Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

The serine-threonine kinase Akt plays a fundamental role in cell survival, metabolism, proliferation, and migration. To keep these essential processes under control, Akt activity and stability must be tightly regulated; otherwise, life-threatening conditions might prevail. Although it is well understood that phosphorylation regulates Akt activity, much remains to be known about how its stability is maintained. Here, we characterize BAG5, a chaperone regulator, as a novel Akt-interactor and substrate that attenuates Akt stability together with Hsp70. BAG5 switches monoubiquitination to polyubiquitination of Akt and increases its degradation caused by Hsp90 inhibition and Hsp70 overexpression. Akt interacts with BAG5 at the linker region that joins the first and second BAG domains and phosphorylates the first BAG domain. The Akt-BAG5 complex is formed in serum-starved conditions and dissociates in response to HGF, coincident with BAG5 phosphorylation. BAG5 knockdown attenuated Akt degradation and facilitated its activation, whereas the opposite effect was caused by BAG5 overexpression. Altogether, our results indicate that Akt stability and signaling are dynamically regulated by BAG5, depending on growth factor availability.

Fibrillar Collagen Type I Participates in the Survival and Aggregation of Primary Hepatocytes Cultured on Soft Hydrogels.

Liver is an essential organ that carries out multiple functions such as glycogen storage, the synthesis of plasma proteins, and the detoxification of xenobiotics. Hepatocytes are the parenchyma that sustain almost all the functions supported by this organ. Hepatocytes and non-parenchymal cells respond to the mechanical alterations that occur in the extracellular matrix (ECM) caused by organogenesis and regenerating processes. Rearrangements of the ECM modify the composition and mechanical properties that result in specific dedifferentiation programs inside the hepatic cells. Quiescent hepatocytes are embedded in the soft ECM, which contains an important concentration of fibrillar collagens in combination with a basement membrane-associated matrix (BM). This work aims to evaluate the role of fibrillar collagens and BM on actin cytoskeleton organization and the function of rat primary hepatocytes cultured on soft elastic polyacrylamide hydrogels (PAA HGs). We used rat tail collagen type I and Matrigel® as references of fibrillar collagens and BM respectively and mixed different percentages of collagen type I in combination with BM. We also used peptides obtained from decellularized liver matrices (dECM). Remarkably, hepatocytes showed a poor adhesion in the absence of collagen on soft PAA HGs. We demonstrated that collagen type I inhibited apoptosis and activated extracellular signal-regulated kinases 1/2 (ERK1/2) in primary hepatocytes cultured on soft hydrogels. Epidermal growth factor (EGF) was not able to rescue cell viability in conjugated BM but affected cell aggregation in soft PAA HGs conjugated with combinations of different proportions of collagen and BM. Interestingly, actin cytoskeleton was localized and preserved close to plasma membrane (cortical actin) and proximal to intercellular ducts (canaliculi-like structures) in soft conditions; however, albumin protein expression was not preserved, even though primary hepatocytes did not remodel their actin cytoskeleton significantly in soft conditions. This investigation highlights the important role of fibrillar collagens on soft hydrogels for the maintenance of survival and aggregation of the hepatocytes. Data suggest evaluating the conditions that allow the establishment of optimal biomimetic environments for physiology and cell biology studies, where the phenotype of primary cells may be preserved for longer periods of time.

Hepatic C9 cells switch their behaviour in short or long exposure to soft substrates.

BACKGROUND INFORMATION: There have been several studies to understand the influence of stiffness of the culture substrates for different types of adherent cells. It is generally accepted that cell proliferation, spreading and focal adhesions increase with higher matrix stiffness. However, what remains unclear is whether this kind of cell behaviour may be reverted by culturing on soft substrates those cell lines that were originally selected or primed on stiff surfaces. RESULTS: Here, we studied the influence of substrate softness on proliferation, adhesion and morphology of the highly proliferative hepatic C9 cell line. We cultured C9 cells on soft and stiff polydimethylsiloxane (PDMS) substrates prepared with two different elastic moduli in the range of 10 and 200 kPa, respectively. Lower cell proliferation was observed on substrates with lower stiffness without affecting cell viability. The proliferation rate of C9 cell line along with extracellular growth-regulated kinase (ERK) phosphorylation was decreased on soft PDMS. Despite this cell line has been described as a hepatic epithelial cell, our characterisation demonstrated that the origin of C9 cells is uncertain, although clearly epithelial, with the expression of markers of several hepatic cells. Surprisingly, consecutive passages of C9 cells on soft PDMS did not alter this mesenchymal phenotype, vimentin expression did not decrease when culturing cells in soft substrates, even though the ERK phosphorylation levels eventually were increased after several passages on soft PDMS, triggering again an increase of cell proliferation. CONCLUSIONS AND SIGNIFICANCE: This study shows that the exposure of C9 cells to soft substrates promoted a decrease of cell proliferation rate, as reported for other types of cells on PDMS, whereas a much longer term exposure caused cells to adapt to softness after trained for several passages, reactivating proliferation. During this phenomenon, the morphology and phenotype of trained cells was modified accompanying the increase of cell proliferation rate contrary to the effect observed in short periods of cell culture. In contrast to previous reports, cell death was not observed during these experiments, discarding a cell selection mechanism and suggesting soft cell adaptation may be limited in time in C9 cells.

