Browsing by Author "Pavalko, Fredrick M."
Now showing 1 - 10 of 14
Results Per Page
Sort Options
Item Direct visualization by FRET-FLIM of a putative mechanosome complex involving Src, Pyk2 and MBD2 in living MLO-Y4 cells(PLOS, 2021-12-23) Day, Richard N.; Day, Kathleen H.; Pavalko, Fredrick M.; Anatomy, Cell Biology and Physiology, School of MedicineEarlier, we proposed the "mechanosome" concept as a testable model for understanding how mechanical stimuli detected by cell surface adhesion molecules are transmitted to modulate gene expression inside cells. Here, for the first time we document a putative mechanosome involving Src, Pyk2 and MBD2 in MLO-Y4 osteocytes with high spatial resolution using FRET-FLIM. Src-Pyk2 complexes were concentrated at the periphery of focal adhesions and the peri-nuclear region. Pyk2-MBD2 complexes were located primarily in the nucleus and peri-nuclear region. Lifetime measurements indicated that Src and MBD2 did not interact directly. Finally, mechanical stimulation by fluid flow induced apparent accumulation of Src-Pyk2 protein complexes in the peri-nuclear/nuclear region, consistent with the proposed behavior of a mechanosome in response to a mechanical stimulus.Item DISTINCT LOCALIZATION OF NADPH OXIDASE FLAVOCYTOCHROME B IN RESTING AND INTERFERON GAMMA ACTIVATED MACROPHAGES(2009-06-22T19:52:25Z) Casbon, Amy Jo; Dinauer, Mary C; Kaplan, Mark H.; Bauer, Margaret E.; Pavalko, Fredrick M.Flavocytochrome b558, the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91phox (NOX2) and a smaller subunit, p22phox. Localization of flavocytochrome b to the phagosome is essential for microbial killing, yet the subcellular distribution of flavocytochrome b in macrophages and how it is incorporated into macrophage phagosomes is not well characterized. In neutrophils, flavocytochrome b localizes primarily to specific granules that are rapidly mobilized to the phagosome upon stimulation. In contrast to neutrophils, macrophages do not contain specific granules, and trafficking of membrane proteins to the phagosome is more dynamic, involving fission and fusion events with endosomal compartments. We hypothesized that in macrophages, flavocytochrome b localizes to both plasma membrane and endosomal compartments that deliver flavocytochrome b to the phagosome. We generated fluorescently tagged versions of both p22phox and gp91phox, and rigorously verified their functionality in Chinese Hamster Ovary cells. Localization of flavocytochrome b was then examined in both RAW 264.7 murine macrophages and primary murine bone marrow derived macrophages (BMDM) in the presence and absence of interferon gamma (IFNg). We found that in “resting” macrophages, flavocytochrome b localizes primarily to the Rab11-positive endosome recycling compartment that recycles to the plasma membrane. In addition, phagocytosis assays showed flavocytochrome b is incorporated into the phagocytic cup and colocalized with Rab11 at the base of the cup, suggesting Rab11-positive endosomes may be involved in trafficking of flavocytochrome b between intracellular membranes and forming or nascent phagosomes. However, in IFNg activated macrophages, flavocytochrome b was localized predominantly in the plasma membrane, with little present in endosomal compartments. This shift in flavocytochrome b distribution occurred following sustained exposure to IFNg and correlated with increased flavocytochrome b protein expression and increased extracellular production of superoxide. Taken together, our results suggest the IFNg-induced redistribution of flavocytochrome b may be important for enhancing the production of superoxide at the cell surface and may be a potential new mechanism by which IFNg enhances antimicrobial activity in macrophages.Item The Effects of Chromium on Skeletal Muscle Membrane/Cytoskeletal Parameters and Insulin Sensitivity(2012-07-03) Hoffman, Nolan John; Elmendorf, Jeffrey S.; Considine, Robert V.; Morral, Nuria; Pavalko, Fredrick M.A recent review of randomized controlled trials found that trivalent chromium (Cr3+) supplementation significantly improved glycemia among patients