Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.     

Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase.

A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs.     

Stress kinase p38 mediates EGFR transactivation by hyperosmolar concentrations of sorbitol

Activation of the epidermal growth factor receptor (EGFR) has been shown to occur by ligand-dependent and ligand-independent mechanisms. Different molecular mechanisms have been found to be responsible for ligand-independent receptor transactivation. Here, we show that hyperosmolar concentrations of sorbitol activate the EGFR in human keratinocytes. Experiments using specific inhibitors of EGFR phosphorylation show that the increased amount of activated receptors is the result of a decreased rate of dephosphorylation. Furthermore, sorbitol treatment results in a strong activation of stress kinase p38. Treatment of the cells with SB203580, a known inhibitor of p38 alpha and beta kinases, results in impairment of receptor activation, indicating that the stress kinase is involved in receptor activation modulation. This is further reinforced by experiments showing that addition of Toxin B, known to be an inhibitor of the small Rho GTPases rac1, cdc42, and Rho A/B, to the cells results in a strong induction of EGFR activation. Our results point, therefore, to a mechanism by which osmotic shock activates EGFR through the small Rho GTPases-p38 stress kinase pathway.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

Mechanisms of endothelin-1-induced contraction in pulmonary arteries from chronically hypoxic rats

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.     

Neuronal responses to myelin are mediated by rho kinase

CNS myelin inhibits axon growth due to the expression of several growth-inhibitory proteins, including myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein and Nogo. Myelin-associated inhibitory proteins activate rho GTPase in responsive neurons. Rho kinase (ROCK) has been implicated as a critical rho effector in this pathway due to the ability of the pharmacological inhibitor Y-27632 to circumvent myelin-dependent inhibition. Y-27632, however, inhibits the activity of additional kinases. Using three independent approaches, we provide direct evidence that ROCKII is activated in response to the myelin-associated inhibitor Nogo. We demonstrate that Nogo treatment enhances ROCKII translocation to the cellular membrane in PC12 cells and enhances ROCKII kinase activity towards an in vitro substrate. In addition, Nogo treatment enhances phosphorylation of myosin light chain II, a known ROCK substrate. Further, we demonstrate that primary dorsal root ganglia neurons can be rendered insensitive to the inhibitory effects of myelin via infection with dominant negative ROCK. Together these data provide direct evidence for a rho-ROCK-myosin light chain-II signaling cascade in response to myelin-associated inhibitors.     

Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions.     

Mechanisms of resistance to BCR-ABL and other kinase inhibitors

In this article, we are reviewing the molecular mechanisms that lead to kinase inhibitor resistance. As the oncogenic BCR-ABL kinase is the target of the first approved small-molecule kinase inhibitor imatinib, we will first focus on the structural and mechanistic basis for imatinib resistance. We will then show ways how next generations of BCR-ABL inhibitors and alternative targeting strategies have helped to offer effective treatment options for imatinib-resistant patients. Based on these insights, we discuss commonalities and further mechanisms that lead to resistance to other kinase inhibitors in solid tumors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Effects of pharmacological inhibition of small GTPases on axon extension and migration of enteric neural crest-derived cells

In the developing enteric nervous system, there is a close association between migrating neural crest-derived cells and the axons of early differentiating neurons. We used pharmacological inhibitors of small GTPases to determine if crest cell migration and axon growth could be uncoupled in cultured intact explants of embryonic mouse gut and slices of embryonic gut grown on collagen gels containing GDNF. Inhibition of the Rho effectors, ROCKI/II, or Rac/Cdc42 inhibited both cell migration and neurite growth in intact explants of embryonic gut. The effects of both ROCKI/II and Rac/Cdc42 inhibitors were more severe on cell migration and axon extension in gut explants from Ret(+/-) mice than in explants from wildtype mice, indicating that Rho GTPases probably act downstream of the receptor tyrosine kinase, Ret. Inhibition of ROCKI/II had different effects on migration and axon extension in gut slices grown on collagen gels containing GDNF from that seen in intact explants of gut. We conclude that ROCKI/II and Rac/Cdc42 are required for both neural crest-derived cell migration and axon growth in the developing gut.     