GIPC proteins negatively modulate Plexind1 signaling during vascular development.

Semaphorins (SEMAs) and their Plexin (PLXN) receptors are central regulators of metazoan cellular communication. SEMA-PLXND1 signaling plays important roles in cardiovascular, nervous, and immune system development, and cancer biology. However, little is known about the molecular mechanisms that modulate SEMA-PLXND1 signaling. As PLXND1 associates with GIPC family endocytic adaptors, we evaluated the requirement for the molecular determinants of their association and PLXND1's vascular role. Zebrafish that endogenously express a Plxnd1 receptor with a predicted impairment in GIPC binding exhibit low penetrance angiogenesis deficits and antiangiogenic drug hypersensitivity. Moreover, gipc mutant fish show angiogenic impairments that are ameliorated by reducing Plxnd1 signaling. Finally, GIPC depletion potentiates SEMA-PLXND1 signaling in cultured endothelial cells. These findings expand the vascular roles of GIPCs beyond those of the Vascular Endothelial Growth Factor (VEGF)-dependent, proangiogenic GIPC1-Neuropilin 1 complex, recasting GIPCs as negative modulators of antiangiogenic PLXND1 signaling and suggest that PLXND1 trafficking shapes vascular development.

Reck enables cerebrovascular development by promoting canonical Wnt signaling.

The cerebral vasculature provides the massive blood supply that the brain needs to grow and survive. By acquiring distinctive cellular and molecular characteristics it becomes the blood-brain barrier (BBB), a selectively permeable and protective interface between the brain and the peripheral circulation that maintains the extracellular milieu permissive for neuronal activity. Accordingly, there is great interest in uncovering the mechanisms that modulate the formation and differentiation of the brain vasculature. By performing a forward genetic screen in zebrafish we isolated no food for thought (nft (y72)), a recessive late-lethal mutant that lacks most of the intracerebral central arteries (CtAs), but not other brain blood vessels. We found that the cerebral vascularization deficit of nft (y72) mutants is caused by an inactivating lesion in reversion-inducing cysteine-rich protein with Kazal motifs [reck; also known as suppressor of tumorigenicity 15 protein (ST15)], which encodes a membrane-anchored tumor suppressor glycoprotein. Our findings highlight Reck as a novel and pivotal modulator of the canonical Wnt signaling pathway that acts in endothelial cells to enable intracerebral vascularization and proper expression of molecular markers associated with BBB formation. Additional studies with cultured endothelial cells suggest that, in other contexts, Reck impacts vascular biology via the vascular endothelial growth factor (VEGF) cascade. Together, our findings have broad implications for both vascular and cancer biology.

Chemotactic and proangiogenic role of calcium sensing receptor is linked to secretion of multiple cytokines and growth factors in breast cancer MDA-MB-231 cells.

