Rho-dependent Manner Research Articles

Bortezomib induces Rho-dependent hyperpermeability of endothelial cells synergistically with inflammatory mediators

BackgroundBortezomib (BTZ), a selective 26 S proteasome inhibitor, is clinically useful in treating multiple myeloma and mantle cell lymphoma. BTZ exerts its antitumor effect by suppressing nuclear factor-B in myeloma cells, promoting endothelial cell apoptosis, and inhibiting angiogenesis. Despite its success, pulmonary complications, such as capillary leak syndrome of the vascular hyperpermeability type, were reported prior to its approval. Although the incidence of these complications has decreased with the use of steroids, the underlying mechanism remains unclear. This study aims to investigate how BTZ influences endothelial cell permeability.MethodsWe examined the impact of BTZ on vascular endothelial cells, focusing on its effects on RhoA and RhoC proteins. Stress fiber formation, a known indicator of increased permeability, was assessed through the Rho/ROCK pathway.ResultsBTZ was found to elevate the protein levels of RhoA and RhoC in vascular endothelial cells, leading to stress fiber formation via the Rho/ROCK pathway. This process resulted in enhanced vascular permeability in a Rho-dependent manner. Furthermore, the stress fiber formation induced by BTZ had synergistic effects with the inflammatory mediator histamine.ConclusionsOur findings suggest that BTZ accumulates RhoA and RhoC proteins in endothelial cells, amplifying the inflammatory mediator-induced increase in the active GTP-bound state of Rho, thereby exaggerating vascular permeability during pulmonary inflammation. This study provides novel insights into the molecular mechanism underlying the pulmonary complications of BTZ, suggesting that BTZ may enhance inflammatory responses in pulmonary endothelial cells by increasing RhoA and RhoC protein levels.

Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells.

Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events-differentiation, proliferation, migration, and adhesion-which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham's F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham's F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 μg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 μg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.

Investigation of Evolutionary History and Origin of the Tre1 Family Suggests a Role in Regulating Hemocytes Cells Infiltration of the Blood-Brain Barrier.

Simple SummaryUnderstanding the evolutionary association between immune cells and the blood–brain barrier (BBB) is vital to develop therapeutic approaches. In Drosophila, glial cells form the BBB that regulates the access of hemocytes to the brain. It is still not known which diapedesis route hemocytes cells follow. In vertebrates, paracellular migration is dependent on PECAM1, while transcellular migration is dependent on the expression of CAV1. The drosophila genome lacks both genes. The Tre1 family (Tre1, moody, and Dmel_CG4313) contribute to regulating transepithelial migration in Drosophila. However, its evolutionary history is not known. We performed phylogenetic analysis to reconstruct the evolutionary history of the Tre1 family. We found Dmel_CG4313 only in insects. Tre1 exists only in invertebrates and is highly conserved. moody evolutionary history is more spread as it appears from Cnidaria up to mammals and is less conserved. The Tre1 family origin seems to be related to opsins. We have identified an SH3 motif in Tre1, moody, and Dmel_CG4313. SH3 regulates actin movement in a Rho-dependent manner in PECAM1. Our results suggest that the Tre1 family could be playing an important role in paracellular diapedesis in Drosophila. Thus, targeting the Tre1 family could help us regulate access to the brain.Understanding the evolutionary relationship between immune cells and the blood–brain barrier (BBB) is important to devise therapeutic strategies. In vertebrates, immune cells follow either a paracellular or a transcellular pathway to infiltrate the BBB. In Drosophila, glial cells form the BBB that regulates the access of hemocytes to the brain. However, it is still not known which diapedesis route hemocytes cells follow. In vertebrates, paracellular migration is dependent on PECAM1, while transcellular migration is dependent on the expression of CAV1. Interestingly Drosophila genome lacks both genes. Tre1 family (Tre1, moody, and Dmel_CG4313) play a diverse role in regulating transepithelial migration in Drosophila. However, its evolutionary history and origin are not yet known. We performed phylogenetic analysis, together with HH search, positive selection, and ancestral reconstruction to investigate the Tre1 family. We found that Tre1 exists in Mollusca, Arthropoda, Ambulacraria, and Scalidophora. moody is shown to be a more ancient protein and it has existed since Cnidaria emergence and has a homolog (e.g., GPCR84) in mammals. The third family member (Dmel_CG4313) seems to only exist in insects. The origin of the family seems to be related to the rhodopsin-like family and in particular family α. We found that opsin is the nearest receptor to have a common ancestor with the Tre1 family that has diverged in sponges. We investigated the positive selection of the Tre1 family using PAML. Tre1 seems to have evolved under negative selection, whereas moody has evolved during positive selection. The sites that we found under positive selection are likely to play a role in the speciation of function in the case of moody. We have identified an SH3 motif, in Tre1 and, moody and Dmel_CG4313. SH3 is known to play a fundamental role in regulating actin movement in a Rho-dependent manner in PECAM1. Our results suggest that the Tre1 family could be playing an important role in paracellular diapedesis in Drosophila.

