The Scribble-SGEF-Dlg1 complex regulates E-cadherin and ZO-1 stability, turnover and transcription in epithelial cells.

SGEF (also known as ARHGEF26), a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of both E-cadherin and ZO-1 (also known as TJP1) protein levels. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble-SGEF-Dlg1 complex. Our results show that the three members of the ternary complex are required to maintain the stability of the apical junctions, ZO-1 protein levels and tight junction (TJ) permeability. In contrast, only SGEF is necessary to regulate E-cadherin levels. The absence of SGEF destabilizes the E-cadherin-catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, ß-catenin signaling and the transcriptional repressor Slug (also known as SNAI2).

Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.

Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance.

ARHGEF26 enhances Salmonella invasion and inflammation in cells and mice.

Salmonella hijack host machinery in order to invade cells and establish infection. While considerable work has described the role of host proteins in invasion, much less is known regarding how natural variation in these invasion-associated host proteins affects Salmonella pathogenesis. Here we leveraged a candidate cellular GWAS screen to identify natural genetic variation in the ARHGEF26 (Rho Guanine Nucleotide Exchange Factor 26) gene that renders lymphoblastoid cells susceptible to Salmonella Typhi and Typhimurium invasion. Experimental follow-up redefined ARHGEF26's role in Salmonella epithelial cell infection. Specifically, we identified complex serovar-by-host interactions whereby ARHGEF26 stimulation of S. Typhi and S. Typhimurium invasion into host cells varied in magnitude and effector-dependence based on host cell type. While ARHGEF26 regulated SopB- and SopE-mediated S. Typhi (but not S. Typhimurium) infection of HeLa cells, the largest effect of ARHGEF26 was observed with S. Typhimurium in polarized MDCK cells through a SopB- and SopE2-independent mechanism. In both cell types, knockdown of the ARHGEF26-associated protein DLG1 resulted in a similar phenotype and serovar specificity. Importantly, we show that ARHGEF26 plays a critical role in S. Typhimurium pathogenesis by contributing to bacterial burden in the enteric fever murine model, as well as inflammation in the colitis infection model. In the enteric fever model, SopB and SopE2 are required for the effects of Arhgef26 deletion on bacterial burden, and the impact of sopB and sopE2 deletion in turn required ARHGEF26. In contrast, SopB and SopE2 were not required for the impacts of Arhgef26 deletion on colitis. A role for ARHGEF26 on inflammation was also seen in cells, as knockdown reduced IL-8 production in HeLa cells. Together, these data reveal pleiotropic roles for ARHGEF26 during infection and highlight that many of the interactions that occur during infection that are thought to be well understood likely have underappreciated complexity.

The 'invisible hand': regulation of RHO GTPases by RHOGDIs.

The 'invisible hand' is a term originally coined by Adam Smith in The Theory of Moral Sentiments to describe the forces of self-interest, competition and supply and demand that regulate the resources in society. This metaphor continues to be used by economists to describe the self-regulating nature of a market economy. The same metaphor can be used to describe the RHO-specific guanine nucleotide dissociation inhibitor (RHOGDI) family, which operates in the background, as an invisible hand, using similar forces to regulate the RHO GTPase cycle.

Evaluation of active Rac1 levels in cancer cells: A case of misleading conclusions from immunofluorescence analysis.

A large number of aggressive cancer cell lines display elevated levels of activated Rac1, a small GTPase widely implicated in cytoskeleton reorganization, cell motility, and metastatic dissemination. A commonly accepted methodological approach for detecting Rac1 activation in cancer cells involves the use of a conformation-sensitive antibody that detects the active (GTP-bound) Rac1 without interacting with the GDP-bound inactive form. This antibody has been extensively used in fixed cell immunofluorescence and immunohistochemistry. Taking advantage of prostate and pancreatic cancer cell models known to have high basal Rac1-GTP levels, here we have established that this antibody does not recognize Rac1 but rather detects the intermediate filament protein vimentin. Indeed, Rac1-null PC3 prostate cancer cells or cancer models with low levels of Rac1 activation still show a high signal with the anti-Rac1-GTP antibody, which is lost upon silencing of vimentin expression. Moreover, this antibody was unable to detect activated Rac1 in membrane ruffles induced by epidermal growth factor stimulation. These results have profound implications for the study of this key GTPase in cancer, particularly because a large number of cancer cell lines with characteristic mesenchymal features show simultaneous up-regulation of vimentin and high basal Rac1-GTP levels when measured biochemically. This misleading correlation can lead to assumptions about the validity of this antibody and inaccurate conclusions that may affect the development of appropriate therapeutic approaches for targeting the Rac1 pathway.

