RhoA/ROCK1 signaling regulates stress granule formation and apoptosis

Cells form stress granules (SGs), in response to unfavorable environments, to avoid apoptosis, but it is unclear whether and how SG formation and cellular apoptosis are coordinately regulated. In this study we detected the small GTPase, Ras homolog gene family member A (RhoA), and its downstream kinase, Rho-associated, coiled-coil containing protein kinase 1 (ROCK1), in SG, and found that their stress-induced activities were important for SG formation and subsequent global translational repression. Importantly, only activated RhoA and ROCK1 were sequestered into SG. Sequestration of activated ROCK1 into SG prevented ROCK1 from interacting with JNK-interacting protein 3 (JIP-3) and its activation of c-Jun N-terminal kinase (JNK), a pathway triggering apoptosis, thereby protecting cells from apoptosis. This study identifies a specific signaling pathway, mediated by RhoA and ROCK1, which determines cell fate by promoting SG formation or initiating apoptosis during stress.     

Akt signaling regulates actin organization via modulation of MMP-2 activity during chondrogenesis of chick wing limb bud mesenchymal cells

Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes. This process is characterized by a strong interdependence of cell shape and cytoskeletal organization accompanying the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. In this study, we hypothesized that the activation of matrix metalloproteinase (MMP)-2 would be involved in the reorganization of the actin cytoskeleton and that this would require an Akt-dependent signaling pathway in chick wing bud mesenchymal cells. The pharmacological inhibition of Akt signaling resulted in decreased glycosaminoglycan synthesis and reduced the level of active MMP-2, leading to suppressed cortical actin organization which is characteristic of differentiated chondrocytes. In addition, the exposure of cells to bafilomycin A1 reversed these chondro-inhibitory effects induced by inhibition of Akt signaling. In conclusion, our data indicate that Akt signaling is involved in the activation of MMP-2 and that this Akt-induced activation of MMP-2 is responsible for reorganization of the actin cytoskeleton into a cortical pattern with parallel rounding of chondrogenic competent cells.     

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton.     

Insulin receptor signaling regulates actin cytoskeletal organization in developing photoreceptors

The insulin receptor (IR) and IR signaling proteins are widely distributed throughout the CNS. IR signaling provides a trophic signal for transformed retinal neurons in culture and we recently reported that deletion of IR in rod photoreceptors by Cre/ lox system resulted in stress-induced photoreceptor degeneration. These studies suggest a neuroprotective role of IR in rod photoreceptor cell function. However, there are no studies available on the role of insulin-induced IR signaling in the development of normal photoreceptors. To examine the role of insulin-induced IR signaling, we analyzed cultured neuronal cells isolated from newborn rodent retinas. In insulin-lacking cultures, photoreceptors from wild-type rat retinas exhibited an abnormal morphology with a wide axon cone and disorganization of the actin and tubulin cytoskeleton. Photoreceptors from IR knockout mouse retinas also exhibited a similar abnormal morphology. A novel finding in this study was that addition of docosahexaenoic acid, a photoreceptor trophic factor, restored normal axonal outgrowth in insulin-lacking cultures. These data suggest that IR signaling pathways regulate actin and tubulin cytoskeletal organization in photoreceptors; they also imply that insulin and docosahexaenoic acid activate at least partially overlapping signaling pathways that are essential for the development of normal photoreceptors.     

AFAP120 regulates actin organization during neuronal differentiation

During development, dynamic changes in the actin cytoskeleton determine both cell motility and morphological differentiation. In most mature tissues, cells are generally minimally motile and have morphologies specialized to their functions. In metastatic cancer, cells generally lose their specialized morphology and become motile. Therefore, proteins that regulate the transition between the motile and morphologically differentiated states can play important roles in determining cancer outcomes. AFAP120 is a neuronal-specific protein that binds Src kinase and protein kinase C (PKC) and cross-links actin filaments. Here we report that expression and tyrosine phosphorylation of AFAP120 are developmentally regulated in the cerebellum. In cerebellar cultures, PKC activation induces Src kinase-dependent phosphorylation of AFAP120, indicating that AFAP120 may be a downstream effector of Src. In neuroblastoma cells induced to differentiate by treatment with a PKC activator, tyrosine phosphorylation of AFAP120 appears to regulate the formation of the lamellar actin structures and subsequent neurite initiation. Together, these results indicate that AFAP120 plays a role in organizing dynamic actin structures during neuronal differentiation and suggest that AFAP120 may help regulate the transition from motile precursor to morphologically differentiated neurons.     

Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells

In this study we describe a novel Rho small GTPase dependent pathway that elicits apoptotic responses controlled by actin reorganization in hormone-sensitive LNCaP- and hormone insensitive DU145-prostate cancer cells stimulated with membrane androgen receptor selective agonists. Using an albumin-conjugated steroid, testosterone-BSA, we now show significant induction of actin polymerization and apoptosis that can be reversed by actin disrupting agents in both cell lines. Testosterone-BSA triggered RhoA/B and Cdc42 activation in DU145 cells followed by stimulation of downstream effectors ROCK, LIMK2 and ADF/destrin. Furthermore, dominant-negative RhoA, RhoB or Cdc42 mutants or pharmacological inhibitors of ROCK inhibited both actin organization and apoptosis in DU145 cells. Activation of RhoA/B and ROCK was also implicated in membrane androgen receptor-dependent actin polymerization and apoptosis in LNCaP cells. Our findings suggest that Rho small GTPases are major membrane androgen receptor effectors controlling actin reorganization and apoptosis in prostate cancer cells.     

Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation

Notch signaling is involved in several cell lineage determination processes during embryonic development. Recently, we have shown that Sox9 is most likely a primary target gene of Notch1 signaling in embryonic stem cells (ESCs). By using our in vitro differentiation protocol for chondrogenesis from ESCs through embryoid bodies (EBs) together with our tamoxifen-inducible system to activate Notch1, we analyzed the function of Notch signaling and its induction of Sox9 during EB differentiation towards the chondrogenic lineage. Temporary activation of Notch1 during early stages of EB, when lineage determination occurs, was accompanied by rapid and transient Sox9 upregulation and resulted in induction of chondrogenic differentiation during later stages of EB cultivation. Using siRNA targeting Sox9, we knocked down and adjusted this early Notch1-induced Sox9 expression peak to non-induced levels, which led to reversion of Notch1-induced chondrogenic differentiation. In contrast, continuous Notch1 activation during EB cultivation resulted in complete inhibition of chondrogenic differentiation. Furthermore, a reduction and delay of cardiac differentiation observed in EBs after early Notch1 activation was not reversed by siRNA-mediated Sox9 knockdown. Our data indicate that Notch1 signaling has an important role during early stages of chondrogenic lineage determination by regulation of Sox9 expression.     

Pur‐alpha regulates RhoA developmental expression and downstream signaling

Pur‐alpha is an essential protein for postnatal brain development which localizes specifically to dendrites where it plays a role in the translation of neuronal RNA. Mice lacking Pur‐alpha display decreased neuronogenesis and impaired neuronal differentiation. Here we examined two Rho GTPases, Rac1 and RhoA, which play opposing roles in neurite outgrowth and are critical for dendritic maturation during mouse brain development in the presence and absence of Pur‐alpha. Pur‐alpha is developmentally regulated in the mouse brain with expression beginning shortly after birth and rapidly increasing to peak during the third week of postnatal development. RhoA levels analyzed by Western blotting rapidly fluctuated in the wild‐type mouse brain, however, in the absence of Pur‐alpha, a decrease in RhoA levels shortly after birth and a delay in the cycling of RhoA regulation was observed leading to reduced basal levels of RhoA after day 10 postnatal. Immunohistochemistry of brain tissues displayed reduced RhoA levels in the cortex and cerebellum and loss of perinuclear cytoplasmic labeling of RhoA within the cortex in the knockout mouse brain. While Rac1 levels remained relatively stable at all time points during development and were similar in both wild‐type and Pur‐alpha knockout mice, changes in subcellular localization of Rac1 were seen in the absence of Pur‐alpha. These findings suggest that Pur‐alpha can regulate RhoA at multiple levels including basal protein levels, subcellular compartmentalization, as well as turnover of active RhoA in order to promote dendritic maturation. J. Cell. Physiol. 228: 65–72, 2013. © 2012 Wiley Periodicals, Inc.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Matrix-bound PAI-1 supports cell blebbing via RhoA/ROCK1 signaling

The microenvironment of a tumor can influence both the morphology and the behavior of cancer cells which, in turn, can rapidly adapt to environmental changes. Increasing evidence points to the involvement of amoeboid cell migration and thus of cell blebbing in the metastatic process; however, the cues that promote amoeboid cell behavior in physiological and pathological conditions have not yet been clearly identified. Plasminogen Activator Inhibitor type-1 (PAI-1) is found in high amount in the microenvironment of aggressive tumors and is considered as an independent marker of bad prognosis. Here we show by immunoblotting, activity assay and immunofluorescence that, in SW620 human colorectal cancer cells, matrix-associated PAI-1 plays a role in the cell behavior needed for amoeboid migration by maintaining cell blebbing, localizing PDK1 and ROCK1 at the cell membrane and maintaining the RhoA/ROCK1/MLC-P pathway activation. The results obtained by modeling PAI-1 deposition around tumors indicate that matrix-bound PAI-1 is heterogeneously distributed at the tumor periphery and that, at certain spots, the elevated concentrations of matrix-bound PAI-1 needed for cancer cells to undergo the mesenchymal-amoeboid transition can be observed. Matrix-bound PAI-1, as a matricellular protein, could thus represent one of the physiopathological requirements to support metastatic formation.     

Phospholipase Cdelta3 regulates RhoA/Rho kinase signaling and neurite outgrowth

Phospholipase Cδ3 (PLCδ3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLCδ3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLCδ3 in neuronal migration and outgrowth. PLCδ3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLCδ3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLCδ3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLCδ3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLCδ3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLCδ3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLCδ3KD cells. We also found that exogenous expression of PLCδ3 down-regulated RhoA protein, and constitutively active PLCδ3 promotes the RhoA down-regulation more significantly than PLCδ3 upon differentiation. These results indicate that PLCδ3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.     

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.