The Crossroads between RAS and RHO Signaling Pathways in Cellular Transformation, Motility and Contraction.

Ras and Rho proteins are GTP-regulated molecular switches that control multiple signaling pathways in eukaryotic cells. Ras was among the first identified oncogenes, and it appears mutated in many forms of human cancer. It mainly promotes proliferation and survival through the MAPK pathway and the PI3K/AKT pathways, respectively. However, the myriad proteins close to the plasma membrane that activate or inhibit Ras make it a major regulator of many apparently unrelated pathways. On the other hand, Rho is weakly oncogenic by itself, but it critically regulates microfilament dynamics; that is, actin polymerization, disassembly and contraction. Polymerization is driven mainly by the Arp2/3 complex and formins, whereas contraction depends on myosin mini-filament assembly and activity. These two pathways intersect at numerous points: from Ras-dependent triggering of Rho activators, some of which act through PI3K, to mechanical feedback driven by actomyosin action. Here, we describe the main points of connection between the Ras and Rho pathways as they coordinately drive oncogenic transformation. We emphasize the biochemical crosstalk that drives actomyosin contraction driven by Ras in a Rho-dependent manner. We also describe possible routes of mechanical feedback through which myosin II activation may control Ras/Rho activation.

A Computational Study of the Dynamics of Cadherin-Catenin Complex Regulated by Actin Cytoskeleton

Cadherin ligations activate many intracellular signaling cascades that govern cell and tissue behaviors. For example, cadherin homophilic interactions lead to structural reorganization of actin networks in a Rho-dependent manner. Interestingly, actin filaments can also affect cadherin dynamics by anchoring the cadherin-catenin complex (CCC) and limiting the diffusion of CCC. Despite experimental efforts, a mechanistic analysis of interactions between CCC and actin structures does not exist because both systems are highly dynamic and complex. In this study, we probed how cadherin dynamics is regulated by actin filaments using a coarse-grained computational model. We found that CCC movement is inhibited to an extent depending on actin concentration and average filament length, resulting in different cluster size and lifetime. We also showed that the number of actin available for CCC binding outcompetes the binding affinity between CCC and actin filaments. This implies the importance of vinculin which can link CCC to a deeper and larger actin pool beneath the cell membrane. We further tested the hypothesis that actin turnover accelerates cadherin cluster disassembly by competing with cis interactions. Our model sheds light on the interactions between the actin cytoskeleton and cell adhesion molecules and also pave the way for future studies with detailed and additional cellular circumstances taken into consideration.

Targeting cell surface GRP78 enhances pancreatic cancer radiosensitivity through YAP/TAZ protein signaling

Ionizing radiation (IR) can promote migration and invasion of cancer cells, but the basis for this phenomenon has not been fully elucidated. IR increases expression of glucose-regulated protein 78kDa (GRP78) on the surface of cancer cells (CS-GRP78), and this up-regulation is associated with more aggressive behavior, radioresistance, and recurrence of cancer. Here, using various biochemical and immunological methods, including flow cytometry, cell proliferation and migration assays, Rho activation and quantitative RT-PCR assays, we investigated the mechanism by which CS-GRP78 contributes to radioresistance in pancreatic ductal adenocarcinoma (PDAC) cells. We found that activated α2-Macroglobulin (α2M*) a ligand of the CS-GRP78 receptor, induces formation of the AKT kinase (AKT)/DLC1 Rho-GTPase-activating protein (DLC1) complex and thereby increases Rho activation. Further, CS-GRP78 activated the transcriptional coactivators Yes-associated protein (YAP) and tafazzin (TAZ) in a Rho-dependent manner, promoting motility and invasiveness of PDAC cells. We observed that radiation-induced CS-GRP78 stimulates the nuclear accumulation of YAP/TAZ and increases YAP/TAZ target gene expressions. Remarkably, targeting CS-GRP78 with C38 monoclonal antibody (Mab) enhanced radiosensitivity and increased the efficacy of radiation therapy by curtailing PDAC cell motility and invasion. These findings reveal that CS-GRP78 acts upstream of YAP/TAZ signaling and promote migration and radiation-resistance in PDAC cells. We therefore conclude that, C38 Mab is a promising candidate for use in combination with radiation therapy to manage PDAC.