Breast cancer metastasis to the bone, potentially facilitated by chemotactic and angiogenic cytokines, contributes to a dramatic osteolytic effect associated with this invasive behavior. Based on the intrinsic ability of calcium sensing receptor (CaSR) to control hormonal secretion and considering its expression in the breast, we hypothesized that CaSR plays a chemotactic and proangiogenic role in highly invasive MDA-MB-231 breast cancer cells by promoting secretion of multiple cytokines. In this study, we show that MDA-MB-231 cells stimulated with R-568 calcimimetic and extracellular calcium secreted multiple cytokines and growth factors that induced endothelial cell migration and in vitro angiogenesis. These effects were dependent on the activity of CaSR as demonstrated by the inhibitory effect of either anti-CaSR blocking monoclonal antibodies or calcilytic NPS-2143. Moreover, CaSR knockdown prevented the proangiogenic effect of CaSR agonists. Importantly, CaSR promoted secretion of pleiotropic molecules like GM-CSF, EGF, MDC/CCL22, FGF-4 and IGFBP2, all known to be chemotactic mediators with putative angiogenic factor properties. In contrast, constitutive secretion of IL-6 and ß-NGF was attenuated by CaSR. In the case of normal mammary cells, secretion of IL-6 was stimulated by CaSR, whereas a constitutive secretion of RANTES, Angiogenin and Oncostatin M was attenuated by this receptor. Taken together, our results indicate that an altered secretion of chemotactic and proangiogenic cytokines in breast cancer cells is modulated by CaSR, which can be considered a potential target in the therapy of metastatic breast cancer.

Calcium-sensing receptor inhibits TGF-ß-signaling by decreasing Smad2 phosphorylation.

Calcium-sensing receptor (CaSR) contributes to maintain homeostatic levels of extracellular calcium. In addition, CaSR controls other cellular activities such as proliferation and migration, particularly in cells not related to extracellular calcium homeostasis, potentially by cross-talking with parallel signaling pathways. Here we report that CaSR attenuates transforming growth factor-ß (TGF-ß)-signaling in hepatic C9 cells and in transfected HEK293 cells. Wild type CaSR interferes with TGF-ß-dependent Smad2 phosphorylation and induces its proteasomal degradation, resulting in a decrease of TGF-ß-dependent transcriptional activity, whereas an inactivating CaSR mutant does not transduce an inhibitory effect of extracellular calcium on TGF-ß signaling. Attenuation of TGF-ß signaling in response to extracellular calcium is linked to Rab11-dependent CaSR-trafficking with the intervention of CaSR carboxyl-terminal tail. Our data suggest that CaSR might regulate TGF-ß-dependent cellular responses mediated by TGF-ß signaling inhibition.

Interferon gamma induces actin polymerization, Rac1 activation and down regulates phagocytosis in human monocytic cells.

IFNγ is a potent activator and IL-10 a powerful inhibitor of macrophage functions. However, neither all cellular functions are enhanced by IFNγ nor IL-10 inhibits all cellular responses. Thus, FcγRs-mediated phagocytosis in monocyte-derived macrophages (MDM) increases after IL-10 treatment, and decreases after treatment with IFNγ, although both IL-10 and IFNγ up regulate FcγRI expression. In this work we investigated the effect of IFNγ and IL-10 on phagocytic signaling by FcγRs in MDM. Treatment with IFNγ diminished phagocytosis of IgG-opsonized SRBC (IgG-SRBC) while treatment with IL-10 increased it. These opposite effects cannot be attributed to changes in FcγR expression induced by each cytokine. Early biochemical responses mediated by FcγRs were distinctly affected by cytokine treatment. Syk phosphorylation and the rise in [Ca(2+)](i) were higher after IL-10 treatment, whereas IFNγ treatment also increased Syk phosphorylation but had no effect on the rise in [Ca(2+)](i). IFNγ treatment led to increased basal levels of F-actin and this effect correlated with the decrease in phagocytosis of both IgG-SRBC and non-opsonized Escherichia coli. IL-10 did not alter F-actin basal levels, and enhanced the phagocytosis of E. coli and IgG-SRBC. The level of F-actin reached after IFNγ treatment was not further increased after stimulation with IgG-SRBC or CCL5, whereas MDM treated with IL-10 showed a slightly higher response than control cells to CCL5. IFNγ increased Rac1-GTP levels. Inhibition of PI3K with LY294002 prevented IFNγ-mediated actin polymerization. Our data suggest that IFNγ induces a higher basal level of F-actin and activation of Rac1, affecting the response to stimuli that induce cytoskeleton rearrangement such as phagocytic or chemotactic stimuli.

Regulation of mTORC1 complex assembly and signaling by GRp58/ERp57.

