation. We show that secondary actin filament branching via the Arp2/3 complex is required for osteogenesis and that preventing actin branching stimulates adipogenesis, as shown by expression profiling of osteogenic and adipogenic biomarkers and unbiased RNA-seq analysis. Mechanistically, Cyto D activates osteoblast master regulators (e.g., Runx2, Sp7, Dlx5) and novel coregulated genes (e.g., Atoh8, Nr4a3, Slfn5). Formin-induced primary actin filament formation is critical for Arp2/3 complex recruitment: osteogenesis is prevente...
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Jan 24, 2017
The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We use... more The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be attenuated via exercise and whether this correlates with gains in bone quantity and quality. C57BL/6 mice were divided into diet-induced obesity (DIO, n = 14) versus low-fat diet (LFD, n = 14). After 3 months, 16-week-old mice were allocated to an exercise intervention (LFD-E, DIO-E) or a control group (LFD, DIO) for 6 weeks (4 groups, n = 7/group). Marrow adipocyte area was 44% higher with obesity (p < 0.0001) and after exercise 33% lower in LFD (p < 0.0001) and 39% lower in DIO (p < 0.0001). In LFD, exercise did not affect adipocyte number; however, in DIO, the adipocyte number was 56% lower (p < 0.0001). MAT was 44% higher in DIO measured by osmium-μCT, whereas exercise associated with reduced MAT (-23% in LFD, -48% in DIO, p < 0.05). MAT was additionally quantified by 9.4TMRI, and correlated with osmium-µC...
Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle ... more Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle lipid is not associated with insulin resistance, a phenomenon known as the athlete's paradox. To understand if exercise altered the phenotype of muscle lipid, female C57BL/6 mice fed CTL or high-fat diet (HFD for 6 or 18 weeks) were further divided into sedentary or exercising groups (CTL-E or HFD-E) with voluntary access to running wheels for the last 6 weeks of experiments, running 6 h/night. Diet did not affect running time or distance. HFD mice weighed more than CTL after 18 weeks (p < 0.01). Quadriceps muscle TG was increased in running animals and in sedentary mice fed HFD for 18 weeks (p < 0.05). In exercised animals, markers of fat, Plin1, aP2, FSP27, and Fasn, were increased significantly in HFD groups. Ucp1 and Pgc1a, markers for brown fat, increased with exercise in the setting of high fat feeding. Fndc5, which encodes irisin, and CytC were sensitive to exercise rega...
Osteocytes modulate bone density through both osteoblast and osteoclast effectors, and " rea... more Osteocytes modulate bone density through both osteoblast and osteoclast effectors, and " reach out " to interact with cells in the marrow compartment. The stellate morphology and spatial organization of osteocytes are optimal for sensing force and transmitting biochemical signals through their interconnected lacuno-canalicular system. In addition to the paracrine effects of sclerostin and RANKL on bone effector cells, osteocytes secrete endocrine signals including DMP1 and FGF23, which alter matrix mineralization and renal phosphate homeostasis. To understand how osteocytes respond to hormonal and physical signals there is a need for cell models that faithfully replicate the in vivo osteocyte phenotype. Current osteocyte cell lines present limitations that inaccurately represent in vivo conditions. We have developed a novel osteocyte model expressing the defining markers of these cells while displaying a stellate morphology within a mineralized matrix. Primary polyclonal m...
βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathw... more βcatenin acts as a primary intracellular signal transducer for mechanical and Wnt signaling pathways to control cell function and fate. Regulation of βcatenin in the cytoplasm has been well studied but βcatenin nuclear trafficking and function remains unclear. In a previous study we showed that, in mesenchymal stem cells (MSC), mechanical blockade of adipogenesis relied on inhibition of βcatenin destruction complex element GSK3β (glycogen synthase kinase 3β) to increase nuclear βcatenin as well as the function of Linker of Cytoskeleton and Nucleoskeleton (LINC) complexes, suggesting that these two mechanisms may be linked. Here we show that shortly after inactivation of GSK3β due to either low intensity vibration (LIV), substrate strain or pharmacologic inhibition, βcatenin associates with the nucleoskeleton, defined as the insoluble nuclear fraction that provides structure to the integrated nuclear envelope, nuclear lamina and chromatin. Co-depleting LINC elements Sun-1 and Sun-2 interfered with both nucleoskeletal association and nuclear entry of βcatenin, resulting in decreased nuclear βcatenin levels. Our findings reveal that the insoluble structural nucleoskeleton actively participates in βcatenin dynamics. As the cytoskeleton transmits applied mechanical force to the nuclear surface to influence the nucleoskeleton and its LINC mediated interaction, our results suggest a pathway by which LINC mediated connectivity may play a role in signaling pathways that depend on nuclear access of βcatenin.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2015
Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and... more Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2008
Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the mole... more Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the molecular events involved in mechanical signal transduction are not well understood. Wnt/ -catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce -catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on -catenin signaling. We found that mechanical strain caused a rapid, transient accumulation of active -catenin in the cytoplasm and its translocation to the nucleus. This was followed by up-regulation of the Wnt/-catenin target genes Wisp1 and Cox2, with peak responses at 4 and 1 h of strain, respectively. The increase of -catenin was temporally related to the activation of Akt and subsequent inactivation of GSK3, and caveolin-1 was not required for these molecular events. Application of Dkk-1, which disrupts canonical Wnt/LRP5 signaling, did not block strain-induced nuclear translocation of -catenin or upregulation of Wisp1 and Cox2 expression. Conditions that increased basal -catenin levels, such as lithium chloride treatment or repression of caveolin-1 expression, were shown to enhance the effects of strain. In summary, mechanical strain activates Akt and inactivates GSK3 to allow -catenin translocation, and Wnt signaling through LRP5 is not required for these strain-mediated responses. Thus, -catenin serves as both a modulator and effector of mechanical signals in bone cells.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2015
A cell's ability to recognize and adapt to the physical environment is central to its survival an... more A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSC), high magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of FAK/mTORC2/Akt signaling generated at focal adhesions [1]. Physiologic systems also rely on a persistent barrage of low level signals to regulate behavior [2]. Exposing MSC to extremely low magnitude mechanical signals (LMS) suppresses adipocyte formation [3] despite the virtual absence of substrate strain (<0.001%) [2], suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating focal adhesion kinase (FAK) and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the focal adhesions, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting LINC (Linker of Nucleoskeleton and Cytoskeleton) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by high magnitude strain (HMS) was unaffected by LINC decoupling, consistent with signal initiation at the focal adhesion
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2011
Mechanical signals can inactivate glycogen synthase kinase 3 (GSK3), resulting in stabilization... more Mechanical signals can inactivate glycogen synthase kinase 3 (GSK3), resulting in stabilization of -catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3 in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3 at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3 at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but straininduced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3 inactivation was prevented, whereas insulin inhibition of GSK3 was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3. Thus, mechanical regulation of GSK3 downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2011
Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, ... more Mechanical loading induces positive changes in the skeleton due to direct effects on bone cells, which may include regulation of transcription factors that support osteoblast differentiation and function. Flow effects on osteoblast transcription factors have generally been evaluated after short exposures. In this work, we assayed flow effects on osteogenic genes at early and late time points in a preosteoblast (CIMC-4) cell line and evaluated both steady and oscillatory flows. Four hours of steady unidirectional flow decreased the level of RANKL mRNA 53 ± 7% below that of nonflowed cells, but increases in Runx2 and osterix mRNA (44 ± 22% and 129 ± 12%, respectively) were significant only after 12-19 h of continuous flow. Late flow effects on RANKL and osterix were also induced by an intermittent flow-rest protocol (four cycles of 1 h on/1 h off + overnight rest). Four hours of oscillatory flow decreased RANKL mRNA at this early time point (63 ± 2%) but did not alter either osterix or Runx2. When oscillatory flow was delivered using the intermittent flow-rest protocol, Runx2 and osterix mRNA increased significantly (85 ± 19% and
The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cell... more The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cells (MSCs), where adipogenic commitment depletes the available pool for osteogenesis. Cell architecture influences lineage decisions, where interfering with cytoskeletal structure promotes adipogenesis. Mechanical strain suppresses MSC adipogenesis partially through RhoA driven enhancement of cytoskeletal structure. To understand the basis of force-driven RhoA activation, we considered critical GEFs (activators) and GAPs (inactivators) on bone marrow MSC lineage fate. Knockdown of LARG accelerated adipogenesis and repressed basal RhoA activity. Importantly, mechanical activation of RhoA was almost entirely inhibited following LARG depletion, and the ability of strain to inhibit adipogenesis was impaired. Knockdown of ARHGAP18 increased basal RhoA activity and actin stress fiber formation, but did not enhance mechanical strain activation of RhoA. ARHGAP18 null MSCs exhibited suppressed adipogenesis assessed by Oil-Red-O staining and Western blot of adipogenic markers. Furthermore, ARHGAP18 knockdown enhanced osteogenic commitment, confirmed by alkaline phosphatase staining and qPCR of Sp7, Alpl, and Bglap genes. This suggests that ARHGAP18 conveys tonic inhibition of MSC cytoskeletal assembly, returning RhoA to an "off state" and affecting cell lineage in the static state. In contrast, LARG is recruited during dynamic mechanical strain, and is necessary for mechanical suppression of adipogenesis. In summary, mechanical activation of RhoA in mesenchymal progenitors is dependent on LARG, while ARHGAP18 limits RhoA delineated cytoskeletal structure in static cultures. Thus, on and off GTP exchangers work through RhoA to influence MSC fate and responses to static and dynamic physical factors in the microenvironment.
Mesenchymal stem cells (MSCs) respond to environmental forces with both cytoskeletal re-structuri... more Mesenchymal stem cells (MSCs) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, β-catenin, and yes-associated protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles × 2%) and static (1 × 2% hold) strain on nuclear translocation of β-catenin and YAP1 at 3 hours after force application. Dynamic strain induced nuclear accumulation of β-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced β-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid did not increase nuclear content of β-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds β-catenin, we considered whether β-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both β-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of β-catenin via mechanisms used for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, β-catenin, and YAP1. Open in a separate windowThe symmetry breaking of autophagic activity in CD133-positive human neuroblastoma cells. During cytokinesis, the recycling endosomal CD133 localizes to the pericentrosomal region asymmetrically to suppress autophagy. The asymmetric distribution of pericentrosomal CD133 endosomes then promotes the localization of β-catenin to the nucleus. β-catenin directly repressed p62/SQSTM1 expression. This suggests that pericentrosomal CD133 and β-catenin cooperatively suppress autophagy. In addition, nuclear β-catenin may inhibit pericentrosomal CD133 degradation to repress p62 gene expression.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2017
The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We use... more The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be attenuated via exercise and whether this correlates with gains in bone quantity and quality. C57BL/6 mice were divided into diet-induced obesity (DIO, n = 14) versus low-fat diet (LFD, n = 14). After 3 months, 16-week-old mice were allocated to an exercise intervention (LFD-E, DIO-E) or a control group (LFD, DIO) for 6 weeks (4 groups, n = 7/group). Marrow adipocyte area was 44% higher with obesity (p<0.0001) and after exercise 33% lower in LFD (p<0.0001) and 39% lower in DIO (p<0.0001). In LFD, exercise did not affect adipocyte number; however, in DIO, the adipocyte number was 56% lower (p<0.0001). MAT was 44% higher in DIO measured by osmium-µCT, whereas exercise associated with reduced MAT (-23% in LFD,-48% in DIO, p<0.05). MAT was additionally quantified by 9.4TMRI, and correlated with osmium-µCT (r = 0.645; p<0.01). Consistent with higher lipid beta oxidation, perilipin 3 (PLIN3) rose with exercise in tibial mRNA (+92% in LFD, +60% in DIO, p<0.05). Tibial µCT-derived trabecular bone volume (BV/TV) was not influenced by DIO but responded to exercise with an increase of 19% (p<0.
Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle ... more Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle lipid is not associated with insulin resistance, a phenomenon known as the athlete's paradox. To understand if exercise altered the phenotype of muscle lipid, female C57BL/6 mice fed CTL or high-fat diet (HFD for 6 or 18 weeks) were further divided into sedentary or exercising groups (CTL-E or HFD-E) with voluntary access to running wheels for the last 6 weeks of experiments, running 6 h/night. Diet did not affect running time or distance. HFD mice weighed more than CTL after 18 weeks ( p < 0.01). Quadriceps muscle TG was increased in running animals and in sedentary mice fed HFD for 18 weeks ( p < 0.05). In exercised animals, markers of fat, Plin1, aP2, FSP27, and Fasn, were increased significantly in HFD groups. Ucp1 and Pgc1a, markers for brown fat, increased with exercise in the setting of high fat feeding. Fndc5, which encodes irisin, and CytC were sensitive to exercise regardless of diet. Plin5 was increased with HFD and unaffected by exercise; the respiratory exchange ratio was 15% lower in the 18-week HFD group compared with CTL ( p < 0.001) and 10% lower in 18 weeks HFD-E compared with CTL-E ( p < 0.001). Increased Ucp1 and Pgc1a in exercised muscle of running mice suggests that a beige/brown fat phenotype develops, which differs from the fat phenotype that induces insulin resistance in high fat feeding. This suggests that increased muscle lipid may develop a "brown" phenotype in the setting of endurance exercise training, a shift that is further promoted by HFD.
Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a ... more Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a condition characterized by paucity of white adipose tissue, with eventual diabetes, and steatosis. We applied a mouse model of global deficiency of Bscl2 (SEIPIN), required for lipid droplet formation. Male twelve-week-old B6 knockouts (KO) and wild type (WT) littermates were assigned six-weeks of voluntary, running exercise (E) versus non-exercise (N=5-8). KO weighed 14% less than WT (p=0.01) and exhibited an absence of epididymal adipose tissue; KO liver Plin1 via qPCR was 9-fold that of WT (p=0.04), consistent with steatosis. Bone marrow adipose tissue (BMAT), unlike white adipose, was measurable, although 40.5% lower in KO vs WT (p=0.0003) via 9.4T MRI/advanced image analysis. SEIPIN ablation’s most notable effect marrow adiposity was in the proximal femoral diaphysis (-56% KO vs WT, p=0.005), with relative preservation in KO-distal-femur. Bone via μCT was preserved in SEIPIN KO, tho...
ABSTRACTUse of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce ... more ABSTRACTUse of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β‐cateninfl/fl in 28‐day‐old male and female Prrx1‐CreER/β‐cateninfl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β‐catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild‐type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose‐dependent manner analyzed by micro–computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) an...
Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In ... more Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentia...
ABSTRACTMarrow adipose tissue (MAT) and its relevance to skeletal health during caloric restricti... more ABSTRACTMarrow adipose tissue (MAT) and its relevance to skeletal health during caloric restriction (CR) is unknown: It remains unclear whether exercise, which is anabolic to bone in a calorie‐replete state, alters bone or MAT in CR. We hypothesized that response of bone and MAT to exercise in CR differs from the calorie‐replete state. Ten‐week‐old female B6 mice fed a regular diet (RD) or 30% CR diet were allocated to sedentary (RD, CR, n = 10/group) or running exercise (RD‐E, CR‐E, n = 7/group). After 6 weeks, CR mice weighed 20% less than RD, p < 0.001; exercise did not affect weight. Femoral bone volume (BV) via 3D MRI was 20% lower in CR versus RD (p < 0.0001). CR was associated with decreased bone by μCT: Tb.Th was 16% less in CR versus RD, p < 0.003, Ct.Th was 5% less, p < 0.07. In CR‐E, Tb.Th was 40% less than RD‐E, p < 0.0001. Exercise increased Tb.Th in RD (+23% RD‐E versus RD, p < 0.003) but failed to do so in CR. Cortical porosity increased after exerci...
The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cell... more The quantity and quality of bone depends on osteoblastic differentiation of mesenchymal stem cells (MSCs), where adipogenic commitment depletes the available pool for osteogenesis. Cell architecture influences lineage decisions, where interfering with cytoskeletal structure promotes adipogenesis. Mechanical strain suppresses MSC adipogenesis partially through RhoA driven enhancement of cytoskeletal structure. To understand the basis of force-driven RhoA activation, we considered critical GEFs (activators) and GAPs (inactivators) on bone marrow MSC lineage fate. Knockdown of LARG accelerated adipogenesis and repressed basal RhoA activity. Importantly, mechanical activation of RhoA was almost entirely inhibited following LARG depletion, and the ability of strain to inhibit adipogenesis was impaired. Knockdown of ARHGAP18 increased basal RhoA activity and actin stress fiber formation, but did not enhance mechanical strain activation of RhoA. ARHGAP18 null MSCs exhibited suppressed adip...