The RhoGEF protein Plekhg5 regulates apical constriction of bottle cells during gastrulation.

Apical constriction regulates epithelial morphogenesis during embryonic development, but how this process is controlled is not understood completely. Here, we identify a Rho guanine nucleotide exchange factor (GEF) gene plekhg5 as an essential regulator of apical constriction of bottle cells during Xenopus gastrulation. plekhg5 is expressed in the blastopore lip and its expression is sufficient to induce ectopic bottle cells in epithelia of different germ layers in a Rho-dependent manner. This activity is not shared by arhgef3, which encodes another organizer-specific RhoGEF. Plekhg5 protein is localized in the apical cell cortex via its pleckstrin homology domain, and the GEF activity enhances its apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation. Knockdown of plekhg5 inhibits activin-induced bottle cell formation and endogenous blastopore lip formation in gastrulating frog embryos. Apical accumulation of actomyosin, apical constriction and bottle cell formation fail to occur in these embryos. Taken together, our data indicate that transcriptional regulation of plekhg5 expression at the blastopore lip determines bottle cell morphology via local polarized activation of Rho by Plekhg5, which stimulates apical actomyosin activity to induce apical constriction.

RhoA activation and nuclearization marks loss of chondrocyte phenotype in crosstalk with Wnt pathway

De-differentiation comprises a major drawback for the use of autologous chondrocytes in cartilage repair. Here, we investigate the role of RhoA and canonical Wnt signaling in chondrocyte phenotype. Chondrocyte de-differentiation is accompanied by an upregulation and nuclear localization of RhoA. Effectors of canonical Wnt signaling including β-catenin and YAP/TAZ are upregulated in de-differentiating chondrocytes in a Rho-dependent manner. Inhibition of Rho activation with C3 transferase inhibits nuclear localization of RhoA, induces expression of chondrogenic markers on 2D and enhances the chondrogenic effect of 3D culturing. Upregulation of chondrogenic markers by Rho inhibition is accompanied by loss of canonical Wnt signaling markers in 3D or on 2D whereas treatment of chondrocytes with Wnt-3a abrogates this effect. However, induction of canonical Wnt signaling inhibits chondrogenic markers on 2D but enhances chondrogenic re-differentiation on 2D with C3 transferase or in 3D. These data provide insights on the context-dependent role of RhoA and Wnt signaling in de-differentiation and on mechanisms to induce chondrogenic markers for therapeutic approaches.

Formin-like2 regulates Rho/ROCK pathway to promote actin assembly and cell invasion of colorectal cancer.

Formin-like2 (FMNL2) is a member of the diaphanous-related formins family, which act as effectors and upstream modulators of Rho GTPases signaling and control the actin-dependent processes, such as cell motility or invasion. FMNL2 has been identified as promoting the motility and metastasis in colorectal carcinoma (CRC). However, whether FMNL2 regulates Rho signaling to promote cancer cell invasion remains unclear. In this study, we demonstrated an essential role for FMNL2 in the activations of Rho/ROCK pathway, SRF transcription or actin assembly, and subsequent CRC cell invasion. FMNL2 could activate Rho/ROCK pathway, and required ROCK to promote CRC cell invasion. Moreover, FMNL2 promoted the formation of filopodia and stress fiber, and activated the SRF transcription in a Rho-dependent manner. We also demonstrated that FMNL2 was necessary for LPA-induced invasion, RhoA/ROCK activation, actin assembly and SRF activation. FMNL2 was an essential component of LPA signal transduction toward RhoA by directly interacting with LARG. LARG silence inhibited RhoA/ROCK pathway and CRC cell invasion. Collectively, these data indicate that FMNL2, acting as upstream of RhoA by interacting with LARG, can promote actin assembly and CRC cell invasion through a Rho/ROCK-dependent mechanism.