with diabetes, consistent with a long-standing appreciation that this micronutrient optimizes carbohydrate metabolism. Nevertheless, a clear limitation in the current evidence is a lack of understanding of Cr3+ action. We tested if increased AMP-activated protein kinase (AMPK) activity, previously observed in Cr3+-treated cells or tissues from Cr3+-supplemented animals, mediates improved glucose transport regulation under insulin-resistant hyperinsulinemic conditions. In L6 myotubes stably expressing the glucose transporter GLUT4 carrying an exofacial myc-epitope tag, acute insulin stimulation increased GLUT4myc translocation by 69% and glucose uptake by 97%. In contrast, the hyperinsulinemic state impaired insulin stimulation of these processes. Consistent with Cr3+’s beneficial effect on glycemic status, chromium picolinate (CrPic) restored insulin’s ability to fully regulate GLUT4myc translocation and glucose transport. Insulin-resistant myotubes did not display impaired insulin signaling, nor did CrPic amplify insulin signaling. However, CrPic normalized elevated membrane cholesterol that impaired cortical filamentous actin (F-actin) structure. Mechanistically, data support that CrPic lowered membrane cholesterol via AMPK. Consistent with this data, siRNA-mediated AMPK silencing blocked CrPic’s beneficial effects on GLUT4 and glucose transport regulation. Furthermore, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) protected against hyperinsulinemia-induced membrane/cytoskeletal defects and GLUT4 dysregulation. To next test Cr3+ action in vivo, we utilized obesity-prone C57Bl/6J mice fed a low fat (LF) or high fat (HF) diet for eight weeks without or with CrPic supplementation administered in the drinking water (8 µg/kg/day). HF feeding increased body weight beginning four weeks after diet intervention regardless of CrPic supplementation and was independent of changes in food consumption. Early CrPic supplementation during a five week acclimation period protected against glucose intolerance induced by the subsequent eight weeks of HF feeding. As observed in other insulin-resistant animal models, skeletal muscle from HF-fed mice displayed membrane cholesterol accrual and loss of F-actin. Skeletal muscle from CrPic-supplemented HF-fed mice showed increased AMPK activity and protection against membrane cholesterol accrual and F-actin loss. Together these data suggest a mechanism by which Cr3+ may positively impact glycemic status, thereby stressing a plausible beneficial action of Cr3+ in glucose homeostasis.Item Estrogen receptor-β regulates mechanical signaling in primary osteoblasts(American Physiological Society (APS), 2014-04-15) Castillo, Alesha B.; Triplett, Jason W.; Pavalko, Fredrick M.; Turner, Charles H.; Department of Cellular & Integrative Physiology, IU School of MedicineMechanical loading is an important regulator in skeletal growth, maintenance, and aging. Estrogen receptors have a regulatory role in mechanically induced bone adaptation. Estrogen receptor-α (ERα) is known to enhance load-induced bone formation, whereas ERβ negatively regulates this process. We hypothesized that ERβ regulates mechanical signaling in osteoblasts. We tested this hypothesis by subjecting primary calvarial cells isolated from wild-type and ERβ-knockout mice (BERKO) to oscillatory fluid flow in the absence or presence of estradiol (E2). We found that the known responses to fluid shear stress, i.e., phosphorylation of the mitogen-activated protein kinase ERK and upregulation of COX-2 expression, were inhibited in BERKO cells in the absence of E2. Flow-induced increase in prostaglandin E2 (PGE2) release was not altered in BERKO cells in the absence of E2, but was increased when E2 was present. Additionally, immunofluorescence analysis and estrogen response element luciferase assays revealed increased ERα expression and flow- and ligand-induced nuclear translocation as well as transcriptional activity in BERKO cells in both the presence and absence of E2. Taken together, these data suggest that ERβ plays both ligand-dependent and ligand-independent roles in mechanical signaling