Syndecan-4/PAR-3 signaling regulates focal adhesion dynamics in mesenchymal cells.

BACKGROUND: Syndecans regulate cell migration thus having key roles in scarring and wound healing processes. Our previous results have shown that Thy-1/CD90 can engage both αvß3 integrin and Syndecan-4 expressed on the surface of astrocytes to induce cell migration. Despite a well-described role of Syndecan-4 during cell movement, information is scarce regarding specific Syndecan-4 partners involved in Thy-1/CD90-stimulated cell migration. METHODS: Mass spectrometry (MS) analysis of complexes precipitated with the Syndecan-4 cytoplasmic tail peptide was used to identify potential Syndecan-4-binding partners. The interactions found by MS were validated by immunoprecipitation and proximity ligation assays. The conducted research employed an array of genetic, biochemical and pharmacological approaches, including: PAR-3, Syndecan-4 and Tiam1 silencing, active Rac1 GEFs affinity precipitation, and video microscopy. RESULTS: We identified PAR-3 as a Syndecan-4-binding protein. Its interaction depended on the carboxy-terminal EFYA sequence present on Syndecan-4. In astrocytes where PAR-3 expression was reduced, Thy-1-induced cell migration and focal adhesion disassembly was impaired. This effect was associated with a sustained Focal Adhesion Kinase activation in the siRNA-PAR-3 treated cells. Our data also show that Thy-1/CD90 activates Tiam1, a PAR-3 effector. Additionally, we found that after Syndecan-4 silencing, Tiam1 activation was decreased and it was no longer recruited to the membrane. Syndecan-4/PAR-3 interaction and the alteration in focal adhesion dynamics were validated in mouse embryonic fibroblast (MEF) cells, thereby identifying this novel Syndecan-4/PAR-3 signaling complex as a general mechanism for mesenchymal cell migration involved in Thy-1/CD90 stimulation. CONCLUSIONS: The newly identified Syndecan-4/PAR-3 signaling complex participates in Thy-1/CD90-induced focal adhesion disassembly in mesenchymal cells. The mechanism involves focal adhesion kinase dephosphorylation and Tiam1 activation downstream of Syndecan-4/PAR-3 signaling complex formation. Additionally, PAR-3 is defined here as a novel adhesome-associated component with an essential role in focal adhesion disassembly during polarized cell migration. These novel findings uncover signaling mechanisms regulating cell migration, thereby opening up new avenues for future research on Syndecan-4/PAR-3 signaling in processes such as wound healing and scarring.

A RhoG-mediated signaling pathway that modulates invadopodia dynamics in breast cancer cells.

One of the hallmarks of cancer is the ability of tumor cells to invade surrounding tissues and metastasize. During metastasis, cancer cells degrade the extracellular matrix, which acts as a physical barrier, by developing specialized actin-rich membrane protrusion structures called invadopodia. The formation of invadopodia is regulated by Rho GTPases, a family of proteins that regulates the actin cytoskeleton. Here, we describe a novel role for RhoG in the regulation of invadopodia disassembly in human breast cancer cells. Our results show that RhoG and Rac1 have independent and opposite roles in the regulation of invadopodia dynamics. We also show that SGEF (also known as ARHGEF26) is the exchange factor responsible for the activation of RhoG during invadopodia disassembly. When the expression of either RhoG or SGEF is silenced, invadopodia are more stable and have a longer lifetime than in control cells. Our findings also demonstrate that RhoG and SGEF modulate the phosphorylation of paxillin, which plays a key role during invadopodia disassembly. In summary, we have identified a novel signaling pathway involving SGEF, RhoG and paxillin phosphorylation, which functions in the regulation of invadopodia disassembly in breast cancer cells.

mDia2 and CXCL12/CXCR4 chemokine signaling intersect to drive tumor cell amoeboid morphological transitions.

Morphological plasticity in response to environmental cues in migrating cancer cells requires F-actin cytoskeletal rearrangements. Conserved formin family proteins play critical roles in cell shape, tumor cell motility, invasion and metastasis, in part, through assembly of non-branched actin filaments. Diaphanous-related formin-2 (mDia2/Diaph3/Drf3/Dia) regulates mesenchymal-to-amoeboid morphological conversions and non-apoptotic blebbing in tumor cells by interacting with its inhibitor diaphanous-interacting protein (DIP), and disrupting cortical F-actin assembly and bundling. F-actin disruption is initiated by a CXCL12-dependent mechanism. Downstream CXCL12 signaling partners inducing mDia2-dependent amoeboid conversions remain enigmatic. We found in MDA-MB-231 tumor cells CXCL12 induces DIP and mDia2 interaction in blebs, and engages its receptor CXCR4 to induce RhoA-dependent blebbing. mDia2 and CXCR4 associate in blebs upon CXCL12 stimulation. Both CXCR4 and RhoA are required for CXCL12-induced blebbing. Neither CXCR7 nor other Rho GTPases that activate mDia2 are required for CXCL12-induced blebbing. The Rho Guanine Nucleotide Exchange Factor (GEF) Net1 is required for CXCL12-driven RhoA activation and subsequent blebbing. These results reveal CXCL12 signaling, through CXCR4, directs a Net1/RhoA/mDia-dependent signaling hub to drive cytoskeleton rearrangements to regulate morphological plasticity in tumor cells. These signaling hubs may be conserved during normal and cancer cells responding to chemotactic cues.

RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells.

The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin ß1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene.

VAV3 mediates resistance to breast cancer endocrine therapy.

INTRODUCTION: Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process. METHODS: A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression. RESULTS: The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy. CONCLUSIONS: This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

Simvastatin inhibits secretion of Th17-polarizing cytokines and antigen presentation by DCs in patients with relapsing remitting multiple sclerosis.

Statins, widely used cholesterol-lowering agents, have also been demonstrated to have antiinflammatory effects. Here, we characterize the capacity of simvastatin to target DCs and modulate T-cell priming and Th17-cell differentiation, in a cohort of patients with relapsing remitting multiple sclerosis (RRMS). We report that simvastatin inhibits IL-1ß, IL-23, TGF-ß, IL-21, IL-12p70, and induces IL-27 secretion from DCs in RRMS patients, providing an inhibitory cytokine milieu for Th17 and Th1-cell differentiation. The effect on DCs is mediated via induction of SOCS1, SOCS3, and SOCS7 gene expression, which are associated with the inhibition of STAT1, STAT3, and ERK1/2 phosphorylation. A geranylgeranyl transferase inhibitor replicated simvastatin's effects on DC cytokine secretion, implicating that simvastatin-induced depletion of isoprenoids mediates this effect. Simvastatin inhibited antigen presentation by DCs via suppression of the MHC class I expression, costimulatory molecules CD80 and CD40, and by inducing a dramatic loss of dendritic processes. The changes in DC morphology were also mediated via inhibition of geranylgeranylation. The therapeutic use of geranylgeranylation inhibitors may provide selective inhibition of key pathogenic cytokines that drive the autoimmune response in MS; their use represents a promising therapeutic approach that requires further clinical testing.

The invasive capacity of HPV transformed cells requires the hDlg-dependent enhancement of SGEF/RhoG activity.

A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity.

The Scribble/SGEF/Dlg1 complex regulates the stability of apical junctions in epithelial cells.

SGEF, a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of the expression of both E-cadherin and ZO-1. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble/SGEF/Dlg1 complex. Our results show that an intact ternary complex is required to maintain the stability of the apical junctions, the expression of ZO-1, and TJ permeability. In contrast, only SGEF is necessary to regulate E-cadherin expression. The absence of SGEF destabilizes the E-cadherin/catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, ß-catenin signaling and the transcriptional repressor Slug.

Macrophage Mechano-Responsiveness Within Three-Dimensional Tissue Matrix upon Mechanotherapy-Associated Strains.

Mechano-rehabilitation, also known as mechanotherapy, represents the forefront of noninvasive treatment for musculoskeletal (MSK) tissue disorders, encompassing conditions affecting tendons, cartilage, ligaments, and muscles. Recent emphasis has underscored the significance of macrophage presence in the healing of MSK tissues. However, a considerable gap still exists in comprehending how mechanical strains associated with mechanotherapy impact both the naïve and pro-inflammatory macrophage phenotypes within the three-dimensional (3D) tissue matrix, as well as whether the shift in macrophage phenotype is contingent on the mechanical strains inherent to mechanotherapy. In this study, we delineated alterations in mechano-adaptation and polarization of both naive and M1 macrophages within 3D matrices, elucidating their response to varying degrees of mechanical strain exposure (3%, 6%, and 12%). To evaluate macrophage mechano-adaptation and mechano-sensitivity within 3D collagen matrices under mechanical loading, we employed structural techniques (scanning electron microscopy, histology), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time polymerase chain reaction. Our data reveal that the response of macrophages to mechanical loading is not only contingent on their specific sub-phenotype but also varies with the amplitude of mechanical strain. Notably, although supra-mechanical loading (12% strain) was requisite to induce a phenotypic shift in naive (M0) macrophages, as little as 3% mechanical strain proved sufficient to prompt phenotypic alterations in pro-inflammatory (M1) macrophages. These findings pave the way for leveraging the macrophage mechanome in customized and targeted applications of mechanical strain within the mechano-therapeutic framework. Considering the prevalence of MSK tissue injuries and their profound societal and economic implications, the development of well-informed and effective clinical mechanotherapy modalities for MSK tissue healing becomes an imperative endeavor. Impact statement Mechanotherapy is a primary noninvasive treatment for musculoskeletal (MSK) tissue injuries, but the effect of mechanical strain on macrophage phenotypes is not fully understood. A recent study found that macrophage response to mechanical loading is both sub-phenotype specific and amplitude-dependent, with even small strains enough to induce phenotypic changes in pro-inflammatory macrophages. These findings could pave the way for using macrophage mechanome in targeted mechanotherapy applications for better MSK tissue healing.