Role of EPS8 in integrin-dependent pancreatic cancer invasion

BackgroundTumour cell motility requires reorganisation of the actin cytoskeleton. The epidermal growth factor receptor pathway substrate 8 (EPS8) regulates actin remodelling, through interactions with numerous intracellular proteins, including integrins. The integrin αvβ6 is overexpressed in many carcinomas, and promotes two key features of pancreatic cancer—aggressive local invasion, and activation of transforming growth factor β1 (TGFβ1) leading to desmoplasia. We aimed to investigate the role of EPS8 in αvβ6-dependent functions in pancreatic cancer. MethodsWe used immunohistochemistry to examine the expression of αvβ6, EPS8, and the EPS8-binding partners SOS1 and ABI1 in human pancreatic cancer specimens using tissue microarrays. Cell motility was investigated using transwell assays and organotypic cultures after RNA interference. Primary pancreatic stellate cells were isolated from fresh tissue to optimise these organotypic cultures. TGFβ1 activation was measured using mink lung epithelial cells expressing a TGFβ1-responsive luciferase construct. Findingsαvβ6, EPS8, SOS1, and ABI1 were upregulated in more than 70% of human pancreatic cancer tissues compared with neighbouring non-cancerous pancreatic tissue. αvβ6-dependent cell motility in panceatic cancer was dependent on EPS8, SOS1, and the small GTPase Rac1. Knockdown of any of these proteins inhibited cell motility. Conversely, EPS8 knockdown promoted αvβ6-dependent TGFβ1 activation, in a Rho-dependent manner. InterpretationEPS8 promotes motility in pancreatic cancer, modulated through αvβ6, yet inhibits αvβ6-dependent TGFβ1 activation. EPS8 might therefore function as a master regulator of cell invasion in pancreatic cancer by acting as a molecular switch, differentially regulating small GTPases (Rac1 and Rho) and thereby altering cell function between cell motility and TGFβ1 activation. FundingUK Medical Research Council.

GAP-independent functions of DLC1 in metastasis

Metastases are responsible for most cancer-related deaths. One of the hallmarks of metastatic cells is increased motility and migration through extracellular matrixes. These processes rely on specific small GTPases, in particular those of the Rho family. Deleted in liver cancer-1 (DLC1) is a tumor suppressor that bears a RhoGAP activity. This protein is lost in most cancers, allowing malignant cells to proliferate and disseminate in a Rho-dependent manner. However, DLC1 is also a scaffold protein involved in alternative pathways leading to tumor and metastasis suppressor activities. Recently, substantial information has been gathered on these mechanisms and this review is aiming at describing the potential and known alternative GAP-independent mechanisms allowing DLC1 to impair migration, invasion, and metastasis formation.

Differences in the Nuclear Export Mechanism between Myocardin and Myocardin-related Transcription Factor A

Myocardin (Mycd), a key factor in smooth muscle cell differentiation, is constitutively located in the nucleus, whereas myocardin-related transcription factors A and B (MRTF-A/B) reside mostly in the cytoplasm and translocate to the nucleus in a Rho-dependent manner. Here, we investigated the nuclear export of Mycd family members. They possess two leucine-rich sequences: L1 in the N terminus and L2 in the Gln-rich domain. Although L2 (but not L1) served as a CRM1-binding site for Mycd, CRM1-mediated nuclear export did not affect its subcellular localization. Serum response factor (SRF) competitively inhibited Mycd/CRM1 interaction. Furthermore, such interaction was autonomously inhibited. The N terminus of Mycd bound intramolecularly to Mycd, resulting in masking L2. In contrast, the CRM1-binding affinity of MRTF-A was much higher than that of Mycd because both L1 and L2 of MRTF-A served as functional CRM1-binding sites, and the autoinhibition observed in the Mycd/CRM1 interaction was absent in the MRTF-A/CRM1 interaction. Additionally, because the SRF-binding affinity of MRTF-A was lower than that of Mycd, the inhibitory effect of SRF on the MRTF-A/CRM1 interaction was weak. Thus, MRTF-A is much more likely to be exported from the nucleus. These differences could be the reason for the distinct subcellular localization of Mycd and MRTF-A.