in osteoblasts. Furthermore, our data suggest that one mechanism by which ERβ regulates mechanotransduction in osteoblasts may result from its inhibitory effect on ERα expression and function. Targeting estrogen receptors (e.g., inhibiting ERβ) may represent an effective approach for prevention and treatment of age-related bone loss.Item Expression of a Degradation‐Resistant β‐Catenin Mutant in Osteocytes Protects the Skeleton From Mechanodeprivation‐Induced Bone Wasting(Wiley, 2019) Bullock, Whitney A.; Hoggatt, April; Horan, Daniel J.; Lewis, Karl; Yokota, Hiroki; Hann, Steven; Warman, Matthew L.; Sebastian, Aimy; Loots, Gabriela G.; Pavalko, Fredrick M.; Robling, Alexander G.; Anatomy and Cell Biology, IU School of MedicineMechanical stimulation is a key regulator of bone mass, maintenance, and turnover. Wnt signaling is a key regulator of mechanotransduction in bone, but the role of β‐catenin—an intracellular signaling node in the canonical Wnt pathway—in disuse mechanotransduction is not defined. Using the β‐catenin exon 3 flox (constitutively active [CA]) mouse model, in conjunction with a tamoxifen‐inducible, osteocyte‐selective Cre driver, we evaluated the effects of degradation‐resistant β‐catenin on bone properties during disuse. We hypothesized that if β‐catenin plays an important role in Wnt‐mediated osteoprotection, then artificial stabilization of β‐catenin in osteocytes would protect the limbs from disuse‐induced bone wasting. Two disuse models were tested: tail suspension, which models fluid shift, and botulinum‐toxin (botox)‐induced muscle paralysis, which models loss of muscle force. Tail suspension was associated with a significant loss of tibial bone mass and density, reduced architectural properties, and decreased bone formation indices in uninduced (control) mice, as assessed by dual‐energy X‐ray absorptiometry (DXA), micro‐computed tomography (µCT), and histomorphometry. Activation of the βcatCA allele in tail‐suspended mice resulted in little to no change in those properties; ie, these mice were protected from bone loss. Similar protective effects were observed among botox‐treated mice when the βcatCA was activated. RNAseq analysis of altered gene regulation in tail‐suspended mice yielded 35 genes, including Wnt11, Gli1, Nell1, Gdf5, and Pgf, which were significantly differentially regulated between tail‐suspended β‐catenin stabilized mice and tail‐suspended nonstabilized mice. Our findings indicate that selectively targeting/blocking of β‐catenin degradation in bone cells could have therapeutic implications in mechanically induced bone disease.Item Genome-Wide Mapping and Interrogation of the Nmp4 Antianabolic Bone Axis(Oxford University Press, 2015-09) Childress, Paul; Stayrook, Keith R.; Alvarez, Marta B.; Wang, Zhiping; Shao, Yu; Hernandez-Buquer, Selene; Mack, Justin K.; Grese, Zachary R.; He, Yongzheng; Horan, Daniel; Pavalko, Fredrick M.; Warden, Stuart J.; Robling, Alexander G.; Yang, Feng-Chun; Allen, Matthew R.; Krishnan, Venkatesh; Liu, Yunlong; Bidwell, Joseph P.; Department of Anatomy & Cell Biology, IU School of MedicinePTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4(-/-) mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8(+) T cells. To determine whether the Nmp4(-/-) phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4(-/-) mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4(-/-) bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4(-/-) mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8(+) T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4(-/-) MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4(-/-) MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.Item Glucocorticoid induced osteoporosis and mechanisms of intervention(2017-03) Sato, Amy Yoshiko; Bellido, Teresita; Plotkin, Lilian I.; Pavalko, Fredrick M.; Robling, Alexander G.Glucocorticoid excess is a leading cause of osteoporosis. The loss of bone mass and strength corresponds to the increase in fractures exhibited after three months of glucocorticoid therapy. Glucocorticoids induce the bone cellular responses of deceased bone formation, increased osteoblast/osteocyte apoptosis, and transient increased bone resorption, which result in rapid bone loss and degradation of bone microarchitecture. The current standard of care for osteoporosis is bisphosphonate treatment; however, these agents further suppress bone formation and increase osteonecrosis and low energy atypical fracture risks. Thus, there is an unmet need for interventions that protect from glucocorticoid therapy. The purpose of these studies was to investigate novel mechanisms that potentially interfere with glucocorticoid-induced bone loss. We chose to explore pathways that regulate endoplasmic reticulum stress, the canonical Wnt pathway, and Pyk2 activity. Pharmacologic reduction of endoplasmic reticulum stress through salubrinal administration protected against glucocorticoid-induced bone loss by preservation of bone formation and osteoblast/osteocyte viability. In contrast, inhibition of Wnt antagonist Sost/sclerostin and inhibition of Pyk2 signaling did not prevent glucocorticoid-induced reductions in bone formation; however, both Sost/sclerostin and Pyk2 deficiency protected against bone loss through inhibition of increases in resorption. Overall, these studies demonstrate the significant contributions of reductions in bone formation, increased osteoblast/osteocyte apoptosis, and elevations in resorption to the rapid 6-12% bone loss exhibited during the first year of glucocorticoid therapy. However, glucocorticoid excess also induces skeletal muscle weakness, which is not reversed by bisphosphonate treatment or the interventions reported here of salubrinal, Sost/sclerostin inhibition, or Pyk2 deficiency. Further, the novel finding of increased E3 ubiquitin ligase atrophy signaling induce by glucocorticoids in both bone and muscle, by tissue-specific upstream mechanisms, provides opportunities for therapeutic combination strategies. Thus, future studies are warranted to investigate the role of E3 ubiquitin ligase signaling in the deleterious glucocorticoid effects of bone and muscle.Item Lrp4 Mediates Bone Homeostasis and Mechanotransduction through Interaction with Sclerostin In Vivo(Elsevier, 2019-10-25) Bullock, Whitney A.; Hoggatt, April M.; Horan, Daniel J.; Elmendorf, Andrew J.; Sato, Amy Y.; Bellido, Teresita; Loots, Gabriela G.; Pavalko, Fredrick M.; Robling, Alexander G.; Anatomy and Cell Biology, School of MedicineWnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse.Item Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy(MDPI, 2012-11-07) Hum, Julia M.; Siegel, Amanda P.; Pavalko, Fredrick M.; Day, Richard N.; Cellular and Integrative Physiology, School of MedicineLive-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.Item Nmp4 restricts bone marrow osteoprogenitors and parathyroid hormone induced bone formation in healthy and estrogen depleted female mice(2014-12) Childress, Paul Jeffrey; Bidwell, Joseph P.; Pavalko, Fredrick M.; Bellido, Teresita; Robling, Alexander G.; Kacena, Melissa A.We have shown that nuclear matrix protein 4 (Nmp4) attenuates the response to intermittent parathyroid hormone (PTH) in healthy and ovariectomized (OVX) female mice using a global knockout of the Nmp4 gene. Additionally, these mice have increased bone marrow osteoprogenitors and CD8+ T-cells which support osteoblast differentiation. The animals were not protected from bone loss following OVX, but retained the hypersensitivity seen in the intact mice. Mesenchymal stem/progenitor cells (osteoprogenitors) demonstrated increased growth rate in culture and showed more robust differentiation into mineralizing bone cells. Chromosome precipitation followed by next generation sequencing and bioinformatics analysis characterized Nmp4 as a negative regulator of synthetic processes and suggested the IGF1/Akt and BMP2/Smad biochemical pathways which are likely targets for Nmp4 regulation. We have experimentally verified these pathways in immortalized bone marrow mesenchymal cells from wild type and Nmp4-KO mice. Disabling Nmp4 in estrogen replete or depleted mice confers an enhanced bone formation from intermittent parathyroid hormone.