Browsing by Subject "RhoA"

Now showing 1 - 5 of 5
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    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 Sciences
    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 (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.
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    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 Sciences
    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.
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    Periostin and matrix stiffness combine to regulate myofibroblast differentiation and fibronectin synthesis during palatal healing
    (Elsevier, 2020) Nikoloudaki, Georgia; Snider, Paige; Simmons, Olga; Conway, Simon J.; Hamilton, Douglas W.; Pediatrics, School of Medicine
    Although the matricellular protein periostin is prominently upregulated in skin and gingival healing, it plays contrasting roles in myofibroblast differentiation and matrix synthesis respectively. Palatal healing is associated with scarring that can alter or restrict maxilla growth, but the expression pattern and contribution of periostin in palatal healing is unknown. Using periostin-knockout (Postn-/-) and wild-type (WT) mice, the contribution of periostin to palatal healing was investigated through 1.5 mm full-thickness excisional wounds in the hard palate. In WT mice, periostin was upregulated 6 days post-wounding, with mRNA levels peaking at day 12. Genetic deletion of periostin significantly reduced wound closure rates compared to WT mice. Absence of periostin reduced mRNA levels of pivotal genes in wound repair, including α-SMA/acta2, fibronectin and βigh3. Recruitment of fibroblasts and inflammatory cells, as visualized by immunofluorescent staining for fibroblast specific factor-1, vimentin, and macrophages markers Arginase-1 and iNOS was also impaired in Postn-/-, but not WT mice. Palatal fibroblasts isolated from the hard palate of mice were cultured on collagen gels and prefabricated silicon substrates with varying stiffness. Postn-/- fibroblasts showed a significantly reduced ability to contract a collagen gel, which was rescued by the exogenous addition of recombinant periostin. As the stiffness increased, Postn-/- fibroblasts increasingly differentiated into myofibroblasts, but not to the same degree as the WT. Pharmacological inhibition of Rac rescued the deficient myofibroblastic phenotype of Postn-/- cells. Low stiffness substrates (0.2 kPa) resulted in upregulation of fibronectin in WT cells, an effect which was significantly reduced in Postn-/- cells. Quantification of immunostaining for vinculin and integrinβ1 adhesions revealed that Periostin is required for the formation of focal and fibrillar adhesions in mPFBs. Our results suggest that periostin modulates myofibroblast differentiation and contraction via integrinβ1/RhoA pathway, and fibronectin synthesis in an ECM stiffness dependent manner in palatal healing.
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    The regulation of the serum response network by the RGS RHOGEFS is critical for YAP1 activity and cell fate decisions
    (2017-07) Lane, Brandon S.; Wells, Clark D.
    The growth of mammary epithelial cells is regulated by interactions with neighboring cells and by exposure to soluble factors including hormones and growth factors. These cues are integrated within the cell, perpetuating changes onto the organization of the actin cytoskeleton, resulting in altered transcriptional programs. Rho family GTPases regulates actin dynamics that facilitate transcriptional reprogramming. In particular, RhoA induces the formation of actin stress fibers to promote the transcriptional co-activator YAP1 to translocate from the cytosol into the nucleus. There, it co-activates TEAD family transcription factors to drive the expression of pro-growth and survival genes. Rho family members are activated by guanine exchange factors (GEF) and inhibited by GTPase activating proteins (GAP). Here, we determined the relative effects of expression of 67 RhoGEFs and RhoGAPs on the activation of TEAD. This revealed that regulator of G-protein signaling (RGS) domain containing ArhGEF1, ArhGEF11 and ArhGEF12 all promoted YAP1 dependent activation of TEAD. These RhoGEFs mediate signaling from heptahelical receptors that are stimulated by lipid mitogens to activate the heterotrimeric G-proteins Gα12 and Gα13. Consistently, loss of expression of ArhGEF12 and to a lesser degree ArhGEF11 prevented actin stress fiber accumulation and activation of YAP1 mediated signaling by serum. Conversely, several complementary experiments revealed that ArhGEF1 dominantly limits Gα13 selective activation of YAP1 and the mitogen activated protein kinase (MAPK) cascades. Furthermore excessive Gα13 activity results in both high levels of filamentous actin and arrest cells in the G1/0 phase of the cell cycle. This is likely due to the systemic inhibition of cell cycle promoting signaling and a loss of protein translation. Further, YAP1 was found to be essential for the survival of ArhGEF1 silenced cells. Together, these studies define a circuit whereby the rgRhoGEFs regulate Gα 12/13-RhoA signaling flux to regulate cellular growth that is promoted by serum factors.
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    RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation
    (Springer, 2016) Wu, Xiangbing; Walker, Chandler L.; Lu, Qingbo; Wu, Wei; Eddelman, Daniel B.; Parish, Jonathan M.; Xu, Xiao-Ming; Department of Neurological Surgery, IU School of Medicine
    Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A2 (cPLA2), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA2. Inhibition of RhoA, Rho kinase and cPLA2 significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA2 activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA2. The immunofluorescence staining showed that ROCK1 or ROCK2, two isoforms of Rho kinase, was co-localized with cPLA2 in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK1 or ROCK2 bonded directly with cPLA2 and phospho-cPLA2. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA2 activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA2 activation.