Actin structure contributes to physiologic events within the nucleus to control mesenchymal strom... more Actin structure contributes to physiologic events within the nucleus to control mesenchymal stromal cell (MSC) differentiation. Continuous cytochalasin D (Cyto D) disruption of the MSC actin cytoskeleton leads to osteogenic or adipogenic differentiation, both requiring mass transfer of actin into the nucleus. Cyto D remains extranuclear, thus intranuclear actin polymerization is potentiated by actin transfer: we asked whether actin structure affects differentiation. We show that secondary actin filament branching via the Arp2/3 complex is required for osteogenesis and that preventing actin branching stimulates adipogenesis, as shown by expression profiling of osteogenic and adipogenic biomarkers and unbiased RNA-seq analysis. Mechanistically, Cyto D activates osteoblast master regulators (e.g., Runx2, Sp7, Dlx5) and novel coregulated genes (e.g., Atoh8, Nr4a3, Slfn5). Formin-induced primary actin filament formation is critical for Arp2/3 complex recruitment: osteogenesis is prevente...
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Jan 24, 2017
The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We use... more The relationship between marrow adipose tissue (MAT) and bone health is poorly understood. We used running exercise to ask whether obesity-associated MAT can be attenuated via exercise and whether this correlates with gains in bone quantity and quality. C57BL/6 mice were divided into diet-induced obesity (DIO, n = 14) versus low-fat diet (LFD, n = 14). After 3 months, 16-week-old mice were allocated to an exercise intervention (LFD-E, DIO-E) or a control group (LFD, DIO) for 6 weeks (4 groups, n = 7/group). Marrow adipocyte area was 44% higher with obesity (p < 0.0001) and after exercise 33% lower in LFD (p < 0.0001) and 39% lower in DIO (p < 0.0001). In LFD, exercise did not affect adipocyte number; however, in DIO, the adipocyte number was 56% lower (p < 0.0001). MAT was 44% higher in DIO measured by osmium-μCT, whereas exercise associated with reduced MAT (-23% in LFD, -48% in DIO, p < 0.05). MAT was additionally quantified by 9.4TMRI, and correlated with osmium-µC...
Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle ... more Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle lipid is not associated with insulin resistance, a phenomenon known as the athlete's paradox. To understand if exercise altered the phenotype of muscle lipid, female C57BL/6 mice fed CTL or high-fat diet (HFD for 6 or 18 weeks) were further divided into sedentary or exercising groups (CTL-E or HFD-E) with voluntary access to running wheels for the last 6 weeks of experiments, running 6 h/night. Diet did not affect running time or distance. HFD mice weighed more than CTL after 18 weeks (p < 0.01). Quadriceps muscle TG was increased in running animals and in sedentary mice fed HFD for 18 weeks (p < 0.05). In exercised animals, markers of fat, Plin1, aP2, FSP27, and Fasn, were increased significantly in HFD groups. Ucp1 and Pgc1a, markers for brown fat, increased with exercise in the setting of high fat feeding. Fndc5, which encodes irisin, and CytC were sensitive to exercise rega...
Osteocytes modulate bone density through both osteoblast and osteoclast effectors, and " rea... more Osteocytes modulate bone density through both osteoblast and osteoclast effectors, and " reach out " to interact with cells in the marrow compartment. The stellate morphology and spatial organization of osteocytes are optimal for sensing force and transmitting biochemical signals through their interconnected lacuno-canalicular system. In addition to the paracrine effects of sclerostin and RANKL on bone effector cells, osteocytes secrete endocrine signals including DMP1 and FGF23, which alter matrix mineralization and renal phosphate homeostasis. To understand how osteocytes respond to hormonal and physical signals there is a need for cell models that faithfully replicate the in vivo osteocyte phenotype. Current osteocyte cell lines present limitations that inaccurately represent in vivo conditions. We have developed a novel osteocyte model expressing the defining markers of these cells while displaying a stellate morphology within a mineralized matrix. Primary polyclonal m...
Uploads
Papers by Zhihui Xie