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Browsing by Author "Wang, Kun"
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Item Fibroblast Growth Factor 23 Does Not Directly Influence Skeletal Muscle Cell Proliferation and Differentiation or Ex Vivo Muscle Contractility(American Physiological Society, 2018-10-01) Avin, Keith G.; Vallejo, Julian A.; Chen, Neal X.; Wang, Kun; Touchberry, Chad D.; Brotto, Marco; Dallas, Sarah L.; Moe, Sharon M.; Wacker, Michael J.; Physical Therapy, School of Health and Rehabilitation SciencesSkeletal muscle dysfunction accompanies the clinical disorders of chronic kidney disease (CKD) and hereditary hypophosphatemic rickets. In both disorders, fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D metabolism, becomes chronically elevated. FGF23 has been shown to play a direct role in cardiac muscle dysfunction; however, it is unknown whether FGF23 signaling can also directly induce skeletal muscle dysfunction. We found expression of potential FGF23 receptors ( Fgfr1-4) and α-Klotho in muscles of two animal models (CD-1 and Cy/+ rat, a naturally occurring rat model of chronic kidney disease-mineral bone disorder) as well as C2C12 myoblasts and myotubes. C2C12 proliferation, myogenic gene expression, oxidative stress marker 8-OHdG, intracellular Ca2+ ([Ca2+]i), and ex vivo contractility of extensor digitorum longus (EDL) or soleus muscles were assessed after treatment with various amounts of FGF23. FGF23 (2-100 ng/ml) did not alter C2C12 proliferation, expression of myogenic genes, or oxidative stress after 24- to 72-h treatment. Acute or prolonged FGF23 treatment up to 6 days did not alter C2C12 [Ca2+]i handling, nor did acute treatment with FGF23 (9-100 ng/ml) affect EDL and soleus muscle contractility. In conclusion, although skeletal muscles express the receptors involved in FGF23-mediated signaling, in vitro FGF23 treatments failed to directly alter skeletal muscle development or function under the conditions tested. We hypothesize that other endogenous substances may be required to act in concert with FGF23 or apart from FGF23 to promote muscle dysfunction in hereditary hypophosphatemic rickets and CKD.Item Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs(Wiley, 2018-06) Lu, Yongbo; Kamel-El Sayed, Suzan A.; Wang, Kun; Tiede-Lewis, LeAnn M.; Grillo, Michael A.; Veno, Patricia A.; Dusevich, Vladimir; Phillips, Charlotte L.; Bonewald, Lynda F.; Dallas, Sarah L.; Anatomy and Cell Biology, IU School of MedicineType I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen.Item A Novel Osteogenic Cell Line that Differentiates into GFP‐Tagged Osteocytes and forms Mineral with a Bone‐like Lacunocanalicular Structure(Wiley, 2019) Wang, Kun; Le, Lisa; Chun, Brad M.; Tiede-Lewis, LeAnn M.; Shiflett, Lora A.; Prideaux, Matthew; Campos, Richard S.; Veno, Patricia A.; Xie, Yixia; Dusevich, Vladimir; Bonewald, Lynda F.; Dallas, Sarah L.; Anatomy and Cell Biology, IU School of MedicineOsteocytes, the most abundant cells in bone, were once thought to be inactive but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane‐targeted GFP driven by the Dmp1‐promoter. One of these clones, OmGFP66, has unique properties compared to previous osteogenic and osteocyte cell models and forms 3‐dimensional mineralized bone‐like structures, containing highly dendritic GFP‐positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone‐like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase, produce a mineralized type‐I‐collagen matrix and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23 and the mature osteocyte marker, Sost. They also express RankL, Opg and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1 and Opg and upregulation of RankL and E11/gp38. Live‐cell imaging revealed the dynamic process by which OmGFP66 bone‐like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule‐like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment.