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Browsing by Author "Uzer, Gunes"
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Item Cell Mechanosensitivity to Extremely Low Magnitude Signals is Enabled by a LINCed Nucleus(Wiley, 2015-06) Uzer, Gunes; Thompson, William R.; Sen, Buer; Xie, Zhihui; Yen, Sherwin S.; Miller, Sean; Bas, Guniz; Styner, Maya; Rubin, Clinton T.; Judex, Stefan; Burridge, Keith; Rubin, Janet; Physical Therapy, School of Health and Rehabilitation SciencesA 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 (MSCs), high-magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low-level signals to regulate behavior. Exposing MSC to extremely low-magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), 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 FAK and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) 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 HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with "outside-in" signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent "inside-inside" signaling.Item Concise Review: Plasma and Nuclear Membranes Convey Mechanical Information to Regulate Mesenchymal Stem Cell Lineage(Wiley, 2016-06) Uzer, Gunes; Fuchs, Robyn K.; Rubin, Janet; Thompson, William R.; Department of Physical Therapy, School of Health and Rehabilitation SciencesNumerous factors including chemical, hormonal, spatial, and physical cues determine stem cell fate. While the regulation of stem cell differentiation by soluble factors is well-characterized, the role of mechanical force in the determination of lineage fate is just beginning to be understood. Investigation of the role of force on cell function has largely focused on “outside-in” signaling, initiated at the plasma membrane. When interfaced with the extracellular matrix, the cell uses integral membrane proteins, such as those found in focal adhesion complexes to translate force into biochemical signals. Akin to these outside-in connections, the internal cytoskeleton is physically linked to the nucleus, via proteins that span the nuclear membrane. Although structurally and biochemically distinct, these two forms of mechanical coupling influence stem cell lineage fate and, when disrupted, often lead to disease. Here we provide an overview of how mechanical coupling occurs at the plasma and nuclear membranes. We also discuss the role of force on stem cell differentiation, with focus on the biochemical signals generated at the cell membrane and the nucleus, and how those signals influence various diseases. While the interaction of stem cells with their physical environment and how they respond to force is complex, an understanding of the mechanical regulation of these cells is critical in the design of novel therapeutics to combat diseases associated with aging, cancer, and osteoporosis.Item LARG GEF and ARHGAP18 orchestrate RhoA activity to control mesenchymal stem cell lineage(Elsevier, 2018-02) Thompson, William R.; Yen, Sherwin S.; Uzer, Gunes; Xie, Zhihui; Sen, Buer; Styner, Maya; Burridge, Keith; Rubin, Janet; Physical Therapy, School of Health and Rehabilitation SciencesThe 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.Item Mechanical suppression of breast cancer cell invasion and paracrine signaling to osteoclasts requires nucleo-cytoskeletal connectivity(Nature, 2020-11-17) Yi, Xin; Wright, Laura E.; Pagnotti, Gabriel M.; Uzer, Gunes; Powell, Katherine M.; Wallace, Joseph M.; Sankar, Uma; Rubin, Clinton T.; Mohammad, Khalid; Guise, Theresa A.; Thompson, William R.; Physical Therapy, School of Health and Human SciencesExercise benefits the musculoskeletal system and reduces the effects of cancer. The effects of exercise are multifactorial, where metabolic changes and tissue adaptation influence outcomes. Mechanical signals, a principal component of exercise, are anabolic to the musculoskeletal system and restrict cancer progression. We examined the mechanisms through which cancer cells sense and respond to low-magnitude mechanical signals introduced in the form of vibration. Low-magnitude, high-frequency vibration was applied to human breast cancer cells in the form of low-intensity vibration (LIV). LIV decreased matrix invasion and impaired secretion of osteolytic factors PTHLH, IL-11, and RANKL. Furthermore, paracrine signals from mechanically stimulated cancer cells, reduced osteoclast differentiation and resorptive capacity. Disconnecting the nucleus by knockdown of SUN1 and SUN2 impaired LIV-mediated suppression of invasion and osteolytic factor secretion. LIV increased cell stiffness; an effect dependent on the LINC complex. These data show that mechanical vibration reduces the metastatic potential of human breast cancer cells, where the nucleus serves as a mechanosensory apparatus to alter cell structure and intercellular signaling.Item Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model(Nature Publishing Group, 2015-06-09) Thompson, William R.; Uzer, Gunes; Brobst, Kaitlyn E.; Xie, Zhihui; Sen, Buer; Yen, Sherwin S.; Styner, Maya; Rubin, Janet; Department of Physical Therapy, IU School of Health and Rehabilitation SciencesStudying osteocyte behavior in culture has proven difficult because these embedded cells require spatially coordinated interactions with the matrix and surrounding cells to achieve the osteocyte phenotype. Using an easily attainable source of bone marrow mesenchymal stem cells, we generated cells with the osteocyte phenotype within two weeks. These "stem cell derived-osteocytes" (SCD-O) displayed stellate morphology and lacunocanalicular ultrastructure. Osteocytic genes Sost, Dmp1, E11, and Fgf23 were maximally expressed at 15 days and responded to PTH and 1,25(OH)2D3. Production of sclerostin mRNA and protein, within 15 days of culture makes the SCD-O model ideal for elucidating regulatory mechanisms. We found sclerostin to be regulated by mechanical factors, where low intensity vibration significantly reduced Sost expression. Additionally, this model recapitulates sclerostin production in response to osteoactive hormones, as PTH or LIV repressed secretion of sclerostin, significantly impacting Wnt-mediated Axin2 expression, via β-catenin signaling. In summary, SCD-O cells produce abundant matrix, rapidly attain the osteocyte phenotype, and secrete functional factors including sclerostin under non-immortalized conditions. This culture model enables ex vivo observations of osteocyte behavior while preserving an organ-like environment. Furthermore, as marrow-derived mesenchymal stem cells can be obtained from transgenic animals; our model enables study of genetic control of osteocyte behaviors.