Mouse EP3 α, β, and γ Receptor Variants Reduce Tumor Cell Proliferation and Tumorigenesis in Vivo

Prostaglandin E(2), which exerts its functions by binding to four G protein-coupled receptors (EP1-4), is implicated in tumorigenesis. Among the four E-prostanoid (EP) receptors, EP3 is unique in that it exists as alternatively spliced variants, characterized by differences in the cytoplasmic C-terminal tail. Although three EP3 variants, alpha, beta, and gamma, have been described in mice, their functional significance in regulating tumorigenesis is unknown. In this study we provide evidence that expressing murine EP3 alpha, beta, and gamma receptor variants in tumor cells reduces to the same degree their tumorigenic potential in vivo. In addition, activation of each of the three mEP3 variants induces enhanced cell-cell contact and reduces cell proliferation in vitro in a Rho-dependent manner. Finally, we demonstrate that EP3-mediated RhoA activation requires the engagement of the heterotrimeric G protein G(12). Thus, our study provides strong evidence that selective activation of each of the three variants of the EP3 receptor suppresses tumor cell function by activating a G(12)-RhoA pathway.

PRL Tyrosine Phosphatases Regulate Rho Family GTPases to Promote Invasion and Motility

Phosphatase found in regenerating liver (PRL)-1, PRL-2, and PRL-3 [also known as PTP4A1, PTP4A2, and PTP4A3, respectively] constitute a unique family of putative protein tyrosine phosphatases (PTPs) modified by farnesylation. PRL-3 is amplified and its message is up-regulated in colorectal carcinoma metastases. Its ectopic expression promotes invasive and metastatic properties, supporting a causal link between PRL-3 and late-stage cancer development. However, neither PRL phosphatase substrates nor their signaling pathways have been defined. To address possible mechanisms for the biological activity of PRL-3, we sought to identify its downstream targets, reasoning that regulators of motility and invasion, such as the Rho family of small GTPases, might be logical candidates. We found that levels of active RhoA and RhoC were increased 4- to 7-fold in SW480 colorectal carcinoma cells expressing exogenous PRL-1 and PRL-3, and that PRL-mediated motility and Matrigel invasion were blocked by pharmacologic inhibition of Rho kinase (ROCK), a key Rho effector. In contrast, the activity of Rac was reduced by PRL PTPs, whereas Cdc42 activity was unaffected. PRL-3 stimulated transcription driven by the serum response element in a Rho-dependent manner. We also confirmed that the ability of PRL PTPs to induce invasion and motility is dependent on farnesylation. Catalytic PRL-3 mutants (C104A or D72A) were impaired in PRL-3-induced invasion and Rho activation, indicating that these properties require phosphatase activity. We conclude that PRL PTPs stimulate Rho signaling pathways to promote motility and invasion. Characterization of PRL activity and regulatory pathways should enhance efforts to understand and interfere with PRL-mediated events in invasion and metastasis.

Fibronectin's Central Cell-binding Domain Supports Focal Adhesion Formation and Rho Signal Transduction

Fibroblast adhesion to fibronectin (FN) induces formation of focal adhesions (FAs), structures that have significant effect on cell migration and signaling. FA formation requires actomyosin-based contractility that is regulated by Rho-dependent myosin light chain (MLC) phosphorylation. Previous studies indicated that the FN central cell-binding (and integrin-binding) domain (CBD) is insufficient for FA formation and that the major heparin-binding domain (HepII) facilitates FA formation in a Rho-dependent manner. We describe here conditions under which FN CBD alone is sufficient for FA formation in both human dermal fibroblasts and the FN-null murine fibroblasts. CBD-mediated FA formation is dependent on its surface adsorption and the adhesion activity of the cells. Attachment of FN-null fibroblasts to CBD elicits the same biphasic regulation of Rho activity as seen on intact FN, whereas adhesion to HepII alone does not activate Rho. Activation of Rho requires high levels of integrin occupancy. However, FN or CBD may induce FAs without increased activation of Rho (i.e. the basal level of GTP-Rho induces sufficient phospho-MLC for FA assembly under this condition). In contrast, adhesion to HepII alone does not sustain MLC phosphorylation. Pulse stimulation of cells on CBD or HepII with lysophosphatidic acid elevates Rho GTP loading to the same level, but the lysophosphatidic acid-stimulated MLC phosphorylation is significantly lower in cells on HepII than on CBD. Coating HepII with suboptimal concentrations of CBD induces FAs without increased activation of Rho. Therefore, FN CBD can support FA formation and generate contraction by activating Rho or by facilitating Rho downstream signaling.