The mammalian target of rapamycin (mTOR) regulates cell growth and survival via two different multiprotein complexes, mTORC1 and mTORC2. The assembly of these serine-threonine kinase multiprotein complexes occurs via poorly understood molecular mechanisms. Here, we demonstrate that GRp58/ERp57 regulates the existence and activity of mTORC1. Endogenous mTOR interacts with GRp58/ERp57 in different mammalian cells. In vitro, recombinant GRp58/ERp57 preferentially interacts with mTORC1. GRp58/ERp57 knockdown reduces mTORC1 levels and phosphorylation of 4E-BP1 and p70(S6K) in response to insulin. In contrast, GRp58/ERp57 overexpression increases mTORC1 levels and activity. A redox-sensitive mechanism that depends on GRp58/ERp57 expression activates mTORC1. Although GRp58/ERp57 is known as an endoplasmic reticulum (ER) resident, we demonstrate its presence at the cytosol, together with mTOR, Raptor, and Rictor as well as a pool of these proteins associated to the ER. In addition, the presence of GRp58/ERp57 at the ER decreases in response to insulin or leucine. Interestingly, a fraction of p70(S6K), but not 4E-BP1, is associated to the ER and phosphorylated in response to serum, insulin, or leucine. Altogether, our results suggest that GRp58/ERp57 is involved in the assembly of mTORC1 and positively regulates mTORC1 signaling at the cytosol and the cytosolic side of the ER.

Phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex-1), a guanine nucleotide exchange factor for Rac, mediates angiogenic responses to stromal cell-derived factor-1/chemokine stromal cell derived factor-1 (SDF-1/CXCL-12) linked to Rac activation, endothelial cell migration, and in vitro angiogenesis.

Stromal cell-derived factor-1 (SDF-1/CXCL-12) and vascular endothelial growth factor (VEGF), which can be secreted by hypoxic tumors, promote the generation of new blood vessels. These potent angiogenic factors stimulate endothelial cell migration via the activation of Rho GTPases and the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. Thus, characterization of guanine nucleotide exchange factors critical in the angiogenic signaling cascades offers the possibility of identifying novel molecular targets. We demonstrated previously that mammalian target of rapamycin, an important effector and regulator of PI3K/AKT, activates phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex1), a Rac guanine nucleotide exchange factor identified as a target of G betagamma and PI3K, via direct interactions. In this study, we tested the hypothesis that P-Rex1 is involved in the angiogenic responses elicited by SDF-1 and VEGF. Using a knockdown approach, we demonstrate that P-Rex1 is indeed required for SDF-1 promoted signaling pathway, because there is decreased Rac activation, cell migration, and in vitro angiogenesis in P-Rex1 knockdown cells stimulated with SDF-1. In contrast, P-Rex1 knockdown does not affect responses to VEGF, and signaling to extracellular signal-regulated kinase in response to either angiogenic factor is not sensitive to P-Rex1 knockdown. We also demonstrate that in endothelial cells, VEGF promotes an increase in the expression of endogenous P-Rex1 and the SDF-1 receptor CXCR4, In addition, VEGF-pretreated cells show an increased migratory and angiogenic response to SDF-1, suggesting that VEGF stimulation can complement SDF-1/CXCR4 signaling to induce angiogenesis. We conclude that P-Rex1 is a key element in SDF-1-induced angiogenic responses and a potential target for therapeutic intervention.

Differential inhibitor of Gbetagamma signaling to AKT and ERK derived from phosducin-like protein: effect on sphingosine 1-phosphate-induced endothelial cell migration and in vitro angiogenesis.

Differential inhibitors of Gbetagamma-effector regions are required to dissect the biological contribution of specific Gbetagamma-initiated signaling pathways. Here, we characterize PhLP-M1-G149, a Gbetagamma-interacting construct derived from phosducin-like protein 1 (PhLP) as a differential inhibitor of Gbetagamma, which, in endothelial cells, prevented sphingosine 1-phosphate-induced phosphorylation of AKT, glycogen synthase kinase 3beta, cell migration, and tubulogenesis, while having no effect on ERK phosphorylation or hepatocyte growth factor-dependent responses. This construct attenuated the recruitment of phosphoinositide 3-kinase gamma (PI3Kgamma) to the plasma membrane and the signaling to AKT in response to Gbetagamma overexpression. In coimmunoprecipitation experiments, PhLP-M1-G149 interfered with the interaction between PI3Kgamma and Gbetagamma. Other PhLP-derived constructs interacted with Gbetagamma but were not effective inhibitors of Gbetagamma signaling to AKT or ERK. Our results indicate that PhLP-M1-G149 is a suitable tool to differentially modulate the Gbetagamma-initiated pathway linking this heterodimer to AKT, endothelial cell migration, and in vitro angiogenesis. It can be also useful to further characterize the molecular determinants of the Gbetagamma-PI3Kgamma interaction.

P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration.

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.