Modulating TRPV4 Channel Activity in Pro-Inflammatory Macrophages within the 3D Tissue Analog.

Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca2+ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca2+ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(-)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson's trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(-) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues.

A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca 2+ which are disrupted when Ca 2+ influx through L-type channels is blocked or internal Ca 2+ stores are depleted. PACAP liberates stored Ca 2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca 2+ mobilization to Ca 2+ influx and supporting Ca 2+ -induced Ca 2+ -release. These Ca 2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ , is regulated by a signaling network that promotes sustained elevations in intracellular Ca 2+ through multiple pathways.

A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases.

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.

Deciphering the Cell-Specific Effect of Osteoblast-Macrophage Crosstalk in Periodontitis.

In periodontitis, the bone remodeling process is disrupted by the prevalent involvement of bacteria-induced proinflammatory macrophage cells and their interaction with osteoblast cells residing within the infected bone tissue. The complex interaction between the cells needs to be deciphered to understand the dominant player in tipping the balance from osteogenesis to osteoclastogenesis. Yet, only a few studies have examined the crosstalk interaction between osteoblasts and macrophages using biomimetic three-dimensional (3D) tissue-like matrices. In this study, we created a cell-laden 3D tissue analog to study indirect crosstalk between these two cell types and their direct synergistic effect when cultured on a 3D scaffold. The cell-specific role of osteoclast differentiation was investigated through osteoblast- and proinflammatory macrophage-specific feedback studies. The results suggested that when macrophages were exposed to osteoblasts-derived conditioned media from the mineralized matrix, the M1 macrophages tended to maintain their proinflammatory phenotype. Further, when osteoblasts were exposed to secretions from proinflammatory macrophages, they demonstrated elevated receptor activator of nuclear factor-κB ligand (RANKL) expression and decreased alkaline phosphate (ALP) activities compared to osteoblasts exposed to only osteogenic media. In addition, the upregulation of tumor necrosis factor-alpha (TNF-α) and c-Fos in proinflammatory macrophages within the 3D matrix indirectly increased the RANKL expression and reduced the ALP activity of osteoblasts, promoting osteoclastogenesis. The contact coculturing with osteoblast and proinflammatory macrophages within the 3D matrix demonstrated that the proinflammatory markers (TNF-α and interleukin-1ß) expressions were upregulated. In contrast, anti-inflammatory markers (c-c motif chemokine ligand 18 [CCL18]) were downregulated, and osteoclastogenic markers (TNF receptor associated factor 6 [TRAF6] and acid phosphatase 5, tartrate resistant [ACP5]) were unchanged. The data suggested that the osteoblasts curbed the osteoclastogenic differentiation of macrophages while macrophages still preserved their proinflammatory lineages. The osteoblast within the 3D coculture demonstrated increased ALP activity and did not express RANKL significantly different than the osteoblast cultured within a 3D collagen matrix without macrophages. Contact coculturing has an anabolic effect on bone tissue in a bacteria-derived inflammatory environment.

ARHGAP17 regulates the spatiotemporal activity of Cdc42 at invadopodia.

Invadopodia formation is regulated by Rho GTPases. However, the molecular mechanisms that control Rho GTPase signaling at invadopodia remain poorly understood. Here, we have identified ARHGAP17, a Cdc42-specific RhoGAP, as a key regulator of invadopodia in breast cancer cells and characterized a novel ARHGAP17-mediated signaling pathway that controls the spatiotemporal activity of Cdc42 during invadopodia turnover. Our results show that during invadopodia assembly, ARHGAP17 localizes to the invadopodia ring and restricts the activity of Cdc42 to the invadopodia core, where it promotes invadopodia growth. Invadopodia disassembly starts when ARHGAP17 translocates from the invadopodia ring to the core, in a process that is mediated by its interaction with the Cdc42 effector CIP4. Once at the core, ARHGAP17 inactivates Cdc42 to promote invadopodia disassembly. Our results in invadopodia provide new insights into the coordinated transition between the activation and inactivation of Rho GTPases.