The Arabidopsis KLUNKER gene controls cell shape changes and encodes the AtSRA1 homolog

The analysis of a group of seven trichome mutants in Arabidopsis, which all show distorted trichomes along with severe actin defects has revealed insight into the role of the actin cytoskeleton in cell shape control. Four of the corresponding genes encode components of a protein complex, the ARP2/3 complex that stimulates the production of 'fine actin' at active growth sites. In this study, we show that another member of the distorted group, KLUNKER (KLK), encodes the AtSRA1 homolog of Arabidopsis and that klk mutants show a similar range of cell shape defects to those of arp2/3 mutants. In animals, SRA1 regulates the activity of the ARP2/3-regulating WAVE-HSPC300 complex in a Rho-dependent manner. Our findings provide evidence that a Rho/ARP2/3 regulation pathway exists in plants.

TIM, a Dbl-related protein, regulates cell shape and cytoskeletal organization in a Rho-dependent manner

The Dbl-like guanine nucleotide exchange factors (GEFs) have been implicated in direct activation of the Rho family of small GTPases. We previously isolated transforming immortalized mammary (TIM) as a Dbl-like protein. Here, we show that, when expressed in cells, TIM was a potent activator of RhoA. Like activated Rho proteins, expression of TIM potentiated the serum response factor (SRF)- and AP-1-regualted transcriptional activities and activated the SAPK/JNK signaling pathway. In NIH 3T3 cells, TIM induced transforming foci, which was inhibited by the ROCK inhibitor Y-27632 or the dominant negative mutants of Rho proteins. Expression of TIM led to pronounced changes in cell shape and organization of the actin cytoskeleton, including the formation of thick stress fibers at the cell periphery and cell rounding. TIM also promoted redistribution of vinculin-enriched focal adhesions at the cell periphery and increased the phosphorylation of myosin light chain (MLC). These results, taken together, suggest that TIM acts as an upstream regulator for the RhoA/ROCK-mediated cellular functions.

Proteinase-Activated Receptor-2 Stimulates Prostaglandin Production in Keratinocytes: Analysis of Prostaglandin Receptors on Human Melanocytes and Effects of PGE2 and PGF2α on Melanocyte Dendricity

Prostaglandins (PG) are key mediators of diverse functions in the skin and several reports suggest that PG mediate post-inflammatory pigmentary changes through modulation of melanocyte dendricity and melanin synthesis. The proteinase-activated receptor 2 (PAR-2) is important for skin pigmentation because activation of keratinocyte PAR-2 stimulates uptake of melanosomes through phagocytosis in a Rho-dependent manner. In this report, we show that activation of keratinocyte PAR-2 stimulates release of PGE(2) and PGF(2alpha) and that PGE(2) and PGF(2alpha) act as paracrine factors that stimulate melanocyte dendricity. We characterized the expression of the EP and FP receptors in human melanocytes and show that human melanocytes express EP1 and EP3, and the FP receptor, but not EP2 and EP4. Treatment of melanocytes with EP1 and EP3 receptor agonists resulted in increased melanocyte dendricity, indicating that both EP1 and EP3 receptor signaling contribute to PGE(2)-mediated melanocyte dendricity. Certain EP3 receptor subtypes have been shown to increase adenosine 3',5'-cyclic monophosphate (cAMP) through coupling to Gs, whereas EP1 is known to couple to Gq to activate phospholipase C with elevation in Ca(2+). The cAMP/protein kinase A system is known to modulate melanocyte dendrite formation through modulation of Rac and Rho activity. Neither PGF(2alpha) or PGE(2) elevated cAMP in human melanocytes showing that dendricity observed in response to PGE(2) and PGF(2alpha) is cAMP-independent. Our data suggest that PAR-2 mediates cutaneous pigmentation both through increased uptake of melanosomes by keratinocytes, as well as by release of PGE(2) and PGF(2alpha) that stimulate melanocyte dendricity through EP1, EP3, and FP receptors.

Mechanisms of internalization and recycling of the chemokine receptor, CCR5.

CCR5 is a G protein-coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV-1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV-1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand-induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine-induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP-1. CCR5 internalization and recycling were inhibited by Toxin B and C(3) exoenzyme treatment in CHO and THP-1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand-induced activation of CCR5 leads to Rho kinase-dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho-dependent manner.

The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.

Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.