Identification of potential small molecule binding pockets on Rho family GTPases

Rho GTPases are conformational switches that control a wide variety of signaling pathways critical for eukaryotic cell development and proliferation. They represent attractive targets for drug design as their aberrant function and deregulated activity is associated with many human diseases including cancer. Extensive high-resolution structures (>100) and recent mutagenesis studies have laid the foundation for the design of new structure-based chemotherapeutic strategies. Although the inhibition of Rho signaling with drug-like compounds is an active area of current research, very little attention has been devoted to directly inhibiting Rho by targeting potential allosteric non-nucleotide binding sites. By avoiding the nucleotide binding site, compounds may minimize the potential for undesirable off-target interactions with other ubiquitous GTP and ATP binding proteins. Here we describe the application of molecular dynamics simulations, principal component analysis, sequence conservation analysis, and ensemble small-molecule fragment mapping to provide an extensive mapping of potential small-molecule binding pockets on Rho family members. Characterized sites include novel pockets in the vicinity of the conformationaly responsive switch regions as well as distal sites that appear to be related to the conformations of the nucleotide binding region. Furthermore the use of accelerated molecular dynamics simulation, an advanced sampling method that extends the accessible time-scale of conventional simulations, is found to enhance the characterization of novel binding sites when conformational changes are important for the protein mechanism.     

Multiple signaling pathways mediate compaction of collagen matrices by EGF-stimulated fibroblasts

Fibroblasts stimulated by EGF within collagen matrices generate contraction forces that are likely of importance to cell migration and matrix compaction during wound healing. We have employed an in vitro fibroblast-embedded collagen matrix compaction assay to ascertain signaling pathway components downstream of EGFR activation leading to generation and transmission of contractile force. EGF compacts this floating collagen matrix to a similar extent as PDGF. We demonstrate that compaction requires EGFR kinase activity, yet is maximal in magnitude at intermediate EGF concentrations. This suggests that transmission of EGFR-induced contractile force to the matrix can be mitigated by consequent anti-adhesive effects of EGFR signaling in a dose-dependent manner. Treatment with pharmacological inhibitors demonstrated involvement of the signaling components extracellular signal-regulated kinase (ERK), Rho kinase, and myosin light chain kinase (MLCK) in the force generation and/or transmission process. Moreover, treatment with the pan-calpain inhibitor ALLN and isoform-specific downregulation of m-calpain (CAPN2) using RNA interference determined m-calpain to be a key component of the EGF-induced force response. ALLN treatment modulated the compaction response in a biphasic manner, enhancing matrix deformation to the greatest extent at intermediate concentrations. Our findings have thus identified key signals downstream of EGFR, which integrate in a complex manner to generate and transmit contractile forces to yield matrix deformation.     

Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability

Neutrophil-induced microvascular leakage is an early event in ischemic and inflammatory heart diseases. The specific signaling paradigm by which neutrophils increase microvascular permeability is not yet established. We investigated whether the small GTPase RhoA and its downstream effector Rho kinase mediate neutrophil-stimulated endothelial hyperpermeability. We assessed the effect of neutrophils on Rho activity in bovine coronary venular endothelial cells (CVEC) with a Rho-GTP pull-down assay. Permeability to FITC-albumin was evaluated using CVEC monolayers. We then tested the role of Rho kinase in the permeability response to neutrophils using two structurally distinct pharmacological inhibitors: Y-27632 and HA-1077. Furthermore, neutrophil-stimulated changes in endothelial F-actin organization were examined with fluorescence microscopy. The results show that C5a-activated neutrophils induced an increase in permeability coupled with RhoA activation in CVEC. Inhibition of Rho kinase with either Y-27632 or HA-1077 attenuated the hyperpermeability response. Rho kinase inhibition also attenuated increases in permeability stimulated by the neutrophil supernatant. In addition, activated neutrophils caused actin stress fiber formation in CVEC, which was diminished by either Y-27632 or HA-1077. These findings suggest that RhoA and Rho kinase are involved in the mediation of neutrophil-induced endothelial actin reorganization and barrier dysfunction.     

Regulation of LPA-promoted myofibroblast contraction: role of Rho, myosin light chain kinase, and myosin light chain phosphatase

Myofibroblasts generate the contractile force responsible for wound healing and pathological tissue contracture. In this paper the stress-relaxed collagen lattice model was used to study lysophosphatidic acid (LPA)-promoted myofibroblast contraction and the role of the small GTPase Rho and its downstream targets Rho kinase and myosin light chain phosphatase (MLCPPase) in regulating myofibroblast contraction. In addition, the regulation of myofibroblast contraction was compared with that of smooth muscle cells. LPA-promoted myofibroblast contraction was inhibited by the myosin light chain kinase (MLCK) inhibitors KT5926 and ML-7; however, in contrast to that observed in smooth muscle cells, elevation of intracellular calcium alone was not sufficient to promote myofibroblast contraction. These results suggest that Ca(2+)-mediated activation of MLCK, while necessary, is not sufficient to promote myofibroblast contraction. The specific Rho inactivator C3-transferase and the Rho kinase inhibitor Y-27632 inhibited LPA-promoted myofibroblast contraction, suggesting that contraction depends on activation of the Rho/Rho kinase pathway. Calyculin, a type 1 phosphatase inhibitor known to inhibit MLCPPase, could promote myofibroblast contraction in the absence of LPA, as well as restore contraction in the presence of C3-transferase or Y-27632. Together these results support a model whereby Rho/Rho kinase-mediated inhibition of MLCPPase is necessary for LPA-promoted myofibroblast contraction, in contrast to smooth muscle cells in which Ca(2+) activation of MLCK alone is sufficient to promote contraction.     

Salicylaldehyde derivatives as new protein kinase CK2 inhibitors

Protein kinase CK2 is a Ser/Thr kinase, with a constitutive activity, that is considered as a promising target for cancer therapy. The currently available CK2 inhibitors lack the potency and the pharmacological properties necessary to be suitable and successful in clinical settings. We report the development of new potent CK2 inhibitors from salicylaldehyde derivatives identified by automated screening of a proprietary small-molecule library. Docking simulations and analysis of the structure-activity relationship for the hits allowed to determine their binding modes on CK2, and to carry out the optimization of their structures. This strategy led to the discovery of potent CK2 inhibitors with novel structures, one of which was able to inhibit CK2 activity in living cells and promote tumor cell death. The essential features required for potent CK2 inhibitory activity of this class of compounds are discussed.     

Regulation of Rho/ROCK signaling in airway smooth muscle by membrane potential and [Ca2+]i

Recently, we have shown that Rho and Rho-activated kinase (ROCK) may become activated by high-millimolar KCl, which had previously been widely assumed to act solely through opening of voltage-dependent Ca(2+) channels. In this study, we explored in more detail the relationship between membrane depolarization, Ca(2+) currents, and activation of Rho/ROCK in bovine tracheal smooth muscle. Ca(2+) currents began to activate at membrane voltages more positive than -40 mV and were maximally activated above 0 mV; at the same time, these underwent time- and voltage-dependent inactivation. Depolarizing intact tissues by KCl challenge evoked contractions that were blocked equally, and in a nonadditive fashion, by nifedipine or by the ROCK inhibitor Y-27632. Other agents that elevate intracellular calcium concentration ([Ca(2+)](i)) by pathways independent of G protein-coupled receptors, namely the SERCA-pump inhibitor cyclopiazonic acid and the Ca(2+) ionophore A-23187, evoked contractions that were also largely reduced by Y-27632. KCl directly increased Rho and ROCK activities in a concentration-dependent fashion that paralleled closely the effect of KCl on tone and [Ca(2+)](i), as well as the voltage-dependent Ca(2+) currents that were measured over the voltage ranges that are evoked by 0-120 mM KCl. Through the use of various pharmacological inhibitors, we ruled out roles for Ca(2+)/calmodulin-dependent CaM kinase II, protein kinase C, and protein kinase A in mediating the KCl-stimulated changes in tone and Rho/ROCK activities. In conclusion, Rho is activated by elevation of [Ca(2+)](i) (although the signal transduction pathway underlying this Ca(2+) dependence is still unclear) and possibly also by membrane depolarization per se.     

Convulxin induces platelet shape change through myosin light chain kinase and Rho kinase.

Once platelets are activated, the first event to occur is a rapid change in shape, associated with Ca2+/calmodulin-dependent myosin light chain (MLC) phosphorylation and with Rho kinase activation. The purpose of this study was to investigate which is the biochemical pathway that leads to platelet shape change in response to convulxin, a selective GpVI activator, and to verify whether MLC phosphorylation is essential for this process. The inhibition of the Ca2+-dependent pathway by means of the Ca2+ chelator BAPTA, the Ca2+/calmodulin inhibitor W-7 or the cAMP enhancing drug iloprost reduced about 50% of platelet shape change in response to convulxin. The treatment with either the Rho kinase inhibitors Y27632 or HA 1077 had no effect on platelet shape change induced by convulxin. When both Ca2+/calmodulin-dependent and Rho kinase-dependent pathways were concomitantly inhibited by the combined use of Y27632 plus BAPTA, W-7 or iloprost, platelet shape change was completely abolished. Our findings suggest that convulxin-induced platelet shape change occurs via both pathways, the Ca2+/calmodulin-dependent, which appears to be more important, and the Rho kinase-dependent one. The pattern of MLC phosphorylation was not modified by Rho kinase inhibitors. Conversely, the inhibition of the Ca2+-dependent pathway caused a strong reduction of MLC phosphorylation in BAPTA-treated platelets, and a total inhibition in W-7 or iloprost-treated platelets. Our results demonstrate that following Rho kinase-dependent pathway platelet shape change can occur without the involvement of MLC phosphorylation.     

Structural Basis for the Selective Inhibition of c-Jun N-Terminal Kinase 1 Determined by Rigid DARPin–DARPin Fusions

To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design.     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.     

Novel atypical PKC inhibitors prevent vascular endothelial growth factor-induced blood-retinal barrier dysfunction

Pro-inflammatory cytokines and growth factors such as VEGF (vascular endothelial growth factor) contribute to the loss of the BRB (blood-retinal barrier) and subsequent macular oedema in various retinal pathologies. VEGF signalling requires PKCβ [conventional PKC (protein kinase C)] activity; however, PKCβ inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability, suggesting the involvement of alternative signalling pathways. In the present study, we provide evidence for the involvement of aPKC (atypical PKC) signalling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small-molecule inhibitors, and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small-molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. The results of the present study suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis, and the BBB (blood-brain barrier) in the presence of brain tumours.     

Centaureidin promotes dendrite retraction of melanocytes by activating Rho

Melanosomes synthesized within melanocytes are transferred to keratinocytes through dendrites, resulting in a constant supply of melanin to the epidermis, and this process determines skin pigmentation. During screening for inhibitors of melanosome transfer, we found a novel reagent, centaureidin, that induces significant morphological changes in normal human epidermal melanocytes and inhibits melanocyte dendrite elongation, resulting in a reduction of melanosome transfer in an in vitro melanocyte-keratinocyte co-culture system. Since members of the Rho family of small GTP-binding proteins act as master regulators of dendrite formation, and activated Rho promotes dendrite retraction, we studied the effects of centaureidin on the small GTPases. In in vitro binding assay, centaureidin activated Rho and furthermore, a Rho inhibitor (C. botulinum C3 exoenzyme), a Rho kinase inhibitor (Y27632) and a small GTPase inhibitor (Toxin B) blocked dendrite retraction induced by centaureidin. These results suggest centaureidin could act via the Rho signaling pathway, and it may directly or indirectly activate Rho. Thus, centaureidin appears to inhibit dendrite outgrowth from melanocytes by activating Rho, resulting in the inhibition of melanosome transfer from melanocytes to keratinocytes.